WO2021029777A1 - Novel assays - Google Patents

Novel assays Download PDF

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Publication number
WO2021029777A1
WO2021029777A1 PCT/NZ2020/050089 NZ2020050089W WO2021029777A1 WO 2021029777 A1 WO2021029777 A1 WO 2021029777A1 NZ 2020050089 W NZ2020050089 W NZ 2020050089W WO 2021029777 A1 WO2021029777 A1 WO 2021029777A1
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salt
polynucleotide
seq
sequence
present
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PCT/NZ2020/050089
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French (fr)
Inventor
Shalen KUMAR
Kelly CAMPEN
Jennifer Pamela SOUNDY
Shiwei LI
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Auramer Bio Limited
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Publication of WO2021029777A1 publication Critical patent/WO2021029777A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/115—Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith ; Nucleic acids binding to non-nucleic acids, e.g. aptamers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
    • G01N33/948—Sedatives, e.g. cannabinoids, barbiturates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6804—Nucleic acid analysis using immunogens
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
    • G01N33/946—CNS-stimulants, e.g. cocaine, amphetamines
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
    • G01N33/9486—Analgesics, e.g. opiates, aspirine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/16—Aptamers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2320/00—Applications; Uses
    • C12N2320/10—Applications; Uses in screening processes

Definitions

  • the present invention is concerned with the detection of illicit and prescription drugs including, for example, methamphetamine, synthetic/cannabis, cocaine, ecstasy, morphine, oxycodone, benzodiazepines and ketamine.
  • the present invention provides novel polynucleotide aptamers, which aptamers fold and selectively bind to a molecule/drug target of interest, as well as test kits, and assay methods for the detection of illicit and prescription drugs in a test sample for use (e.g.) in road-side testing or in work-place monitoring.
  • RNA or DNA single-stranded polynucleotides
  • Aptamers are single-stranded polynucleotides (RNA or DNA) that possess the ability to bind targets with high affinity and specificity (Tuerk, C., and L. Gold. Science, 1990, 249(4968), 505-10; Ellington, A. D. & Szostak, J. W. Nature, 1990, 346, 818-822).
  • Aptamers are developed in vitro using a process known as systematic evolution of ligands by exponential enrichment (SELEX).
  • Aptamers are selected from a random oligonucleotide library based on their ability to bind a specific target over multiple selection rounds. Aptamers have several advantages over antibodies. First, aptamers can be selected against any target, even those that are toxic or that have low immunogenicity. Second, aptamers are generally robust to temperature and can be synthesized at high volumes with little batch-to-batch variation. Third, the biophysical characteristics of aptamer-target binding offers greater flexibility in biosensor design (Song, S. et al. Trends in Analytical Chemistry, 2008, 27(2), 108-17).
  • a polynucleotide or salt thereof consisting in the sequence AT ACGAGCTTGTTCAATA[X] n TGAT AGT AAGAGCAATC (SEQ ID NO: 565), where X is selected from A, T, U, G or C and n is an integer of between about 10 and about 120, in particular between about 35 and about 45.
  • a polynucleotide or salt thereof comprising the sequence ATACGAGCTTGTTCAATA[X] n TGATAGTAAGAGCAATC (SEQ ID NO: 565), where X is selected from A, T, U, G or C and n is an integer of between about 10 and about 120, in particular between about 35 and about 45, which polynuceotide or salt thereof selectively binds to methamphetamine.
  • a polynucleotide or salt thereof comprising the sequence AT ACGAGCTTGTTCAATA[Y]TGAT AGT AAGAGCAATC (SEQ ID NO: 566), where Y is any sequence comprising a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to any one of SEQ ID Nos: 2, 4, 6, 8, 10, 12,
  • polynuceotide or salt thereof selectively binds to methamphetamine.
  • a polynucleotide or salt thereof comprising the sequence AT ACGAGCTTGTTCAATA[Y]TGAT AGT AAGAGCAATC (SEQ ID NO: 567), where Y is any sequence comprising any one of SEQ ID Nos: 2, 4, 6, 8, 10, 12, 14,
  • a polynucleotide or salt thereof comprising the sequence ATACGAGCTTGTTCAATA[Y]TGATAGTAAGAGCAATC (SEQ ID NO: 566), where Y is any sequence consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to any one of SEQ ID Nos: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72 or 74, which polynuceotide or salt thereof selectively binds to methamphetamine.
  • a polynucleotide or salt thereof comprising the sequence ATACGAGCTTGTTCAATA[Y]TGATAGTAAGAGCAATC (SEQ ID NO: 567), where Y is any sequence consisting in a sequence of any one of SEQ ID Nos: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72 or 74, which polynuceotide or salt thereof selectively binds to methamphetamine.
  • a polynucleotide or salt threreof comprising a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to any one of SEQ ID Nos: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71 and 73, which polynucleotide of salt thereof selectively binds to methamphetamine.
  • polynucleotide or salt threreof comprising a sequence of any one of SEQ ID Nos: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19,
  • polynucleotide or salt thereof consisting in a sequence of any one of SEQ ID Nos: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19,
  • a method for detecting the presence of methamphetamine in a test sample comprising the steps of:
  • test kit or article of manufacture for detecting the presence of methamphetamine in a test sample, the test kit or article of manufacture comprising a polynucleotide or salt thereof comprising a sequence defined by any one of SEQ ID Nos: 1-74 together with instructions for how to detect the presence of methamphetamine in the test sample.
  • a polynucleotide or salt thereof comprising the sequence ATACGAGCTTGTTCAATA[X] n TGATAGTAAGAGCAATC (SEQ ID NO: 568), where X is selected from A, T, U, G or C and n is an integer of between about 10 and about 120, in particular between about 35 and about 45, which polynuceotide or salt thereof selectively binds to tetrahydrocannabinol.
  • a polynucleotide or salt thereof comprising the sequence ATACGAGCTTGTTCAATA[Y]TGATAGTAAGAGCAATC (SEQ ID NO: 569), where Y is any sequence comprising a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to any one of SEQ ID Nos: 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134 and 136, which polynuceotide or salt thereof selectively binds to tetrahydrocannabinol.
  • a polynucleotide or salt thereof comprising the sequence ATACGAGCTTGTTCAATA[Y]TGATAGTAAGAGCAATC (SEQ ID NO: 570), where Y is any sequence comprising any one of SEQ ID Nos: 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134 and 136, which polynuceotide or salt thereof selectively binds to tetrahydrocannabinol.
  • a polynucleotide or salt thereof comprising the sequence ATACGAGCTTGTTCAATA[Y]TGATAGTAAGAGCAATC (SEQ ID NO: 569), where Y is any sequence consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to any one of SEQ ID Nos: 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134 and 136, which polynuceotide or salt thereof selectively binds to tetrahydrocannabinol.
  • a polynucleotide or salt thereof comprising the sequence ATACGAGCTTGTTCAATA[Y]TGATAGTAAGAGCAATC (SEQ ID NO: 570), where Y is any sequence consisting in a sequence of any one of SEQ ID Nos: 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134 and 136, which polynuceotide or salt thereof selectively binds to tetrahydrocannabinol.
  • a polynucleotide or salt threreof comprising a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to any one of SEQ ID Nos: 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 133 and 135, which polynucleotide of salt thereof selectively binds to tetrahydrocannabinol.
  • a polynucleotide or salt threreof comprising a sequence of any one of SEQ ID Nos: 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 133 and 135, which polynucleotide of salt thereof selectively binds to tetrahydrocannabinol.
  • a polynucleotide or salt thereof consisting in a sequence of any one of SEQ ID Nos: 75, 77, 79, 81, 83, 85, 87, 89,
  • a method for detecting the presence of tetrahydrocannabinol in a test sample comprising the steps of:
  • test kit or article of manufacture for detecting the presence of tetrahydrocannabinol in a test sample, the test kit or article of manufacture comprising a polynucleotide or salt thereof comprising a sequence defined by any one of SEQ ID Nos: 75-136 together with instructions for how to detect the presence of tetrahydrocannabinol in the test sample.
  • a polynucleotide or salt thereof comprising the sequence ATACGAGCTTGTTCAATA[X] n TGATAGTAAGAGCAATC (SEQ ID NO: 571), where X is selected from A, T, U, G or C and n is an integer of between about 10 and about 120, in particular between about 35 and about 45, which polynuceotide or salt thereof selectively binds to cocaine.
  • a polynucleotide or salt thereof comprising the sequence ATACGAGCTTGTTCAATA[Y]TGATAGTAAGAGCAATC (SEQ ID NO: 572), where Y is any sequence comprising a sequence that has at least 90, 91,
  • a polynucleotide or salt thereof comprising the sequence ATACGAGCTTGTTCAATA[Y]TGATAGTAAGAGCAATC (SEQ ID NO: 573), where Y is any sequence comprising any one of SEQ ID Nos: 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170 and 172, which polynuceotide or salt thereof selectively binds to cocaine.
  • a polynucleotide or salt thereof comprising the sequence ATACGAGCTTGTTCAATA[Y]TGATAGTAAGAGCAATC (SEQ ID NO: 572), where Y is any sequence consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to any one of SEQ ID Nos: 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170 and 172, which polynuceotide or salt thereof selectively binds to cocaine.
  • a polynucleotide or salt thereof comprising the sequence ATACGAGCTTGTTCAATA[Y]TGATAGTAAGAGCAATC (SEQ ID NO: 573), where Y is any sequence consisting in a sequence of any one of SEQ ID Nos: 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170 and 172, which polynuceotide or salt thereof selectively binds to cocaine.
  • a polynucleotide or salt threreof comprising a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to any one of SEQ ID Nos: 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169 and 171, which polynucleotide of salt thereof selectively binds to cocaine.
  • a polynucleotide or salt threreof comprising a sequence of any one of SEQ ID Nos: 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169 and 171, which polynucleotide of salt thereof selectively binds to cocaine.
  • a polynucleotide or salt thereof consisting in a sequence of any one of SEQ ID Nos: 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169 and 171, which polynucleotide of salt thereof selectively binds to cocaine.
  • test kit or article of manufacture for detecting the presence of cocaine in a test sample, the test kit or article of manufacture comprising a polynucleotide or salt thereof comprising a sequence defined by any one of SEQ ID Nos: 137-172 together with instructions for how to detect the presence of cocaine in the test sample.
  • a polynucleotide or salt thereof comprising the sequence ATACGAGCTTGTTCAATA[X] n TGATAGTAAGAGCAATC (SEQ ID NO: 574), where X is selected from A, T, U, G or C and n is an integer of between about 10 and about 120, in particular between about 35 and about 45, which polynuceotide or salt thereof selectively binds to 3,4-methylenedioxymethamphetamine.
  • a polynucleotide or salt thereof comprising the sequence ATACGAGCTTGTTCAATA[Y]TGATAGTAAGAGCAATC (SEQ ID NO: 575), where Y is any sequence comprising a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to any one of SEQ ID Nos: 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212,
  • polynuceotide or salt thereof selectively binds to 3,4-methylenedioxymethamphetamine.
  • a polynucleotide or salt thereof comprising the sequence ATACGAGCTTGTTCAATA[Y]TGATAGTAAGAGCAATC (SEQ ID NO: 576), where Y is any sequence comprising any one of SEQ ID Nos: 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212,
  • polynuceotide or salt thereof selectively binds to 3,4-methylenedioxymethamphetamine.
  • a polynucleotide or salt thereof comprising the sequence ATACGAGCTTGTTCAATA[Y]TGATAGTAAGAGCAATC (SEQ ID NO: 575), where Y is any sequence consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to any one of SEQ ID Nos: 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212,
  • polynuceotide or salt thereof selectively binds to 3,4-methylenedioxymethamphetamine.
  • a polynucleotide or salt thereof comprising the sequence ATACGAGCTTGTTCAATA[Y]TGATAGTAAGAGCAATC (SEQ ID NO: 576), where Y is any sequence consisting in a sequence of any one of SEQ ID Nos: 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206,
  • a polynucleotide or salt threreof comprising a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to any one of SEQ ID Nos: 173, 175, 177, 179, 181, 183, 185, 187,
  • a polynucleotide or salt threreof comprising a sequence of any one of SEQ ID Nos: 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243 and 245, which polynucleotide of salt thereof selectively binds to 3,4-methylenedioxymethamphetamine.
  • a polynucleotide or salt thereof consisting in a sequence of any one of SEQ ID Nos: 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243 and 245, which polynucleotide of salt thereof selectively binds to 3,4-methylenedioxymethamphetamine.
  • test kit or article of manufacture for detecting the presence of 3,4-methylenedioxymethamphetamine in a test sample
  • the test kit or article of manufacture comprising a polynucleotide or salt thereof comprising a sequence defined by any one of SEQ ID Nos: 173-246 together with instructions for how to detect the presence of 3,4-methylenedioxymethamphetamine in the test sample.
  • a polynucleotide or salt thereof comprising the sequence ATACGAGCTTGTTCAATA[X] n TGATAGTAAGAGCAATC (SEQ ID NO: 577), where X is selected from A, T, U, G or C and n is an integer of between about 10 and about 120, in particular between about 35 and about 45, which polynuceotide or salt thereof selectively binds to morphine.
  • a polynucleotide or salt thereof comprising the sequence ATACGAGCTTGTTCAATA[Y]TGATAGTAAGAGCAATC (SEQ ID NO: 578), where Y is any sequence comprising a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to any one of SEQ ID Nos: 249, 251, 253, 255, 257, 259, 261, 263, 264, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289 and 291, which polynuceotide or salt thereof selectively binds to morphine.
  • a polynucleotide or salt thereof comprising the sequence ATACGAGCTTGTTCAATA[Y]TGATAGTAAGAGCAATC (SEQ ID NO: 579), where Y is any sequence comprising any one of SEQ ID Nos: 249, 251, 253, 255, 257, 259, 261, 263, 264, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289 and 291, which polynuceotide or salt thereof selectively binds to morphine.
  • a polynucleotide or salt thereof comprising the sequence ATACGAGCTTGTTCAATA[Y]TGATAGTAAGAGCAATC (SEQ ID NO: 578), where Y is any sequence consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to any one of SEQ ID Nos: 249, 251, 253, 255,
  • a polynucleotide or salt thereof comprising the sequence ATACGAGCTTGTTCAATA[Y]TGATAGTAAGAGCAATC (SEQ ID NO: 579), where Y is any sequence consisting in a sequence of any one of SEQ ID Nos: 249, 251, 253, 255, 257, 259, 261, 263, 264, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289 and 291, which polynuceotide or salt thereof selectively binds to morphine.
  • a polynucleotide or salt threreof comprising a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to any one of SEQ ID Nos: 247, 248, 250, 252, 254, 256, 258, 260, 262, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288 and 290, which polynucleotide of salt thereof selectively binds to morphine.
  • polynucleotide or salt threreof comprising a sequence of any one of SEQ ID Nos: 247, 248, 250, 252, 254, 256,
  • polynucleotide or salt thereof consisting in a sequence of any one of SEQ ID Nos: 247, 248, 250, 252, 254, 256, 258, 260, 262, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288 and 290, which polynucleotide of salt thereof selectively binds to morphine.
  • a method for detecting the presence of morphine in a test sample comprising the steps of:
  • test kit or article of manufacture for detecting the presence of morphine in a test sample, the test kit or article of manufacture comprising a polynucleotide or salt thereof comprising a sequence defined by any one of SEQ ID Nos: 247-291 together with instructions for how to detect the presence of morphine in the test sample.
  • a polynucleotide or salt thereof comprising the sequence ATACGAGCTTGTTCAATA[X] n TGATAGTAAGAGCAATC (SEQ ID NO: 580), where X is selected from A, T, U, G or C and n is an integer of between about 10 and about 120, in particular between about 35 and about 45, which polynuceotide or salt thereof selectively binds to diazepam.
  • a polynucleotide or salt thereof comprising the sequence ATACGAGCTTGTTCAATA[Y]TGATAGTAAGAGCAATC (SEQ ID NO: 581), where Y is any sequence comprising a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to any one of SEQ ID Nos: 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317 and 319, which polynuceotide or salt thereof selectively binds to diazepam.
  • a polynucleotide or salt thereof comprising the sequence ATACGAGCTTGTTCAATA[Y]TGATAGTAAGAGCAATC (SEQ ID NO: 582), where Y is any sequence comprising any one of SEQ ID Nos: 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317 and 319, which polynuceotide or salt thereof selectively binds to diazepam.
  • a polynucleotide or salt thereof comprising the sequence ATACGAGCTTGTTCAATA[Y]TGATAGTAAGAGCAATC (SEQ ID NO: 581), where Y is any sequence consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to any one of SEQ ID Nos: 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317 and 319, which polynuceotide or salt thereof selectively binds to diazepam.
  • a polynucleotide or salt thereof comprising the sequence ATACGAGCTTGTTCAATA[Y]TGATAGTAAGAGCAATC (SEQ ID NO: 582), where Y is any sequence consisting in a sequence of any one of SEQ ID Nos: 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317 and 319, which polynuceotide or salt thereof selectively binds to diazepam.
  • a polynucleotide or salt threreof comprising a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to any one of SEQ ID Nos: 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316 and 318, which polynucleotide of salt thereof selectively binds to diazepam.
  • a polynucleotide or salt threreof comprising a sequence of any one of SEQ ID Nos: 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316 and 318, which polynucleotide of salt thereof selectively binds to diazepam.
  • a polynucleotide or salt thereof consisting in a sequence of any one of SEQ ID Nos: 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316 and 318, which polynucleotide of salt thereof selectively binds to diazepam.
  • a method for detecting the presence of diazepam in a test sample comprising the steps of:
  • test kit or article of manufacture for detecting the presence of diazepam in a test sample, the test kit or article of manufacture comprising a polynucleotide or salt thereof comprising a sequence defined by any one of SEQ ID Nos: 292-319 together with instructions for how to detect the presence of diazepam in the test sample.
  • a polynucleotide or salt thereof comprising the sequence ATACGAGCTTGTTCAATA[X] n TGATAGTAAGAGCAATC (SEQ ID NO: 583), where X is selected from A, T, U, G or C and n is an integer of between about 10 and about 120, in particular between about 35 and about 45, which polynuceotide or salt thereof selectively binds to AMB-FUBINACA.
  • a polynucleotide or salt thereof comprising the sequence ATACGAGCTTGTTCAATA[Y]TGATAGTAAGAGCAATC (SEQ ID NO: 584), where Y is any sequence comprising a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to any one of SEQ ID Nos: 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 355, 357, 359, 361, 363, 365, 367, 369 and 371, which polynuceotide or salt thereof selectively binds to AMB- FUBINACA.
  • a polynucleotide or salt thereof comprising the sequence ATACGAGCTTGTTCAATA[Y]TGATAGTAAGAGCAATC (SEQ ID NO: 585), where Y is any sequence comprising any one of SEQ ID Nos: 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 355, 357, 359, 361, 363, 365, 367, 369 and 371, which polynuceotide or salt thereof selectively binds to AMB- FUBINACA.
  • a polynucleotide or salt thereof comprising the sequence ATACGAGCTTGTTCAATA[Y]TGATAGTAAGAGCAATC (SEQ ID NO: 584), where Y is any sequence consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to any one of SEQ ID Nos: 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 355, 357, 359, 361, 363, 365, 367, 369 and 371, which polynuceotide or salt thereof selectively binds to AMB- FUBINACA.
  • a polynucleotide or salt thereof comprising the sequence ATACGAGCTTGTTCAATA[Y]TGATAGTAAGAGCAATC (SEQ ID NO: 585), where Y is any sequence consisting in a sequence of any one of SEQ ID Nos: 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 355, 357, 359, 361, 363, 365, 367, 369 and 371, which polynuceotide or salt thereof selectively binds to AMB-FUBINACA.
  • a polynucleotide or salt threreof comprising a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to any one of SEQ ID Nos: 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368 and 370, which polynucleotide of salt thereof selectively binds to AMB-FUBINACA.
  • a polynucleotide or salt threreof comprising a sequence of any one of SEQ ID Nos: 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368 and 370, which polynucleotide of salt thereof selectively binds to AMB-FUBINACA.
  • a polynucleotide or salt thereof consisting in a sequence of any one of SEQ ID Nos: 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368 and 370, which polynucleotide of salt thereof selectively binds to AMB-FUBINACA.
  • a method for detecting the presence of AMB-FUBINACA in a test sample comprising the steps of:
  • test kit or article of manufacture for detecting the presence of AMB-FUBINACA in a test sample
  • the test kit or article of manufacture comprising a polynucleotide or salt thereof comprising a sequence defined by any one of SEQ ID Nos: 320-371 together with instructions for how to detect the presence of AMB-FUBINACA in the test sample.
  • a polynucleotide or salt thereof comprising the sequence ATACGAGCTTGTTCAATA[X] n TGATAGTAAGAGCAATC (SEQ ID NO: 586), where X is selected from A, T, U, G or C and n is an integer of between about 10 and about 120, in particular between about 35 and about 45, which polynuceotide or salt thereof selectively binds to AM2201.
  • a polynucleotide or salt thereof comprising the sequence ATACGAGCTTGTTCAATA[Y]TGATAGTAAGAGCAATC (SEQ ID NO: 587), where Y is any sequence comprising a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to any one of SEQ ID Nos: 373, 375, 377, 379, 381, 383, 385, 387, 389, 391, 393, 395, 397, 399, 401, 403, 405, 407, 409, 411, 413, 415, 417 and 419, which polynuceotide or salt thereof selectively binds to AM2201.
  • a polynucleotide or salt thereof comprising the sequence ATACGAGCTTGTTCAATA[Y]TGATAGTAAGAGCAATC (SEQ ID NO: 588), where Y is any sequence comprising any one of SEQ ID Nos: 373, 375, 377, 379, 381, 383, 385, 387, 389, 391, 393, 395, 397, 399, 401, 403, 405, 407, 409, 411, 413, 415, 417 and 419, which polynuceotide or salt thereof selectively binds to AM2201.
  • a polynucleotide or salt thereof comprising the sequence ATACGAGCTTGTTCAATA[Y]TGATAGTAAGAGCAATC (SEQ ID NO: 587), where Y is any sequence consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to any one of SEQ ID Nos: 373, 375, 377, 379, 381, 383, 385, 387, 389, 391, 393, 395, 397, 399, 401, 403, 405, 407, 409, 411, 413, 415, 417 and 419, which polynuceotide or salt thereof selectively binds to AM2201.
  • a polynucleotide or salt thereof comprising the sequence ATACGAGCTTGTTCAATA[Y]TGATAGTAAGAGCAATC (SEQ ID NO: 588), where Y is any sequence consisting in a sequence of any one of SEQ ID Nos: 373, 375, 377, 379, 381, 383, 385, 387, 389, 391, 393, 395, 397, 399, 401, 403, 405, 407, 409, 411, 413, 415, 417 and 419, which polynuceotide or salt thereof selectively binds to AM2201.
  • a polynucleotide or salt threreof comprising a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to any one of SEQ ID Nos: 372, 374, 376, 378, 380, 382, 384, 386, 388, 390 ,392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416 and 418, which polynucleotide of salt thereof selectively binds to AM2201.
  • a polynucleotide or salt threreof comprising a sequence of any one of SEQ ID Nos: 372, 374, 376, 378, 380, 382, 384, 386, 388, 390 ,392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416 and 418, which polynucleotide of salt thereof selectively binds to AM2201.
  • a polynucleotide or salt thereof consisting in a sequence of any one of SEQ ID Nos: 372, 374, 376, 378, 380, 382, 384, 386, 388, 390 ,392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416 and 418 and 277, which polynucleotide of salt thereof selectively binds to AM2201.
  • a method for detecting the presence of AM2201 in a test sample comprising the steps of:
  • test kit or article of manufacture for detecting the presence of AM2201 in a test sample, the test kit or article of manufacture comprising a polynucleotide or salt thereof comprising a sequence defined by any one of SEQ ID Nos: 372-419 together with instructions for how to detect the presence of AM2201 in the test sample.
  • a polynucleotide or salt thereof comprising the sequence ATACGAGCTTGTTCAATA[X] n TGATAGTAAGAGCAATC (SEQ ID NO: 589), where X is selected from A, T, U, G or C and n is an integer of between about 10 and about 120, in particular between about 35 and about 45, which polynuceotide or salt thereof selectively binds to oxycodone.
  • a polynucleotide or salt thereof comprising the sequence ATACGAGCTTGTTCAATA[Y]TGATAGTAAGAGCAATC (SEQ ID NO: 590), where Y is any sequence comprising a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to any one of SEQ ID Nos: 421, 423, 425, 427, 429, 431, 433, 435, 437, 439, 441, 443, 445, 447, 449, 451, 453, 455, 457, 459, 461, 463, 465, which polynuceotide or salt thereof selectively binds to oxycodone.
  • a polynucleotide or salt thereof comprising the sequence ATACGAGCTTGTTCAATA[Y]TGATAGTAAGAGCAATC (SEQ ID NO: 591), where Y is any sequence comprising any one of SEQ ID Nos: 421, 423, 425, 427, 429, 431, 433, 435, 437, 439, 441, 443, 445, 447, 449, 451, 453, 455, 457, 459, 461, 463, 465, which polynuceotide or salt thereof selectively binds to oxycodone.
  • a polynucleotide or salt thereof comprising the sequence ATACGAGCTTGTTCAATA[Y]TGATAGTAAGAGCAATC (SEQ ID NO: 590), where Y is any sequence consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to any one of SEQ ID Nos: 421, 423, 425, 427, 429, 431, 433, 435, 437, 439, 441, 443, 445, 447, 449, 451, 453, 455, 457, 459, 461, 463, 465, which polynuceotide or salt thereof selectively binds to oxycodone.
  • a polynucleotide or salt thereof comprising the sequence ATACGAGCTTGTTCAATA[Y]TGATAGTAAGAGCAATC (SEQ ID NO: 591), where Y is any sequence consisting in a sequence of any one of SEQ ID Nos: 421, 423, 425, 427, 429, 431, 433, 435, 437, 439, 441, 443, 445, 447, 449, 451, 453, 455, 457, 459, 461, 463, 465, which polynuceotide or salt thereof selectively binds to oxycodone.
  • a polynucleotide or salt threreof comprising a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to any one of SEQ ID Nos: 420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440, 442, 444, 446, 448, 450, 452, 454, 456, 458, 460, 462, 464 and 466, which polynucleotide of salt thereof selectively binds to oxycodone.
  • a polynucleotide or salt threreof comprising a sequence of any one of SEQ ID Nos: 420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440, 442, 444, 446, 448, 450, 452, 454, 456, 458, 460, 462, 464 and 466, which polynucleotide of salt thereof selectively binds to oxycodone.
  • a polynucleotide or salt thereof consisting in a sequence of any one of SEQ ID Nos: 420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440, 442, 444, 446, 448, 450, 452, 454, 456, 458, 460, 462, 464 and 466, which polynucleotide of salt thereof selectively binds to oxycodone.
  • a method for detecting the presence of oxycodone in a test sample comprising the steps of:
  • test kit or article of manufacture for detecting the presence of oxycodone in a test sample, the test kit or article of manufacture comprising a polynucleotide or salt thereof comprising a sequence defined by any one of SEQ ID Nos: 420-467 together with instructions for how to detect the presence of oxycodone in the test sample.
  • a polynucleotide or salt thereof comprising the sequence ATACGAGCTTGTTCAATA[X] n TGATAGTAAGAGCAATC (SEQ ID NO: 592), where X is selected from A, T, U, G or C and n is an integer of between about 10 and about 120, in particular between about 35 and about 45, which polynuceotide or salt thereof selectively binds to ketamine.
  • a polynucleotide or salt thereof comprising the sequence ATACGAGCTTGTTCAATA[Y]TGATAGTAAGAGCAATC (SEQ ID NO: 593), where Y is any sequence comprising a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to any one of SEQ ID Nos: 469, 471, 473, 475, 477, 479, 481, 483, 485, 487, 489, 491, 493, 495, 497, 499, 501, 503, 505, 507, 509, 511, 513, 515, 517, 519, 521 and 523, which polynuceotide or salt thereof selectively binds to ketamine.
  • a polynucleotide or salt thereof comprising the sequence ATACGAGCTTGTTCAATA[Y]TGATAGTAAGAGCAATC (SEQ ID NO: 594), where Y is any sequence comprising any one of SEQ ID Nos: 469, 471, 473, 475, 477, 479, 481, 483, 485, 487, 489, 491, 493, 495, 497, 499, 501, 503, 505, 507, 509, 511, 513, 515, 517, 519, 521 and 523, which polynuceotide or salt thereof selectively binds to ketamine.
  • a polynucleotide or salt thereof comprising the sequence ATACGAGCTTGTTCAATA[Y]TGATAGTAAGAGCAATC (SEQ ID NO: 593), where Y is any sequence consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to any one of SEQ ID Nos: 469, 471, 473, 475, 477, 479, 481, 483, 485, 487, 489, 491, 493, 495, 497, 499, 501, 503, 505, 507, 509, 511, 513, 515, 517, 519, 521 and 523, which polynuceotide or salt thereof selectively binds to ketamine.
  • a polynucleotide or salt thereof comprising the sequence ATACGAGCTTGTTCAATA[Y]TGATAGTAAGAGCAATC (SEQ ID NO: 594), where Y is any sequence consisting in a sequence of any one of SEQ ID Nos: 469, 471, 473, 475, 477, 479, 481, 483, 485, 487, 489, 491, 493, 495, 497, 499, 501, 503, 505, 507, 509, 511, 513, 515, 517, 519, 521 and 523, which polynuceotide or salt thereof selectively binds to ketamine.
  • a polynucleotide or salt threreof comprising a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to any one of SEQ ID Nos: 468, 470, 472, 474, 476, 478, 480, 482,
  • polynucleotide or salt threreof comprising a sequence of any one of SEQ ID Nos: 468, 470, 472, 474, 476, 478,
  • a polynucleotide or salt thereof consisting in a sequence of any one of SEQ ID Nos: 468, 470, 472, 474, 476, 478, 480, 482, 484, 486, 488, 490, 492, 494, 496, 498, 500, 502, 504, 506, 508, 510, 512, 514, 516, 518, 520 and 522, which polynucleotide of salt thereof selectively binds to ketamine.
  • a method for detecting the presence of ketamine in a test sample comprising the steps of:
  • test kit or article of manufacture for detecting the presence of ketamine in a test sample, the test kit or article of manufacture comprising a polynucleotide or salt thereof comprising a sequence defined by any one of SEQ ID Nos: 468-523 together with instructions for how to detect the presence of ketamine in the test sample.
  • polynucleotides described herein may need direct modification embedded e.g. redox active molecules, fluorophores and bioactice molecules and may also need to be modifed and/or embedded with chemical functional groups for chemical conjugation to a structure e.g. electrodes (gold, platnium, carbon based, or conducting ploymer based), particles with and without magnetic properties, and peptides and/or proteins but not limited to.
  • a structure e.g. electrodes (gold, platnium, carbon based, or conducting ploymer based), particles with and without magnetic properties, and peptides and/or proteins but not limited to.
  • the polynucleotides described herein may be hybridised with complementary probes with and without any of the aforementioned modification examples to then be adopted onto an analytical and separation platforms.
  • the polynucleotides described herein may be used individually or collectively in an analytical format where the collective data can be utilised to deduce the presence of a drug in the sample tested e.g. blood, urine, body fluid sample etc.
  • Figure 1 shows the structure of methamphetamine.
  • Figure 2 shows a phylogenetic tree generated using Clustal Omega, depicting the structural relationship of the aptamer sequences which selectively bind to methamphetamine, as disclosed herein. Sequence distance scores are indicated against each sequence name.
  • Figure 4 shows the structure of (-)-trans-D 9 - tetrahydrocannabinol (THC), the active molecule found in cannabis.
  • Figure 5 shows the structure of a phylogenetic tree generated using Clustal Omega, depicting the structural relationship of the aptamer sequences which selectively bind to (-)-trans-D 9 -tetrahydrocannabinol (THC), as disclosed herein. Sequence distance scores are indicated against each sequence name.
  • Figure 6 shows dissociation constants for aptamer sequences
  • Figure 7 shows the structure of cocaine.
  • Figure 8 shows a phylogenetic tree generated using Clustal Omega, depicting the structural relationship of the aptamer sequences which selectively bind to cocaine, as disclosed herein. Sequence distance scores are indicated against each sequence name.
  • Figure 9 shows the structure of 3,4-methylenedioxymethamphetamine (MDMA).
  • Figure 10 shows a phylogenetic tree generated using Clustal Omega, depicting the structural relationship of the aptamer sequences which selectively bind to 3,4- methylenedioxymethamphetamine (MDMA), as disclosed herein. Sequence distance scores are indicated against each sequence name.
  • MDMA 3,4- methylenedioxymethamphetamine
  • Figure 11 shows the structure of morphine.
  • Figure 12 shows a phylogenetic tree generated using Clustal Omega, depicting the structural relationship of the aptamer sequences which selectively bind to morphine, as disclosed herein. Sequence distance scores are indicated against each sequence name.
  • Figure 13 shows dissociation constants for aptamer sequences
  • Figure 14 shows the structure of benzodiazepine.
  • Figure 15 shows a phylogenetic tree generated using Clustal Omega, depicting the structural relationship of the aptamer sequences which selectively bind to diazepam, as disclosed herein. Sequence distance scores are indicated against each sequence name.
  • Figure 16 shows the structure of AMB-FUBINACA (a synthetic cannabis).
  • Figure 17 shows a phylogenetic tree generated using Clustal Omega, depicting the structural relationship of the aptamer sequences which selectively bind to AMB-FUBINACA (a synthetic cannabis), as disclosed herein. Sequence distance scores are indicated against each sequence name.
  • FIG 18 shows the structure of AM2201 (a synthetic cannabis).
  • Figure 19 shows a phylogenetic tree generated using Clustal Omega, depicting the structural relationship of the aptamer sequences which selectively bind to AM2201 (a synthetic cannabis), as disclosed herein. Sequence distance scores are indicated against each sequence name.
  • Figure 20 shows the structure of oxycodone.
  • Figure 21 shows a phylogenetic tree generated using Clustal Omega, depicting the structural relationship of the aptamer sequences which selectively bind to oxycodone, as disclosed herein. Sequence distance scores are indicated against each sequence name.
  • Figure 22 shows the structure of ketamine.
  • Figure 23 shows a phylogenetic tree generated using Clustal Omega, depicting the structural relationship of the aptamer sequences which selectively bind to ketamine, as disclosed herein. Sequence distance scores are indicated against each sequence name.
  • a or “an” refers to one or more than one of the entity specified; for example, "a receptor” or “a nucleic acid molecule” may refer to one or more receptor or nucleic acid molecule, or at least one receptor or nucleic acid molecule. As such, the terms “a” or “an”, “one or more” and “at least one” can be used interchangeably herein.
  • composition of matter, group of steps or group of compositions of matter shall be taken to encompass one and a plurality (i.e. one or more) of those steps, compositions of matter, groups of steps or group of compositions of matter.
  • fragment as used herein is used interchangeably with the term “functional fragment” and means the same thing.
  • the term "functional fragment” as used herein means any part of a polynucleotide of the inventions disclosed herein that retains its ability to selectively bind to the same drug target as the polynucleotide from which it is derived.
  • test kit refers to an article of manufacture comprising various components to perform the assays and methods according to the inventions descibed herein.
  • THC tetrahydrocannabinol and includes, for example, (-)-trans-D 9 -tetrahydrocannabinol.
  • MDMA 3,4-methylenedioxymethamphetamine.
  • AMB-FUBINACA refers to a synthetic form of cannabis, the structure for which is illustrated in Figure 16.
  • AM2201 as used herein also refers to a synthetic form of cannabis, the structure for which is illustrated in Figure 18.
  • polynucleotide refers to a deoxyribose nucleic acid (DNA) sequence, a ribose nucleic acid sequence (RNA), messenger ribose nucleic acid (mRNA) and complementary DNA (cDNA), and is comprised of a continuous sequence of two or more nucleotides, also referred to as "a nucleic acid sequence”.
  • ligand refers generally to any molecule that binds to a receptor, and includes without limitation, a polypeptide, a protein, a vitamin, a carbohydrate, a glycoprotein, a therapeutic agent, a drug, a glycosaminoglycan, or any combination thereof.
  • ligand may include a drug or molecule associated with a drug, and in particular drugs of abuse including, without limitation, methamphetamine, cannabis, synthetic cannabis, cocaine, ecstasy, morphine, oxycodone and benzodiazepines.
  • sample refers to any sample for which it is desired to test for the presence of a drug of abuse.
  • reference threshold means the level of assay activity measured in the absence of a test sample. In certain examples according to the inventions described herein, the reference threshold is determined using ethanol in place of test sample.
  • detection means refers to any apparatus, equipment or configuration adapted to detect the binding interaction between an aptamer/polynucleotide and its drug target.
  • detection means include, but are not limited to, optical methods, spectroscopy, visible spectroscopy, Raman spectroscopy, UV spectroscopy, surface plasmon resonance, electrochemical methods, impedance, resistance, capacitance, mechanical sensing by changes in mass, changes in mechanical resonance, electrophoresis, gel electrophoresis, gel retardation, imaging, fluorescence and fluorescence resonance energy transfer, polymerase chain reaction etc.
  • the present invention provides polynucloetides, test kits and methods useful for the detection of drugs of abuse including, without limitation, detection of methamphetamine, cannabis, synthetic cannabis, cocaine, ecstasy, morphine, benzodiazepines, and oxycodone.
  • polynucloetides, test kits and methods according to the present invention are particularly useful for road-side testing or in work-place monitoring to detect drug abuse by individuals that places themselves or others in the community in danger. For example, in the operation of a motor vehicle or work-place machinary while under the influence of a drug of abuse, creating a potential for serious harm, permanent injury or death.
  • Polynucleotides/Aptamers are particularly useful for road-side testing or in work-place monitoring to detect drug abuse by individuals that places themselves or others in the community in danger. For example, in the operation of a motor vehicle or work-place machinary while under the influence of a drug of abuse, creating a potential for serious harm, permanent injury or death.
  • the polynucleotides of the present invention spontaneously fold to form aptamers having secondary structure features that promote selective binding to a target molecule, in particular a target associated with an illicit drug.
  • the polynuceotides selectively bind to illicit drug targets including, without limitation, methamphetamine, tetrahydrocannabinol, cocaine, 3,4-methylenedioxymethamphetamine, diazepam, morphine, AMB-FUNIBACA, AM2201, oxycodone and ketamine.
  • polynucleotide or salt thereof comprising or consisting in the sequence
  • ATACGAGCTTGTTCAATA[X]nTGATAGTAAGAGCAATC where X is selected from A, T, U, G or C and n is an integer of between about 10 and about 120, in particular between about 35 and about 45.
  • aptamer sequences which are highly selective for methamphetamine.
  • these aptamers display highly selective binding affinity for methamphetamine, with minimal cross reactivity to other drug targets.
  • Figures 1-3 Applicant developed aptamer sequences which selectively bind to methamphetamine, the structure of which is depicted in Figure 1, as well as structural analogs of methamphetamine.
  • a polynucleotide or salt thereof comprising the sequence ATACGAGCTTGTTCAATA[Y]TGATAGTAAGAGCAATC (SEQ ID NO: 566), where Y is any sequence comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to any one of SEQ ID Nos: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72 and 74, which polynuceotide or salt thereof selectively binds to methamphetamine.
  • a polynucleotide or salt thereof comprising the sequence ATACGAGCTTGTTCAATA[Y]TGATAGTAAGAGCAATC (SEQ ID NO: 567), where Y is any sequence comprising or consisting in a sequence selected from any one of SEQ ID Nos: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72 and 74, which polynuceotide or salt thereof selectively binds to methamphetamine.
  • a polynucleotide or salt thereof which selectively binds to methamphetamine comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 1 or a fragment thereof which retains selective binding for methamphetamine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 2.
  • a polynucleotide or salt thereof which selectively binds to methamphetamine comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 3 or a fragment thereof which retains selective binding for methamphetamine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 4.
  • a polynucleotide or salt thereof which selectively binds to methamphetamine comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 5 or a fragment thereof which retains selective binding for methamphetamine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 6.
  • a polynucleotide or salt thereof which selectively binds to methamphetamine comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 7 or a fragment thereof which retains selective binding for methamphetamine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 8.
  • a polynucleotide or salt thereof which selectively binds to methamphetamine comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 9 or a fragment thereof which retains selective binding for methamphetamine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 10.
  • a polynucleotide or salt thereof which selectively binds to methamphetamine comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 11 or a fragment thereof which retains selective binding for methamphetamine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 12.
  • a polynucleotide or salt thereof which selectively binds to methamphetamine comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 13 or a fragment thereof which retains selective binding for methamphetamine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 14.
  • a polynucleotide or salt thereof which selectively binds to methamphetamine comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 15 or a fragment thereof which retains selective binding for methamphetamine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 16.
  • a polynucleotide or salt thereof which selectively binds to methamphetamine comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 17 or a fragment thereof which retains selective binding for methamphetamine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 18.
  • a polynucleotide or salt thereof which selectively binds to methamphetamine comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 19 or a fragment thereof which retains selective binding for methamphetamine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 20.
  • a polynucleotide or salt thereof which selectively binds to methamphetamine comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 21 or a fragment thereof which retains selective binding for methamphetamine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 22.
  • a polynucleotide or salt thereof which selectively binds to methamphetamine comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 23 or a fragment thereof which retains selective binding for methamphetamine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 24.
  • a polynucleotide or salt thereof which selectively binds to methamphetamine comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 25 or a fragment thereof which retains selective binding for methamphetamine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 26.
  • a polynucleotide or salt thereof which selectively binds to methamphetamine comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 27 or a fragment thereof which retains selective binding for methamphetamine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 28.
  • a polynucleotide or salt thereof which selectively binds to methamphetamine comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 29 or a fragment thereof which retains selective binding for methamphetamine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 30.
  • a polynucleotide or salt thereof which selectively binds to methamphetamine comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 31 or a fragment thereof which retains selective binding for methamphetamine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 32.
  • a polynucleotide or salt thereof which selectively binds to methamphetamine comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 33 or a fragment thereof which retains selective binding for methamphetamine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 34.
  • a polynucleotide or salt thereof which selectively binds to methamphetamine comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 35 or a fragment thereof which retains selective binding for methamphetamine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 36.
  • a polynucleotide or salt thereof which selectively binds to methamphetamine comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 37 or a fragment thereof which retains selective binding for methamphetamine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 38.
  • a polynucleotide or salt thereof which selectively binds to methamphetamine comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 39 or a fragment thereof which retains selective binding for methamphetamine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 40.
  • a polynucleotide or salt thereof which selectively binds to methamphetamine comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 41 or a fragment thereof which retains selective binding for methamphetamine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 42.
  • a polynucleotide or salt thereof which selectively binds to methamphetamine comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 43 or a fragment thereof which retains selective binding for methamphetamine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 44.
  • a polynucleotide or salt thereof which selectively binds to methamphetamine comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 45 or a fragment thereof which retains selective binding for methamphetamine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 46.
  • a polynucleotide or salt thereof which selectively binds to methamphetamine comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 47 or a fragment thereof which retains selective binding for methamphetamine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 48.
  • a polynucleotide or salt thereof which selectively binds to methamphetamine comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 49 or a fragment thereof which retains selective binding for methamphetamine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 50.
  • a polynucleotide or salt thereof which selectively binds to methamphetamine comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 51 or a fragment thereof which retains selective binding for methamphetamine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 52.
  • a polynucleotide or salt thereof which selectively binds to methamphetamine comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 53 or a fragment thereof which retains selective binding for methamphetamine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 54.
  • a polynucleotide or salt thereof which selectively binds to methamphetamine comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 55 or a fragment thereof which retains selective binding for methamphetamine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 56.
  • a polynucleotide or salt thereof which selectively binds to methamphetamine comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 57 or a fragment thereof which retains selective binding for methamphetamine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 58.
  • a polynucleotide or salt thereof which selectively binds to methamphetamine comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 59 or a fragment thereof which retains selective binding for methamphetamine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 60.
  • a polynucleotide or salt thereof which selectively binds to methamphetamine comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 61 or a fragment thereof which retains selective binding for methamphetamine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 62.
  • a polynucleotide or salt thereof which selectively binds to methamphetamine comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 63 or a fragment thereof which retains selective binding for methamphetamine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 64.
  • a polynucleotide or salt thereof which selectively binds to methamphetamine comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 65 or a fragment thereof which retains selective binding for methamphetamine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 66.
  • a polynucleotide or salt thereof which selectively binds to methamphetamine comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 67 or a fragment thereof which retains selective binding for methamphetamine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 68.
  • a polynucleotide or salt thereof which selectively binds to methamphetamine comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 69 or a fragment thereof which retains selective binding for methamphetamine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 70.
  • a polynucleotide or salt thereof which selectively binds to methamphetamine comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 71 or a fragment thereof which retains selective binding for methamphetamine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 72.
  • a polynucleotide or salt thereof which selectively binds to methamphetamine comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 73 or a fragment thereof which retains selective binding for methamphetamine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 74.
  • these aptamers display highly selective binding affinity for methamphetamine.
  • the polynucleotides encoding methamphetamine aptamers described herein including without limitation SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71 and 73, display a binding affinity for methamphetamine which is between about 1 nM and about 5 mM, including between about 1 nM and about 2 mM, or about 1 nM and about 1500 nM, or about 1 nM and about 1000 nM, or about 1 nM and about 500 nM.
  • the term "between about 1 nM and about 500 nM” includes a binding dissociation constant that is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 55, 50, 65, 60, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 405, 410, 415, 420, 425, 430, 435, 440, 445, 450, 452, 454, 456, 458, 460, 462, 464, 468, 470, 472, 474, 476, 478, 480, 482, 484, 486, 488, 490, 491, 492, 493,
  • the Geneious Prime pairwise alignments successfuly identified four discrete methamphetamine aptamer consensus sequences defined in Tables 3-6 below and identified as SEQ ID NO: 524, SEQ ID NO: 525, SEQ ID NO: 526 and SEQ ID NO: 527.
  • a polynuceotide or salt thereof which comprises or consists in a consensus sequence defined by SEQ ID NO: 524, which polynucleotide or salt thereof selectively binds methamphetamine.
  • the polynucleotide or salt thereof which selectively binds methamphetamine comprises or consists in a sequence selected from SEQ ID Nos: 13, 73, 45, 47, 49, 51, 55, and 9.
  • the polynucleotide or salt thereof comprises or consists in SEQ ID No: 13.
  • the Gibbs Free Energy (DG) associated with each methamphetamine aptamer disclosed herein is reflected in Table 1.
  • the quantum of Gibbs Free Energy is reflective of aptamer stability; a more negative DG value reflects an aptamer having a higher degree of overall stability compared to an aptamer with a less negative DG value.
  • Information concerning Gibbs Free Energy may be an important consideration when selecting an aptamer for use in a particular assay format. For example, it is preferable to employ an aptamer which displays a higher degree of stability for use in a colorimetric/nanoparticle detection assay, such as that described in Applicant's earlier work (WO 2015/174863). In contrast, other analyte detection assays involving aptamer chemistry require aptamers which are less stable for the reason that the presence of a target molecule triggers a conformational change in the aptamer structure, and it is a physical property associated with this structural change which is used to detect the presence of a target molecule in a sample under investigation.
  • polynucleotide or salt thereof which comprises or consists in a consensus sequence defined by SEQ ID NO: 525, which polynucleotide or salt thereof selectively binds methamphetamine.
  • the polynucleotide or salt thereof which selectively binds methamphetamine comprises or consists in a sequence selected from SEQ ID Nos: 1, 5, 27, 29, 3, 17, 19, 21, 23, 25, 31, 33 and 39.
  • the polynucleotide or salt thereof comprises or consists in a sequence selected from SEQ ID Nos: 1, 5, 27 and 29.
  • polynucleotide or salt thereof which comprises or consists in a consensus sequence defined by SEQ ID NO: 526, which polynucleotide or salt thereof selectively binds methamphetamine.
  • the polynucleotide or salt thereof which selectively binds methamphetamine comprises or consists in a sequence selected from SEQ ID Nos: 71, 67, 41, 43, 53, 7, 11, 15 and 65.
  • the polynucleotide or salt thereof comprises or consists in a sequence selected from SEQ ID Nos: 71 and 67.
  • DG -2.21 to -6.71.
  • a polynucleotide or salt thereof which comprises or consists in a consensus sequence defined by SEQ ID NO: 527, which polynucleotide or salt thereof selectively binds methamphetamine.
  • the polynucleotide or salt thereof which selectively binds methamphetamine comprises or consists in a sequence selected from SEQ ID Nos: 69, 57, 35 and 37.
  • aptamer sequences which are highly selective for tetrahydrocannabinol.
  • these aptamers display highly selective binding affinity for tetrahydrocannabinol, with minimal cross-reactivity to other drug targets.
  • Y is any sequence comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to any one of SEQ ID Nos: 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134 and 136, which polynuceotide or salt thereof selectively binds to tetrahydrocannabinol.
  • a polynucleotide or salt thereof comprising the sequence ATACGAGCTTGTTCAATA[Y]TGATAGTAAGAGCAATC (SEQ ID NO: 570), where Y is any sequence comprising or consisting in a sequence selected from any one of SEQ ID Nos: 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134 and 136, which polynuceotide or salt thereof selectively binds to tetrahydrocannabinol.
  • a polynucleotide or salt thereof which selectively binds to tetrahydrocannabinol comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 75 or a fragment thereof which retains selective binding for tetrahydrocannabinol.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 76.
  • a polynucleotide or salt thereof which selectively binds to tetrahydrocannabinol comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 77 or a fragment thereof which retains selective binding for tetrahydrocannabinol.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 78.
  • a polynucleotide or salt thereof which selectively binds to tetrahydrocannabinol comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 79 or a fragment thereof which retains selective binding for tetrahydrocannabinol.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 80.
  • a polynucleotide or salt thereof which selectively binds to tetrahydrocannabinol comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 81 or a fragment thereof which retains selective binding for tetrahydrocannabinol.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 82.
  • a polynucleotide or salt thereof which selectively binds to tetrahydrocannabinol comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 83 or a fragment thereof which retains selective binding for tetrahydrocannabinol.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 84.
  • a polynucleotide or salt thereof which selectively binds to tetrahydrocannabinol comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 85 or a fragment thereof which retains selective binding for tetrahydrocannabinol.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 86.
  • a polynucleotide or salt thereof which selectively binds to tetrahydrocannabinol comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 87 or a fragment thereof which retains selective binding for tetrahydrocannabinol.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 88.
  • a polynucleotide or salt thereof which selectively binds to tetrahydrocannabinol comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 89 or a fragment thereof which retains selective binding for tetrahydrocannabinol.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 90.
  • a polynucleotide or salt thereof which selectively binds to tetrahydrocannabinol comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 91 or a fragment thereof which retains selective binding for tetrahydrocannabinol.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 92.
  • a polynucleotide or salt thereof which selectively binds to tetrahydrocannabinol comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 93 or a fragment thereof which retains selective binding for tetrahydrocannabinol.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 94.
  • a polynucleotide or salt thereof which selectively binds to tetrahydrocannabinol comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 95 or a fragment thereof which retains selective binding for tetrahydrocannabinol.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 96.
  • a polynucleotide or salt thereof which selectively binds to tetrahydrocannabinol comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 97 or a fragment thereof which retains selective binding for tetrahydrocannabinol.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 98.
  • a polynucleotide or salt thereof which selectively binds to tetrahydrocannabinol comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 99 or a fragment thereof which retains selective binding for tetrahydrocannabinol.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 100.
  • a polynucleotide or salt thereof which selectively binds to tetrahydrocannabinol comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 101 or a fragment thereof which retains selective binding for tetrahydrocannabinol.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 102
  • a polynucleotide or salt thereof which selectively binds to tetrahydrocannabinol comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 103 or a fragment thereof which retains selective binding for tetrahydrocannabinol.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 104.
  • a polynucleotide or salt thereof which selectively binds to tetrahydrocannabinol comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 105 or a fragment thereof which retains selective binding for tetrahydrocannabinol.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 106.
  • a polynucleotide or salt thereof which selectively binds to tetrahydrocannabinol comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 107 or a fragment thereof which retains selective binding for tetrahydrocannabinol.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 108.
  • a polynucleotide or salt thereof which selectively binds to tetrahydrocannabinol comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 109 or a fragment thereof which retains selective binding for tetrahydrocannabinol.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 110.
  • a polynucleotide or salt thereof which selectively binds to tetrahydrocannabinol comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 111 or a fragment thereof which retains selective binding for tetrahydrocannabinol.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 112.
  • a polynucleotide or salt thereof which selectively binds to tetrahydrocannabinol comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 113 or a fragment thereof which retains selective binding for tetrahydrocannabinol.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 114.
  • a polynucleotide or salt thereof which selectively binds to tetrahydrocannabinol comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 115 or a fragment thereof which retains selective binding for tetrahydrocannabinol.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 116.
  • a polynucleotide or salt thereof which selectively binds to tetrahydrocannabinol comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 117 or a fragment thereof which retains selective binding for tetrahydrocannabinol.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 118.
  • a polynucleotide or salt thereof which selectively binds to tetrahydrocannabinol comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 119 or a fragment thereof which retains selective binding for tetrahydrocannabinol.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 120.
  • a polynucleotide or salt thereof which selectively binds to tetrahydrocannabinol comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 121 or a fragment thereof which retains selective binding for tetrahydrocannabinol.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 122.
  • a polynucleotide or salt thereof which selectively binds to tetrahydrocannabinol comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 123 or a fragment thereof which retains selective binding for tetrahydrocannabinol.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 124.
  • a polynucleotide or salt thereof which selectively binds to tetrahydrocannabinol comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 125 or a fragment thereof which retains selective binding for tetrahydrocannabinol.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 126.
  • a polynucleotide or salt thereof which selectively binds to tetrahydrocannabinol comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 127 or a fragment thereof which retains selective binding for tetrahydrocannabinol.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 128.
  • a polynucleotide or salt thereof which selectively binds to tetrahydrocannabinol comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 129 or a fragment thereof which retains selective binding for tetrahydrocannabinol.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 130.
  • a polynucleotide or salt thereof which selectively binds to tetrahydrocannabinol comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 131 or a fragment thereof which retains selective binding for tetrahydrocannabinol.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 132.
  • a polynucleotide or salt thereof which selectively binds to tetrahydrocannabinol comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 133 or a fragment thereof which retains selective binding for tetrahydrocannabinol.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 134.
  • a polynucleotide or salt thereof which selectively binds to tetrahydrocannabinol comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 135 or a fragment thereof which retains selective binding for tetrahydrocannabinol.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 136.
  • THC-30 and THC-32 share a higher degree of structural similarity compared to (e.g.) THC-01 and THC-09.
  • THC-30, THC-32, THC-01 and THC-09 collectively share a higher degree of structural similarity compared to (e.g.) THCR9-S6, THCR9-S1 and THCR9-S10.
  • Figures 6A-6C provide dissociation constants for THC-01 (SEQ ID NO: 133) of 6916 nM, THCR9-S6 (SEQ ID NO: 91) of 573 nM and THCR9-S9 (SEQ ID NO: 97) of 284 nM. These data would indicate that THCR9-S6 and THCR9-S9 are more selective for tetrahydrocannabinol than THC-01. With dissociation constants in the high nM range, these aptamers display highly selective binding affinity for tetrahydrocannabinol.
  • the polynucleotides encoding tetrahydrocannabinol aptamers described herein including without limitation SEQ ID NOs: 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 133 and 135, display a binding affinity for tetrahydrocannabinol which is between about 1 nM and about 1000 nM, including between about about 1 nM and about 750 nM, and about 1 nM and 300 nM.
  • the term "between about 1 nM and about 300 nM” includes a binding dissociation constant that is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 55, 50, 65, 60, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299 and
  • a polynucleotide or salt thereof which comprises or consists in a consensus sequence defined by SEQ ID NO: 528, which polynucleotide or salt thereof selectively binds tetrahydrocannabinol.
  • the polynucleotide or salt thereof which selectively binds tetrahydrocannabinol comprises or consists in a sequence selected from SEQ ID Nos: 91, 83, 95 and 85.
  • the polynucleotide or salt thereof comprises or consists in SEQ ID No: 91.
  • a polynucleotide or salt thereof which comprises or consists in a consensus sequence defined by SEQ ID NO: 529, which polynucleotide or salt thereof selectively binds tetrahydrocannabinol.
  • the polynucleotide or salt thereof which selectively binds tetrahydrocannabinol comprises or consists in a sequence selected from SEQ ID Nos: 105, 117, 79, 81, 87, 89 and 93.
  • a polynucleotide or salt thereof which comprises or consists in a consensus sequence defined by SEQ ID NO: 530, which polynucleotide or salt thereof selectively binds tetrahydrocannabinol.
  • the polynucleotide or salt thereof which selectively binds tetrahydrocannabinol comprises or consists in a sequence selected from SEQ ID Nos: 97, 99, 101, 119, 75, 77 and 77.
  • a polynucleotide or salt thereof which comprises or consists in a consensus sequence defined by SEQ ID NO: 531, which polynucleotide or salt thereof selectively binds tetrahydrocannabinol.
  • the polynucleotide or salt thereof which selectively binds tetrahydrocannabinol comprises or consists in a sequence selected from SEQ ID Nos: 133, 135, 107, 125, 127, 129, 131.
  • the polynucleotide or salt thereof comprises or consists in a sequence selected from SEQ ID No: 133 and SEQ ID NO: 135.
  • aptamer sequences which are highly selective for cocaine.
  • these aptamers display highly selective binding affinity for cocaine, with minimal cross-reactivity to other drug targets.
  • a polynucleotide or salt thereof comprising the sequence ATACGAGCTTGTTCAATA[Y]TGATAGTAAGAGCAATC (SEQ ID NO: 572), where Y is any sequence comprising a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to any one of SEQ ID Nos: 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170 and 172, which polynuceotide or salt thereof selectively binds to cocaine.
  • a polynucleotide or salt thereof comprising the sequence ATACGAGCTTGTTCAATA[Y]TGATAGTAAGAGCAATC (SEQ ID NO: 573, where Y is any sequence comprising any one of SEQ ID Nos: 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170 and 172, which polynuceotide or salt thereof selectively binds to cocaine.
  • a polynucleotide or salt thereof which selectively binds to cocaine comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 137 or a fragment thereof which retains selective binding for cocaine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 138
  • a polynucleotide or salt thereof which selectively binds to cocaine comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 139 or a fragment thereof which retains selective binding for cocaine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 140.
  • a polynucleotide or salt thereof which selectively binds to cocaine comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 141 or a fragment thereof which retains selective binding for cocaine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 142.
  • a polynucleotide or salt thereof which selectively binds to cocaine comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 143 or a fragment thereof which retains selective binding for cocaine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 144.
  • a polynucleotide or salt thereof which selectively binds to cocaine comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 145 or a fragment thereof which retains selective binding for cocaine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 146.
  • a polynucleotide or salt thereof which selectively binds to cocaine comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 147 or a fragment thereof which retains selective binding for cocaine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 148.
  • a polynucleotide or salt thereof which selectively binds to cocaine comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 149 or a fragment thereof which retains selective binding for cocaine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 150.
  • a polynucleotide or salt thereof which selectively binds to cocaine comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 151 or a fragment thereof which retains selective binding for cocaine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 152.
  • a polynucleotide or salt thereof which selectively binds to cocaine comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 153 or a fragment thereof which retains selective binding for cocaine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 154.
  • a polynucleotide or salt thereof which selectively binds to cocaine comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 155 or a fragment thereof which retains selective binding for cocaine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 156.
  • a polynucleotide or salt thereof which selectively binds to cocaine comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 157 or a fragment thereof which retains selective binding for cocaine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 158.
  • a polynucleotide or salt thereof which selectively binds to cocaine comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 159 or a fragment thereof which retains selective binding for cocaine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 160.
  • a polynucleotide or salt thereof which selectively binds to cocaine comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 161 or a fragment thereof which retains selective binding for cocaine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 162.
  • a polynucleotide or salt thereof which selectively binds to cocaine comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 163 or a fragment thereof which retains selective binding for cocaine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 164.
  • a polynucleotide or salt thereof which selectively binds to cocaine comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 165 or a fragment thereof which retains selective binding for cocaine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 166.
  • a polynucleotide or salt thereof which selectively binds to cocaine comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 167 or a fragment thereof which retains selective binding for cocaine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 168.
  • a polynucleotide or salt thereof which selectively binds to cocaine comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 169 or a fragment thereof which retains selective binding for cocaine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 170.
  • a polynucleotide or salt thereof which selectively binds to cocaine comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 171 or a fragment thereof which retains selective binding for cocaine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 172.
  • a polynucleotide or salt thereof which comprises or consists in a consensus sequence defined by SEQ ID NO: 532, which polynucleotide or salt thereof selectively binds cocaine.
  • the polynucleotide or salt thereof which selectively binds cocaine comprises or consists in a sequence selected from SEQ ID Nos: 153, 157, 161 and 165.
  • a polynucleotide or salt thereof which comprises or consists in a consensus sequence defined by SEQ ID NO: 533, which polynucleotide or salt thereof selectively binds cocaine.
  • the polynucleotide or salt thereof which selectively binds cocaine comprises or consists in a sequence selected from SEQ ID Nos: 151, 163, 167 and 169.
  • a polynucleotide or salt thereof which comprises or consists in a consensus sequence defined by SEQ ID NO: 534, which polynucleotide or salt thereof selectively binds cocaine.
  • the polynucleotide or salt thereof which selectively binds cocaine comprises or consists in a sequence selected from SEQ ID Nos: 139, 141 and 137.
  • a polynucleotide or salt thereof which comprises or consists in a consensus sequence defined by SEQ ID NO: 535, which polynucleotide or salt thereof selectively binds cocaine.
  • aptamer sequences which are highly selective for 3,4-methylenedioxymethamphetamine (MDMA).
  • MDMA 3,4-methylenedioxymethamphetamine
  • a polynucleotide or salt thereof comprising the sequence ATACGAGCTTGTTCAATA[Y]TGATAGTAAGAGCAATC (SEQ ID NO: 575), where Y is any sequence comprising a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to any one of SEQ ID Nos: 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226,
  • a polynucleotide or salt thereof comprising the sequence ATACGAGCTTGTTCAATA[Y]TGATAGTAAGAGCAATC (SEQ ID NO: 576), where Y is any sequence comprising any one of SEQ ID Nos: 174, 176,
  • polynuceotide or salt thereof selectively binds to 3,4- methylenedioxymethamphetamine.
  • a polynucleotide or salt thereof which selectively binds to 3,4-methylenedioxymethamphetamine
  • the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 173 or a fragment thereof which retains selective binding for 3,4-methylenedioxymethamphetamine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 174.
  • a polynucleotide or salt thereof which selectively binds to 3,4-methylenedioxymethamphetamine
  • the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 175 or a fragment thereof which retains selective binding for 3,4-methylenedioxymethamphetamine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 176.
  • a polynucleotide or salt thereof which selectively binds to 3,4-methylenedioxymethamphetamine
  • the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 177 or a fragment thereof which retains selective binding for 3,4-methylenedioxymethamphetamine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 178.
  • a polynucleotide or salt thereof which selectively binds to 3,4-methylenedioxymethamphetamine
  • the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 179 or a fragment thereof which retains selective binding for 3,4-methylenedioxymethamphetamine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 180.
  • a polynucleotide or salt thereof which selectively binds to 3,4-methylenedioxymethamphetamine comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 181 or a fragment thereof which retains selective binding for 3,4-methylenedioxymethamphetamine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 182.
  • a polynucleotide or salt thereof which selectively binds to 3,4-methylenedioxymethamphetamine
  • the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 183 or a fragment thereof which retains selective binding for 3,4-methylenedioxymethamphetamine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 184.
  • a polynucleotide or salt thereof which selectively binds to 3,4-methylenedioxymethamphetamine
  • the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 185 or a fragment thereof which retains selective binding for 3,4-methylenedioxymethamphetamine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 186.
  • a polynucleotide or salt thereof which selectively binds to 3,4-methylenedioxymethamphetamine
  • the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 187 or a fragment thereof which retains selective binding for 3,4-methylenedioxymethamphetamine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 188.
  • a polynucleotide or salt thereof which selectively binds to 3,4-methylenedioxymethamphetamine
  • the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 189 or a fragment thereof which retains selective binding for 3,4-methylenedioxymethamphetamine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 190.
  • a polynucleotide or salt thereof which selectively binds to 3,4-methylenedioxymethamphetamine
  • the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 191 or a fragment thereof which retains selective binding for 3,4-methylenedioxymethamphetamine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 192.
  • a polynucleotide or salt thereof which selectively binds to 3,4-methylenedioxymethamphetamine comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 193 or a fragment thereof which retains selective binding for 3,4-methylenedioxymethamphetamine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 194.
  • a polynucleotide or salt thereof which selectively binds to 3,4-methylenedioxymethamphetamine
  • the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 195 or a fragment thereof which retains selective binding for 3,4-methylenedioxymethamphetamine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 196.
  • a polynucleotide or salt thereof which selectively binds to 3,4-methylenedioxymethamphetamine
  • the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 197 or a fragment thereof which retains selective binding for 3,4-methylenedioxymethamphetamine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 198.
  • a polynucleotide or salt thereof which selectively binds to 3,4-methylenedioxymethamphetamine
  • the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 199 or a fragment thereof which retains selective binding for 3,4-methylenedioxymethamphetamine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 200.
  • a polynucleotide or salt thereof which selectively binds to 3,4-methylenedioxymethamphetamine
  • the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 201 or a fragment thereof which retains selective binding for 3,4-methylenedioxymethamphetamine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 202.
  • a polynucleotide or salt thereof which selectively binds to 3,4-methylenedioxymethamphetamine
  • the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 203 or a fragment thereof which retains selective binding for 3,4-methylenedioxymethamphetamine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 204.
  • a polynucleotide or salt thereof which selectively binds to 3,4-methylenedioxymethamphetamine comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 205 or a fragment thereof which retains selective binding for 3,4-methylenedioxymethamphetamine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 206.
  • a polynucleotide or salt thereof which selectively binds to 3,4-methylenedioxymethamphetamine comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 207 or a fragment thereof which retains selective binding for 3,4-methylenedioxymethamphetamine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 208.
  • a polynucleotide or salt thereof which selectively binds to 3,4-methylenedioxymethamphetamine comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 209 or a fragment thereof which retains selective binding for 3,4-methylenedioxymethamphetamine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 210.
  • a polynucleotide or salt thereof which selectively binds to 3,4-methylenedioxymethamphetamine comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 211 or a fragment thereof which retains selective binding for 3,4-methylenedioxymethamphetamine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 212.
  • a polynucleotide or salt thereof which selectively binds to 3,4-methylenedioxymethamphetamine
  • the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 213 or a fragment thereof which retains selective binding for 3,4-methylenedioxymethamphetamine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 214.
  • a polynucleotide or salt thereof which selectively binds to 3,4-methylenedioxymethamphetamine
  • the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 215 or a fragment thereof which retains selective binding for 3,4-methylenedioxymethamphetamine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 216.
  • a polynucleotide or salt thereof which selectively binds to 3,4-methylenedioxymethamphetamine
  • the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 217 or a fragment thereof which retains selective binding for 3,4-methylenedioxymethamphetamine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 218.
  • a polynucleotide or salt thereof which selectively binds to 3,4-methylenedioxymethamphetamine
  • the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 219 or a fragment thereof which retains selective binding for 3,4-methylenedioxymethamphetamine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 220.
  • a polynucleotide or salt thereof which selectively binds to 3,4-methylenedioxymethamphetamine
  • the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 221 or a fragment thereof which retains selective binding for 3,4-methylenedioxymethamphetamine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 222.
  • a polynucleotide or salt thereof which selectively binds to 3,4-methylenedioxymethamphetamine
  • the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 223 or a fragment thereof which retains selective binding for 3,4-methylenedioxymethamphetamine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 224.
  • a polynucleotide or salt thereof which selectively binds to 3,4-methylenedioxymethamphetamine
  • the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 225 or a fragment thereof which retains selective binding for 3,4-methylenedioxymethamphetamine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 226.
  • a polynucleotide or salt thereof which selectively binds to 3,4-methylenedioxymethamphetamine
  • the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 227 or a fragment thereof which retains selective binding for 3,4-methylenedioxymethamphetamine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 228.
  • a polynucleotide or salt thereof which selectively binds to 3,4-methylenedioxymethamphetamine
  • the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 229 or a fragment thereof which retains selective binding for 3,4-methylenedioxymethamphetamine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 230.
  • a polynucleotide or salt thereof which selectively binds to 3,4-methylenedioxymethamphetamine
  • the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 231 or a fragment thereof which retains selective binding for 3,4-methylenedioxymethamphetamine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 232.
  • a polynucleotide or salt thereof which selectively binds to 3,4-methylenedioxymethamphetamine comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 233 or a fragment thereof which retains selective binding for 3,4-methylenedioxymethamphetamine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 234.
  • a polynucleotide or salt thereof which selectively binds to 3,4-methylenedioxymethamphetamine
  • the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 235 or a fragment thereof which retains selective binding for 3,4-methylenedioxymethamphetamine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 236.
  • a polynucleotide or salt thereof which selectively binds to 3,4-methylenedioxymethamphetamine
  • the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 237 or a fragment thereof which retains selective binding for 3,4-methylenedioxymethamphetamine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 238.
  • a polynucleotide or salt thereof which selectively binds to 3,4-methylenedioxymethamphetamine comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 239 or a fragment thereof which retains selective binding for 3,4-methylenedioxymethamphetamine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 240.
  • a polynucleotide or salt thereof which selectively binds to 3,4-methylenedioxymethamphetamine
  • the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 241 or a fragment thereof which retains selective binding for 3,4-methylenedioxymethamphetamine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 242.
  • a polynucleotide or salt thereof which selectively binds to 3,4-methylenedioxymethamphetamine comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 243 or a fragment thereof which retains selective binding for 3,4-methylenedioxymethamphetamine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 244.
  • a polynucleotide or salt thereof which selectively binds to 3,4-methylenedioxymethamphetamine
  • the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 245 or a fragment thereof which retains selective binding for 3,4-methylenedioxymethamphetamine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 246.
  • MDMA aptamers Cluster analysis was also performed on the MDMA aptamers disclosed herein using Clustal Omega. The results from these analyses is presented in Figure 10 where the level of structural (i.e. sequence) dis/similarity between the different aptamers in this drug class is pictorily illustrated.
  • the MDMA aptamers designated MD10 and MD24 share a higher degree of structural similarity compared to (e.g.) MD31, MD98 and MD65.
  • MD10, MD24, MD31, MD98 and MD65 collectively share a higher degree of structural similarity compared to (e.g.) MD95, MD97, MD52 and MD310.
  • a polynucleotide or salt thereof which comprises or consists in a consensus sequence defined by SEQ ID NO: 536, which polynucleotide or salt thereof selectively binds MDMA.
  • the polynucleotide or salt thereof which selectively binds MDMA comprises or consists in a sequence selected from SEQ ID Nos: 229, 235, 237, 239 and 245.
  • a polynucleotide or salt thereof which comprises or consists in a consensus sequence defined by SEQ ID NO: 537, which polynucleotide or salt thereof selectively binds MDMA.
  • the polynucleotide or salt thereof which selectively binds cocaine comprises or consists in a sequence selected from SEQ ID Nos: 219, 241 and 243.
  • a polynucleotide or salt thereof which comprises or consists in a consensus sequence defined by SEQ ID NO: 538, which polynucleotide or salt thereof selectively binds MDMA.
  • the polynucleotide or salt thereof which selectively binds cocaine comprises or consists in a sequence selected from SEQ ID Nos: 203, 195, 199 and 213.
  • a polynucleotide or salt thereof which comprises or consists in a consensus sequence defined by SEQ ID NO: 539, which polynucleotide or salt thereof selectively binds MDMA.
  • the polynucleotide or salt thereof which selectively binds cocaine comprises or consists in a sequence selected from SEQ ID Nos: 225, 211 and 207.
  • aptamer sequences which are highly selective for morphine.
  • these aptamers display highly selective binding affinity for morphine, with minimal cross-reactivity to other drug targets.
  • a polynucleotide or salt thereof comprising the sequence ATACGAGCTTGTTCAATA[Y]TGATAGTAAGAGCAATC (SEQ ID NO: 578), where Y is any sequence comprising a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to any one of SEQ ID Nos: 249, 251, 253, 255, 257, 259, 261, 263, 264, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289 and 291, which polynuceotide or salt thereof selectively binds to morphine.
  • a polynucleotide or salt thereof comprising the sequence ATACGAGCTTGTTCAATA[Y]TGATAGTAAGAGCAATC (SEQ ID NO: 579), where Y is any sequence comprising any one of SEQ ID Nos: 249, 251, 253, 255, 257, 259, 261, 263, 264, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289 and 291, which polynuceotide or salt thereof selectively binds to morphine.
  • a polynucleotide or salt thereof which selectively binds to morphine comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 247 or a fragment thereof which retains selective binding for morphine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 265.
  • a polynucleotide or salt thereof which selectively binds to morphine comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 248 or a fragment thereof which retains selective binding for morphine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 249.
  • a polynucleotide or salt thereof which selectively binds to morphine comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 250 or a fragment thereof which retains selective binding for morphine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 251.
  • a polynucleotide or salt thereof which selectively binds to morphine comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 252 or a fragment thereof which retains selective binding for morphine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 253.
  • a polynucleotide or salt thereof which selectively binds to morphine comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 254 or a fragment thereof which retains selective binding for morphine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 255.
  • a polynucleotide or salt thereof which selectively binds to morphine comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 256 or a fragment thereof which retains selective binding for morphine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 257.
  • a polynucleotide or salt thereof which selectively binds to morphine comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 258 or a fragment thereof which retains selective binding for morphine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 259.
  • a polynucleotide or salt thereof which selectively binds to morphine comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 260 or a fragment thereof which retains selective binding for morphine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 261.
  • a polynucleotide or salt thereof which selectively binds to morphine comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 262 or a fragment thereof which retains selective binding for morphine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 263.
  • a polynucleotide or salt thereof which selectively binds to morphine comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 266 or a fragment thereof which retains selective binding for morphine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 267.
  • a polynucleotide or salt thereof which selectively binds to morphine comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 268 or a fragment thereof which retains selective binding for morphine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 269.
  • a polynucleotide or salt thereof which selectively binds to morphine comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 270 or a fragment thereof which retains selective binding for morphine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 271.
  • a polynucleotide or salt thereof which selectively binds to morphine comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 272 or a fragment thereof which retains selective binding for morphine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 273.
  • a polynucleotide or salt thereof which selectively binds to morphine comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 274 or a fragment thereof which retains selective binding for morphine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 275.
  • a polynucleotide or salt thereof which selectively binds to morphine comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 276 or a fragment thereof which retains selective binding for morphine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 277.
  • a polynucleotide or salt thereof which selectively binds to morphine comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 278 or a fragment thereof which retains selective binding for morphine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 279.
  • a polynucleotide or salt thereof which selectively binds to morphine comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 280 or a fragment thereof which retains selective binding for morphine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 281.
  • a polynucleotide or salt thereof which selectively binds to morphine comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 282 or a fragment thereof which retains selective binding for morphine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 283 or SEQ ID NO: 264.
  • a polynucleotide or salt thereof which selectively binds to morphine comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 284 or a fragment thereof which retains selective binding for morphine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 285.
  • a polynucleotide or salt thereof which selectively binds to morphine comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 286 or a fragment thereof which retains selective binding for morphine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 287.
  • a polynucleotide or salt thereof which selectively binds to morphine comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 288 or a fragment thereof which retains selective binding for morphine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 289.
  • a polynucleotide or salt thereof which selectively binds to morphine comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 290 or a fragment thereof which retains selective binding for morphine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 291.
  • the polynucleotides encoding morphine aptamers described herein include without limitation SEQ ID NOs: 247, 248, 250, 252, 254, 256, 258, 260, 262, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288 and 290, display a binding affinity for morphine which is between about 1 nM and about 1000 nM, including between about about 1 nM and about 750 nM, and about 1 nM and 300 nM.
  • a polynucleotide or salt thereof which comprises or consists in a consensus sequence defined by SEQ ID NO: 540, which polynucleotide or salt thereof selectively binds morphine.
  • the polynucleotide or salt thereof which selectively binds morphine comprises or consists in a sequence selected from SEQ ID Nos: 288, 290, 280, 282, 254, 266, 272 and 262.
  • the polynucleotide or salt thereof comprises or consists in a sequence selected from SEQ ID NOs: 288, 290, 280 and 282.
  • polynucleotide or salt thereof which comprises or consists in a consensus sequence defined by SEQ ID NO: 541, which polynucleotide or salt thereof selectively binds morphine.
  • the polynucleotide or salt thereof which selectively binds morphine comprises or consists in a sequence selected from SEQ ID Nos: 268, 247, 248, 250, 252, 256, 270, 274, 276, 258, 260, 278, 284, 286.
  • the polynucleotide or salt thereof comprises or consists in SEQ ID NO: 268.
  • aptamer sequences which are highly selective for benzodiazepine.
  • these aptamers display highly selective binding affinity for benzodiazepine, with minimal cross reactivity to other drug targets.
  • a polynucleotide or salt thereof comprising the sequence ATACGAGCTTGTTCAATA[Y]TGATAGTAAGAGCAATC (SEQ ID NO: 581), where Y is any sequence comprising a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to any one of SEQ ID Nos: 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317 and 319, which polynuceotide or salt thereof selectively binds to benzodiazepine.
  • a polynucleotide or salt thereof comprising the sequence ATACGAGCTTGTTCAATA[Y]TGATAGTAAGAGCAATC (SEQ ID NO: 582), where Y is any sequence comprising any one of SEQ ID Nos: 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317 and 319, which polynuceotide or salt thereof selectively binds to benzodiazepine.
  • a polynucleotide or salt thereof which selectively binds to diazepam comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 292 or a fragment thereof which retains selective binding for benzodiazepine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 293.
  • a polynucleotide or salt thereof which selectively binds to diazepam comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 294 or a fragment thereof which retains selective binding for benzodiazepine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 295.
  • a polynucleotide or salt thereof which selectively binds to diazepam comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 296 or a fragment thereof which retains selective binding for benzodiazepine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 297.
  • a polynucleotide or salt thereof which selectively binds to diazepam comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 298 or a fragment thereof which retains selective binding for benzodiazepine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 299.
  • a polynucleotide or salt thereof which selectively binds to diazepam comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 300 or a fragment thereof which retains selective binding for benzodiazepine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 301.
  • a polynucleotide or salt thereof which selectively binds to diazepam comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 302 or a fragment thereof which retains selective binding for benzodiazepine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 303.
  • a polynucleotide or salt thereof which selectively binds to diazepam comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 304 or a fragment thereof which retains selective binding for benzodiazepine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 305.
  • a polynucleotide or salt thereof which selectively binds to diazepam comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 306 or a fragment thereof which retains selective binding for benzodiazepine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 307.
  • a polynucleotide or salt thereof which selectively binds to diazepam comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 308 or a fragment thereof which retains selective binding for benzodiazepine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 309.
  • a polynucleotide or salt thereof which selectively binds to diazepam comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 310 or a fragment thereof which retains selective binding for benzodiazepine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 311.
  • a polynucleotide or salt thereof which selectively binds to diazepam comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 312 or a fragment thereof which retains selective binding for benzodiazepine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 313.
  • a polynucleotide or salt thereof which selectively binds to diazepam comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 314 or a fragment thereof which retains selective binding for benzodiazepine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 315.
  • a polynucleotide or salt thereof which selectively binds to diazepam comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 316 or a fragment thereof which retains selective binding for benzodiazepine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 317.
  • a polynucleotide or salt thereof which selectively binds to diazepam comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 318 or a fragment thereof which retains selective binding for benzodiazepine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 319.
  • a polynucleotide or salt thereof which comprises or consists in a consensus sequence defined by SEQ ID NO: 542, which polynucleotide or salt thereof selectively binds benzodiazepine.
  • the polynucleotide or salt thereof which selectively binds benzodiazepine comprises or consists in a sequence selected from SEQ ID Nos: 292, 308, 312, 314, 316 and 318.
  • a polynucleotide or salt thereof which comprises or consists in a consensus sequence defined by SEQ ID NO: 543, which polynucleotide or salt thereof selectively binds benzodiazepine.
  • the polynucleotide or salt thereof which selectively binds benzodiazepine comprises or consists in a sequence selected from SEQ ID Nos: 294, 296, 298, 300 and 306.
  • a polynucleotide or salt thereof which comprises or consists in a consensus sequence defined by SEQ ID NO: 544, which polynucleotide or salt thereof selectively binds benzodiazepine.
  • the polynucleotide or salt thereof which selectively binds benzodiazepine comprises or consists in a sequence selected from SEQ ID Nos: 302, 304 and 310.
  • aptamer sequences which are highly selective for AMB-FUBINACA.
  • these aptamers display highly selective binding affinity for AMB-FUBINACA, with minimal cross- reactivity to other drug targets.
  • a polynucleotide or salt thereof comprising the sequence ATACGAGCTTGTTCAATA[Y]TGATAGTAAGAGCAATC (SEQ ID NO: 584), where Y is any sequence comprising a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to any one of SEQ ID Nos: 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 355, 357, 359, 361, 363, 365, 367, 369 and 371, which polynuceotide or salt thereof selectively binds to AMB-FUBINACA.
  • a polynucleotide or salt thereof comprising the sequence ATACGAGCTTGTTCAATA[Y]TGATAGTAAGAGCAATC (SEQ ID NO: 585), where Y is any sequence comprising any one of SEQ ID Nos: 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 355, 357, 359, 361, 363, 365, 367, 369 and 371, which polynuceotide or salt thereof selectively binds to AMB-FUBINACA.
  • a polynucleotide or salt thereof which selectively binds to AMB-FUBINACA comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 320 or a fragment thereof which retains selective binding for AMB-FUBINACA.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 321.
  • a polynucleotide or salt thereof which selectively binds to AMB-FUBINACA comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 322 or a fragment thereof which retains selective binding for AMB-FUBINACA.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 323.
  • a polynucleotide or salt thereof which selectively binds to AMB-FUBINACA comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 324 or a fragment thereof which retains selective binding for AMB-FUBINACA.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 325.
  • a polynucleotide or salt thereof which selectively binds to AMB-FUBINACA comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 326 or a fragment thereof which retains selective binding for AMB-FUBINACA.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 327.
  • a polynucleotide or salt thereof which selectively binds to AMB-FUBINACA comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 328 or a fragment thereof which retains selective binding for AMB-FUBINACA.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 329.
  • a polynucleotide or salt thereof which selectively binds to AMB-FUBINACA comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 330 or a fragment thereof which retains selective binding for AMB-FUBINACA.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 331.
  • a polynucleotide or salt thereof which selectively binds to AMB-FUBINACA comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 332 or a fragment thereof which retains selective binding for AMB-FUBINACA.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 333.
  • a polynucleotide or salt thereof which selectively binds to AMB-FUBINACA comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 334 or a fragment thereof which retains selective binding for AMB-FUBINACA.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 335.
  • a polynucleotide or salt thereof which selectively binds to AMB-FUBINACA comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 336 or a fragment thereof which retains selective binding for AMB-FUBINACA.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 337.
  • a polynucleotide or salt thereof which selectively binds to AMB-FUBINACA comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 338 or a fragment thereof which retains selective binding for AMB-FUBINACA.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 339.
  • a polynucleotide or salt thereof which selectively binds to AMB-FUBINACA comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 340 or a fragment thereof which retains selective binding for AMB-FUBINACA.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 341.
  • a polynucleotide or salt thereof which selectively binds to AMB-FUBINACA comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 342 or a fragment thereof which retains selective binding for AMB-FUBINACA.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 343.
  • a polynucleotide or salt thereof which selectively binds to AMB-FUBINACA comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 344 or a fragment thereof which retains selective binding for AMB-FUBINACA.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 345.
  • a polynucleotide or salt thereof which selectively binds to AMB-FUBINACA comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 346 or a fragment thereof which retains selective binding for AMB-FUBINACA.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 347.
  • a polynucleotide or salt thereof which selectively binds to AMB-FUBINACA comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 348 or a fragment thereof which retains selective binding for AMB-FUBINACA.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 349.
  • a polynucleotide or salt thereof which selectively binds to AMB-FUBINACA comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 350 or a fragment thereof which retains selective binding for AMB-FUBINACA.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 351.
  • a polynucleotide or salt thereof which selectively binds to AMB-FUBINACA comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 352 or a fragment thereof which retains selective binding for AMB-FUBINACA.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 353.
  • a polynucleotide or salt thereof which selectively binds to AMB-FUBINACA comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 354 or a fragment thereof which retains selective binding for AMB-FUBINACA.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 355.
  • a polynucleotide or salt thereof which selectively binds to AMB-FUBINACA comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 356 or a fragment thereof which retains selective binding for AMB-FUBINACA.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 357.
  • a polynucleotide or salt thereof which selectively binds to AMB-FUBINACA comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 358 or a fragment thereof which retains selective binding for AMB-FUBINACA.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 359.
  • a polynucleotide or salt thereof which selectively binds to AMB-FUBINACA comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 360 or a fragment thereof which retains selective binding for AMB-FUBINACA.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 361.
  • a polynucleotide or salt thereof which selectively binds to AMB-FUBINACA comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 362 or a fragment thereof which retains selective binding for AMB-FUBINACA.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 363.
  • a polynucleotide or salt thereof which selectively binds to AMB-FUBINACA comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 364 or a fragment thereof which retains selective binding for AMB-FUBINACA.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 365.
  • a polynucleotide or salt thereof which selectively binds to AMB-FUBINACA comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 366 or a fragment thereof which retains selective binding for AMB-FUBINACA.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 367.
  • a polynucleotide or salt thereof which selectively binds to AMB-FUBINACA comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 368 or a fragment thereof which retains selective binding for AMB-FUBINACA.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 369.
  • a polynucleotide or salt thereof which selectively binds to AMB-FUBINACA comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 370 or a fragment thereof which retains selective binding for AMB-FUBINACA.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 371.
  • a polynucleotide or salt thereof which comprises or consists in a consensus sequence defined by SEQ ID NO: 545, which polynucleotide or salt thereof selectively binds AMB-FUBINACA.
  • the polynucleotide or salt thereof which selectively binds AMB-FUBINACA comprises or consists in a sequence selected from SEQ ID Nos: 330, 338, 340, 342, 344 and 366.
  • a polynucleotide or salt thereof which comprises or consists in a consensus sequence defined by SEQ ID NO: 546, which polynucleotide or salt thereof selectively binds AMB-FUBINACA.
  • the polynucleotide or salt thereof which selectively binds AMB-FUBINACA comprises or consists in a sequence selected from SEQ ID Nos: 322, 354, 356, 358 and 368.
  • a polynucleotide or salt thereof which comprises or consists in a consensus sequence defined by SEQ ID NO: 547, which polynucleotide or salt thereof selectively binds AMB-FUBINACA.
  • the polynucleotide or salt thereof which selectively binds AMB-FUBINACA comprises or consists in a sequence selected from SEQ ID Nos: 326, 328, 334, 360, 362 and 364.
  • a polynucleotide or salt thereof which comprises or consists in a consensus sequence defined by SEQ ID NO: 548, which polynucleotide or salt thereof selectively binds AMB-FUBINACA.
  • the polynucleotide or salt thereof which selectively binds AMB-FUBINACA comprises or consists in a sequence selected from SEQ ID Nos: 332, 348, 350, 352 and 370.
  • a polynucleotide or salt thereof which comprises or consists in a consensus sequence defined by SEQ ID NO: 549, which polynucleotide or salt thereof selectively binds AMB-FUBINACA.
  • the polynucleotide or salt thereof which selectively binds AMB-FUBINACA comprises or consists in a sequence selected from SEQ ID Nos: 336, 324 and 346.
  • aptamer sequences which are highly selective for AM2201.
  • these aptamers display highly selective binding affinity for AM2201, with minimal cross-reactivity to other drug targets.
  • a polynucleotide or salt thereof comprising the sequence ATACGAGCTTGTTCAATA[Y]TGATAGTAAGAGCAATC (SEQ ID NO: 587), where Y is any sequence comprising a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to any one of SEQ ID Nos: 373, 375, 377, 379, 381, 383, 385, 387, 389, 391, 393, 395, 397, 399, 401, 403, 405, 407, 409, 411, 413, 415, 417 and 419, which polynuceotide or salt thereof selectively binds to AM2201.
  • a polynucleotide or salt thereof comprising the sequence ATACGAGCTTGTTCAATA[Y]TGATAGTAAGAGCAATC (SEQ ID NO: 588), where Y is any sequence comprising any one of SEQ ID Nos: 373, 375, 377, 379, 381, 383, 385, 387, 389, 391, 393, 395, 397, 399, 401, 403, 405, 407, 409, 411, 413, 415, 417 and 419, which polynuceotide or salt thereof selectively binds to AM2201.
  • a polynucleotide or salt thereof which selectively binds to AM2201 comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 372 or a fragment thereof which retains selective binding for AM2201.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 373.
  • a polynucleotide or salt thereof which selectively binds to AM2201 comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 374 or a fragment thereof which retains selective binding for AM2201.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 375.
  • a polynucleotide or salt thereof which selectively binds to AM2201 comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 376 or a fragment thereof which retains selective binding for AM2201.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 377.
  • a polynucleotide or salt thereof which selectively binds to AM2201 comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 378 or a fragment thereof which retains selective binding for AM2201.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 379.
  • a polynucleotide or salt thereof which selectively binds to AM2201 comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 380 or a fragment thereof which retains selective binding for AM2201.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 381.
  • a polynucleotide or salt thereof which selectively binds to AM2201 comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 382 or a fragment thereof which retains selective binding for AM2201.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 383.
  • a polynucleotide or salt thereof which selectively binds to AM2201 comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 384 or a fragment thereof which retains selective binding for AM2201.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 385.
  • a polynucleotide or salt thereof which selectively binds to AM2201 comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 386 or a fragment thereof which retains selective binding for AM2201.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 387.
  • a polynucleotide or salt thereof which selectively binds to AM2201 comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 388 or a fragment thereof which retains selective binding for AM2201.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 389.
  • a polynucleotide or salt thereof which selectively binds to AM2201 comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 390 or a fragment thereof which retains selective binding for AM2201.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 391.
  • a polynucleotide or salt thereof which selectively binds to AM2201 comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 392 or a fragment thereof which retains selective binding for AM2201.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 393.
  • a polynucleotide or salt thereof which selectively binds to AM2201 comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 394 or a fragment thereof which retains selective binding for AM2201.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 395.
  • a polynucleotide or salt thereof which selectively binds to AM2201 comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 396 or a fragment thereof which retains selective binding for AM2201.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 397.
  • a polynucleotide or salt thereof which selectively binds to AM2201 comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 398 or a fragment thereof which retains selective binding for AM2201.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 399.
  • a polynucleotide or salt thereof which selectively binds to AM2201 comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 400 or a fragment thereof which retains selective binding for AM2201.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 401.
  • a polynucleotide or salt thereof which selectively binds to AM2201 comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 402 or a fragment thereof which retains selective binding for AM2201.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 403.
  • a polynucleotide or salt thereof which selectively binds to AM2201 comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 404 or a fragment thereof which retains selective binding for AM2201.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 405.
  • a polynucleotide or salt thereof which selectively binds to AM2201 comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 406 or a fragment thereof which retains selective binding for AM2201.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 407.
  • a polynucleotide or salt thereof which selectively binds to AM2201 comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 408 or a fragment thereof which retains selective binding for AM2201.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 409.
  • a polynucleotide or salt thereof which selectively binds to AM2201 comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 410 or a fragment thereof which retains selective binding for AM2201.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 411.
  • a polynucleotide or salt thereof which selectively binds to AM2201 comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 412 or a fragment thereof which retains selective binding for AM2201.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 413.
  • a polynucleotide or salt thereof which selectively binds to AM2201 comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 414 or a fragment thereof which retains selective binding for AM2201.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 415.
  • a polynucleotide or salt thereof which selectively binds to AM2201 comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 416 or a fragment thereof which retains selective binding for AM2201.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 417.
  • a polynucleotide or salt thereof which selectively binds to AM2201 comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 418 or a fragment thereof which retains selective binding for AM2201.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 419.
  • AM2201 aptamers disclosed herein using Clustal Omega. The results from these analyses is presented in Figure 19 where the level of structural (i.e. sequence) dis/similarity between the different aptamers in this drug class is pictorily illustrated.
  • the AM2201 aptamer designated AM01 and AM06 share a higher degree of structural similarity compared to (e.g.) AM09.
  • AM01, AM06 and AM09 collectively share a higher degree of structural similarity compared to (e.g.) AM11, AM12, AM13, AM02 and AM14.
  • a polynucleotide or salt thereof which comprises or consists in a consensus sequence defined by SEQ ID NO: 550, which polynucleotide or salt thereof selectively binds AM2201.
  • the polynucleotide or salt thereof which selectively binds AM2201 comprises or consists in a sequence selected from SEQ ID Nos: 374, 380, 382, 386, 388 and 418.
  • polynucleotide or salt thereof which comprises or consists in a consensus sequence defined by SEQ ID NO: 551, which polynucleotide or salt thereof selectively binds AM2201.
  • the polynucleotide or salt thereof which selectively binds AM2201 comprises or consists in a sequence selected from SEQ ID Nos: 384, 406, 408 and 410.
  • polynucleotide or salt thereof which comprises or consists in a consensus sequence defined by SEQ ID NO: 552, which polynucleotide or salt thereof selectively binds AM2201.
  • the polynucleotide or salt thereof which selectively binds AM2201 comprises or consists in a sequence selected from SEQ ID Nos: 390, 412, 414 and 416.
  • a polynucleotide or salt thereof which comprises or consists in a consensus sequence defined by SEQ ID NO: 553, which polynucleotide or salt thereof selectively binds AM2201.
  • the polynucleotide or salt thereof which selectively binds AM2201 comprises or consists in a sequence selected from SEQ ID Nos: 378, 400, 402 and 404.
  • a polynucleotide or salt thereof which comprises or consists in a consensus sequence defined by SEQ ID NO: 554, which polynucleotide or salt thereof selectively binds AM2201.
  • the polynucleotide or salt thereof which selectively binds AM2201 comprises or consists in a sequence selected from SEQ ID Nos: 376, 392, 394, 396 and 398.
  • aptamer sequences which are highly selective for oxycodone.
  • these aptamers display highly selective binding affinity for oxycodone, with minimal cross-reactivity to other drug targets.
  • a polynucleotide or salt thereof comprising the sequence ATACGAGCTTGTTCAATA[Y]TGATAGTAAGAGCAATC (SEQ ID NO: 590), where Y is any sequence comprising a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to any one of SEQ ID Nos: 421, 423, 425, 427, 429, 431, 433, 435, 437, 439, 441, 443, 445, 447, 449, 451, 453, 455, 457, 459, 461, 463, 465 and 467, which polynuceotide or salt thereof selectively binds to oxycodone.
  • a polynucleotide or salt thereof comprising the sequence ATACGAGCTTGTTCAATA[Y]TGATAGTAAGAGCAATC (SEQ ID NO: 591), where Y is any sequence comprising any one of SEQ ID Nos: 421, 423, 425, 427, 429, 431, 433, 435, 437, 439, 441, 443, 445, 447, 449, 451, 453, 455, 457, 459,
  • a polynucleotide or salt thereof which selectively binds to oxycodone comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 279 or a fragment thereof which retains selective binding for oxycodone.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 280.
  • a polynucleotide or salt thereof which selectively binds to oxycodone comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 420 or a fragment thereof which retains selective binding for oxycodone.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 421.
  • a polynucleotide or salt thereof which selectively binds to oxycodone comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 422 or a fragment thereof which retains selective binding for oxycodone.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 423.
  • a polynucleotide or salt thereof which selectively binds to oxycodone comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 424 or a fragment thereof which retains selective binding for oxycodone.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 425.
  • a polynucleotide or salt thereof which selectively binds to oxycodone comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 426 or a fragment thereof which retains selective binding for oxycodone.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 427.
  • a polynucleotide or salt thereof which selectively binds to oxycodone comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 428 or a fragment thereof which retains selective binding for oxycodone.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 429.
  • a polynucleotide or salt thereof which selectively binds to oxycodone comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 430 or a fragment thereof which retains selective binding for oxycodone.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 431.
  • a polynucleotide or salt thereof which selectively binds to oxycodone comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 432 or a fragment thereof which retains selective binding for oxycodone.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 433.
  • a polynucleotide or salt thereof which selectively binds to oxycodone comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 434 or a fragment thereof which retains selective binding for oxycodone.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 435.
  • a polynucleotide or salt thereof which selectively binds to oxycodone comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 436 or a fragment thereof which retains selective binding for oxycodone.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 437.
  • a polynucleotide or salt thereof which selectively binds to oxycodone comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 438 or a fragment thereof which retains selective binding for oxycodone.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 439.
  • a polynucleotide or salt thereof which selectively binds to oxycodone comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 440 or a fragment thereof which retains selective binding for oxycodone.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 441.
  • a polynucleotide or salt thereof which selectively binds to oxycodone comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 442 or a fragment thereof which retains selective binding for oxycodone.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 443.
  • a polynucleotide or salt thereof which selectively binds to oxycodone comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 444 or a fragment thereof which retains selective binding for oxycodone.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 445.
  • a polynucleotide or salt thereof which selectively binds to oxycodone comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 446 or a fragment thereof which retains selective binding for oxycodone.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 447.
  • a polynucleotide or salt thereof which selectively binds to oxycodone comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 448 or a fragment thereof which retains selective binding for oxycodone.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 449.
  • a polynucleotide or salt thereof which selectively binds to oxycodone comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 450 or a fragment thereof which retains selective binding for oxycodone.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 451.
  • a polynucleotide or salt thereof which selectively binds to oxycodone comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 452 or a fragment thereof which retains selective binding for oxycodone.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 453.
  • a polynucleotide or salt thereof which selectively binds to oxycodone comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 454 or a fragment thereof which retains selective binding for oxycodone.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 455.
  • a polynucleotide or salt thereof which selectively binds to oxycodone comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 456 or a fragment thereof which retains selective binding for oxycodone.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 457.
  • a polynucleotide or salt thereof which selectively binds to oxycodone comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 458 or a fragment thereof which retains selective binding for oxycodone.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 459.
  • a polynucleotide or salt thereof which selectively binds to oxycodone comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 460 or a fragment thereof which retains selective binding for oxycodone.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 461.
  • a polynucleotide or salt thereof which selectively binds to oxycodone comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 462 or a fragment thereof which retains selective binding for oxycodone.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 463.
  • a polynucleotide or salt thereof which selectively binds to oxycodone comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 464 or a fragment thereof which retains selective binding for oxycodone.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 465.
  • a polynucleotide or salt thereof which selectively binds to oxycodone comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 466 or a fragment thereof which retains selective binding for oxycodone.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 467.
  • a polynucleotide or salt thereof which comprises or consists in a consensus sequence defined by SEQ ID NO: 555, which polynucleotide or salt thereof selectively binds oxycodone.
  • the polynucleotide or salt thereof which selectively binds oxycodone comprises or consists in a sequence selected from SEQ ID Nos: 420, 462, 422, 424, 426, 428 and 440.
  • a polynucleotide or salt thereof which comprises or consists in a consensus sequence defined by SEQ ID NO: 556, which polynucleotide or salt thereof selectively binds oxycodone.
  • the polynucleotide or salt thereof which selectively binds oxycodone comprises or consists in a sequence selected from SEQ ID Nos: 466, 450, 452, 454, 456, 458 and 460.
  • a polynucleotide or salt thereof which comprises or consists in a consensus sequence defined by SEQ ID NO: 557, which polynucleotide or salt thereof selectively binds oxycodone.
  • the polynucleotide or salt thereof which selectively binds oxycodone comprises or consists in a sequence selected from SEQ ID Nos: 464, 442, 444, 446 and 448.
  • a polynucleotide or salt thereof which comprises or consists in a consensus sequence defined by SEQ ID NO: 558, which polynucleotide or salt thereof selectively binds oxycodone.
  • the polynucleotide or salt thereof which selectively binds oxycodone comprises or consists in a sequence selected from SEQ ID Nos: 430, 434 and 438.
  • aptamer sequences which are highly selective for ketamine.
  • these aptamers display highly selective binding affinity for ketamine, with minimal cross-reactivity to other drug targets.
  • a polynucleotide or salt thereof comprising the sequence ATACGAGCTTGTTCAATA[Y]TGATAGTAAGAGCAATC (SEQ ID NO: 593), where Y is any sequence comprising a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to any one of SEQ ID Nos: 469, 471, 473, 475, 477, 479, 481, 483, 485, 487, 489, 491, 493, 495, 497, 499, 501, 503, 505, 507, 509, 511, 513, 515, 517, 519, 521 and 523, which polynuceotide or salt thereof selectively binds to ketamine.
  • a polynucleotide or salt thereof comprising the sequence ATACGAGCTTGTTCAATA[Y]TGATAGTAAGAGCAATC (SEQ ID NO: 594), where Y is any sequence comprising any one of SEQ ID Nos: 469, 471, 473, 475, 477, 479, 481, 483, 485, 487, 489, 491, 493, 495, 497, 499, 501, 503, 505, 507, 509, 511, 513, 515, 517, 519, 521 and 523, which polynuceotide or salt thereof selectively binds to ketamine.
  • a polynucleotide or salt thereof which selectively binds to ketamine comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 468 or a fragment thereof which retains selective binding for ketamine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 469.
  • a polynucleotide or salt thereof which selectively binds to ketamine comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 468 or a fragment thereof which retains selective binding for ketamine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 469.
  • a polynucleotide or salt thereof which selectively binds to ketamine comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 470 or a fragment thereof which retains selective binding for ketamine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 471.
  • a polynucleotide or salt thereof which selectively binds to ketamine comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 472 or a fragment thereof which retains selective binding for ketamine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 473.
  • a polynucleotide or salt thereof which selectively binds to ketamine comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 474 or a fragment thereof which retains selective binding for ketamine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 475.
  • a polynucleotide or salt thereof which selectively binds to ketamine comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 476 or a fragment thereof which retains selective binding for ketamine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 477.
  • a polynucleotide or salt thereof which selectively binds to ketamine comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 478 or a fragment thereof which retains selective binding for ketamine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 479.
  • a polynucleotide or salt thereof which selectively binds to ketamine comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 480 or a fragment thereof which retains selective binding for ketamine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 481.
  • a polynucleotide or salt thereof which selectively binds to ketamine comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 482 or a fragment thereof which retains selective binding for ketamine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 483.
  • a polynucleotide or salt thereof which selectively binds to ketamine comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 484 or a fragment thereof which retains selective binding for ketamine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 485.
  • a polynucleotide or salt thereof which selectively binds to ketamine comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 486 or a fragment thereof which retains selective binding for ketamine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 487.
  • a polynucleotide or salt thereof which selectively binds to ketamine comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 488 or a fragment thereof which retains selective binding for ketamine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 489.
  • a polynucleotide or salt thereof which selectively binds to ketamine comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 490 or a fragment thereof which retains selective binding for ketamine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 491.
  • a polynucleotide or salt thereof which selectively binds to ketamine comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 492 or a fragment thereof which retains selective binding for ketamine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 493.
  • a polynucleotide or salt thereof which selectively binds to ketamine comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 494 or a fragment thereof which retains selective binding for ketamine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 495.
  • a polynucleotide or salt thereof which selectively binds to ketamine comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 496 or a fragment thereof which retains selective binding for ketamine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 497.
  • a polynucleotide or salt thereof which selectively binds to ketamine comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 498 or a fragment thereof which retains selective binding for ketamine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 499.
  • a polynucleotide or salt thereof which selectively binds to ketamine comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 500 or a fragment thereof which retains selective binding for ketamine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 501.
  • a polynucleotide or salt thereof which selectively binds to ketamine comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 502 or a fragment thereof which retains selective binding for ketamine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 503.
  • a polynucleotide or salt thereof which selectively binds to ketamine comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 504 or a fragment thereof which retains selective binding for ketamine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 505.
  • a polynucleotide or salt thereof which selectively binds to ketamine comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 506 or a fragment thereof which retains selective binding for ketamine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 507.
  • a polynucleotide or salt thereof which selectively binds to ketamine comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 508 or a fragment thereof which retains selective binding for ketamine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 509.
  • a polynucleotide or salt thereof which selectively binds to ketamine comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 510 or a fragment thereof which retains selective binding for ketamine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 511.
  • a polynucleotide or salt thereof which selectively binds to ketamine comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 512 or a fragment thereof which retains selective binding for ketamine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 513.
  • a polynucleotide or salt thereof which selectively binds to ketamine comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 514 or a fragment thereof which retains selective binding for ketamine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 515.
  • a polynucleotide or salt thereof which selectively binds to ketamine comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 516 or a fragment thereof which retains selective binding for ketamine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 517.
  • a polynucleotide or salt thereof which selectively binds to ketamine comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 518 or a fragment thereof which retains selective binding for ketamine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 519.
  • a polynucleotide or salt thereof which selectively binds to ketamine comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 520 or a fragment thereof which retains selective binding for ketamine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 521.
  • a polynucleotide or salt thereof which selectively binds to ketamine comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 522 or a fragment thereof which retains selective binding for ketamine.
  • the fragment comprises or consists in a sequence defined by SEQ ID NO: 523.
  • a polynucleotide or salt thereof which comprises or consists in a consensus sequence defined by SEQ ID NO: 559, which polynucleotide or salt thereof selectively binds ketamine.
  • the polynucleotide or salt thereof which selectively binds ketamine comprises or consists in a sequence selected from SEQ ID Nos: 500, 506, 510, 512, 514 and 522.
  • a polynucleotide or salt thereof which comprises or consists in a consensus sequence defined by SEQ ID NO: 560, which polynucleotide or salt thereof selectively binds ketamine.
  • the polynucleotide or salt thereof which selectively binds ketamine comprises or consists in a sequence selected from SEQ ID Nos: 486, 488, 492 and 508.
  • a polynucleotide or salt thereof which comprises or consists in a consensus sequence defined by SEQ ID NO: 561, which polynucleotide or salt thereof selectively binds ketamine.
  • the polynucleotide or salt thereof which selectively binds ketamine comprises or consists in a sequence selected from SEQ ID Nos: 472, 480 and 496.
  • a polynucleotide or salt thereof which comprises or consists in a consensus sequence defined by SEQ ID NO: 562, which polynucleotide or salt thereof selectively binds ketamine.
  • the polynucleotide or salt thereof which selectively binds ketamine comprises or consists in a sequence selected from SEQ ID Nos: 490, 498 and 516.
  • a polynucleotide or salt thereof which comprises or consists in a consensus sequence defined by SEQ ID NO: 563, which polynucleotide or salt thereof selectively binds ketamine.
  • the polynucleotide or salt thereof which selectively binds ketamine comprises or consists in a sequence selected from SEQ ID Nos: 468, 474 and 476.
  • a polynucleotide or salt thereof which comprises or consists in a consensus sequence defined by SEQ ID NO: 564, which polynucleotide or salt thereof selectively binds ketamine.
  • the polynucleotide or salt thereof which selectively binds ketamine comprises or consists in a sequence selected from SEQ ID Nos: 470, 478 and 482.
  • the aptamer/polynucleotide sequences disclosed herein may be useful in performing assay methods for the detection of illicit drugs. This may include, for example, determining the drug status of an individual at a road-side testing station or in quality care governance for employees operating heavy machinary, (e.g.) in the mining industry.
  • a method for determining the drug status of an individual comprising combining a test sample obtained from the individual with a test kit or article of manufacture described herein, and using the results obtained from performance of the test kits to determine the drug status of an individual.
  • the test kit is configured to detect multiple drug targets from discrete drug classes to determine concurrent drug abuse. For example, detection of tetrahydrocannabinol in respect of cannabis use, tested alongside cocaine. Both are widely used recreational drugs, which are often abused by an individual at the same time.
  • the method comprising detecting an illicit drug from a test sample.
  • the illicit drug includes, but is not limited to, methamphetamine, tetrahydrocannabinol, cocaine, morphine, 3,4-methylenedioxymethamphetamine, benzodiazepine, AMB-FUBINACA, AM2201, oxycodone and ketamine, and includes structural analogs of methamphetamine, tetrahydrocannabinol, cocaine, morphine, 3,4-methylenedioxymethamphetamine, benzodiazepine, AMB-FUBINACA, AM2201, oxycodone and ketamine.
  • detection of drugs of abuse is typically achieved by contacting a test sample with a polynucleotide (aptamer) selective for a drug target, and measuring a binding interaction between the aptamer and drug target, if present in the test sample.
  • aptamer polynucleotide
  • a method for detecting the presence of methamphetamine in a test sample comprising the steps of:
  • a method for detecting the presence of tetrahydrocannabinol in a test sample comprising the steps of:
  • a method for detecting the presence of morphine in a test sample comprising the steps of:
  • a method for detecting the presence of benzodiazepine in a test sample comprising the steps of: (i) contacting a test sample with a polynucleotide or salt thereof comprising a sequence defined by any one of SEQ ID Nos: 292-319; and
  • a method for detecting the presence of AMB-FUBINACA in a test sample comprising the steps of:
  • a method for detecting the presence of AM2201 in a test sample comprising the steps of:
  • a method for detecting the presence of oxycodone in a test sample comprising the steps of:
  • a method for detecting the presence of ketamine in a test sample comprising the steps of:
  • a binding interaction between the polynucleotide and drug target is measured using a detection means.
  • detection means include, but are not limited, optical methods, spectroscopy, visible spectroscopy, Raman spectroscopy, UV spectroscopy, surface plasmon resonance, electrochemical methods, impedance, resistance, capacitance, mechanical sensing by changes in mass, changes in mechanical resonance, electrophoresis, gel electrophoresis, gel retardation, imaging, fluorescence, fluorescence resonance energy transfer.
  • test kit or article of manufacture suitable for performing a method or assay described herein, in particular for the detection of an illicit drug from a test sample.
  • test kit or article of manufacture for detecting the presence of methamphetamine in a test sample, the test kit or article of manufacture comprising a polynucleotide or salt thereof comprising a sequence defined by any one of SEQ ID Nos: 1-74, and optionally instructions for how to detect the presence of methamphetamine in the test sample.
  • test kit or article of manufacture for detecting the presence of tetrahydrocannabinol in a test sample, the test kit or article of manufacture comprising a polynucleotide or salt thereof comprising a sequence defined by any one of SEQ ID Nos: 75-136, and optionally instructions for how to detect the presence of tetrahydrocannabinol in the test sample.
  • test kit or article of manufacture for detecting the presence of cocaine in a test sample, the test kit or article of manufacture comprising a polynucleotide or salt thereof comprising a sequence defined by any one of SEQ ID Nos: 137-172, and optionally instructions for how to detect the presence of cocaine in the test sample.
  • test kit or article of manufacture for detecting the presence of 3,4-methylenedioxymethamphetamine in a test sample
  • the test kit or article of manufacture comprising a polynucleotide or salt thereof comprising a sequence defined by any one of SEQ ID Nos: 173-246, and optionally instructions for how to detect the presence of 3,4-methylenedioxymethamphetamine in the test sample.
  • test kit or article of manufacture for detecting the presence of morphine in a test sample, the test kit or article of manufacture comprising a polynucleotide or salt thereof comprising a sequence defined by any one of SEQ ID Nos: 247-291, and optionally instructions for how to detect the presence of morphine in the test sample.
  • test kit or article of manufacture for detecting the presence of benzodiazepine in a test sample, the test kit or article of manufacture comprising a polynucleotide or salt thereof comprising a sequence defined by any one of SEQ ID Nos: 292-319, and optionally instructions for how to detect the presence of benzodiazepine in the test sample.
  • test kit or article of manufacture for detecting the presence of AMB-FUBINACA in a test sample, the test kit or article of manufacture comprising a polynucleotide or salt thereof comprising a sequence defined by any one of SEQ ID Nos: 320-371, and optionally instructions for how to detect the presence of AMB-FUBINACA in the test sample.
  • test kit or article of manufacture for detecting the presence of AM2201 in a test sample, the test kit or article of manufacture comprising a polynucleotide or salt thereof comprising a sequence defined by any one of SEQ ID Nos: 372-419, and optionally instructions for how to detect the presence of AM2201 in the test sample.
  • test kit or article of manufacture for detecting the presence of oxycodone in a test sample, the test kit or article of manufacture comprising a polynucleotide or salt thereof comprising a sequence defined by any one of SEQ ID Nos: 420-467, and optionally instructions for how to detect the presence of oxycodone in the test sample.
  • test kit or article of manufacture for detecting the presence of oxycodone in a test sample, the test kit or article of manufacture comprising a polynucleotide or salt thereof comprising a sequence defined by any one of SEQ ID Nos: 420-467, and optionally instructions for how to detect the presence of oxycodone in the test sample.
  • test kit or article of manufacture for detecting the presence of ketamine in a test sample, the test kit or article of manufacture comprising a polynucleotide or salt thereof comprising a sequence defined by any one of SEQ ID Nos: 468-523, and optionally instructions for how to detect the presence of oxycodone in the test sample.
  • test kits or articles of manufacture optionally include instructions for how to perform the detection of an illicit drug from the test sample interrogated.
  • test kits and methods described herein may be performed on any test sample.
  • the test sample is derived from biological material selected from the group consisting of urine, saliva, stool, hair, tissues including, but not limited to, blood (plasma and serum), muscle, tumors, semen, etc.
  • the biological sample is derived from a human.
  • the test sample is derived from a food selected from the group consisting of vegetable, meat, beverage including but not limited to sports drink and milk, supplements including, but not limited to, food supplements and sports supplements, nutritional supplements, herbal extracts, etc.
  • the test sample is derived from a medication including, without limitation, a drug, a tonic, a syrup, a pill, a lozenge, a cream, a spray and a gel.
  • the sample is derived from the environment including, without limitation, a liquid, water, plastics and a mineral.
  • polynucleotide aptamers disclosed herein can reduce exonuclease degradation, increase half-life for diagnostic or therapeutic applications, and/or for other purposes.
  • Modification of the 3'end of the aptamer with inverted thymidine, deoxythymidine nucleotide, and polyethylene glycol (PEG) can reduce degradation of the oligonucleotide aptamer and increases stability of the aptamer.
  • PEG has an average molecular weight from about 20 to 80 kDa.
  • the phosphodiester linkages of the deoxyribose-phosphate backbone of the aptamer can also be modified to improve stability.
  • the aptamer is a polynucleotide comprising repeating units of the structure shown in Formula 1. Wavy lines demarcate one nucleotide and/or repeat unit from a neighboring nucleotide and/or repeat unit.
  • each repeat unit of Formula 1 has a deoxyribose moiety linked to one of the common nucleotide bases (B) , such as, adenosine, cytidine, guanosine, thymidine, and uridine.
  • the base (B) for each repeating unit is independent from the other repeat units.
  • the nucleotide sequences disclosed herein describe the order of appearance of bases (B) in an aptamer from the repeat unit on the 5'end of the aptamer to the 3'end of the aptamer.
  • L is a linker group that links the deoxyribose moiety of adjacent repeat units.
  • the L group is a phosphate group PO 4 H, which can exist as a salt or in a neutral protonated form.
  • the deoxyribose moiety together with the linker group forms the backbone of the aptamer, where the nucleotide base "B” varies independently of bonds and linkers between repeat units.
  • the majority of the linker groups (L) forming the repeat units of Formula 1 in the aptamer are phosphate groups.
  • a majority of the backbone of the aptamer can be referred to as a deoxyribose-phosphate backbone.
  • linker groups "L" other than phosphate can be incorporated into an oligonucleotide or aptamer to reduce or prevent degradation by nucleases.
  • L can be replaced with a group as shown in Formula 2, where X 1-4 are independently O or S.
  • X 2 and X 3 can be bonded to either the 3'carbon or the 5'carbon of a deoxyribose moiety.
  • X 1 is O and X 4 is O that can be either protonated or unprotonated.
  • one or more of X 2 and/or X 3 is S and X 1 and X 4 are O, where O can be either protonated or unprotonated.
  • the aptamer can be referred to as having a thioester linkage in the deoxyribose-phosphate backbone.
  • the linker group "L” is an amide-containing group as shown in Formula 3, where R can be selected from hydrogen substituted or unsubstituted C 1 -C 10 hydrocarbyl group.
  • a “hydrocarbon” or “hydrocarbyl” refers to organic compounds or radicals consisting exclusively of the elements carbon and hydrogen. Hydrocarbyl includes alkyl, alkenyl, alkynyl, and aryl moieties. Hydrocarbyl also includes alkyl, alkenyl, alkynyl, and aryl moieties substituted with other aliphatic, cyclic or aryl hydrocarbon groups, such as alkaryl, alkenaryl and alkynaryl.
  • the linker group "L” is a group having Formula 3, and the aptamer comprises amide linkage (s) in the deoxyribose-phosphate backbone.
  • the "NR" group of Formula 3 can be bonded to either the 3'carbon or the 5'carbon of a deoxyribose moiety.
  • R is methoxymethyl or methoxyethyl.
  • the aptamer has from about 14 to about 100 nucleotide bases and/or repeat units, or any number of nucleotide bases or repeating units between 14 to 100, such as between 20 to 50 nucleotide bases and/or repeating units. In some examples, the aptamer has from about 14 to about 50 nucleotide bases and/or repeat units. In some examples, the aptamer has from about 30 to about 35 nucleotide bases and/or repeat units.
  • the aptamer has from 14 to 100, 14 to 95, 14 to 90, 14 to 85, 14 to 80, 14 to 75, 14 to 70, 14 to 65, 14 to 60, 14 to 55, 14 to 50, 14 to 45, 14 to 40, 14 to 35, 14 to 30, 14 to 25, 14 to 20, 14 to 18, 14 to 16, 14 to 15 nucleotide bases and/or repeating units, inclusive.
  • the aptamer has from 20 to 100, 20 to 95, 20 to 90, 20 to 85, 20 to 80, 20 to 75, 20 to 70, 20 to 65, 20 to 60, 20 to 55, 20 to 50, 20 to 45, 20 to 40, 20 to 35, 20 to 30, 20 to 25 nucleotide bases and/or repeating units, inclusive. In some examples, the aptamer has from 25 to 100, 25 to 95, 25 to 90, 25 to 85, 25 to 80, 25 to 75, 25 to 70,
  • the aptamer has from 30 to 100, 30 to
  • the aptamer has from 35 to 100, 35 to 95, 35 to 90, 35 to 85, 35 to 80, 35 to 75, 35 to 70, 35 to 65, 35 to 60, 35 to 55, 35 to 50, 35 to 45, 35 to 40 nucleotide bases and/or repeating units, inclusive. In some examples, the aptamer has from 40 to 100, 40 to 95, 40 to 90, 40 to 85, 40 to 80, 40 to 75, 40 to 70, 40 to 65, 40 to 60, 40 to 55, 40 to 50, 40 to 45 nucleotide bases and/or repeating units, inclusive.
  • the aptamer has from 45 to 100, 45 to 95, 45 to 90, 45 to 85, 45 to 80, 45 to 75, 45 to 70, 45 to 65, 45 to 60, 45 to 55, 45 to 50 nucleotide bases and/or repeating units, inclusive. In some examples, the aptamer has from 50 to 100, 50 to 95, 50 to 90, 50 to 85, 50 to 80, 50 to 75, 50 to 70, 50 to 65, 50 to 60, 50 to 55 nucleotide bases and/or repeating units, inclusive.
  • the aptamer has from 55 to 100, 55 to 95, 55 to 90, 55 to 85, 55 to 80, 55 to 75, 55 to 70, 55 to 65, 55 to 60 nucleotide bases and/or repeating units, inclusive. In some examples, the aptamer has from 60 to 100, 60 to 95, 60 to 90, 60 to 85, 60 to 80, 60 to 75, 60 to 70, 60 to 65 nucleotide bases and/or repeating units, inclusive. In some examples, the aptamer has 14, 15, 16, 18, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 nucleotide bases and/or repeating units.
  • the aptamer comprises one or more repeating units having the linker "L” selected independently from Formulae 2 and 3.
  • the number of repeating units having the linker “L” selected independently from Formulae 2 and 3 is between 1 to 15 (inclusive), or any number there between, such as, for example, 1 to 10, 2 to 8, and 3 to 5, inclusive.
  • the number of repeating units having the linker "L” selected independently from Formulae 2 and 3 is between 1 to 15, 1 to 14, 1 to 12, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, inclusive.
  • the number of repeating units having the linker "L" selected independently from Formulae 2 and 3 is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, or 15.
  • Linker groups in repeat units not selected from Formulae 2 or 3 are phosphate.
  • the aptamer In some examples of the aptamer, about 10% to about 100% (or any percentages there between, such as about 20%to about 90%, about 30%to about 50%) of the repeat units have the linker "L" selected independently from Formulae 2 and 3. In some examples of the aptamer, about 10% to about 70%of the repeat units have the linker "L” selected independently from Formulae 2 and 3. In some examples of the aptamer, about 10% to about 50% of the repeat units have the linker "L” selected independently from Formulae 2 and 3. In some examples of the aptamer, about 10% to about 30% of the repeat units have the linker "L” selected independently from Formulae 2 and 3. In some examples of the aptamer, about 10% to about 20% of the repeat units have the linker "L” selected independently from Formulae 2 and 3. Linker groups in repeat units not selected from Formulae 2 or 3 are phosphate.
  • nucleases are exonucleases that degrade oligonucleotides from the 5' or 3'end.
  • a linker group L selected from Formulae 2 or 3 is located within about 5 repeat units from the 5' or the 3'end of the aptamer.
  • a linker group L selected from Formulae 2 or 3 is located within about 3 repeat units from the 5' or the 3'end of the aptamer.
  • a linker group L selected from Formulae 2 or 3 is located within 3 repeat units from the 5' or the 3'end of the aptamer.
  • a linker group L selected from Formulae 2 or 3 is located within 2 repeat units from the 5' or the 3' end of the aptamer. In some examples, a linker group L selected from Formulae 2 or 3 is part of the repeat unit on the 5' or the 3'end of the aptamer.
  • Degradation of the aptamers can also be reduced by the inclusion of modified nucleotide bases (B) .
  • the pyrimidine nucleotide bases, cytosine, thymine and uracil can be replaced with alkylated pyrimidines.
  • alkylated pyrimidines examples include pseudoisocytosine; N4, N4-ethanocytosine; 4-acetylcytosine, 5- (carboxyhydroxylmethyl) uracil; 5-fluorouracil; 5-bromouracil; 5-carboxymethylaminomethyl-2-thiouracil; 5- carboxymethylaminomethyl uracil; dihydrouracil; 1-methylpseudouracil; 3-methylcytosine; 5-methylcytosine; 5-methylaminomethyl uracil; 5-methoxy amino methyl-2-thiouracil; 5- methoxycarbonylmethyluracil; 5-methoxyuracil; uracil-5-oxyacetic acid methyl ester; pseudouracil; 2-thiocytosine; 5-methyl-2 thiouracil, 2-thiouracil; 4-thiouracil; 5-methyluracil; N-uracil-5-oxyacetic acid methylester; uracil 5-oxyacetic acid; 2-thiocyto
  • the purine nucleotide bases, adenine and guanine can be replaced by alkylated purines.
  • alkylated purines include 8-hydroxy-N6-methyladenine; inosine; N6-isopentyl-adenine; 1-methyladenine; 1- methylguanine; 2, 2-dimethylguanine; 2-methyladenine; 2-methylguanine; N6- methyladenine; 7-methylguanine; 2-methylthio-N6-isopentenyladenine; and 1- methylguanine.
  • At least one deoxyribose or ribose of the nucleic acid aptamer is replaced with a morpholine ring.
  • at least one phosphorothioate or phosphodiester linkage of the nucleic acid aptamer is replaced with phosphorodiamidate.
  • the nucleotides that can be substituted for natural nucleotides of DNA have a base moiety such as inosine, 5-fluorouracil, 5-bromouracil, hypoxanthine, 1-methylguanine, 5- methylcytosine, or tritylated bases.
  • the sugar moiety of the nucleotide in a sequence can also be modified with a group such as arabinose, xylulose, or hexose.
  • the adenine, cytosine, guanine, thymine, and uracil bases of the nucleotides can be modified with acetyl, methyl, hydroxyl, and thio groups. Sequences containing nucleotide substitutions, deletions, and/or insertions can be prepared and tested using standard techniques known in the art.
  • the disclosed polynucleotide aptamers may be modified to promote an association with a substrate (e.g. particle or solid surface).
  • a substrate e.g. particle or solid surface
  • the disclosed polynucleotide aptamers may be coupled or conjugated to one or more chemical entities or moieties to aid functionalization (e.g.) in an illicit drug detection assay involving a substrate including but not limited to a particle or solid surface.
  • detection labels may be directly associated with or coupled to a polynucleotide aptamer.
  • a detection label is any molecule that can be associated with an aptamer, directly or indirectly, and which results in a measurable, detectable signal, either directly or indirectly. Many such labels for are known to those of skill in the art.
  • detection labels include radioisotopes (e.g., 3 H, 14 C, 35 S, 125 I, 131 I), fluorescent labels (e.g., methylene blue, FITC, rhodamine, lanthanide phosphors), luminescent labels such as luminol; enzymatic labels (e.g., horseradish peroxidase, beta-galactosidase, luciferase, alkaline phosphatase, acetylcholinestease), biotinyl groups (which can be detected by marked avidin, e.g., streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or calorimetric methods), predetermined polypeptide epitopes recognized by a secondary reporter (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags).
  • a secondary reporter e.g., leucine zipper pair sequences, binding sites for secondary antibodies,
  • the detection labels associated with the aptamers contemplated by the present invention can be part of, and detectable with, enzyme-linked detection systems.
  • Enzyme- linked detection generally involves an enzyme as a label or tag on a component where the presence of the enzyme (and thus of the analyte with which the enzyme is associated) is detected by having the enzyme convert an enzymatic substrate into a form that produces a detectable signal.
  • analytes labeled or associated with alkaline phosphatase can be detected by adding the chemiluminescent substrate CSPD (Tropix, Inc). The fluorescent reaction product can then be detected.
  • Preferred forms of detection labels are enzymes, such as alkaline phosphatases and peroxidases, for use in an enzyme-linked detection system.
  • fluorescent labels examples include fluorescein (FITC), 5, 6-carboxy methyl fluorescein, Texas red, nitrobenz-2-oxa-1, 3-diazol-4-yl (NBD), coumarin, dansyl chloride, rhodamine, 4'-6-diamidino-2-phenylinodole (DAPI), and the cyanine dyes Cy3, Cy3.5, Cy5, Cy5.5 and Cy7.
  • Preferred fluorescent labels are fluorescein (5-carboxyfluorescein-N- hydroxysuccinimide ester) and rhodamine (5, 6-tetramethyl rhodamine) .
  • Preferred fluorescent labels for combinatorial multicolor coding are FITC and the cyanine dyes Cy3, Cy3.5, Cy5, Cy5.5 and Cy7.
  • the absorption and emission maxima, respectively, for these fluors are: FITC (490 nm; 520 nm), Cy3 (554 nm; 568 nm), Cy3.5 (581 nm; 588 nm), Cy5 (652 nm: 672 nm), Cy5.5 (682 nm; 703 nm) and Cy7 (755 nm; 778 nm), thus allowing their simultaneous detection.
  • Detection labels such as biotin can be subsequently detected using sensitive methods well-known in the art.
  • biotin can be detected using streptavidin-alkaline phosphatase conjugate (Tropix, Inc), which is bound to the biotin and subsequently detected by chemiluminescence of suitable substrates (for example, chemiluminescent substrate CSPD: disodium, 3- (4-methoxyspiro- [1, 2, -dioxetane-3-2'- (5'-chloro) tricyclo [3.3.1.1 3, 7] decane] -4-yl) phenyl phosphate; Tropix, Inc. ).
  • suitable substrates for example, chemiluminescent substrate CSPD: disodium, 3- (4-methoxyspiro- [1, 2, -dioxetane-3-2'- (5'-chloro) tricyclo [3.3.1.1 3, 7] decane] -4-yl) phenyl phosphate; Tropix, Inc. ).
  • detection labels that combine two or more of these detection labels are also contemplated by the present invention. Any of the known detection labels can be used with the disclosed detection agents. Methods for detecting and measuring signals generated by detection labels are also known to those of skill in the art. For example, radioactive isotopes can be detected by scintillation counting or direct visualization; fluorescent molecules can be detected with fluorescent spectrophotometers; phosphorescent molecules can be detected with a spectrophotometer or directly visualized with a camera; enzymes can be detected by detection or visualization of the product of a reaction catalyzed by the enzyme; antibodies can be detected by detecting a secondary detection element coupled to the antibody. Such methods can be used directly in the disclosed method of amplification and detection. As used herein, detection agents are molecules which interact with amplified nucleic acid and to which one or more detection labels are coupled.
  • Aptamers are nucleic acid species that have been engineered through repeated rounds of in vitro selection equivalently, SELEX (systematic evolution of ligands by exponential enrichment) to bind to various molecular targets such as small molecules, proteins, nucleic acids, and even cells, tissues and organisms.
  • Aptamers offer molecular binding and recognition equivalent to antibodies.
  • aptamers offer advantages over antibodies as they can be engineered completely in vitro, are readily produced by chemical synthesis, possess desirable storage properties, and elicit little or no immunogenicity in therapeutic applications.
  • the aptamer is a monomer (one unit).
  • the aptamer is a multimeric aptamer.
  • the multimeric aptamer may comprise a plurality of aptamer units (mers). Each of the plurality of units of the aptamer may be identical. In such a case the multimeric aptamer is a homomultimer having a single specificity but enhanced avidity (multivalent aptamer).
  • the multimeric aptamer may comprise two or more aptameric monomers, wherein at least two mers of the multimeric aptamer are non-identical in structure, nucleic acid sequence or both.
  • a multimeric aptamer is referred to herein as a heteromultimer.
  • the heteromultimer may be directed to a single binding site i.e., monospecific (such as to avoid steric hindrance).
  • the heteromultimer may be directed to a plurality of binding sites i.e., multispecific.
  • the heteromultimer may be directed to a plurality of binding sites on different analytes, including for different drugs of abuse Further description of the multimeric aptamer is provided below.
  • a plurality of multimeric aptamers may be conjugated to form a conjugate of multimeric aptamers.
  • the multimeric aptamer may comprise, two (dimer), three (trimer), four (tetramer), five (pentamer), six (hexamer), and even more units.
  • Aptamers of the invention can be synthesized and screened by any suitable methods known in the art.
  • aptamers can be screened and identified from a random aptamer library by SELEX (systematic evolution of ligands by exponential enrichment). Aptamers that bind to an antigen of interest can be suitably screened and selected by a modified selection method herein referred to as cell-SELEX or cellular-SELEX. In other examples, aptamers that bind to a cell surface target molecule can be screened by capillary electrophoresis and enriched by SELEX based on the observation that aptamer-target molecule complexes exhibited retarded migration rate in native polyacrylamide gel electrophoresis as compared to unbound aptamers.
  • SELEX systematic evolution of ligands by exponential enrichment
  • Aptamers that bind to an antigen of interest can be suitably screened and selected by a modified selection method herein referred to as cell-SELEX or cellular-SELEX.
  • aptamers that bind to a cell surface target molecule can be screened
  • a random aptamer library can be created that contains monomeric, dimeric, trimeric, tetrameric or other higher multimeric aptamers.
  • a random aptamer library (either ssDNA or RNA) can be modified by including oligonucleotide linkers to link individual aptamer monomers to form multimeric aptamer fusion molecules.
  • a random oligonucleotide library is synthesized with randomized 45 nt sequences flanked by defined 20 nt sequences both upstream and downstream of the random sequence, i.e., known as 5'-arm and 3'-arm, which are used for the amplification of selected aptamers.
  • a linking oligonucleotide i.e., linker
  • linker is designed to contain sequences complementary to both 5'-arm and 3'-arm regions of random aptamers to form dimeric aptamers.
  • a small trimeric or tetrameric (i.e., a Holiday junction-like) DNA nanostructure is engineered to include sequences complementary to the 3'-arm region of the random aptamers, therefore creating multimeric aptamer fusion through hybridization.
  • 3-5 or 5-10 dT rich nucleotides can be engineered into the linker polynucleotides as a single stranded region between the aptamer-binding motifs, which offers flexibility and freedom of multiple aptamers to coordinate and synergize multivalent interactions with cellular ligands or receptors.
  • multimeric aptamers can also be formed by mixing biotinylated aptamers with streptavidin.
  • Multimeric aptamers may be multivalent but be of single binding specificity (i.e., homomultimeric aptamers).
  • the multimeric aptamer may be multivalent and multi- specific (i.e., heteromultimeric aptamers).
  • each monomer of the homomultimeric aptamer binds the target protein (e.g., selected drug target) in an identical manner.
  • all monomeric components of the homomultimeric aptamer are identical.
  • a heteromultimeric aptamer comprises a plurality of monomeric aptamers at least two of which bind different sites on a single target protein or bind at least two different target proteins.
  • a suitable nucleotide length for an aptamer ranges from about 15 to about 100 nucleotide (nt), and in various other examples, 12-30, 14-30, 15-30 nt, 30-100 nt, 30-60 nt, 25-70 nt, 25-60 nt, 40-60 nt, or 40-70 nt in length.
  • the aptamer has affinity at the range of 10-100 nM, which, after binding of the aptamer to a molecule, permits dissociation of the aptamer from the target molecule, which leads to the release and recycle of the aptamer nucleic acid nanostructure.
  • the affinity of individual aptamers can be increased by 4-50 fold by constructing multimeric aptamers linked together by covalent or non-covalent linkages. Methods of multimerizing aptamers are further described below.
  • the desirable affinity of an aptamer to an analyte of interest can be fine-tuned by adjusting the multiplexity of the monomeric aptamer.
  • Multimerization can be done at the library level as follows.
  • a linker polynucleotide has a length between about 5 nucleotides (nt) and about 100 nt; in various examples, 10-30 nt, 20-30 nt, 25-35 nt, 30-50 nt, 40-50 nt, 50-60 nt, 55-65 nt, 50-80 nt, or 80-100 nt. It is within the ability of one of skill in the art to adjust the length of the linker polynucleotide to accommodate each monomeric aptamer in the multimeric structure.
  • the multimeric aptamers can be identified and screened from a random multimeric aptamer library as described herein.
  • the monomeric aptamers are linked to each other by one or a plurality of linker polynucleotides to form multimeric aptamers.
  • Monomeric aptamers can be linked to form multimeric aptamers by any suitable means and in any configurations.
  • Multimers can be linearly linked by continuous linear synthesis of DNA without spacers or with nucleic acid spacers. Aptamer synthesis usually relies on standard solid phase phosphoramitide chemistry.
  • dimers, trimers and tetramers or higher oligomeric structures can be linked by a polymeric spacer.
  • the aptamers are further modified to protect the aptamers from nuclease and other enzymatic activities.
  • the aptamer sequence can be modified by any suitable methods known in the art. For example, phosphorothioate can be incorporated into the backbone, and 5'-modified pyrimidine can be included in 5' end of ssDNA for DNA aptamer.
  • modified nucleotides such as substitutions of the 2'-OH groups of the ribose backbone, e.g., with 2'-deoxy-NTP or - fluoro-NTP, can be incorporated into the RNA molecule using T7 RNA polymerase mutants.
  • the resistance of these modified aptamers to nuclease can be tested by incubating them with either purified nucleases or nuclease from mouse serum, and the integrity of aptamers can be analyzed by gel electrophoresis.
  • the monomeric or multimeric aptamer of the invention can be further attached or conjugated to a detectable or therapeutic moiety (i.e., a pharmaceutical moiety).
  • a diagnostic moiety such as a detectable moiety e.g., label (e.g., His tag, flag tag), fluorescent, radioactive, biotin/avidin etc., can be bound to the aptamer, and imaging, immunohistochemistry, for target identification.
  • label e.g., His tag, flag tag
  • fluorescent e.g., radioactive
  • biotin/avidin e.g., biotin/avidin
  • a polynucleotide is an oligonucleotide or a single strand of RNA or DNA.
  • the polynucleotide or functional fragment thereof e.g. including a target molecule/ligand binding domain
  • salt of either is an aptamer.
  • aptamer refers to a polynucleotide or functional fragment thereof, or salt of either that specifically binds a target molecule.
  • specifically binds is used interchangeably herein with “selectively binds” and means the same thing.
  • the terms “specifically binds” and “selectively binds” in reference to an aptamer describe the binding of an aptamer to a target molecule and mean that aptamer binding to the target molecule does not involve the formation of nucleotide base pairs between the aptamer and the target molecule.
  • the skilled person would recognize it is well-known in the art that the polynucleotide sequence of an aptamer may include base pairs that are not required for specific binding of the aptamer to a given target molecule, and that smaller fragments of an aptamer, even fragments having below 50% sequence identity may still be capable of effectively binding to a target molecule (Alsager, Omar A., et al. "Ultrasensitive Colorimetric Detection of 17b-Estradiol: The Effect of Shortening DNA Aptamer Sequences.” Analytical chemistry 87.8 (2015): 4201-4209).
  • an aptamer exerts an inhibitory effect on a target, e.g., by binding of the target, by catalytically altering the target, by reacting with the target in a way which modifies the target or the functional activity of the target, by ionically or covalently attaching to the target or by facilitating the reaction between the target and another molecule.
  • the aptamer can comprise a ribonucleotide, deoxyribonucleotide, or other type of nucleic acid, or two or more different types of nucleic acids.
  • An aptamer can also comprise one or more modified bases, sugars, polyethylene glycol spacers or phosphate backbone units.
  • the aptamer comprises one or more 2' sugar modifications, such as a 2'-0- alkyl (e.g., 2'-0-methyl or 2'-0-methoxyethyl) or a 2'-fluoro modification.
  • 2' sugar modifications such as a 2'-0- alkyl (e.g., 2'-0-methyl or 2'-0-methoxyethyl) or a 2'-fluoro modification.
  • an aptamer is a polynucleotide of about 80, about 75, about 70, about 65, about 60, about 55, about 50, about 45, about 40, about 35, about 30, about 25, about 20, about 15 nucleotides in length.
  • an aptamer is a polynucleotide of less than 80, less than 75, less than 70, less than 65, less than 60, less than 55, less than 50, less than 45, less than 40, less than 35, less than 30, less than 25, less than 20, less than 15 nucleotides in length.
  • an aptamer is a polynucleotide of about 70 to 80, is about 60 to 70, is about 50 to 60, is about 40 to 50, is about 30 to 40, is about 20 to 30, is about 10 to 20 nucleotides in length.
  • an aptamer is a polynucleotide of about 75 to 85, is about 65 to 75, is about 55 to 65, is about 45 to 55, is about 35 to 45, is about 25 to 35, is about 15 to 25 nucleotides in length.
  • an aptamer is a polynucleotide of 70 to 80, is 60 to 70, is 50 to 60, is 40 to 50, is 30 to 40, is 20 to 30, is 10 to 20 nucleotides in length.
  • an aptamer is a polynucleotide of 75 to 85, is 65 to 75, is 55 to 65, is 45 to 55, is 35 to 45, is 25 to 35, is 15 to 25 nucleotides in length. In some examples the aptamer is about 73 to about 77 or about 74 to about 76 nucleotides in length.
  • an aptamer may be any length polynucleotide that falls within the size parameters set out herein.
  • an aptamer may be about 76, about 61, about 54, about 43, about 29 or about 27 nucleotides in length or may be 77, 62, 55, 44, 28 or 26 nucleotides in length. What is important is that the aptamer specifically binds the target molecule.
  • an aptamer of the invention is a polynucleotide of about 80, about 75, about 70, about 65, about 60, about 55, about 50, about 45, about 40, about 35, about 30, about 25, about 20, about 15 nucleotides in length that selectively binds a target molecule as described herein.
  • an aptamer is a polynucleotide of less than 80, less than 75, less than 70, less than 65, less than 60, less than 55, less than 50, less than 45, less than 40, less than 35, less than 30, less than 25, less than 20, less than 15 nucleotides in length that selectively binds a target molecule as described herein.
  • an aptamer is a polynucleotide of about 70 to 80, is about 60 to 70, is about 50 to 60, is about 40 to 50, is about 30 to 40, is about 20 to 30, is about 10 to 20 nucleotides in length that selectively binds a target molecule as described herein.
  • an aptamer is a polynucleotide of about 75 to 85, is about 65 to 75, is about 55 to 65, is about 45 to 55, is about 35 to 45, is about 25 to 35, is about 15 to 25 nucleotides in length that selectively binds a target molecule as described herein.
  • an aptamer is a polynucleotide of 70 to 80, is 60 to 70, is 50 to 60, is 40 to 50, is 30 to 40, is 20 to 30, is 10 to 20 nucleotides in length that selectively binds a target molecule as described herein.
  • an aptamer is a polynucleotide of 75 to 85, is 65 to 75, is 55 to 65, is 45 to 55, is 35 to 45, is 25 to 35, is 15 to 25 nucleotides in length that selectively binds a target molecule as described herein.
  • salt includes a non-toxic salt of an inorganic or organic acid, including, but not limited to, a halide (chloride, bromide, iodide, fluoride), acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptanoate, hydrochloride, hydrobromide, hydroiodide, 2- hydroxyethanesulfonate, lactate, maleate, malonate, methanesulfonate, nitrate, oxalate, persulfate, phosphate, picrate, pivalate, propionate, p-toluenesulfonate, salicylate
  • chloride
  • salt also includes a non-toxic salt of an organic or inorganic base, including, but not limited to, sodium (Na + ), potassium (K + ), calcium (Ca 2+ ), magnesium (Mg 2+ ), and lithium (Li + ) salts.
  • the target molecule is a small molecule.
  • the small molecule has a molecular weight (MW) of from about 60 to about 2000 g mol -1 . In other examples, the small molecule has a MW of from about 100 to about 500 g mol -1 . In other examples, the small molecule has a MW of from about 150 to about 350 g mol -1 .
  • MW molecular weight
  • the molecular weight of such small molecules and the calculation of their molecular weight are well known to those of skill in the art.
  • the illicit drug is a naturally occurring or synthetic opiate or opioid, or derivative or metabolite thereof (e.g., morphine, oxycodone, hydromorphone, propoxyphene, nicomorphine, dihydrocodeine, diamorphine, papavereturn, codeine, ethylmorphine, phenylpiperidine and derivates thereof, methadone, dextropropoxyphene, buprenorphine, pentazocine, tilidine, tramadol, hydrocodone, meperidine, oxymorphone, alphaprodine, anileridine, dextromoramide, metopone, levorphanol, phenazocine, etoheptazine, propiram, profadol, phenampromide, thiambuten, pholcodeine, codeine, dihydrocodeinon, fentanyl, 3-trans-dimethylamino-4-phenyl-4-trans
  • the small molecule is cocaine, heroin, tetrahydrocannabinol (THC), or any derivative or metabolite thereof.
  • the small molecule is methamphetamine.
  • aptamers described herein were generated/selected using affinity matrix-based systematic evolution of ligands by exponential enrichment (SELEX), capture SELEX, or in silico SELEX methodologies.
  • SELEX is a process by which a random single stranded polynucleotide library is iteratively evolved to produce sequences that can bind to a specific target.
  • affinity matrix-based SELEX methodology is where a target for selection is immobilised onto a solid phase (often, sepharose beads).
  • the immobilisation steps usually include EDC/NHS or epoxy activation chemistry, depending on the structure of the target molecule.
  • the beads are incubated with a single-stranded random polynucleotide library at room temperature in binding and washing buffer (BWB; 20 mM Tris-HCI, pH 7.5, 100 mM NaCI, 5 mM KCI, 2 mM MgCl 2 , 1 mM CaCl 2 , and between 0% and 0.1% IGEPAL) for up to 18 hours.
  • binding and washing buffer BWB; 20 mM Tris-HCI, pH 7.5, 100 mM NaCI, 5 mM KCI, 2 mM MgCl 2 , 1 mM CaCl 2 , and between 0% and 0.1% IGEPAL
  • the beads are washed with 5 mL BWB, and unbound aptamers are discarded in the supernatant while target binding aptamers are retained with the beads and processed for the next round of selection .
  • the sequences that are bound to the beads are amplified by standard amplification techniques such as polymerase chain reaction (PCR).
  • PCR is performed using the HotMasterTM Taq kit with 200 mM dNTPs, 220 nM of forward primer, 220 nM or biotin- labelled forward primer, and 1.5 U HotMaster Taq in a final volume of 50 pL
  • the amplified sequences are incubated with 0.3 NaOH to separate the double stranded sequences into single stranded sequences, and the biotin-labelled strand is removed using streptavidin magnetic beads.
  • the single stranded sequences are then incubated with the beads, as described above. This process can be repeated iteratively until the required target affinity is reached.
  • the incubation, elution, and PCR conditions can be adjusted at each round of SELEX to ensure the sequences retain the desired binding characteristics.
  • Capture SELEX methodology is where target for selection is free in solution and the ssDNA library is immobilised onto beads. Briefly, a polynucleotide probe is conjugated via NHS/EDC chemistry to magnetic beads functionalised with carboxylic acid groups on the surface. A random polynucleotide library is attached to the beads by hybridization with the complementary probe. The ssDNA-beads are added to the solution containing the target and incubated at room temperature in BWB for up to 18 hours. The target-bound sequences dissociate from the probe and remain in the supernatant, while the unbound sequences remain bound to the beads which are discarded.
  • the target-bound sequences are amplified by standard amplification techniques such as polymerase chain reaction (PCR), as described above.
  • PCR polymerase chain reaction
  • the amplified sequences are incubated with 0.3 NaOH to separate the double stranded sequences into single stranded sequences, and the biotin-labelled strand is removed using streptavidin magnetic beads.
  • the resulting single stranded sequences are then hybridised to the complementary probe attached to the beads, and the next round of SELEX is carried out. This process can be repeated iteratively until the required target affinity is reached.
  • EXAMPLE 2 MEASUREMENT OF APTAMER BINDING AFFINITIES
  • a fluorescent imaging assay a Sybr green assay, or a fluorescent microscale thermophoresis (MST) binding assay were used.
  • the fluorescent imaging assay may be used to determine Kd when the affinity matrix SELEX method is used for aptamer generation and selection.
  • a serial dilution of hexachloro-fluorescein (HEX)-labelled aptamer is prepared with BWB.
  • the beads with the target immobilised on the surface are washed twice with BWB before being incubated for up to 2 hours at room temperature with the fluorescently labelled aptamer candidate. Beads are then washed twice with BWB to remove loosely bound aptamers and the beads are imaged with a fluorescent microscope at 535/556 nm excitation/emission. Average fluorescence values of at least 20 beads per concentration are computed and plotted against the aptamer concentration to determine Kd.
  • HEX hexachloro-fluorescein
  • the Sybr Green I assay may be used to determine Kd when the capture SELEX method is used for aptamer generation and selection.
  • Sybr Green I is a fluorescent nucleic acid dye that preferentially binds to double stranded DNA. Binding of an aptamer to its target may elicit a conformational change in the aptamer structure, thereby changing the amount of double stranded regions to which the Sybr Green I dye may bind.
  • a serial dilution of the target is prepared in BWB and added to a constant concentration of the unlabelled aptamer.
  • Sybr Green I After incubation at room temperature for up to 2 hours, a constant concentration of Sybr Green I is added and incubated for up to 2 more hours. The final concentration of aptamer and Sybr Green I are optimised to maximise the difference between a high concentration of target and no target. The fluorescence is measured at excitation/emission 480 nm/524 nm and plotted against target concentration to determine Kd.
  • Sybr Green I assay An alternative approach to the Sybr Green I assay may be used, whereby a complementary probe is hybridised to the aptamer sequence. Addition of the target induces the aptamer to preferentially bind to the target, thereby displacing the probe and reducing the number of double stranded DNA to which the Sybr Green I dye may bind.
  • the aptamer is hybridised with the complementary probe in BWB at room temperature for up to 2 hours.
  • a serial dilution of the target is prepared and is added to the aptamer-probe mixture and incubated at room temperature for up to 2 hours.
  • Sybr Green I is added and incubated for up to 2 hours at room temperature. The fluorescence is measured at excitation/emission 480 nm/524 nm and plotted against target concentration to determine Kd.
  • the fluorescent MST binding assay may be used to determine Kd regardless of the aptamer SELEX method used.
  • Microscale thermophoresis is based on two principles. First, the temperature related intensity changes of a fluorophore, and second, the movement of molecules along a temperature gradient. Briefly, a serial dilution of the target is prepared in BWB before being added to a Cy5-labelled aptamer. The solution is loaded into the capillaries and analysed using a Monolith NT.115. The fluorescence was analysed using a 1: 1 binding model to determine the Kd.

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Abstract

The present invention is concerned with the detection of illicit drugs. The present invention provides polynucleotide/aptamer sequences which spontaneously fold to form secondary structure features which promote selective binding to a drug target, including but not limited to, methamphetamine, cannabis, synthetic cannabis, cocaine, ecstasy, morphine, benzodiazepines, oxycodone and ketamine. Described herein are polynucloetides, test kits and methods configured to detect one or more illicit drugs from a test sample (e.g.) a biological sample such as saliva, blood or urine. Multiplexed systems for simultaneous or concurrent drug detection are also contemplated. Accordingly, the polynucleotides, test kits and methods according to the present invention have utility in road-side testing or in work-place monitoring to detect illicit drug use by individuals who place themselves or other bystanders in the community in danger.

Description

NOVEL ASSAYS
TECHNICAL FIELD
The present invention is concerned with the detection of illicit and prescription drugs including, for example, methamphetamine, synthetic/cannabis, cocaine, ecstasy, morphine, oxycodone, benzodiazepines and ketamine. The present invention provides novel polynucleotide aptamers, which aptamers fold and selectively bind to a molecule/drug target of interest, as well as test kits, and assay methods for the detection of illicit and prescription drugs in a test sample for use (e.g.) in road-side testing or in work-place monitoring.
BACKGROUND OF THE INVENTION
The abuse of potentially impairing drugs, including newly emerging drugs, poses a serious threat to public health, not to mention a great challenge to the health care system. In the past several years, the incidence of drug abuse and drug overdose deaths have rapidly increased, reaching epidemic levels. The number of deaths caused by drug overdose has surpassed that caused by motor vehicles accidents and firearms since 2008, becoming the leading cause of injury death. In 2017 in the United States, the Centers for Disease Control and Prevention (CDC) reported 73,990 drug-induced deaths, a 9.6% increase from 2016. Of these drug-induced deaths, 70,237 occurred as a result of overdose, with the 15 drugs most frequently involved in overdose belonging to the opioid, stimulant, and benzodiazepine drug classes (Hedegaard H, Bastian BA, Trinidad JP, Spencer MR, Warner M. Regional differences in the drugs most frequently involved in drug overdose deaths: United States, 2017. National Vital Statistics Reports; vol 68 no 12. Hyattsville, MD: National Center for Health Statistics. 2019).
There is an ongoing need for the development of rapid and portable tests for drugs of abuse, particularly at the roadside, workplace, and emergency room. A 2013 - 2014 survey by the United States Department of Transportation found that 15.1% of drivers tested positive for illegal drugs, while 4.9% of drivers tested positive for potentially impairing medications (Berning, A., Compton, R., 8i Wochinger, K. (2015, February). Results of the 2013-2014 National Roadside Survey of alcohol and drug use by drivers. (Traffic Safety Facts Research Note. Report No. DOT HS 812 118). Washington, DC: National Highway Traffic Safety Administration).
Current portable detection methods utilise antibodies for detection of illicit drugs, despite the significant limitations of antibodies for in-field testing. In particular, the temperature sensitivity, difficulty in generating antibodies against small molecule targets, and batch-to-batch variability limit the usefulness of antibodies for sensing methods. Aptamers are single-stranded polynucleotides (RNA or DNA) that possess the ability to bind targets with high affinity and specificity (Tuerk, C., and L. Gold. Science, 1990, 249(4968), 505-10; Ellington, A. D. & Szostak, J. W. Nature, 1990, 346, 818-822). Aptamers are developed in vitro using a process known as systematic evolution of ligands by exponential enrichment (SELEX). Aptamers are selected from a random oligonucleotide library based on their ability to bind a specific target over multiple selection rounds. Aptamers have several advantages over antibodies. First, aptamers can be selected against any target, even those that are toxic or that have low immunogenicity. Second, aptamers are generally robust to temperature and can be synthesized at high volumes with little batch-to-batch variation. Third, the biophysical characteristics of aptamer-target binding offers greater flexibility in biosensor design (Song, S. et al. Trends in Analytical Chemistry, 2008, 27(2), 108-17).
SUMMARY OF THE INVENTION
The inventions described and claimed herein have many attributes and examples including, but not limited to, those set forth or described or referenced in this Summary of the Invention. It is not intended to be all-inclusive and the inventions described and claimed herein are not limited to or by the features or examples identified in this Summary of the Invention, which is included for purposes of illustration only and not restriction.
In an aspect of the present invention there is provided a polynucleotide or salt thereof consisting in the sequence AT ACGAGCTTGTTCAATA[X] nTGAT AGT AAGAGCAATC (SEQ ID NO: 565), where X is selected from A, T, U, G or C and n is an integer of between about 10 and about 120, in particular between about 35 and about 45.
In another aspect of the present invention there is provided a polynucleotide or salt thereof comprising the sequence ATACGAGCTTGTTCAATA[X]nTGATAGTAAGAGCAATC (SEQ ID NO: 565), where X is selected from A, T, U, G or C and n is an integer of between about 10 and about 120, in particular between about 35 and about 45, which polynuceotide or salt thereof selectively binds to methamphetamine.
In another aspect of the present invention there is provided a polynucleotide or salt thereof comprising the sequence AT ACGAGCTTGTTCAATA[Y]TGAT AGT AAGAGCAATC (SEQ ID NO: 566), where Y is any sequence comprising a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to any one of SEQ ID Nos: 2, 4, 6, 8, 10, 12,
14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60,
62, 64, 66, 68, 70, 72 or 74, which polynuceotide or salt thereof selectively binds to methamphetamine.
In another aspect of the present invention there is provided a polynucleotide or salt thereof comprising the sequence AT ACGAGCTTGTTCAATA[Y]TGAT AGT AAGAGCAATC (SEQ ID NO: 567), where Y is any sequence comprising any one of SEQ ID Nos: 2, 4, 6, 8, 10, 12, 14,
16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62,
64, 66, 68, 70, 72 or 74, which polynuceotide or salt thereof selectively binds to methamphetamine. In another aspect of the present invention there is provided a polynucleotide or salt thereof comprising the sequence ATACGAGCTTGTTCAATA[Y]TGATAGTAAGAGCAATC (SEQ ID NO: 566), where Y is any sequence consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to any one of SEQ ID Nos: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72 or 74, which polynuceotide or salt thereof selectively binds to methamphetamine.
In another aspect of the present invention there is provided a polynucleotide or salt thereof comprising the sequence ATACGAGCTTGTTCAATA[Y]TGATAGTAAGAGCAATC (SEQ ID NO: 567), where Y is any sequence consisting in a sequence of any one of SEQ ID Nos: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72 or 74, which polynuceotide or salt thereof selectively binds to methamphetamine.
In another aspect of the present invention there is provided a polynucleotide or salt threreof comprising a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to any one of SEQ ID Nos: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71 and 73, which polynucleotide of salt thereof selectively binds to methamphetamine.
In another aspect of the present invention there is provided a polynucleotide or salt threreof comprising a sequence of any one of SEQ ID Nos: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19,
21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67,
69, 71 and 73, which polynucleotide of salt thereof selectively binds to methamphetamine.
In another aspect of the present invention there is provided a polynucleotide or salt thereof consisting in a sequence of any one of SEQ ID Nos: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19,
21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67,
69, 71 and 73, which polynucleotide of salt thereof selectively binds to methamphetamine.
In a further aspect of the present invention there is provided a method for detecting the presence of methamphetamine in a test sample, the method comprising the steps of:
(i) contacting a test sample with a polynucleotide or salt thereof comprising a sequence defined by any one of SEQ ID Nos: 1-74; and
(ii) measuring binding between the polynucleotide and methamphetamine from the test sample, wherein, a measured binding interaction between the polynucleotide and methamphetamine reflects the presence of methamphetamine in the test sample.
In yet a further aspect of the present invention there is provided a test kit or article of manufacture for detecting the presence of methamphetamine in a test sample, the test kit or article of manufacture comprising a polynucleotide or salt thereof comprising a sequence defined by any one of SEQ ID Nos: 1-74 together with instructions for how to detect the presence of methamphetamine in the test sample.
In a further aspect of the present invention there is provided a polynucleotide or salt thereof comprising the sequence ATACGAGCTTGTTCAATA[X]nTGATAGTAAGAGCAATC (SEQ ID NO: 568), where X is selected from A, T, U, G or C and n is an integer of between about 10 and about 120, in particular between about 35 and about 45, which polynuceotide or salt thereof selectively binds to tetrahydrocannabinol.
In a further aspect of the present invention there is provided a polynucleotide or salt thereof comprising the sequence ATACGAGCTTGTTCAATA[Y]TGATAGTAAGAGCAATC (SEQ ID NO: 569), where Y is any sequence comprising a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to any one of SEQ ID Nos: 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134 and 136, which polynuceotide or salt thereof selectively binds to tetrahydrocannabinol.
In a further aspect of the present invention there is provided a polynucleotide or salt thereof comprising the sequence ATACGAGCTTGTTCAATA[Y]TGATAGTAAGAGCAATC (SEQ ID NO: 570), where Y is any sequence comprising any one of SEQ ID Nos: 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134 and 136, which polynuceotide or salt thereof selectively binds to tetrahydrocannabinol.
In a further aspect of the present invention there is provided a polynucleotide or salt thereof comprising the sequence ATACGAGCTTGTTCAATA[Y]TGATAGTAAGAGCAATC (SEQ ID NO: 569), where Y is any sequence consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to any one of SEQ ID Nos: 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134 and 136, which polynuceotide or salt thereof selectively binds to tetrahydrocannabinol.
In a further aspect of the present invention there is provided a polynucleotide or salt thereof comprising the sequence ATACGAGCTTGTTCAATA[Y]TGATAGTAAGAGCAATC (SEQ ID NO: 570), where Y is any sequence consisting in a sequence of any one of SEQ ID Nos: 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134 and 136, which polynuceotide or salt thereof selectively binds to tetrahydrocannabinol.
In a further aspect of the present invention there is provided a polynucleotide or salt threreof comprising a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to any one of SEQ ID Nos: 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 133 and 135, which polynucleotide of salt thereof selectively binds to tetrahydrocannabinol. In a further aspect of the present invention there is provided a polynucleotide or salt threreof comprising a sequence of any one of SEQ ID Nos: 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 133 and 135, which polynucleotide of salt thereof selectively binds to tetrahydrocannabinol.
In a further aspect of the present invention there is provided a polynucleotide or salt thereof consisting in a sequence of any one of SEQ ID Nos: 75, 77, 79, 81, 83, 85, 87, 89,
91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127,
129, 133 and 135, which polynucleotide of salt thereof selectively binds to tetrahydrocannabinol.
In a further aspect of the present invention there is provided a method for detecting the presence of tetrahydrocannabinol in a test sample, the method comprising the steps of:
(i) contacting a test sample with a polynucleotide or salt thereof comprising a sequence defined by any one of SEQ ID Nos: 75-136; and
(ii) measuring binding between the polynucleotide and tetrahydrocannabinol from the test sample, wherein, a measured binding interaction between the polynucleotide and tetrahydrocannabinol reflects the presence of tetrahydrocannabinol in the test sample.
In yet a further aspect of the present invention there is provided a test kit or article of manufacture for detecting the presence of tetrahydrocannabinol in a test sample, the test kit or article of manufacture comprising a polynucleotide or salt thereof comprising a sequence defined by any one of SEQ ID Nos: 75-136 together with instructions for how to detect the presence of tetrahydrocannabinol in the test sample.
In yet another aspect of the present invention there is provided a polynucleotide or salt thereof comprising the sequence ATACGAGCTTGTTCAATA[X]nTGATAGTAAGAGCAATC (SEQ ID NO: 571), where X is selected from A, T, U, G or C and n is an integer of between about 10 and about 120, in particular between about 35 and about 45, which polynuceotide or salt thereof selectively binds to cocaine.
In yet another aspect of the present invention there is provided a polynucleotide or salt thereof comprising the sequence ATACGAGCTTGTTCAATA[Y]TGATAGTAAGAGCAATC (SEQ ID NO: 572), where Y is any sequence comprising a sequence that has at least 90, 91,
92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to any one of SEQ ID Nos: 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170 and 172, which polynuceotide or salt thereof selectively binds to cocaine.
In yet another aspect of the present invention there is provided a polynucleotide or salt thereof comprising the sequence ATACGAGCTTGTTCAATA[Y]TGATAGTAAGAGCAATC (SEQ ID NO: 573), where Y is any sequence comprising any one of SEQ ID Nos: 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170 and 172, which polynuceotide or salt thereof selectively binds to cocaine. In yet another aspect of the present invention there is provided a polynucleotide or salt thereof comprising the sequence ATACGAGCTTGTTCAATA[Y]TGATAGTAAGAGCAATC (SEQ ID NO: 572), where Y is any sequence consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to any one of SEQ ID Nos: 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170 and 172, which polynuceotide or salt thereof selectively binds to cocaine.
In yet another aspect of the present invention there is provided a polynucleotide or salt thereof comprising the sequence ATACGAGCTTGTTCAATA[Y]TGATAGTAAGAGCAATC (SEQ ID NO: 573), where Y is any sequence consisting in a sequence of any one of SEQ ID Nos: 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170 and 172, which polynuceotide or salt thereof selectively binds to cocaine.
In yet another aspect of the present invention there is provided a polynucleotide or salt threreof comprising a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to any one of SEQ ID Nos: 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169 and 171, which polynucleotide of salt thereof selectively binds to cocaine.
In yet another aspect of the present invention there is provided a polynucleotide or salt threreof comprising a sequence of any one of SEQ ID Nos: 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169 and 171, which polynucleotide of salt thereof selectively binds to cocaine.
In yet another aspect of the present invention there is provided a polynucleotide or salt thereof consisting in a sequence of any one of SEQ ID Nos: 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169 and 171, which polynucleotide of salt thereof selectively binds to cocaine.
In a further aspect of the present invention there is provided a method for detecting the presence of cocaine in a test sample, the method comprising the steps of:
(i) contacting a test sample with a polynucleotide or salt thereof comprising a sequence defined by any one of SEQ ID Nos: 137-172; and
(ii) measuring binding between the polynucleotide and cocaine from the test sample, wherein, a measured binding interaction between the polynucleotide and cocaine reflects the presence of cocaine in the test sample.
In yet a further aspect of the present invention there is provided a test kit or article of manufacture for detecting the presence of cocaine in a test sample, the test kit or article of manufacture comprising a polynucleotide or salt thereof comprising a sequence defined by any one of SEQ ID Nos: 137-172 together with instructions for how to detect the presence of cocaine in the test sample. In yet a further aspect of the present invention there is provided a polynucleotide or salt thereof comprising the sequence ATACGAGCTTGTTCAATA[X]nTGATAGTAAGAGCAATC (SEQ ID NO: 574), where X is selected from A, T, U, G or C and n is an integer of between about 10 and about 120, in particular between about 35 and about 45, which polynuceotide or salt thereof selectively binds to 3,4-methylenedioxymethamphetamine.
In yet a further aspect of the present invention there is provided a polynucleotide or salt thereof comprising the sequence ATACGAGCTTGTTCAATA[Y]TGATAGTAAGAGCAATC (SEQ ID NO: 575), where Y is any sequence comprising a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to any one of SEQ ID Nos: 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212,
214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242 and 246, which polynuceotide or salt thereof selectively binds to 3,4-methylenedioxymethamphetamine.
In yet a further aspect of the present invention there is provided a polynucleotide or salt thereof comprising the sequence ATACGAGCTTGTTCAATA[Y]TGATAGTAAGAGCAATC (SEQ ID NO: 576), where Y is any sequence comprising any one of SEQ ID Nos: 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212,
214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242 and 246, which polynuceotide or salt thereof selectively binds to 3,4-methylenedioxymethamphetamine.
In yet a further aspect of the present invention there is provided a polynucleotide or salt thereof comprising the sequence ATACGAGCTTGTTCAATA[Y]TGATAGTAAGAGCAATC (SEQ ID NO: 575), where Y is any sequence consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to any one of SEQ ID Nos: 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212,
214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242 and 246, which polynuceotide or salt thereof selectively binds to 3,4-methylenedioxymethamphetamine.
In yet a further aspect of the present invention there is provided a polynucleotide or salt thereof comprising the sequence ATACGAGCTTGTTCAATA[Y]TGATAGTAAGAGCAATC (SEQ ID NO: 576), where Y is any sequence consisting in a sequence of any one of SEQ ID Nos: 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206,
208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242 and 246, which polynuceotide or salt thereof selectively binds to 3,4- methylenedioxymethamphetamine.
In yet a further aspect of the present invention there is provided a polynucleotide or salt threreof comprising a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to any one of SEQ ID Nos: 173, 175, 177, 179, 181, 183, 185, 187,
189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223,
225, 227, 229, 231, 233, 235, 237, 239, 241, 243 and 245, which polynucleotide of salt thereof selectively binds to 3,4-methylenedioxymethamphetamine. In yet a further aspect of the present invention there is provided a polynucleotide or salt threreof comprising a sequence of any one of SEQ ID Nos: 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243 and 245, which polynucleotide of salt thereof selectively binds to 3,4-methylenedioxymethamphetamine.
In yet a further aspect of the present invention there is provided a polynucleotide or salt thereof consisting in a sequence of any one of SEQ ID Nos: 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243 and 245, which polynucleotide of salt thereof selectively binds to 3,4-methylenedioxymethamphetamine.
In a further aspect of the present invention there is provided a method for detecting the presence of 3,4-methylenedioxymethamphetamine in a test sample, the method comprising the steps of:
(i) contacting a test sample with a polynucleotide or salt thereof comprising a sequence defined by any one of SEQ ID Nos: 173-246; and
(ii) measuring binding between the polynucleotide and 3,4- methylenedioxymethamphetamine from the test sample, wherein, a measured binding interaction between the polynucleotide and 3,4- methylenedioxymethamphetamine reflects the presence of 3,4- methylenedioxymethamphetamine in the test sample.
In yet a further aspect of the present invention there is provided a test kit or article of manufacture for detecting the presence of 3,4-methylenedioxymethamphetamine in a test sample, the test kit or article of manufacture comprising a polynucleotide or salt thereof comprising a sequence defined by any one of SEQ ID Nos: 173-246 together with instructions for how to detect the presence of 3,4-methylenedioxymethamphetamine in the test sample.
In another aspect of the present invention there is provided a polynucleotide or salt thereof comprising the sequence ATACGAGCTTGTTCAATA[X]nTGATAGTAAGAGCAATC (SEQ ID NO: 577), where X is selected from A, T, U, G or C and n is an integer of between about 10 and about 120, in particular between about 35 and about 45, which polynuceotide or salt thereof selectively binds to morphine.
In another aspect of the present invention there is provided a polynucleotide or salt thereof comprising the sequence ATACGAGCTTGTTCAATA[Y]TGATAGTAAGAGCAATC (SEQ ID NO: 578), where Y is any sequence comprising a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to any one of SEQ ID Nos: 249, 251, 253, 255, 257, 259, 261, 263, 264, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289 and 291, which polynuceotide or salt thereof selectively binds to morphine.
In another aspect of the present invention there is provided a polynucleotide or salt thereof comprising the sequence ATACGAGCTTGTTCAATA[Y]TGATAGTAAGAGCAATC (SEQ ID NO: 579), where Y is any sequence comprising any one of SEQ ID Nos: 249, 251, 253, 255, 257, 259, 261, 263, 264, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289 and 291, which polynuceotide or salt thereof selectively binds to morphine.
In another aspect of the present invention there is provided a polynucleotide or salt thereof comprising the sequence ATACGAGCTTGTTCAATA[Y]TGATAGTAAGAGCAATC (SEQ ID NO: 578), where Y is any sequence consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to any one of SEQ ID Nos: 249, 251, 253, 255,
257, 259, 261, 263, 264, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289 and 291, which polynuceotide or salt thereof selectively binds to morphine.
In another aspect of the present invention there is provided a polynucleotide or salt thereof comprising the sequence ATACGAGCTTGTTCAATA[Y]TGATAGTAAGAGCAATC (SEQ ID NO: 579), where Y is any sequence consisting in a sequence of any one of SEQ ID Nos: 249, 251, 253, 255, 257, 259, 261, 263, 264, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289 and 291, which polynuceotide or salt thereof selectively binds to morphine.
In another aspect of the present invention there is provided a polynucleotide or salt threreof comprising a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to any one of SEQ ID Nos: 247, 248, 250, 252, 254, 256, 258, 260, 262, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288 and 290, which polynucleotide of salt thereof selectively binds to morphine.
In another aspect of the present invention there is provided a polynucleotide or salt threreof comprising a sequence of any one of SEQ ID Nos: 247, 248, 250, 252, 254, 256,
258, 260, 262, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288 and 290, which polynucleotide of salt thereof selectively binds to morphine.
In another aspect of the present invention there is provided a polynucleotide or salt thereof consisting in a sequence of any one of SEQ ID Nos: 247, 248, 250, 252, 254, 256, 258, 260, 262, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288 and 290, which polynucleotide of salt thereof selectively binds to morphine.
In a further aspect of the present invention there is provided a method for detecting the presence of morphine in a test sample, the method comprising the steps of:
(i) contacting a test sample with a polynucleotide or salt thereof comprising a sequence defined by any one of SEQ ID Nos: 247-291; and
(ii) measuring binding between the polynucleotide and morphine from the test sample, wherein, a measured binding interaction between the polynucleotide and morphine reflects the presence of morphine in the test sample.
In yet a further aspect of the present invention there is provided a test kit or article of manufacture for detecting the presence of morphine in a test sample, the test kit or article of manufacture comprising a polynucleotide or salt thereof comprising a sequence defined by any one of SEQ ID Nos: 247-291 together with instructions for how to detect the presence of morphine in the test sample.
In a further aspect of the present invention there is provided a polynucleotide or salt thereof comprising the sequence ATACGAGCTTGTTCAATA[X]nTGATAGTAAGAGCAATC (SEQ ID NO: 580), where X is selected from A, T, U, G or C and n is an integer of between about 10 and about 120, in particular between about 35 and about 45, which polynuceotide or salt thereof selectively binds to diazepam.
In a further aspect of the present invention there is provided a polynucleotide or salt thereof comprising the sequence ATACGAGCTTGTTCAATA[Y]TGATAGTAAGAGCAATC (SEQ ID NO: 581), where Y is any sequence comprising a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to any one of SEQ ID Nos: 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317 and 319, which polynuceotide or salt thereof selectively binds to diazepam.
In a further aspect of the present invention there is provided a polynucleotide or salt thereof comprising the sequence ATACGAGCTTGTTCAATA[Y]TGATAGTAAGAGCAATC (SEQ ID NO: 582), where Y is any sequence comprising any one of SEQ ID Nos: 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317 and 319, which polynuceotide or salt thereof selectively binds to diazepam.
In a further aspect of the present invention there is provided a polynucleotide or salt thereof comprising the sequence ATACGAGCTTGTTCAATA[Y]TGATAGTAAGAGCAATC (SEQ ID NO: 581), where Y is any sequence consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to any one of SEQ ID Nos: 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317 and 319, which polynuceotide or salt thereof selectively binds to diazepam.
In a further aspect of the present invention there is provided a polynucleotide or salt thereof comprising the sequence ATACGAGCTTGTTCAATA[Y]TGATAGTAAGAGCAATC (SEQ ID NO: 582), where Y is any sequence consisting in a sequence of any one of SEQ ID Nos: 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317 and 319, which polynuceotide or salt thereof selectively binds to diazepam.
In a further aspect of the present invention there is provided a polynucleotide or salt threreof comprising a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to any one of SEQ ID Nos: 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316 and 318, which polynucleotide of salt thereof selectively binds to diazepam.
In a further aspect of the present invention there is provided a polynucleotide or salt threreof comprising a sequence of any one of SEQ ID Nos: 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316 and 318, which polynucleotide of salt thereof selectively binds to diazepam. In a further aspect of the present invention there is provided a polynucleotide or salt thereof consisting in a sequence of any one of SEQ ID Nos: 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316 and 318, which polynucleotide of salt thereof selectively binds to diazepam.
In a further aspect of the present invention there is provided a method for detecting the presence of diazepam in a test sample, the method comprising the steps of:
(i) contacting a test sample with a polynucleotide or salt thereof comprising a sequence defined by any one of SEQ ID Nos: 292-319; and
(ii) measuring binding between the polynucleotide and diazepam from the test sample, wherein, a measured binding interaction between the polynucleotide and diazepam reflects the presence of diazepam in the test sample.
In yet a further aspect of the present invention there is provided a test kit or article of manufacture for detecting the presence of diazepam in a test sample, the test kit or article of manufacture comprising a polynucleotide or salt thereof comprising a sequence defined by any one of SEQ ID Nos: 292-319 together with instructions for how to detect the presence of diazepam in the test sample.
In yet a further aspect of the present invention there is provided a polynucleotide or salt thereof comprising the sequence ATACGAGCTTGTTCAATA[X]nTGATAGTAAGAGCAATC (SEQ ID NO: 583), where X is selected from A, T, U, G or C and n is an integer of between about 10 and about 120, in particular between about 35 and about 45, which polynuceotide or salt thereof selectively binds to AMB-FUBINACA.
In yet a further aspect of the present invention there is provided a polynucleotide or salt thereof comprising the sequence ATACGAGCTTGTTCAATA[Y]TGATAGTAAGAGCAATC (SEQ ID NO: 584), where Y is any sequence comprising a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to any one of SEQ ID Nos: 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 355, 357, 359, 361, 363, 365, 367, 369 and 371, which polynuceotide or salt thereof selectively binds to AMB- FUBINACA.
In yet a further aspect of the present invention there is provided a polynucleotide or salt thereof comprising the sequence ATACGAGCTTGTTCAATA[Y]TGATAGTAAGAGCAATC (SEQ ID NO: 585), where Y is any sequence comprising any one of SEQ ID Nos: 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 355, 357, 359, 361, 363, 365, 367, 369 and 371, which polynuceotide or salt thereof selectively binds to AMB- FUBINACA.
In yet a further aspect of the present invention there is provided a polynucleotide or salt thereof comprising the sequence ATACGAGCTTGTTCAATA[Y]TGATAGTAAGAGCAATC (SEQ ID NO: 584), where Y is any sequence consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to any one of SEQ ID Nos: 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 355, 357, 359, 361, 363, 365, 367, 369 and 371, which polynuceotide or salt thereof selectively binds to AMB- FUBINACA.
In yet a further aspect of the present invention there is provided a polynucleotide or salt thereof comprising the sequence ATACGAGCTTGTTCAATA[Y]TGATAGTAAGAGCAATC (SEQ ID NO: 585), where Y is any sequence consisting in a sequence of any one of SEQ ID Nos: 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 355, 357, 359, 361, 363, 365, 367, 369 and 371, which polynuceotide or salt thereof selectively binds to AMB-FUBINACA.
In yet a further aspect of the present invention there is provided a polynucleotide or salt threreof comprising a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to any one of SEQ ID Nos: 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368 and 370, which polynucleotide of salt thereof selectively binds to AMB-FUBINACA.
In yet a further aspect of the present invention there is provided a polynucleotide or salt threreof comprising a sequence of any one of SEQ ID Nos: 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368 and 370, which polynucleotide of salt thereof selectively binds to AMB-FUBINACA.
In yet a further aspect of the present invention there is provided a polynucleotide or salt thereof consisting in a sequence of any one of SEQ ID Nos: 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368 and 370, which polynucleotide of salt thereof selectively binds to AMB-FUBINACA.
In a further aspect of the present invention there is provided a method for detecting the presence of AMB-FUBINACA in a test sample, the method comprising the steps of:
(i) contacting a test sample with a polynucleotide or salt thereof comprising a sequence defined by any one of SEQ ID Nos: 320-371; and
(ii) measuring binding between the polynucleotide and AMB-FUBINACA from the test sample, wherein, a measured binding interaction between the polynucleotide and AMB- FUBINACA reflects the presence of AMB-FUBINACA in the test sample.
In yet a further aspect of the present invention there is provided a test kit or article of manufacture for detecting the presence of AMB-FUBINACA in a test sample, the test kit or article of manufacture comprising a polynucleotide or salt thereof comprising a sequence defined by any one of SEQ ID Nos: 320-371 together with instructions for how to detect the presence of AMB-FUBINACA in the test sample.
In yet another aspect of the present invention there is provided a polynucleotide or salt thereof comprising the sequence ATACGAGCTTGTTCAATA[X]nTGATAGTAAGAGCAATC (SEQ ID NO: 586), where X is selected from A, T, U, G or C and n is an integer of between about 10 and about 120, in particular between about 35 and about 45, which polynuceotide or salt thereof selectively binds to AM2201.
In yet another aspect of the present invention there is provided a polynucleotide or salt thereof comprising the sequence ATACGAGCTTGTTCAATA[Y]TGATAGTAAGAGCAATC (SEQ ID NO: 587), where Y is any sequence comprising a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to any one of SEQ ID Nos: 373, 375, 377, 379, 381, 383, 385, 387, 389, 391, 393, 395, 397, 399, 401, 403, 405, 407, 409, 411, 413, 415, 417 and 419, which polynuceotide or salt thereof selectively binds to AM2201.
In yet another aspect of the present invention there is provided a polynucleotide or salt thereof comprising the sequence ATACGAGCTTGTTCAATA[Y]TGATAGTAAGAGCAATC (SEQ ID NO: 588), where Y is any sequence comprising any one of SEQ ID Nos: 373, 375, 377, 379, 381, 383, 385, 387, 389, 391, 393, 395, 397, 399, 401, 403, 405, 407, 409, 411, 413, 415, 417 and 419, which polynuceotide or salt thereof selectively binds to AM2201.
In yet another aspect of the present invention there is provided a polynucleotide or salt thereof comprising the sequence ATACGAGCTTGTTCAATA[Y]TGATAGTAAGAGCAATC (SEQ ID NO: 587), where Y is any sequence consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to any one of SEQ ID Nos: 373, 375, 377, 379, 381, 383, 385, 387, 389, 391, 393, 395, 397, 399, 401, 403, 405, 407, 409, 411, 413, 415, 417 and 419, which polynuceotide or salt thereof selectively binds to AM2201.
In yet another aspect of the present invention there is provided a polynucleotide or salt thereof comprising the sequence ATACGAGCTTGTTCAATA[Y]TGATAGTAAGAGCAATC (SEQ ID NO: 588), where Y is any sequence consisting in a sequence of any one of SEQ ID Nos: 373, 375, 377, 379, 381, 383, 385, 387, 389, 391, 393, 395, 397, 399, 401, 403, 405, 407, 409, 411, 413, 415, 417 and 419, which polynuceotide or salt thereof selectively binds to AM2201.
In yet another aspect of the present invention there is provided a polynucleotide or salt threreof comprising a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to any one of SEQ ID Nos: 372, 374, 376, 378, 380, 382, 384, 386, 388, 390 ,392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416 and 418, which polynucleotide of salt thereof selectively binds to AM2201.
In yet another aspect of the present invention there is provided a polynucleotide or salt threreof comprising a sequence of any one of SEQ ID Nos: 372, 374, 376, 378, 380, 382, 384, 386, 388, 390 ,392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416 and 418, which polynucleotide of salt thereof selectively binds to AM2201.
In yet another aspect of the present invention there is provided a polynucleotide or salt thereof consisting in a sequence of any one of SEQ ID Nos: 372, 374, 376, 378, 380, 382, 384, 386, 388, 390 ,392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416 and 418 and 277, which polynucleotide of salt thereof selectively binds to AM2201.
In a further aspect of the present invention there is provided a method for detecting the presence of AM2201 in a test sample, the method comprising the steps of:
(i) contacting a test sample with a polynucleotide or salt thereof comprising a sequence defined by any one of SEQ ID Nos: 372-419; and
(ii) measuring binding between the polynucleotide and AM2201 from the test sample, wherein, a measured binding interaction between the polynucleotide and AM2201 reflects the presence of AM2201 in the test sample.
In yet a further aspect of the present invention there is provided a test kit or article of manufacture for detecting the presence of AM2201 in a test sample, the test kit or article of manufacture comprising a polynucleotide or salt thereof comprising a sequence defined by any one of SEQ ID Nos: 372-419 together with instructions for how to detect the presence of AM2201 in the test sample.
In yet a further aspect of the present invention there is provided a polynucleotide or salt thereof comprising the sequence ATACGAGCTTGTTCAATA[X]nTGATAGTAAGAGCAATC (SEQ ID NO: 589), where X is selected from A, T, U, G or C and n is an integer of between about 10 and about 120, in particular between about 35 and about 45, which polynuceotide or salt thereof selectively binds to oxycodone.
In yet a further aspect of the present invention there is provided a polynucleotide or salt thereof comprising the sequence ATACGAGCTTGTTCAATA[Y]TGATAGTAAGAGCAATC (SEQ ID NO: 590), where Y is any sequence comprising a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to any one of SEQ ID Nos: 421, 423, 425, 427, 429, 431, 433, 435, 437, 439, 441, 443, 445, 447, 449, 451, 453, 455, 457, 459, 461, 463, 465, which polynuceotide or salt thereof selectively binds to oxycodone.
In yet a further aspect of the present invention there is provided a polynucleotide or salt thereof comprising the sequence ATACGAGCTTGTTCAATA[Y]TGATAGTAAGAGCAATC (SEQ ID NO: 591), where Y is any sequence comprising any one of SEQ ID Nos: 421, 423, 425, 427, 429, 431, 433, 435, 437, 439, 441, 443, 445, 447, 449, 451, 453, 455, 457, 459, 461, 463, 465, which polynuceotide or salt thereof selectively binds to oxycodone.
In yet a further aspect of the present invention there is provided a polynucleotide or salt thereof comprising the sequence ATACGAGCTTGTTCAATA[Y]TGATAGTAAGAGCAATC (SEQ ID NO: 590), where Y is any sequence consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to any one of SEQ ID Nos: 421, 423, 425, 427, 429, 431, 433, 435, 437, 439, 441, 443, 445, 447, 449, 451, 453, 455, 457, 459, 461, 463, 465, which polynuceotide or salt thereof selectively binds to oxycodone. In yet a further aspect of the present invention there is provided a polynucleotide or salt thereof comprising the sequence ATACGAGCTTGTTCAATA[Y]TGATAGTAAGAGCAATC (SEQ ID NO: 591), where Y is any sequence consisting in a sequence of any one of SEQ ID Nos: 421, 423, 425, 427, 429, 431, 433, 435, 437, 439, 441, 443, 445, 447, 449, 451, 453, 455, 457, 459, 461, 463, 465, which polynuceotide or salt thereof selectively binds to oxycodone.
In yet a further aspect of the present invention there is provided a polynucleotide or salt threreof comprising a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to any one of SEQ ID Nos: 420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440, 442, 444, 446, 448, 450, 452, 454, 456, 458, 460, 462, 464 and 466, which polynucleotide of salt thereof selectively binds to oxycodone.
In yet a further aspect of the present invention there is provided a polynucleotide or salt threreof comprising a sequence of any one of SEQ ID Nos: 420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440, 442, 444, 446, 448, 450, 452, 454, 456, 458, 460, 462, 464 and 466, which polynucleotide of salt thereof selectively binds to oxycodone.
In yet a further aspect of the present invention there is provided a polynucleotide or salt thereof consisting in a sequence of any one of SEQ ID Nos: 420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440, 442, 444, 446, 448, 450, 452, 454, 456, 458, 460, 462, 464 and 466, which polynucleotide of salt thereof selectively binds to oxycodone.
In a further aspect of the present invention there is provided a method for detecting the presence of oxycodone in a test sample, the method comprising the steps of:
(i) contacting a test sample with a polynucleotide or salt thereof comprising a sequence defined by any one of SEQ ID Nos: 420-467; and
(ii) measuring binding between the polynucleotide and oxycodone from the test sample, wherein, a measured binding interaction between the polynucleotide and oxycodone reflects the presence of oxycodone in the test sample.
In yet a further aspect of the present invention there is provided a test kit or article of manufacture for detecting the presence of oxycodone in a test sample, the test kit or article of manufacture comprising a polynucleotide or salt thereof comprising a sequence defined by any one of SEQ ID Nos: 420-467 together with instructions for how to detect the presence of oxycodone in the test sample.
In yet a further aspect of the present invention there is provided a polynucleotide or salt thereof comprising the sequence ATACGAGCTTGTTCAATA[X]nTGATAGTAAGAGCAATC (SEQ ID NO: 592), where X is selected from A, T, U, G or C and n is an integer of between about 10 and about 120, in particular between about 35 and about 45, which polynuceotide or salt thereof selectively binds to ketamine. In yet a further aspect of the present invention there is provided a polynucleotide or salt thereof comprising the sequence ATACGAGCTTGTTCAATA[Y]TGATAGTAAGAGCAATC (SEQ ID NO: 593), where Y is any sequence comprising a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to any one of SEQ ID Nos: 469, 471, 473, 475, 477, 479, 481, 483, 485, 487, 489, 491, 493, 495, 497, 499, 501, 503, 505, 507, 509, 511, 513, 515, 517, 519, 521 and 523, which polynuceotide or salt thereof selectively binds to ketamine.
In yet a further aspect of the present invention there is provided a polynucleotide or salt thereof comprising the sequence ATACGAGCTTGTTCAATA[Y]TGATAGTAAGAGCAATC (SEQ ID NO: 594), where Y is any sequence comprising any one of SEQ ID Nos: 469, 471, 473, 475, 477, 479, 481, 483, 485, 487, 489, 491, 493, 495, 497, 499, 501, 503, 505, 507, 509, 511, 513, 515, 517, 519, 521 and 523, which polynuceotide or salt thereof selectively binds to ketamine.
In yet a further aspect of the present invention there is provided a polynucleotide or salt thereof comprising the sequence ATACGAGCTTGTTCAATA[Y]TGATAGTAAGAGCAATC (SEQ ID NO: 593), where Y is any sequence consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to any one of SEQ ID Nos: 469, 471, 473, 475, 477, 479, 481, 483, 485, 487, 489, 491, 493, 495, 497, 499, 501, 503, 505, 507, 509, 511, 513, 515, 517, 519, 521 and 523, which polynuceotide or salt thereof selectively binds to ketamine.
In yet a further aspect of the present invention there is provided a polynucleotide or salt thereof comprising the sequence ATACGAGCTTGTTCAATA[Y]TGATAGTAAGAGCAATC (SEQ ID NO: 594), where Y is any sequence consisting in a sequence of any one of SEQ ID Nos: 469, 471, 473, 475, 477, 479, 481, 483, 485, 487, 489, 491, 493, 495, 497, 499, 501, 503, 505, 507, 509, 511, 513, 515, 517, 519, 521 and 523, which polynuceotide or salt thereof selectively binds to ketamine.
In yet a further aspect of the present invention there is provided a polynucleotide or salt threreof comprising a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to any one of SEQ ID Nos: 468, 470, 472, 474, 476, 478, 480, 482,
484, 486, 488, 490, 492, 494, 496, 498, 500, 502, 504, 506, 508, 510, 512, 514, 516, 518,
520 and 522, which polynucleotide of salt thereof selectively binds to ketamine.
In yet a further aspect of the present invention there is provided a polynucleotide or salt threreof comprising a sequence of any one of SEQ ID Nos: 468, 470, 472, 474, 476, 478,
480, 482, 484, 486, 488, 490, 492, 494, 496, 498, 500, 502, 504, 506, 508, 510, 512, 514,
516, 518, 520 and 522, which polynucleotide of salt thereof selectively binds to ketamine.
In yet a further aspect of the present invention there is provided a polynucleotide or salt thereof consisting in a sequence of any one of SEQ ID Nos: 468, 470, 472, 474, 476, 478, 480, 482, 484, 486, 488, 490, 492, 494, 496, 498, 500, 502, 504, 506, 508, 510, 512, 514, 516, 518, 520 and 522, which polynucleotide of salt thereof selectively binds to ketamine.
In a further aspect of the present invention there is provided a method for detecting the presence of ketamine in a test sample, the method comprising the steps of:
(i) contacting a test sample with a polynucleotide or salt thereof comprising a sequence defined by any one of SEQ ID Nos: 468-523; and
(ii) measuring binding between the polynucleotide and ketamine from the test sample, wherein, a measured binding interaction between the polynucleotide and ketamine reflects the presence of ketamine in the test sample.
In yet a further aspect of the present invention there is provided a test kit or article of manufacture for detecting the presence of ketamine in a test sample, the test kit or article of manufacture comprising a polynucleotide or salt thereof comprising a sequence defined by any one of SEQ ID Nos: 468-523 together with instructions for how to detect the presence of ketamine in the test sample.
In a further aspect of the present invention there is provision for the polynucleotides described herein to be utilised to develop novel complete analytical or separation systems. The analytical systems may utilise platforms such as electrochemical, colorimetric, and amprometric but not limited to. The polynucleotides according to the present invention may need direct modification embedded e.g. redox active molecules, fluorophores and bioactice molecules and may also need to be modifed and/or embedded with chemical functional groups for chemical conjugation to a structure e.g. electrodes (gold, platnium, carbon based, or conducting ploymer based), particles with and without magnetic properties, and peptides and/or proteins but not limited to. Indirectly, the polynucleotides described herein may be hybridised with complementary probes with and without any of the aforementioned modification examples to then be adopted onto an analytical and separation platforms. The polynucleotides described herein may be used individually or collectively in an analytical format where the collective data can be utilised to deduce the presence of a drug in the sample tested e.g. blood, urine, body fluid sample etc.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1 shows the structure of methamphetamine.
Figure 2 shows a phylogenetic tree generated using Clustal Omega, depicting the structural relationship of the aptamer sequences which selectively bind to methamphetamine, as disclosed herein. Sequence distance scores are indicated against each sequence name.
Figure 3 shows dissociation constants for aptamer sequences (A) METH_01 (SEQ ID NO: 57), Kd = 520 nM; (B) METH_02 (SEQ ID NO: 71), Kd = 602 nM; and (C) METH_03 (SEQ ID NO: 27), Kd = 892 nM. Figure 4 shows the structure of (-)-trans-D9- tetrahydrocannabinol (THC), the active molecule found in cannabis.
Figure 5 shows the structure of a phylogenetic tree generated using Clustal Omega, depicting the structural relationship of the aptamer sequences which selectively bind to (-)-trans-D9 -tetrahydrocannabinol (THC), as disclosed herein. Sequence distance scores are indicated against each sequence name.
Figure 6 shows dissociation constants for aptamer sequences (A) THC_B1 (SEQ ID NO: 133), Kd = 6916 nM; (B) THC_S6 (SEQ ID NO: 91), Kd = 573 nM; and (C) THC_S9 (SEQ ID NO: 97), Kd = 284 nM.
Figure 7 shows the structure of cocaine.
Figure 8 shows a phylogenetic tree generated using Clustal Omega, depicting the structural relationship of the aptamer sequences which selectively bind to cocaine, as disclosed herein. Sequence distance scores are indicated against each sequence name.
Figure 9 shows the structure of 3,4-methylenedioxymethamphetamine (MDMA).
Figure 10 shows a phylogenetic tree generated using Clustal Omega, depicting the structural relationship of the aptamer sequences which selectively bind to 3,4- methylenedioxymethamphetamine (MDMA), as disclosed herein. Sequence distance scores are indicated against each sequence name.
Figure 11 shows the structure of morphine.
Figure 12 shows a phylogenetic tree generated using Clustal Omega, depicting the structural relationship of the aptamer sequences which selectively bind to morphine, as disclosed herein. Sequence distance scores are indicated against each sequence name.
Figure 13 shows dissociation constants for aptamer sequences (A) MOR-S15 (SEQ ID NO: 282), Kd = 248 nM; (B) MOR-S13 (SEQ ID NO: 280), Kd = 552 nM; (C) MOR-S06 (SEQ ID NO: 258), Kd = 374 nM; (D) MOR-B11 (SEQ ID NO: 268), Kd = 603 nM; and (E) MOR-B10 (SEQ ID NO: 288), Kd = 567 nM.
Figure 14 shows the structure of benzodiazepine.
Figure 15 shows a phylogenetic tree generated using Clustal Omega, depicting the structural relationship of the aptamer sequences which selectively bind to diazepam, as disclosed herein. Sequence distance scores are indicated against each sequence name.
Figure 16 shows the structure of AMB-FUBINACA (a synthetic cannabis).
Figure 17 shows a phylogenetic tree generated using Clustal Omega, depicting the structural relationship of the aptamer sequences which selectively bind to AMB-FUBINACA (a synthetic cannabis), as disclosed herein. Sequence distance scores are indicated against each sequence name.
Figure 18 shows the structure of AM2201 (a synthetic cannabis).
Figure 19 shows a phylogenetic tree generated using Clustal Omega, depicting the structural relationship of the aptamer sequences which selectively bind to AM2201 (a synthetic cannabis), as disclosed herein. Sequence distance scores are indicated against each sequence name.
Figure 20 shows the structure of oxycodone.
Figure 21 shows a phylogenetic tree generated using Clustal Omega, depicting the structural relationship of the aptamer sequences which selectively bind to oxycodone, as disclosed herein. Sequence distance scores are indicated against each sequence name.
Figure 22 shows the structure of ketamine.
Figure 23 shows a phylogenetic tree generated using Clustal Omega, depicting the structural relationship of the aptamer sequences which selectively bind to ketamine, as disclosed herein. Sequence distance scores are indicated against each sequence name.
GENERAL DEFINITIONS
Unless specifically defined otherwise, all technical and scientific terms used herein are to be understood as having the same meanings as is understood by one of ordinary skill in the relevant art to which this disclosure pertains. Examples of definitions of common terms in medicine, molecular biology and biochemistry a can be found in Dictionary of Microbiology and Molecular Biology, Singleton et al, (2d ed. (1994); The Encyclopedia of Molecular Biology, Kendrew et al. (eds.), Blackwell Science Ltd., (1994); Molecular Biology and Biotechnology: a Comprehensive Desk Reference, Robert A. Meyers (ed.), VCH Publishers, Inc., (1995); The Dictionary of Cell & Molecular Biology, 4th Edition. Lackie. J (Ed.), Academic Press Inc. (2007), and The Oxford Dictionary of Biochemistry and Molecular Biology, 2nd edition, Cammack et al. (Eds.), Oxford University Press Inc. (2006).
It is also believed that practice of various examples of, encompassed by, and falling within the scope of the present invention can be performed using standard molecular biology and biochemistry protocols and procedures as known in the art, and as described, for example in "Current Protocols in Nucleic Acid Chemistry, Wiley Online Library, Various; Molecular Cloning: A Laboratory Manual, Maniatis et al., Cold Spring Harbor Laboratory Press, (1982); Molecular Cloning: A Laboratory Manual (2 ed.), Sambrook et al., Cold Spring Harbor Laboratory Press, (1989); Guide to Molecular Cloning Techniques Vol.152, S. L. Berger and A. R. Kimmerl (Eds.), Academic Press Inc., (1987); Protein Synthesis and Ribosome Structure: Translating the Genome. Nierhaus, K and Wilson D (eds.), Wiley-VCH Inc. (2004); Synthetic Peptides: A User's Guide (Advances in Molecular Biology) 2nd edition, Grant G. (Ed.), Oxford University Press (2002); Remington: The Science and Practice of Pharmacy 21st edition, Beringer, P (Ed.), Lippincott Williams & Wilkins, (2005), pp. 2393; and other commonly available reference materials relevant in the art to which this disclosure pertains, and which are all incorporated by reference herein in their entireties.
It is intended that reference to a range of numbers disclosed herein (e.g. 1 to 10) also incorporates reference to all related numbers within that range (e.g. 1, 1.1, 2, 3, 3.9, 4, 5, 6, 6.5, 7, 8, 9 and 10) and also any range of rational numbers within that range (for example 2 to 8, 1.5 to 5.5 and 3.1 to 4.7) and, therefore, all sub-ranges of all ranges expressly disclosed herein are expressly disclosed. These are only examples of what is specifically intended and all possible combinations of numerical values between the lowest value and the highest value enumerated are to be considered to be expressly stated in this application in a similar manner.
The term "and/or", e.g., "X and/or Y" shall be understood to mean either "X and Y" or "X or Y" and shall be taken to provide explicit support for both meanings or for either meaning.
The term "a" or "an" refers to one or more than one of the entity specified; for example, "a receptor" or "a nucleic acid molecule" may refer to one or more receptor or nucleic acid molecule, or at least one receptor or nucleic acid molecule. As such, the terms "a" or "an", "one or more" and "at least one" can be used interchangeably herein.
Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.
Throughout this specification, unless specifically stated otherwise or the context requires otherwise, reference to a single step, composition of matter, group of steps or group of compositions of matter shall be taken to encompass one and a plurality (i.e. one or more) of those steps, compositions of matter, groups of steps or group of compositions of matter.
SELECTED DEFINITIONS
For the purposes of the present invention, the following terms shall have the following meanings.
The term "about" when used in connection with a referenced numeric indication means the referenced numeric indication plus or minus up to 10% of that referenced numeric indication. For example, "about 100" means from 90 to 110 and "about six" means from 5.4 to 6.6.
The term "fragment" as used herein is used interchangeably with the term "functional fragment" and means the same thing.
The term "functional fragment" as used herein means any part of a polynucleotide of the inventions disclosed herein that retains its ability to selectively bind to the same drug target as the polynucleotide from which it is derived.
The term "test kit" as used herein refers to an article of manufacture comprising various components to perform the assays and methods according to the inventions descibed herein.
The term "THC" as used herein tetrahydrocannabinol and includes, for example, (-)-trans-D9-tetrahydrocannabinol. The term "MDMA" as used herein means 3,4-methylenedioxymethamphetamine.
The term "AMB-FUBINACA" as used herein refers to a synthetic form of cannabis, the structure for which is illustrated in Figure 16.
The term "AM2201" as used herein also refers to a synthetic form of cannabis, the structure for which is illustrated in Figure 18.
The term "polynucleotide" as used herein refers to a deoxyribose nucleic acid (DNA) sequence, a ribose nucleic acid sequence (RNA), messenger ribose nucleic acid (mRNA) and complementary DNA (cDNA), and is comprised of a continuous sequence of two or more nucleotides, also referred to as "a nucleic acid sequence".
The term "ligand" refers generally to any molecule that binds to a receptor, and includes without limitation, a polypeptide, a protein, a vitamin, a carbohydrate, a glycoprotein, a therapeutic agent, a drug, a glycosaminoglycan, or any combination thereof. As used herein, "ligand" may include a drug or molecule associated with a drug, and in particular drugs of abuse including, without limitation, methamphetamine, cannabis, synthetic cannabis, cocaine, ecstasy, morphine, oxycodone and benzodiazepines.
The term "sample" as used herein refers to any sample for which it is desired to test for the presence of a drug of abuse.
The term "reference threshold" as used herein means the level of assay activity measured in the absence of a test sample. In certain examples according to the inventions described herein, the reference threshold is determined using ethanol in place of test sample.
The term "detection means" as used herein refers to any apparatus, equipment or configuration adapted to detect the binding interaction between an aptamer/polynucleotide and its drug target. Examples of detection means include, but are not limited to, optical methods, spectroscopy, visible spectroscopy, Raman spectroscopy, UV spectroscopy, surface plasmon resonance, electrochemical methods, impedance, resistance, capacitance, mechanical sensing by changes in mass, changes in mechanical resonance, electrophoresis, gel electrophoresis, gel retardation, imaging, fluorescence and fluorescence resonance energy transfer, polymerase chain reaction etc.
DETAILED DESCRIPTION
The present invention provides polynucloetides, test kits and methods useful for the detection of drugs of abuse including, without limitation, detection of methamphetamine, cannabis, synthetic cannabis, cocaine, ecstasy, morphine, benzodiazepines, and oxycodone.
The polynucloetides, test kits and methods according to the present invention are particularly useful for road-side testing or in work-place monitoring to detect drug abuse by individuals that places themselves or others in the community in danger. For example, in the operation of a motor vehicle or work-place machinary while under the influence of a drug of abuse, creating a potential for serious harm, permanent injury or death. Polynucleotides/Aptamers
The polynucleotides of the present invention spontaneously fold to form aptamers having secondary structure features that promote selective binding to a target molecule, in particular a target associated with an illicit drug. According to the inventions described herein, the polynuceotides selectively bind to illicit drug targets including, without limitation, methamphetamine, tetrahydrocannabinol, cocaine, 3,4-methylenedioxymethamphetamine, diazepam, morphine, AMB-FUNIBACA, AM2201, oxycodone and ketamine.
In one aspect of the present invention there is provided a polynucleotide or salt thereof comprising or consisting in the sequence
ATACGAGCTTGTTCAATA[X]nTGATAGTAAGAGCAATC, where X is selected from A, T, U, G or C and n is an integer of between about 10 and about 120, in particular between about 35 and about 45. Methamphetamine Aptamers
In reference to the Examples and Figures which follow, Applicant has developed aptamer sequences which are highly selective for methamphetamine. Advantageously, these aptamers display highly selective binding affinity for methamphetamine, with minimal cross reactivity to other drug targets. By way of illustration only, in reference to Tables 1-6 below, read in conjunction with
Figures 1-3, Applicant developed aptamer sequences which selectively bind to methamphetamine, the structure of which is depicted in Figure 1, as well as structural analogs of methamphetamine.
Figure imgf000024_0001
Figure imgf000025_0001
Figure imgf000026_0001
Accordingly, in an aspect of the present invention there is provided a polynucleotide or salt thereof comprising the sequence ATACGAGCTTGTTCAATA[Y]TGATAGTAAGAGCAATC (SEQ ID NO: 566), where Y is any sequence comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to any one of SEQ ID Nos: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72 and 74, which polynuceotide or salt thereof selectively binds to methamphetamine.
In another aspect of the present invention there is provided a polynucleotide or salt thereof comprising the sequence ATACGAGCTTGTTCAATA[Y]TGATAGTAAGAGCAATC (SEQ ID NO: 567), where Y is any sequence comprising or consisting in a sequence selected from any one of SEQ ID Nos: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72 and 74, which polynuceotide or salt thereof selectively binds to methamphetamine.
In another aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to methamphetamine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 1 or a fragment thereof which retains selective binding for methamphetamine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 2.
In another aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to methamphetamine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 3 or a fragment thereof which retains selective binding for methamphetamine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 4.
In another aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to methamphetamine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 5 or a fragment thereof which retains selective binding for methamphetamine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 6.
In another aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to methamphetamine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 7 or a fragment thereof which retains selective binding for methamphetamine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 8. In another aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to methamphetamine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 9 or a fragment thereof which retains selective binding for methamphetamine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 10.
In another aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to methamphetamine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 11 or a fragment thereof which retains selective binding for methamphetamine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 12.
In another aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to methamphetamine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 13 or a fragment thereof which retains selective binding for methamphetamine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 14.
In another aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to methamphetamine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 15 or a fragment thereof which retains selective binding for methamphetamine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 16.
In another aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to methamphetamine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 17 or a fragment thereof which retains selective binding for methamphetamine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 18.
In another aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to methamphetamine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 19 or a fragment thereof which retains selective binding for methamphetamine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 20.
In another aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to methamphetamine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 21 or a fragment thereof which retains selective binding for methamphetamine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 22.
In another aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to methamphetamine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 23 or a fragment thereof which retains selective binding for methamphetamine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 24.
In another aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to methamphetamine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 25 or a fragment thereof which retains selective binding for methamphetamine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 26.
In another aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to methamphetamine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 27 or a fragment thereof which retains selective binding for methamphetamine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 28.
In another aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to methamphetamine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 29 or a fragment thereof which retains selective binding for methamphetamine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 30.
In another aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to methamphetamine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 31 or a fragment thereof which retains selective binding for methamphetamine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 32.
In another aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to methamphetamine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 33 or a fragment thereof which retains selective binding for methamphetamine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 34.
In another aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to methamphetamine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 35 or a fragment thereof which retains selective binding for methamphetamine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 36.
In another aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to methamphetamine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 37 or a fragment thereof which retains selective binding for methamphetamine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 38.
In another aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to methamphetamine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 39 or a fragment thereof which retains selective binding for methamphetamine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 40.
In another aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to methamphetamine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 41 or a fragment thereof which retains selective binding for methamphetamine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 42.
In another aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to methamphetamine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 43 or a fragment thereof which retains selective binding for methamphetamine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 44.
In another aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to methamphetamine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 45 or a fragment thereof which retains selective binding for methamphetamine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 46. In another aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to methamphetamine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 47 or a fragment thereof which retains selective binding for methamphetamine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 48.
In another aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to methamphetamine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 49 or a fragment thereof which retains selective binding for methamphetamine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 50.
In another aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to methamphetamine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 51 or a fragment thereof which retains selective binding for methamphetamine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 52.
In another aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to methamphetamine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 53 or a fragment thereof which retains selective binding for methamphetamine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 54.
In another aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to methamphetamine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 55 or a fragment thereof which retains selective binding for methamphetamine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 56.
In another aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to methamphetamine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 57 or a fragment thereof which retains selective binding for methamphetamine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 58.
In another aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to methamphetamine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 59 or a fragment thereof which retains selective binding for methamphetamine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 60.
In another aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to methamphetamine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 61 or a fragment thereof which retains selective binding for methamphetamine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 62.
In another aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to methamphetamine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 63 or a fragment thereof which retains selective binding for methamphetamine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 64.
In another aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to methamphetamine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 65 or a fragment thereof which retains selective binding for methamphetamine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 66.
In another aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to methamphetamine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 67 or a fragment thereof which retains selective binding for methamphetamine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 68.
In another aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to methamphetamine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 69 or a fragment thereof which retains selective binding for methamphetamine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 70.
In another aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to methamphetamine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 71 or a fragment thereof which retains selective binding for methamphetamine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 72.
In another aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to methamphetamine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 73 or a fragment thereof which retains selective binding for methamphetamine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 74.
Cluster analysis was performed on the methamphetamine aptamers disclosed herein using Clustal Omega Phylogenetic Trees. For further information refer to https//www. ebi.ac.uk/Tools/msa/clustalo/. The results from these analyses is presented in Figure 2 where the level of structural (i.e. sequence) dis/similarity between the different aptamers in this drug class is pictorily illustrated. By way of exemplification only, the methamphetamine aptamers designated MB07, MB04 and MB06 share a higher degree of structural similarity to (e.g.) MB08 and MB02. However, MB07, MB04, MB06, MB08 and MB02 collectively share a higher degree of structural similarity compared to (e.g.) MS04, MS05 and MS06.
Figures 3A-3C provide dissociation constants for the aptamers designated MS01 (SEQ ID NO: 57), MS03 (SEQ ID NO: 71) and MS-02 (SEQ ID NO: 27) of Kd = 520 nM, 602 nM and 892 nM, respectively, as measured using the methodologies described in the Examples. Additional binding affinity data for the methamphetamine aptamers is disclosed in Table 1. Specifically, the dissociation constant for MB-01 (SEQ ID NO: 1): Kd = 1043 nM; MB-06 (SEQ ID NO: 5): Kd = 1580 nM; ME-R9-S7 (SEQ ID NO: 13): Kd = 580 nM; MS-04 (SEQ ID NO: 31): Kd = 3864 nM; MS10 (SEQ ID NO: 67): Kd = 3835 nM; and MS07 (SEQ ID NO: 73): Kd = 580 nM. With dissociation constants in the high nM range, these aptamers display highly selective binding affinity for methamphetamine.
Accordingly, the polynucleotides encoding methamphetamine aptamers described herein, including without limitation SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71 and 73, display a binding affinity for methamphetamine which is between about 1 nM and about 5 mM, including between about 1 nM and about 2 mM, or about 1 nM and about 1500 nM, or about 1 nM and about 1000 nM, or about 1 nM and about 500 nM.
By way of illustration only, the term "between about 1 nM and about 500 nM" includes a binding dissociation constant that is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 55, 50, 65, 60, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 405, 410, 415, 420, 425, 430, 435, 440, 445, 450, 452, 454, 456, 458, 460, 462, 464, 468, 470, 472, 474, 476, 478, 480, 482, 484, 486, 488, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499 and 500 nM. The skilled person would, however, appreciate that "between about 1 nM and about 500 nM" extends beyond the definition presented above to includee any whole integer within this range including, without limitation 53, 101, 207, 274, 318, 423, 479 nM etc. In addition, Geneious Prime pairwise alignments were performed on the various sequence clusters identified using the Clustal Omega Phylogenetic Tree analysis to identify consensus sequences of interest. Geneious uses IUPAC ambiguity codes, as shown in Table 2, below. Geneious will identify 'consensus' at a particular nucleotide position in the alignment if >50% of sequences agree, while ambiguity codes are used if that agreement threshold is not met.
Figure imgf000034_0001
The Geneious Prime pairwise alignments successfuly identified four discrete methamphetamine aptamer consensus sequences defined in Tables 3-6 below and identified as SEQ ID NO: 524, SEQ ID NO: 525, SEQ ID NO: 526 and SEQ ID NO: 527.
Accordingly, in yet a further aspect of the present invention there is provided a polynuceotide or salt thereof which comprises or consists in a consensus sequence defined by SEQ ID NO: 524, which polynucleotide or salt thereof selectively binds methamphetamine. In an example according to this aspect of the present invention, the polynucleotide or salt thereof which selectively binds methamphetamine comprises or consists in a sequence selected from SEQ ID Nos: 13, 73, 45, 47, 49, 51, 55, and 9. In a further related example according to this aspect of the present invention, the polynucleotide or salt thereof comprises or consists in SEQ ID No: 13.
Figure imgf000035_0001
Figure imgf000036_0001
Figure imgf000037_0001
The Gibbs Free Energy (DG) associated with each methamphetamine aptamer disclosed herein is reflected in Table 1. The quantum of Gibbs Free Energy is reflective of aptamer stability; a more negative DG value reflects an aptamer having a higher degree of overall stability compared to an aptamer with a less negative DG value.
Information concerning Gibbs Free Energy may be an important consideration when selecting an aptamer for use in a particular assay format. For example, it is preferable to employ an aptamer which displays a higher degree of stability for use in a colorimetric/nanoparticle detection assay, such as that described in Applicant's earlier work (WO 2015/174863). In contrast, other analyte detection assays involving aptamer chemistry require aptamers which are less stable for the reason that the presence of a target molecule triggers a conformational change in the aptamer structure, and it is a physical property associated with this structural change which is used to detect the presence of a target molecule in a sample under investigation.
According to the information presented in Table 3, the range in Gibbs Free Energy associated with Methamphetamine Aptamer Cluster 1 ("ME1"; SEQ ID NO: 524) is DG = - 3.32 to -6.37.
In another aspect of the present invention there is provided a polynucleotide or salt thereof which comprises or consists in a consensus sequence defined by SEQ ID NO: 525, which polynucleotide or salt thereof selectively binds methamphetamine.
In an example according to this aspect of the present invention, the polynucleotide or salt thereof which selectively binds methamphetamine comprises or consists in a sequence selected from SEQ ID Nos: 1, 5, 27, 29, 3, 17, 19, 21, 23, 25, 31, 33 and 39. In a further related example according to this aspect of the present invention, the polynucleotide or salt thereof comprises or consists in a sequence selected from SEQ ID Nos: 1, 5, 27 and 29.
According to the information presented in Table 4, the range in Gibbs Free Energy associated with the Methamphetamine Aptamer Cluster 2 ("ME2"; SEQ ID NO: 525) is DG = -4.42 to -7.48.
In yet another aspect of the present invention there is provided a polynucleotide or salt thereof which comprises or consists in a consensus sequence defined by SEQ ID NO: 526, which polynucleotide or salt thereof selectively binds methamphetamine.
In an example according to this aspect of the present invention, the polynucleotide or salt thereof which selectively binds methamphetamine comprises or consists in a sequence selected from SEQ ID Nos: 71, 67, 41, 43, 53, 7, 11, 15 and 65. In a further related example according to this aspect of the present invention, the polynucleotide or salt thereof comprises or consists in a sequence selected from SEQ ID Nos: 71 and 67.
According to the information presented in Table 5, the range in Gibbs Free Energy associated with the Methamphetamine Aptamer Cluster 3 ("ME3"; SEQ ID NO: 526) is DG = -2.21 to -6.71. In yet another aspect of the present invention there is provided a polynucleotide or salt thereof which comprises or consists in a consensus sequence defined by SEQ ID NO: 527, which polynucleotide or salt thereof selectively binds methamphetamine.
In an example according to this aspect of the present invention, the polynucleotide or salt thereof which selectively binds methamphetamine comprises or consists in a sequence selected from SEQ ID Nos: 69, 57, 35 and 37.
According to the information presented in Table 6, the range in Gibbs Free Energy associated with the Methamphetamine Aptamer Cluster 4 ("ME4"; SEQ ID NO: 527) is DG = -1.87 to -8.71.
Tetrahydrocannabinol (THC) Aptamers
In reference to the Examples and Figures which follow, Applicant has also developed aptamer sequences which are highly selective for tetrahydrocannabinol. Advantageously, these aptamers display highly selective binding affinity for tetrahydrocannabinol, with minimal cross-reactivity to other drug targets.
By way of illustration only, in reference to Tables 7-11 below, read in conjunction with Figures 4-6, Applicant developed aptamer sequences which selectively bind to tetrahydrocannabinol, the structure of which is depicted in Figure 4, as well as structural analogs of tetrahydrocannabinol.
Accordingly, in yet a further aspect of the present invention there is provided a polynucleotide or salt thereof comprising or consisting in the sequence
ATACGAGCTTGTTCAATA[Y]TGATAGTAAGAGCAATC (SEQ ID NO: 569), where Y is any sequence comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to any one of SEQ ID Nos: 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134 and 136, which polynuceotide or salt thereof selectively binds to tetrahydrocannabinol.
In a further aspect of the present invention there is provided a polynucleotide or salt thereof comprising the sequence ATACGAGCTTGTTCAATA[Y]TGATAGTAAGAGCAATC (SEQ ID NO: 570), where Y is any sequence comprising or consisting in a sequence selected from any one of SEQ ID Nos: 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134 and 136, which polynuceotide or salt thereof selectively binds to tetrahydrocannabinol.
In a further aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to tetrahydrocannabinol, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 75 or a fragment thereof which retains selective binding for tetrahydrocannabinol. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 76.
Figure imgf000041_0001
Figure imgf000042_0001
Figure imgf000043_0001
In a further aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to tetrahydrocannabinol, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 77 or a fragment thereof which retains selective binding for tetrahydrocannabinol. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 78.
In a further aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to tetrahydrocannabinol, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 79 or a fragment thereof which retains selective binding for tetrahydrocannabinol. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 80.
In a further aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to tetrahydrocannabinol, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 81 or a fragment thereof which retains selective binding for tetrahydrocannabinol. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 82.
In a further aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to tetrahydrocannabinol, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 83 or a fragment thereof which retains selective binding for tetrahydrocannabinol. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 84.
In a further aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to tetrahydrocannabinol, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 85 or a fragment thereof which retains selective binding for tetrahydrocannabinol. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 86.
In a further aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to tetrahydrocannabinol, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 87 or a fragment thereof which retains selective binding for tetrahydrocannabinol. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 88.
In a further aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to tetrahydrocannabinol, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 89 or a fragment thereof which retains selective binding for tetrahydrocannabinol. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 90.
In a further aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to tetrahydrocannabinol, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 91 or a fragment thereof which retains selective binding for tetrahydrocannabinol. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 92.
In a further aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to tetrahydrocannabinol, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 93 or a fragment thereof which retains selective binding for tetrahydrocannabinol. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 94.
In a further aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to tetrahydrocannabinol, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 95 or a fragment thereof which retains selective binding for tetrahydrocannabinol. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 96.
In a further aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to tetrahydrocannabinol, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 97 or a fragment thereof which retains selective binding for tetrahydrocannabinol. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 98.
In a further aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to tetrahydrocannabinol, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 99 or a fragment thereof which retains selective binding for tetrahydrocannabinol. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 100.
In a further aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to tetrahydrocannabinol, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 101 or a fragment thereof which retains selective binding for tetrahydrocannabinol. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 102
In a further aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to tetrahydrocannabinol, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 103 or a fragment thereof which retains selective binding for tetrahydrocannabinol. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 104.
In a further aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to tetrahydrocannabinol, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 105 or a fragment thereof which retains selective binding for tetrahydrocannabinol. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 106.
In a further aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to tetrahydrocannabinol, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 107 or a fragment thereof which retains selective binding for tetrahydrocannabinol. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 108.
In a further aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to tetrahydrocannabinol, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 109 or a fragment thereof which retains selective binding for tetrahydrocannabinol. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 110.
In a further aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to tetrahydrocannabinol, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 111 or a fragment thereof which retains selective binding for tetrahydrocannabinol. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 112.
In a further aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to tetrahydrocannabinol, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 113 or a fragment thereof which retains selective binding for tetrahydrocannabinol. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 114. In a further aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to tetrahydrocannabinol, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 115 or a fragment thereof which retains selective binding for tetrahydrocannabinol. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 116.
In a further aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to tetrahydrocannabinol, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 117 or a fragment thereof which retains selective binding for tetrahydrocannabinol. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 118.
In a further aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to tetrahydrocannabinol, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 119 or a fragment thereof which retains selective binding for tetrahydrocannabinol. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 120.
In a further aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to tetrahydrocannabinol, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 121 or a fragment thereof which retains selective binding for tetrahydrocannabinol. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 122.
In a further aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to tetrahydrocannabinol, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 123 or a fragment thereof which retains selective binding for tetrahydrocannabinol. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 124.
In a further aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to tetrahydrocannabinol, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 125 or a fragment thereof which retains selective binding for tetrahydrocannabinol. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 126.
In a further aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to tetrahydrocannabinol, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 127 or a fragment thereof which retains selective binding for tetrahydrocannabinol. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 128.
In a further aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to tetrahydrocannabinol, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 129 or a fragment thereof which retains selective binding for tetrahydrocannabinol. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 130.
In a further aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to tetrahydrocannabinol, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 131 or a fragment thereof which retains selective binding for tetrahydrocannabinol. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 132.
In a further aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to tetrahydrocannabinol, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 133 or a fragment thereof which retains selective binding for tetrahydrocannabinol. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 134.
In a further aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to tetrahydrocannabinol, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 135 or a fragment thereof which retains selective binding for tetrahydrocannabinol. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 136.
Cluster analysis was also performed on the tetrahydrocannabinol aptamers disclosed herein using Clustal Omega. The results from these analyses is presented in Figure 5 where the level of structural (i.e. sequence) dis/similarity between the different aptamers in this drug class is pictorily illustrated. By way of exemplification only, the tetrahydrocannabinol aptamers designated THC-30 and THC-32 share a higher degree of structural similarity compared to (e.g.) THC-01 and THC-09. However, THC-30, THC-32, THC-01 and THC-09 collectively share a higher degree of structural similarity compared to (e.g.) THCR9-S6, THCR9-S1 and THCR9-S10.
Figures 6A-6C provide dissociation constants for THC-01 (SEQ ID NO: 133) of 6916 nM, THCR9-S6 (SEQ ID NO: 91) of 573 nM and THCR9-S9 (SEQ ID NO: 97) of 284 nM. These data would indicate that THCR9-S6 and THCR9-S9 are more selective for tetrahydrocannabinol than THC-01. With dissociation constants in the high nM range, these aptamers display highly selective binding affinity for tetrahydrocannabinol.
Accordingly, the polynucleotides encoding tetrahydrocannabinol aptamers described herein, including without limitation SEQ ID NOs: 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 133 and 135, display a binding affinity for tetrahydrocannabinol which is between about 1 nM and about 1000 nM, including between about about 1 nM and about 750 nM, and about 1 nM and 300 nM.
By way of illustration only, the term "between about 1 nM and about 300 nM" includes a binding dissociation constant that is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 55, 50, 65, 60, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299 and 300. The skilled person would, however, appreciate that "between about 1 nM and about 300 nM" extends beyond the definition presented above to include any whole integer within this range including, without limitation 53, 101, 207, 247, 273 nM etc.
Geneious Prime pairwise alignments successfuly identified four discrete tetrahydrocannabinol aptamer consensus sequences defined in Tables 8-11 below and identified as SEQ ID NO: 528, SEQ ID NO: 529, SEQ ID NO: 530 and SEQ ID NO: 531.
Accordingly, in yet a further aspect of the present invention there is provided a polynucleotide or salt thereof which comprises or consists in a consensus sequence defined by SEQ ID NO: 528, which polynucleotide or salt thereof selectively binds tetrahydrocannabinol.
In an example according to this aspect of the present invention, the polynucleotide or salt thereof which selectively binds tetrahydrocannabinol comprises or consists in a sequence selected from SEQ ID Nos: 91, 83, 95 and 85. In a further related example according to this aspect of the present invention, the polynucleotide or salt thereof comprises or consists in SEQ ID No: 91.
According to the information presented in Table 8, the range in Gibbs Free Energy associated with the Tetrahydrocannabinol Aptamer Cluster 1 ("THC1"; SEQ ID NO: 528) is DG = -2.60 to -4.43.
In yet a further aspect of the present invention there is provided a polynucleotide or salt thereof which comprises or consists in a consensus sequence defined by SEQ ID NO: 529, which polynucleotide or salt thereof selectively binds tetrahydrocannabinol. In an example according to this aspect of the present invention, the polynucleotide or salt thereof which selectively binds tetrahydrocannabinol comprises or consists in a sequence selected from SEQ ID Nos: 105, 117, 79, 81, 87, 89 and 93.
According to the information presented in Table 9, the range in Gibbs Free Energy associated with the Tetrahydrocannabinol Aptamer Cluster 2 ("THC2"; SEQ ID NO: 529) is ΔG = -2.87 to -5.98.
In yet a further aspect of the present invention there is provided a polynucleotide or salt thereof which comprises or consists in a consensus sequence defined by SEQ ID NO: 530, which polynucleotide or salt thereof selectively binds tetrahydrocannabinol. In an example according to this aspect of the present invention, the polynucleotide or salt thereof which selectively binds tetrahydrocannabinol comprises or consists in a sequence selected from SEQ ID Nos: 97, 99, 101, 119, 75, 77 and 77. In a further related example according to this aspect of the present invention, the polynucleotide or salt thereof comprises or consists in SEQ ID No: 97. According to the information presented in Table 10, the range in Gibbs Free Energy associated with the Tetrahydrocannabinol Aptamer Cluster 3 ("THC3"; SEQ ID NO: 530) is ΔG = -4.50 to -8.70.
In yet a further aspect of the present invention there is provided a polynucleotide or salt thereof which comprises or consists in a consensus sequence defined by SEQ ID NO: 531, which polynucleotide or salt thereof selectively binds tetrahydrocannabinol.
In an example according to this aspect of the present invention, the polynucleotide or salt thereof which selectively binds tetrahydrocannabinol comprises or consists in a sequence selected from SEQ ID Nos: 133, 135, 107, 125, 127, 129, 131. In a further related example according to this aspect of the present invention, the polynucleotide or salt thereof comprises or consists in a sequence selected from SEQ ID No: 133 and SEQ ID NO: 135.
According to the information presented in Table 11, the range in Gibbs Free Energy associated with the Tetrahydrocannabinol Aptamer Cluster 4 ("THC4"; SEQ ID NO: 531) is ΔG = -1.87 to -9.11.
Figure imgf000051_0001
Figure imgf000052_0001
Cocaine Aptamers
In reference to the Examples and Figures which follow, Applicant has further developed aptamer sequences which are highly selective for cocaine. Advantageously, these aptamers display highly selective binding affinity for cocaine, with minimal cross-reactivity to other drug targets.
By way of illustration only, in reference to Tables 12-16 below, read in conjunction with Figures 7-8, Applicant developed aptamer sequences which selectively bind to cocaine, the structure of which is depicted in Figure 7, as well as structural analogs of cocaine.
Accordingly, in yet a further aspect of the present invention there is provided a polynucleotide or salt thereof comprising the sequence ATACGAGCTTGTTCAATA[Y]TGATAGTAAGAGCAATC (SEQ ID NO: 572), where Y is any sequence comprising a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to any one of SEQ ID Nos: 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170 and 172, which polynuceotide or salt thereof selectively binds to cocaine.
In yet a further aspect of the present invention there is provided a polynucleotide or salt thereof comprising the sequence ATACGAGCTTGTTCAATA[Y]TGATAGTAAGAGCAATC (SEQ ID NO: 573, where Y is any sequence comprising any one of SEQ ID Nos: 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170 and 172, which polynuceotide or salt thereof selectively binds to cocaine.
In yet a further aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to cocaine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 137 or a fragment thereof which retains selective binding for cocaine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 138
In yet a further aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to cocaine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 139 or a fragment thereof which retains selective binding for cocaine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 140.
In yet a further aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to cocaine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 141 or a fragment thereof which retains selective binding for cocaine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 142.
Figure imgf000054_0001
Figure imgf000055_0001
In yet a further aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to cocaine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 143 or a fragment thereof which retains selective binding for cocaine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 144.
In yet a further aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to cocaine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 145 or a fragment thereof which retains selective binding for cocaine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 146.
In yet a further aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to cocaine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 147 or a fragment thereof which retains selective binding for cocaine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 148.
In yet a further aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to cocaine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 149 or a fragment thereof which retains selective binding for cocaine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 150.
In yet a further aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to cocaine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 151 or a fragment thereof which retains selective binding for cocaine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 152.
In yet a further aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to cocaine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 153 or a fragment thereof which retains selective binding for cocaine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 154.
In yet a further aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to cocaine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 155 or a fragment thereof which retains selective binding for cocaine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 156.
In yet a further aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to cocaine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 157 or a fragment thereof which retains selective binding for cocaine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 158.
In yet a further aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to cocaine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 159 or a fragment thereof which retains selective binding for cocaine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 160.
In yet a further aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to cocaine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 161 or a fragment thereof which retains selective binding for cocaine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 162.
In yet a further aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to cocaine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 163 or a fragment thereof which retains selective binding for cocaine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 164.
In yet a further aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to cocaine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 165 or a fragment thereof which retains selective binding for cocaine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 166.
In yet a further aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to cocaine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 167 or a fragment thereof which retains selective binding for cocaine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 168.
In yet a further aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to cocaine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 169 or a fragment thereof which retains selective binding for cocaine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 170.
In yet a further aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to cocaine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 171 or a fragment thereof which retains selective binding for cocaine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 172.
Cluster analysis was also performed on the cocaine aptamers disclosed herein using Clustal Omega. The results from these analyses is presented in Figure 8 where the level of structural (i.e. sequence) dis/similarity between the different aptamers in this drug class is pictorily illustrated. By way of exemplification only, the cocaine aptamers designated CO5A3 and CO5A6 share a higher degree of structural similarity compared to (e.g.) CO45. However, CO5A3, CO5A6 and CO45 collectively share a higher degree of structural similarity compared to (e.g.) CO1, CO40, CO70, CO50 and CO60.
Geneious Prime pairwise alignments successfuly identified four discrete cocaine aptamer consensus sequences defined in Tables 13-16 below and identified as SEQ ID NO: 532, SEQ ID NO: 533, SEQ ID NO: 534 and SEQ ID NO: 535.
Accordingly, in yet a further aspect of the present invention there is provided a polynucleotide or salt thereof which comprises or consists in a consensus sequence defined by SEQ ID NO: 532, which polynucleotide or salt thereof selectively binds cocaine.
In an example according to this aspect of the present invention, the polynucleotide or salt thereof which selectively binds cocaine comprises or consists in a sequence selected from SEQ ID Nos: 153, 157, 161 and 165.
According to the information presented in Table 13, the range in Gibbs Free Energy associated with the Cocaine Aptamer Cluster 1 (" CO1"; SEQ ID NO: 532) is DG = -4.05 to - 9.44.
In yet a further aspect of the present invention there is provided a polynucleotide or salt thereof which comprises or consists in a consensus sequence defined by SEQ ID NO: 533, which polynucleotide or salt thereof selectively binds cocaine. In an example according to this aspect of the present invention, the polynucleotide or salt thereof which selectively binds cocaine comprises or consists in a sequence selected from SEQ ID Nos: 151, 163, 167 and 169.
According to the information presented in Table 14, the range in Gibbs Free Energy associated with the Cocaine Aptamer Cluster 2 ("CO2"; SEQ ID NO: 533) is ΔG = -3.24 to - 7.86.
In yet a further aspect of the present invention there is provided a polynucleotide or salt thereof which comprises or consists in a consensus sequence defined by SEQ ID NO: 534, which polynucleotide or salt thereof selectively binds cocaine. In an example according to this aspect of the present invention, the polynucleotide or salt thereof which selectively binds cocaine comprises or consists in a sequence selected from SEQ ID Nos: 139, 141 and 137.
According to the information presented in Table 15, the range in Gibbs Free Energy associated with the Cocaine Aptamer Cluster 3 ("CO3"; SEQ ID NO: 534) is ΔG = -1.93 to - 4.35.
In yet a further aspect of the present invention there is provided a polynucleotide or salt thereof which comprises or consists in a consensus sequence defined by SEQ ID NO: 535, which polynucleotide or salt thereof selectively binds cocaine.
In an example according to this aspect of the present invention, the polynucleotide or salt thereof which selectively binds cocaine comprises or consists in a sequence selected from SEQ ID Nos: 147, 149 and 159. According to the information presented in Table 16, the range in Gibbs Free Energy associated with the Cocaine Aptamer Cluster 4 ("CO4"; SEQ ID NO: 535) is ΔG = -3.33 to -5.80.
Figure imgf000060_0001
Figure imgf000061_0001
MDMA Aptamers
In reference to the Examples and Figures which follow, Applicant has further developed aptamer sequences which are highly selective for 3,4-methylenedioxymethamphetamine (MDMA). Advantageously, these aptamers display highly selective binding affinity for MDMA, with minimal cross-reactivity to other drug targets.
By way of illustration only, in reference to Tables 17-21 below, read in conjunction with Figures 9-10, Applicant developed aptamer sequences which selectively bind to MDMA, the structure of which is depicted in Figure 9, as well as structural analogs of MDMA. Accordingly, in yet another aspect of the present invention there is provided a polynucleotide or salt thereof comprising the sequence ATACGAGCTTGTTCAATA[Y]TGATAGTAAGAGCAATC (SEQ ID NO: 575), where Y is any sequence comprising a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to any one of SEQ ID Nos: 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226,
228, 230, 232, 234, 236, 238, 240, 242, 244 and 246, which polynuceotide or salt thereof selectively binds to 3,4-methylenedioxymethamphetamine.
In yet another aspect of the present invention there is provided a polynucleotide or salt thereof comprising the sequence ATACGAGCTTGTTCAATA[Y]TGATAGTAAGAGCAATC (SEQ ID NO: 576), where Y is any sequence comprising any one of SEQ ID Nos: 174, 176,
178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212,
214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244 and 246, which polynuceotide or salt thereof selectively binds to 3,4- methylenedioxymethamphetamine.
Figure imgf000063_0001
Figure imgf000064_0001
Figure imgf000065_0001
In yet another aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to 3,4-methylenedioxymethamphetamine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 173 or a fragment thereof which retains selective binding for 3,4-methylenedioxymethamphetamine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 174.
In yet another aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to 3,4-methylenedioxymethamphetamine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 175 or a fragment thereof which retains selective binding for 3,4-methylenedioxymethamphetamine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 176.
In yet another aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to 3,4-methylenedioxymethamphetamine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 177 or a fragment thereof which retains selective binding for 3,4-methylenedioxymethamphetamine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 178.
In yet another aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to 3,4-methylenedioxymethamphetamine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 179 or a fragment thereof which retains selective binding for 3,4-methylenedioxymethamphetamine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 180.
In yet another aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to 3,4-methylenedioxymethamphetamine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 181 or a fragment thereof which retains selective binding for 3,4-methylenedioxymethamphetamine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 182.
In yet another aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to 3,4-methylenedioxymethamphetamine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 183 or a fragment thereof which retains selective binding for 3,4-methylenedioxymethamphetamine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 184.
In yet another aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to 3,4-methylenedioxymethamphetamine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 185 or a fragment thereof which retains selective binding for 3,4-methylenedioxymethamphetamine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 186.
In yet another aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to 3,4-methylenedioxymethamphetamine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 187 or a fragment thereof which retains selective binding for 3,4-methylenedioxymethamphetamine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 188.
In yet another aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to 3,4-methylenedioxymethamphetamine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 189 or a fragment thereof which retains selective binding for 3,4-methylenedioxymethamphetamine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 190.
In yet another aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to 3,4-methylenedioxymethamphetamine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 191 or a fragment thereof which retains selective binding for 3,4-methylenedioxymethamphetamine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 192.
In yet another aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to 3,4-methylenedioxymethamphetamine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 193 or a fragment thereof which retains selective binding for 3,4-methylenedioxymethamphetamine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 194.
In yet another aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to 3,4-methylenedioxymethamphetamine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 195 or a fragment thereof which retains selective binding for 3,4-methylenedioxymethamphetamine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 196.
In yet another aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to 3,4-methylenedioxymethamphetamine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 197 or a fragment thereof which retains selective binding for 3,4-methylenedioxymethamphetamine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 198.
In yet another aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to 3,4-methylenedioxymethamphetamine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 199 or a fragment thereof which retains selective binding for 3,4-methylenedioxymethamphetamine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 200.
In yet another aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to 3,4-methylenedioxymethamphetamine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 201 or a fragment thereof which retains selective binding for 3,4-methylenedioxymethamphetamine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 202.
In yet another aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to 3,4-methylenedioxymethamphetamine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 203 or a fragment thereof which retains selective binding for 3,4-methylenedioxymethamphetamine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 204. In yet another aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to 3,4-methylenedioxymethamphetamine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 205 or a fragment thereof which retains selective binding for 3,4-methylenedioxymethamphetamine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 206.
In yet another aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to 3,4-methylenedioxymethamphetamine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 207 or a fragment thereof which retains selective binding for 3,4-methylenedioxymethamphetamine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 208.
In yet another aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to 3,4-methylenedioxymethamphetamine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 209 or a fragment thereof which retains selective binding for 3,4-methylenedioxymethamphetamine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 210.
In yet another aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to 3,4-methylenedioxymethamphetamine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 211 or a fragment thereof which retains selective binding for 3,4-methylenedioxymethamphetamine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 212.
In yet another aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to 3,4-methylenedioxymethamphetamine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 213 or a fragment thereof which retains selective binding for 3,4-methylenedioxymethamphetamine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 214.
In yet another aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to 3,4-methylenedioxymethamphetamine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 215 or a fragment thereof which retains selective binding for 3,4-methylenedioxymethamphetamine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 216.
In yet another aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to 3,4-methylenedioxymethamphetamine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 217 or a fragment thereof which retains selective binding for 3,4-methylenedioxymethamphetamine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 218.
In yet another aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to 3,4-methylenedioxymethamphetamine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 219 or a fragment thereof which retains selective binding for 3,4-methylenedioxymethamphetamine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 220.
In yet another aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to 3,4-methylenedioxymethamphetamine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 221 or a fragment thereof which retains selective binding for 3,4-methylenedioxymethamphetamine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 222.
In yet another aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to 3,4-methylenedioxymethamphetamine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 223 or a fragment thereof which retains selective binding for 3,4-methylenedioxymethamphetamine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 224.
In yet another aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to 3,4-methylenedioxymethamphetamine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 225 or a fragment thereof which retains selective binding for 3,4-methylenedioxymethamphetamine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 226.
In yet another aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to 3,4-methylenedioxymethamphetamine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 227 or a fragment thereof which retains selective binding for 3,4-methylenedioxymethamphetamine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 228.
In yet another aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to 3,4-methylenedioxymethamphetamine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 229 or a fragment thereof which retains selective binding for 3,4-methylenedioxymethamphetamine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 230.
In yet another aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to 3,4-methylenedioxymethamphetamine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 231 or a fragment thereof which retains selective binding for 3,4-methylenedioxymethamphetamine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 232.
In yet another aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to 3,4-methylenedioxymethamphetamine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 233 or a fragment thereof which retains selective binding for 3,4-methylenedioxymethamphetamine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 234.
In yet another aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to 3,4-methylenedioxymethamphetamine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 235 or a fragment thereof which retains selective binding for 3,4-methylenedioxymethamphetamine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 236. In yet another aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to 3,4-methylenedioxymethamphetamine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 237 or a fragment thereof which retains selective binding for 3,4-methylenedioxymethamphetamine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 238.
In yet another aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to 3,4-methylenedioxymethamphetamine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 239 or a fragment thereof which retains selective binding for 3,4-methylenedioxymethamphetamine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 240.
In yet another aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to 3,4-methylenedioxymethamphetamine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 241 or a fragment thereof which retains selective binding for 3,4-methylenedioxymethamphetamine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 242.
In yet another aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to 3,4-methylenedioxymethamphetamine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 243 or a fragment thereof which retains selective binding for 3,4-methylenedioxymethamphetamine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 244.
In yet another aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to 3,4-methylenedioxymethamphetamine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 245 or a fragment thereof which retains selective binding for 3,4-methylenedioxymethamphetamine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 246.
Cluster analysis was also performed on the MDMA aptamers disclosed herein using Clustal Omega. The results from these analyses is presented in Figure 10 where the level of structural (i.e. sequence) dis/similarity between the different aptamers in this drug class is pictorily illustrated. By way of exemplification only, the MDMA aptamers designated MD10 and MD24 share a higher degree of structural similarity compared to (e.g.) MD31, MD98 and MD65. However, MD10, MD24, MD31, MD98 and MD65 collectively share a higher degree of structural similarity compared to (e.g.) MD95, MD97, MD52 and MD310.
Geneious Prime pairwise alignments successfuly identified four discrete MDMA aptamer consensus sequences defined in Tables 18-21 below and identified as SEQ ID NO: 536, SEQ ID NO: 537, SEQ ID NO: 538 and SEQ ID NO: 539.
Accordingly, in yet a further aspect of the present invention there is provided a polynucleotide or salt thereof which comprises or consists in a consensus sequence defined by SEQ ID NO: 536, which polynucleotide or salt thereof selectively binds MDMA.
In an example according to this aspect of the present invention, the polynucleotide or salt thereof which selectively binds MDMA comprises or consists in a sequence selected from SEQ ID Nos: 229, 235, 237, 239 and 245.
According to the information presented in Table 18, the range in Gibbs Free Energy associated with the MDMA Aptamer Cluster 1 ("MD1"; SEQ ID NO: 536) is DG = -2.54 to - 10.18.
In yet a further aspect of the present invention there is provided a polynucleotide or salt thereof which comprises or consists in a consensus sequence defined by SEQ ID NO: 537, which polynucleotide or salt thereof selectively binds MDMA.
In an example according to this aspect of the present invention, the polynucleotide or salt thereof which selectively binds cocaine comprises or consists in a sequence selected from SEQ ID Nos: 219, 241 and 243.
According to the information presented in Table 19, the range in Gibbs Free Energy associated with the MDMA Aptamer Cluster 2 ("MD2"; SEQ ID NO: 537) is DG = -5.77 to - 8.12.
In yet a further aspect of the present invention there is provided a polynucleotide or salt thereof which comprises or consists in a consensus sequence defined by SEQ ID NO: 538, which polynucleotide or salt thereof selectively binds MDMA.
In an example according to this aspect of the present invention, the polynucleotide or salt thereof which selectively binds cocaine comprises or consists in a sequence selected from SEQ ID Nos: 203, 195, 199 and 213.
According to the information presented in Table 20, the range in Gibbs Free Energy associated with the MDMA Aptamer Cluster 3 ("MD3"; SEQ ID NO: 538) is DG = -6.93 to - 8.94.
In yet a further aspect of the present invention there is provided a polynucleotide or salt thereof which comprises or consists in a consensus sequence defined by SEQ ID NO: 539, which polynucleotide or salt thereof selectively binds MDMA. In an example according to this aspect of the present invention, the polynucleotide or salt thereof which selectively binds cocaine comprises or consists in a sequence selected from SEQ ID Nos: 225, 211 and 207.
According to the information presented in Table 21, the range in Gibbs Free Energy associated with the MDMA Aptamer Cluster 4 ("MD4"; SEQ ID NO: 539) is DG = -6.40 to - 7.24.
Morphine Aptamers
In reference to the Examples and Figures which follow, Applicant has further developed aptamer sequences which are highly selective for morphine. Advantageously, these aptamers display highly selective binding affinity for morphine, with minimal cross-reactivity to other drug targets.
By way of illustration only, in reference to Table 22-24 below read in conjunction with Figures 11-13, Applicants have developed aptamer sequences which selectively bind to morphine, the structure of which is depicted in Figure 11, as well as structural analogs of morphine.
Accordingly, in yet another aspect of the present invention there is provided a polynucleotide or salt thereof comprising the sequence ATACGAGCTTGTTCAATA[Y]TGATAGTAAGAGCAATC (SEQ ID NO: 578), where Y is any sequence comprising a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to any one of SEQ ID Nos: 249, 251, 253, 255, 257, 259, 261, 263, 264, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289 and 291, which polynuceotide or salt thereof selectively binds to morphine.
In another aspect of the present invention there is provided a polynucleotide or salt thereof comprising the sequence ATACGAGCTTGTTCAATA[Y]TGATAGTAAGAGCAATC (SEQ ID NO: 579), where Y is any sequence comprising any one of SEQ ID Nos: 249, 251, 253, 255, 257, 259, 261, 263, 264, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289 and 291, which polynuceotide or salt thereof selectively binds to morphine.
In another aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to morphine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 247 or a fragment thereof which retains selective binding for morphine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 265.
Figure imgf000075_0001
Figure imgf000076_0001
Figure imgf000077_0001
Figure imgf000078_0001
In another aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to morphine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 248 or a fragment thereof which retains selective binding for morphine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 249.
In another aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to morphine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 250 or a fragment thereof which retains selective binding for morphine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 251.
In another aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to morphine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 252 or a fragment thereof which retains selective binding for morphine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 253.
In another aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to morphine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 254 or a fragment thereof which retains selective binding for morphine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 255.
In another aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to morphine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 256 or a fragment thereof which retains selective binding for morphine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 257.
In another aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to morphine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 258 or a fragment thereof which retains selective binding for morphine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 259.
In another aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to morphine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 260 or a fragment thereof which retains selective binding for morphine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 261.
In another aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to morphine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 262 or a fragment thereof which retains selective binding for morphine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 263.
In another aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to morphine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 266 or a fragment thereof which retains selective binding for morphine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 267.
In another aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to morphine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 268 or a fragment thereof which retains selective binding for morphine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 269.
In another aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to morphine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 270 or a fragment thereof which retains selective binding for morphine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 271.
In another aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to morphine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 272 or a fragment thereof which retains selective binding for morphine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 273.
In another aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to morphine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 274 or a fragment thereof which retains selective binding for morphine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 275.
In another aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to morphine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 276 or a fragment thereof which retains selective binding for morphine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 277.
In another aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to morphine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 278 or a fragment thereof which retains selective binding for morphine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 279.
In another aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to morphine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 280 or a fragment thereof which retains selective binding for morphine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 281.
In another aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to morphine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 282 or a fragment thereof which retains selective binding for morphine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 283 or SEQ ID NO: 264.
In another aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to morphine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 284 or a fragment thereof which retains selective binding for morphine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 285.
In another aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to morphine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 286 or a fragment thereof which retains selective binding for morphine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 287. In another aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to morphine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 288 or a fragment thereof which retains selective binding for morphine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 289.
In another aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to morphine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 290 or a fragment thereof which retains selective binding for morphine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 291.
Cluster analysis was also performed on the morphine aptamers disclosed herein using Clustal Omega. The results from these analyses is presented in Figure 12 where the level of structural (i.e. sequence) dis/similarity between the different aptamers in this drug class is pictorily illustrated. By way of exemplification only, the morphine aptamer designated MOR- B12 and MOR-S15 share a higher degree of structural similarity compared to (e.g.) MOR-B07. However, MOR-B12, MOR-S15 and MOR-B07 collectively share a higher degree of structural similarity compared to (e.g.) MOR-S07, MOR-S02 and MOR-S17.
Figures 13A-13E provide dissociation constants for MOR-S15 (SEQ ID NO: 282), MOR- S13 (SEQ ID NO:280), MOR-S06 (SEQ ID NO: 258), MOR-B11 (SEQ ID NO: 268) and MOR- B10 (SEQ ID NO: 288) of Kd = 248 nM, 552 nM and 374 nM, 603 nM and 567 nM, respectively. Additional binding affinity data for MOR-B12 (SEQ ID NO: 90) is disclosed in Table 22 with a Kd = 284 nM. These data collectively indicate that these particular aptamer sequences all display highly selective binding affinity for morphine.
Accordingly, the polynucleotides encoding morphine aptamers described herein, including without limitation SEQ ID NOs: 247, 248, 250, 252, 254, 256, 258, 260, 262, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288 and 290, display a binding affinity for morphine which is between about 1 nM and about 1000 nM, including between about about 1 nM and about 750 nM, and about 1 nM and 300 nM.
Geneious Prime pairwise alignments successfuly identified two discrete morphine aptamer consensus sequences defined in Tables 23-24 below and identified as SEQ ID NO: 540 and SEQ ID NO: 541.
Accordingly, in yet a further aspect of the present invention there is provided a polynucleotide or salt thereof which comprises or consists in a consensus sequence defined by SEQ ID NO: 540, which polynucleotide or salt thereof selectively binds morphine.
Figure imgf000083_0001
Figure imgf000084_0001
In an example according to this aspect of the present invention, the polynucleotide or salt thereof which selectively binds morphine comprises or consists in a sequence selected from SEQ ID Nos: 288, 290, 280, 282, 254, 266, 272 and 262. In a further related example according to this aspect of the present invention, the polynucleotide or salt thereof comprises or consists in a sequence selected from SEQ ID NOs: 288, 290, 280 and 282.
According to the information presented in Table 23, the range in Gibbs Free Energy associated with the Morphine Aptamer Cluster 1 ("MORI"; SEQ ID NO: 540) is DG = -2.54 to -8.38.
In yet a further aspect of the present invention there is provided a polynucleotide or salt thereof which comprises or consists in a consensus sequence defined by SEQ ID NO: 541, which polynucleotide or salt thereof selectively binds morphine.
In an example according to this aspect of the present invention, the polynucleotide or salt thereof which selectively binds morphine comprises or consists in a sequence selected from SEQ ID Nos: 268, 247, 248, 250, 252, 256, 270, 274, 276, 258, 260, 278, 284, 286. In a further related example according to this aspect of the present invention, the polynucleotide or salt thereof comprises or consists in SEQ ID NO: 268.
According to the information presented in Table 24, the range in Gibbs Free Energy associated with the Morphine Aptamer Cluster 2 ("MOR2"; SEQ ID NO: 541) is DG = -3.46 to -10.15.
Benzodiazepine Aptamers
In reference to the Examples and Figures which follow, Applicant has further developed aptamer sequences which are highly selective for benzodiazepine. Advantageously, these aptamers display highly selective binding affinity for benzodiazepine, with minimal cross reactivity to other drug targets.
By way of illustration only, in reference to Tables 25-28 below read in conjunction with Figures 14-15, Applicants have developed aptamer sequences which selectively bind to benzodiazepine, the structure of which is depicted in Figure 14, as well as structural analogs of benzodiazepine.
Accordingly, in yet another aspect of the present invention there is provided a polynucleotide or salt thereof comprising the sequence ATACGAGCTTGTTCAATA[Y]TGATAGTAAGAGCAATC (SEQ ID NO: 581), where Y is any sequence comprising a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to any one of SEQ ID Nos: 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317 and 319, which polynuceotide or salt thereof selectively binds to benzodiazepine.
Figure imgf000086_0001
In yet another aspect of the present invention there is provided a polynucleotide or salt thereof comprising the sequence ATACGAGCTTGTTCAATA[Y]TGATAGTAAGAGCAATC (SEQ ID NO: 582), where Y is any sequence comprising any one of SEQ ID Nos: 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317 and 319, which polynuceotide or salt thereof selectively binds to benzodiazepine.
In yet another aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to diazepam, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 292 or a fragment thereof which retains selective binding for benzodiazepine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 293.
In yet another aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to diazepam, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 294 or a fragment thereof which retains selective binding for benzodiazepine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 295.
In yet another aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to diazepam, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 296 or a fragment thereof which retains selective binding for benzodiazepine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 297.
In yet another aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to diazepam, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 298 or a fragment thereof which retains selective binding for benzodiazepine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 299.
In yet another aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to diazepam, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 300 or a fragment thereof which retains selective binding for benzodiazepine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 301.
In yet another aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to diazepam, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 302 or a fragment thereof which retains selective binding for benzodiazepine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 303.
In yet another aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to diazepam, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 304 or a fragment thereof which retains selective binding for benzodiazepine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 305.
In yet another aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to diazepam, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 306 or a fragment thereof which retains selective binding for benzodiazepine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 307.
In yet another aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to diazepam, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 308 or a fragment thereof which retains selective binding for benzodiazepine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 309.
In yet another aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to diazepam, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 310 or a fragment thereof which retains selective binding for benzodiazepine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 311.
In yet another aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to diazepam, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 312 or a fragment thereof which retains selective binding for benzodiazepine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 313.
In yet another aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to diazepam, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 314 or a fragment thereof which retains selective binding for benzodiazepine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 315.
In yet another aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to diazepam, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 316 or a fragment thereof which retains selective binding for benzodiazepine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 317.
In yet another aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to diazepam, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 318 or a fragment thereof which retains selective binding for benzodiazepine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 319.
Cluster analysis was also performed on the benzodiazepine aptamers disclosed herein using Clustal Omega. The results from these analyses is presented in Figure 15 where the level of structural (i.e. sequence) dis/similarity between the different aptamers in this drug class is pictorily illustrated. By way of exemplification only, the benzodiazepine aptamer designated DI-21 and DI-15 share a higher degree of structural similarity compared to (e.g.) DI-29. However, DI-21, DI-15 and DI-29 collectively share a higher degree of structural similarity compared to (e.g.) DI-71, DI-67 and DI-50.
Geneious Prime pairwise alignments successfuly identified three discrete benzodiazepine aptamer consensus sequences defined in Tables 26-28 below and identified as SEQ ID NO: 542, SEQ ID NO: 543 and SEQ ID NO: 544.
Accordingly, in yet a further aspect of the present invention there is provided a polynucleotide or salt thereof which comprises or consists in a consensus sequence defined by SEQ ID NO: 542, which polynucleotide or salt thereof selectively binds benzodiazepine.
In an example according to this aspect of the present invention, the polynucleotide or salt thereof which selectively binds benzodiazepine comprises or consists in a sequence selected from SEQ ID Nos: 292, 308, 312, 314, 316 and 318.
According to the information presented in Table 26, the range in Gibbs Free Energy associated with the Benzodiazepine Aptamer Cluster 1 ("Dl1"; SEQ ID NO: 542) is DG = - 1.15 to -8.42.
In yet a further aspect of the present invention there is provided a polynucleotide or salt thereof which comprises or consists in a consensus sequence defined by SEQ ID NO: 543, which polynucleotide or salt thereof selectively binds benzodiazepine. In an example according to this aspect of the present invention, the polynucleotide or salt thereof which selectively binds benzodiazepine comprises or consists in a sequence selected from SEQ ID Nos: 294, 296, 298, 300 and 306.
According to the information presented in Table 27, the range in Gibbs Free Energy associated with the Benzodiazepine Aptamer Cluster 2 ("DI2"; SEQ ID NO: 543) is ΔG = - 3.24 to -9.96.
In yet a further aspect of the present invention there is provided a polynucleotide or salt thereof which comprises or consists in a consensus sequence defined by SEQ ID NO: 544, which polynucleotide or salt thereof selectively binds benzodiazepine. In an example according to this aspect of the present invention, the polynucleotide or salt thereof which selectively binds benzodiazepine comprises or consists in a sequence selected from SEQ ID Nos: 302, 304 and 310.
According to the information presented in Table 28, the range in Gibbs Free Energy associated with the Benzodiazepine Aptamer Cluster 3 ("DI3"; SEQ ID NO: 544) is ΔG = - 3.92 to -6.80.
Figure imgf000091_0001
Figure imgf000092_0001
AMB-FUBINACA Aptamers
In reference to the Examples and Figures which follow, Applicant has further developed aptamer sequences which are highly selective for AMB-FUBINACA. Advantageously, these aptamers display highly selective binding affinity for AMB-FUBINACA, with minimal cross- reactivity to other drug targets.
By way of illustration only, in reference to Tables 29-34 below read in conjunction with Figures 16-17, Applicants have developed aptamer sequences which selectively bind to AMB- FUBINACA, the structure of which is depicted in Figure 16, as well as structural analogs of AMB-FUBINACA.
Accordingly, in another aspect of the present invention there is provided a polynucleotide or salt thereof comprising the sequence ATACGAGCTTGTTCAATA[Y]TGATAGTAAGAGCAATC (SEQ ID NO: 584), where Y is any sequence comprising a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to any one of SEQ ID Nos: 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 355, 357, 359, 361, 363, 365, 367, 369 and 371, which polynuceotide or salt thereof selectively binds to AMB-FUBINACA.
In another aspect of the present invention there is provided a polynucleotide or salt thereof comprising the sequence ATACGAGCTTGTTCAATA[Y]TGATAGTAAGAGCAATC (SEQ ID NO: 585), where Y is any sequence comprising any one of SEQ ID Nos: 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 355, 357, 359, 361, 363, 365, 367, 369 and 371, which polynuceotide or salt thereof selectively binds to AMB-FUBINACA.
Figure imgf000094_0001
Figure imgf000095_0001
In another aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to AMB-FUBINACA, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 320 or a fragment thereof which retains selective binding for AMB-FUBINACA. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 321.
In another aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to AMB-FUBINACA, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 322 or a fragment thereof which retains selective binding for AMB-FUBINACA. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 323.
In another aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to AMB-FUBINACA, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 324 or a fragment thereof which retains selective binding for AMB-FUBINACA. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 325.
In another aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to AMB-FUBINACA, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 326 or a fragment thereof which retains selective binding for AMB-FUBINACA. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 327.
In another aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to AMB-FUBINACA, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 328 or a fragment thereof which retains selective binding for AMB-FUBINACA. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 329.
In another aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to AMB-FUBINACA, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 330 or a fragment thereof which retains selective binding for AMB-FUBINACA. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 331.
In another aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to AMB-FUBINACA, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 332 or a fragment thereof which retains selective binding for AMB-FUBINACA. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 333.
In another aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to AMB-FUBINACA, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 334 or a fragment thereof which retains selective binding for AMB-FUBINACA. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 335.
In another aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to AMB-FUBINACA, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 336 or a fragment thereof which retains selective binding for AMB-FUBINACA. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 337.
In another aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to AMB-FUBINACA, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 338 or a fragment thereof which retains selective binding for AMB-FUBINACA. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 339.
In another aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to AMB-FUBINACA, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 340 or a fragment thereof which retains selective binding for AMB-FUBINACA. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 341.
In another aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to AMB-FUBINACA, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 342 or a fragment thereof which retains selective binding for AMB-FUBINACA. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 343.
In another aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to AMB-FUBINACA, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 344 or a fragment thereof which retains selective binding for AMB-FUBINACA. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 345.
In another aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to AMB-FUBINACA, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 346 or a fragment thereof which retains selective binding for AMB-FUBINACA. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 347.
In another aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to AMB-FUBINACA, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 348 or a fragment thereof which retains selective binding for AMB-FUBINACA. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 349.
In another aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to AMB-FUBINACA, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 350 or a fragment thereof which retains selective binding for AMB-FUBINACA. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 351.
In another aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to AMB-FUBINACA, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 352 or a fragment thereof which retains selective binding for AMB-FUBINACA. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 353.
In another aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to AMB-FUBINACA, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 354 or a fragment thereof which retains selective binding for AMB-FUBINACA. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 355.
In another aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to AMB-FUBINACA, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 356 or a fragment thereof which retains selective binding for AMB-FUBINACA. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 357. In another aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to AMB-FUBINACA, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 358 or a fragment thereof which retains selective binding for AMB-FUBINACA. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 359.
In another aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to AMB-FUBINACA, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 360 or a fragment thereof which retains selective binding for AMB-FUBINACA. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 361.
In another aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to AMB-FUBINACA, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 362 or a fragment thereof which retains selective binding for AMB-FUBINACA. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 363.
In another aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to AMB-FUBINACA, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 364 or a fragment thereof which retains selective binding for AMB-FUBINACA. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 365.
In another aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to AMB-FUBINACA, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 366 or a fragment thereof which retains selective binding for AMB-FUBINACA. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 367.
In another aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to AMB-FUBINACA, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 368 or a fragment thereof which retains selective binding for AMB-FUBINACA. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 369.
In another aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to AMB-FUBINACA, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 370 or a fragment thereof which retains selective binding for AMB-FUBINACA. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 371.
Cluster analysis was also performed on the AMB-FUBINACA aptamers disclosed herein using Clustal Omega. The results from these analyses is presented in Figure 17 where the level of structural (i.e. sequence) dis/similarity between the different aptamers in this drug class is pictorily illustrated. By way of exemplification only, the AMB-FUBINACA aptamers designated FM03 and FM-09 share a higher degree of structural similarity compared to (e.g.) FM13. However, FM03, FM-09 and FM13 collectively share a higher degree of structural similarity compared to (e.g.) FM05-FM015 and FM18.
Geneious Prime pairwise alignments successfuly identified five discrete AMB- FUBINACA aptamer consensus sequences defined in Tables 30-34 below and identified as SEQ ID NO: 545, SEQ ID NO: 546, SEQ ID NO: 547, SEQ ID NO: 548 and SEQ ID NO: 549.
Accordingly, in yet a further aspect of the present invention there is provided a polynucleotide or salt thereof which comprises or consists in a consensus sequence defined by SEQ ID NO: 545, which polynucleotide or salt thereof selectively binds AMB-FUBINACA.
In an example according to this aspect of the present invention, the polynucleotide or salt thereof which selectively binds AMB-FUBINACA comprises or consists in a sequence selected from SEQ ID Nos: 330, 338, 340, 342, 344 and 366.
According to the information presented in Table 30, the range in Gibbs Free Energy associated with the AMB-FUBINACA Aptamer Cluster 1 ("FM1"; SEQ ID NO: 545) is DG = - 2.37 to -4.78.
In yet a further aspect of the present invention there is provided a polynucleotide or salt thereof which comprises or consists in a consensus sequence defined by SEQ ID NO: 546, which polynucleotide or salt thereof selectively binds AMB-FUBINACA.
In an example according to this aspect of the present invention, the polynucleotide or salt thereof which selectively binds AMB-FUBINACA comprises or consists in a sequence selected from SEQ ID Nos: 322, 354, 356, 358 and 368.
According to the information presented in Table 31, the range in Gibbs Free Energy associated with the AMB-FUBINACA Aptamer Cluster 2 ("FM2"; SEQ ID NO: 546) is DG = - 3.97 to -9.39.
In yet a further aspect of the present invention there is provided a polynucleotide or salt thereof which comprises or consists in a consensus sequence defined by SEQ ID NO: 547, which polynucleotide or salt thereof selectively binds AMB-FUBINACA.
In an example according to this aspect of the present invention, the polynucleotide or salt thereof which selectively binds AMB-FUBINACA comprises or consists in a sequence selected from SEQ ID Nos: 326, 328, 334, 360, 362 and 364.
Figure imgf000101_0001
Figure imgf000102_0001
Figure imgf000103_0001
According to the information presented in Table 32, the range in Gibbs Free Energy associated with the AMB-FUBINACA Aptamer Cluster 3 ("FM3"; SEQ ID NO: 547) is DG = - 2.09 to -5.42.
In yet a further aspect of the present invention there is provided a polynucleotide or salt thereof which comprises or consists in a consensus sequence defined by SEQ ID NO: 548, which polynucleotide or salt thereof selectively binds AMB-FUBINACA.
In an example according to this aspect of the present invention, the polynucleotide or salt thereof which selectively binds AMB-FUBINACA comprises or consists in a sequence selected from SEQ ID Nos: 332, 348, 350, 352 and 370.
According to the information presented in Table 33, the range in Gibbs Free Energy associated with the AMB-FUBINACA Aptamer Cluster 4 ("FM4"; SEQ ID NO: 548) is DG = - 7.35 to -10.29.
In yet a further aspect of the present invention there is provided a polynucleotide or salt thereof which comprises or consists in a consensus sequence defined by SEQ ID NO: 549, which polynucleotide or salt thereof selectively binds AMB-FUBINACA.
In an example according to this aspect of the present invention, the polynucleotide or salt thereof which selectively binds AMB-FUBINACA comprises or consists in a sequence selected from SEQ ID Nos: 336, 324 and 346.
According to the information presented in Table 34, the range in Gibbs Free Energy associated with the AMB-FUBINACA Aptamer Cluster 5 ("FM5"; SEQ ID NO: 549) is DG = - 3.58 to -11.00.
AM2201 Aptamers
In reference to the Examples and Figures which follow, Applicant has further developed aptamer sequences which are highly selective for AM2201. Advantageously, these aptamers display highly selective binding affinity for AM2201, with minimal cross-reactivity to other drug targets.
By way of illustration only, in reference to Tables 35-39 below read in conjunction with Figures 18-19, Applicants have developed aptamer sequences which selectively bind to AM2201, the structure of which is depicted in Figure 18, as well as structural analogs of AM2201.
Accordingly, in a further aspect of the present invention there is provided a polynucleotide or salt thereof comprising the sequence ATACGAGCTTGTTCAATA[Y]TGATAGTAAGAGCAATC (SEQ ID NO: 587), where Y is any sequence comprising a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to any one of SEQ ID Nos: 373, 375, 377, 379, 381, 383, 385, 387, 389, 391, 393, 395, 397, 399, 401, 403, 405, 407, 409, 411, 413, 415, 417 and 419, which polynuceotide or salt thereof selectively binds to AM2201.
Figure imgf000105_0001
Figure imgf000106_0001
In a further aspect of the present invention there is provided a polynucleotide or salt thereof comprising the sequence ATACGAGCTTGTTCAATA[Y]TGATAGTAAGAGCAATC (SEQ ID NO: 588), where Y is any sequence comprising any one of SEQ ID Nos: 373, 375, 377, 379, 381, 383, 385, 387, 389, 391, 393, 395, 397, 399, 401, 403, 405, 407, 409, 411, 413, 415, 417 and 419, which polynuceotide or salt thereof selectively binds to AM2201.
In a further aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to AM2201, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 372 or a fragment thereof which retains selective binding for AM2201. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 373.
In a further aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to AM2201, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 374 or a fragment thereof which retains selective binding for AM2201. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 375.
In a further aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to AM2201, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 376 or a fragment thereof which retains selective binding for AM2201. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 377.
In a further aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to AM2201, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 378 or a fragment thereof which retains selective binding for AM2201. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 379.
In a further aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to AM2201, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 380 or a fragment thereof which retains selective binding for AM2201. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 381.
In a further aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to AM2201, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 382 or a fragment thereof which retains selective binding for AM2201. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 383.
In a further aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to AM2201, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 384 or a fragment thereof which retains selective binding for AM2201. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 385.
In a further aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to AM2201, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 386 or a fragment thereof which retains selective binding for AM2201. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 387.
In a further aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to AM2201, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 388 or a fragment thereof which retains selective binding for AM2201. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 389.
In a further aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to AM2201, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 390 or a fragment thereof which retains selective binding for AM2201. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 391.
In a further aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to AM2201, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 392 or a fragment thereof which retains selective binding for AM2201. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 393.
In a further aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to AM2201, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 394 or a fragment thereof which retains selective binding for AM2201. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 395.
In a further aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to AM2201, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 396 or a fragment thereof which retains selective binding for AM2201. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 397.
In a further aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to AM2201, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 398 or a fragment thereof which retains selective binding for AM2201. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 399.
In a further aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to AM2201, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 400 or a fragment thereof which retains selective binding for AM2201. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 401.
In a further aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to AM2201, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 402 or a fragment thereof which retains selective binding for AM2201. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 403.
In a further aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to AM2201, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 404 or a fragment thereof which retains selective binding for AM2201. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 405.
In a further aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to AM2201, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 406 or a fragment thereof which retains selective binding for AM2201. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 407. In a further aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to AM2201, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 408 or a fragment thereof which retains selective binding for AM2201. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 409.
In a further aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to AM2201, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 410 or a fragment thereof which retains selective binding for AM2201. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 411.
In a further aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to AM2201, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 412 or a fragment thereof which retains selective binding for AM2201. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 413.
In a further aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to AM2201, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 414 or a fragment thereof which retains selective binding for AM2201. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 415.
In a further aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to AM2201, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 416 or a fragment thereof which retains selective binding for AM2201. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 417.
In a further aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to AM2201, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 418 or a fragment thereof which retains selective binding for AM2201. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 419.
Cluster analysis was also performed on the AM2201 aptamers disclosed herein using Clustal Omega. The results from these analyses is presented in Figure 19 where the level of structural (i.e. sequence) dis/similarity between the different aptamers in this drug class is pictorily illustrated. By way of exemplification only, the AM2201 aptamer designated AM01 and AM06 share a higher degree of structural similarity compared to (e.g.) AM09. However, AM01, AM06 and AM09 collectively share a higher degree of structural similarity compared to (e.g.) AM11, AM12, AM13, AM02 and AM14.
Geneious Prime pairwise alignments successfuly identified five discrete AM2201 aptamer consensus sequences defined in Tables 35-39 below and identified as SEQ ID NO: 550, SEQ ID NO: 551, SEQ ID NO: 552, SEQ ID NO: 553 and SEQ ID NO: 554.
Accordingly, in yet a further aspect of the present invention there is provided a polynucleotide or salt thereof which comprises or consists in a consensus sequence defined by SEQ ID NO: 550, which polynucleotide or salt thereof selectively binds AM2201.
In an example according to this aspect of the present invention, the polynucleotide or salt thereof which selectively binds AM2201 comprises or consists in a sequence selected from SEQ ID Nos: 374, 380, 382, 386, 388 and 418.
According to the information presented in Table 36, the range in Gibbs Free Energy associated with the AM2201 Aptamer Cluster 1 ("AMI"; SEQ ID NO: 550) is DG = -2.98 to - 15.23.
In yet a further aspect of the present invention there is provided a polynucleotide or salt thereof which comprises or consists in a consensus sequence defined by SEQ ID NO: 551, which polynucleotide or salt thereof selectively binds AM2201.
In an example according to this aspect of the present invention, the polynucleotide or salt thereof which selectively binds AM2201 comprises or consists in a sequence selected from SEQ ID Nos: 384, 406, 408 and 410.
According to the information presented in Table 37, the range in Gibbs Free Energy associated with the AM2201 Aptamer Cluster 2 ("AM2"; SEQ ID NO: 551) is DG = -2.37 to - 7.88.
In yet a further aspect of the present invention there is provided a polynucleotide or salt thereof which comprises or consists in a consensus sequence defined by SEQ ID NO: 552, which polynucleotide or salt thereof selectively binds AM2201.
In an example according to this aspect of the present invention, the polynucleotide or salt thereof which selectively binds AM2201 comprises or consists in a sequence selected from SEQ ID Nos: 390, 412, 414 and 416.
According to the information presented in Table 38, the range in Gibbs Free Energy associated with the AM2201 Aptamer Cluster 3 ("AM3"; SEQ ID NO: 552) is DG = -5.79 to - 8.50.
In yet a further aspect of the present invention there is provided a polynucleotide or salt thereof which comprises or consists in a consensus sequence defined by SEQ ID NO: 553, which polynucleotide or salt thereof selectively binds AM2201. In an example according to this aspect of the present invention, the polynucleotide or salt thereof which selectively binds AM2201 comprises or consists in a sequence selected from SEQ ID Nos: 378, 400, 402 and 404.
According to the information presented in Table 39, the range in Gibbs Free Energy associated with the AM2201 Aptamer Cluster 4 ("AM4"; SEQ ID NO: 553) is DG = -3.80 to - 6.80.
In yet a further aspect of the present invention there is provided a polynucleotide or salt thereof which comprises or consists in a consensus sequence defined by SEQ ID NO: 554, which polynucleotide or salt thereof selectively binds AM2201. In an example according to this aspect of the present invention, the polynucleotide or salt thereof which selectively binds AM2201 comprises or consists in a sequence selected from SEQ ID Nos: 376, 392, 394, 396 and 398.
According to the information presented in Table 40, the range in Gibbs Free Energy associated with the AM2201 Aptamer Cluster 5 ("AM5"; SEQ ID NO: 554) is DG = -4.10 to - 13.26.
Figure imgf000113_0001
Figure imgf000114_0001
Oxycodone Aptamers
In reference to the Examples and Figures which follow, Applicant has further developed aptamer sequences which are highly selective for oxycodone. Advantageously, these aptamers display highly selective binding affinity for oxycodone, with minimal cross-reactivity to other drug targets.
By way of illustration only, in reference to Tables 40-44 below read in conjunction with Figures 20-21, Applicants have developed aptamer sequences which selectively bind to oxycodone, the structure of which is depicted in Figure 21, as well as structual analogs of oxycodone. Accordingly, in yet a further aspect of the present invention there is provided a polynucleotide or salt thereof comprising the sequence ATACGAGCTTGTTCAATA[Y]TGATAGTAAGAGCAATC (SEQ ID NO: 590), where Y is any sequence comprising a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to any one of SEQ ID Nos: 421, 423, 425, 427, 429, 431, 433, 435, 437, 439, 441, 443, 445, 447, 449, 451, 453, 455, 457, 459, 461, 463, 465 and 467, which polynuceotide or salt thereof selectively binds to oxycodone.
In yet a further aspect of the present invention there is provided a polynucleotide or salt thereof comprising the sequence ATACGAGCTTGTTCAATA[Y]TGATAGTAAGAGCAATC (SEQ ID NO: 591), where Y is any sequence comprising any one of SEQ ID Nos: 421, 423, 425, 427, 429, 431, 433, 435, 437, 439, 441, 443, 445, 447, 449, 451, 453, 455, 457, 459,
461, 463, 465 and 467, which polynuceotide or salt thereof selectively binds to oxycodone.
In yet a further aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to oxycodone, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 279 or a fragment thereof which retains selective binding for oxycodone. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 280.
Figure imgf000116_0001
Figure imgf000117_0001
In yet a further aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to oxycodone, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 420 or a fragment thereof which retains selective binding for oxycodone. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 421.
In yet a further aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to oxycodone, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 422 or a fragment thereof which retains selective binding for oxycodone. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 423.
In yet a further aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to oxycodone, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 424 or a fragment thereof which retains selective binding for oxycodone. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 425.
In yet a further aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to oxycodone, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 426 or a fragment thereof which retains selective binding for oxycodone. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 427.
In yet a further aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to oxycodone, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 428 or a fragment thereof which retains selective binding for oxycodone. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 429.
In yet a further aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to oxycodone, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 430 or a fragment thereof which retains selective binding for oxycodone. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 431.
In yet a further aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to oxycodone, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 432 or a fragment thereof which retains selective binding for oxycodone. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 433.
In yet a further aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to oxycodone, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 434 or a fragment thereof which retains selective binding for oxycodone. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 435.
In yet a further aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to oxycodone, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 436 or a fragment thereof which retains selective binding for oxycodone. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 437.
In yet a further aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to oxycodone, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 438 or a fragment thereof which retains selective binding for oxycodone. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 439.
In yet a further aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to oxycodone, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 440 or a fragment thereof which retains selective binding for oxycodone. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 441.
In yet a further aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to oxycodone, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 442 or a fragment thereof which retains selective binding for oxycodone. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 443.
In yet a further aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to oxycodone, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 444 or a fragment thereof which retains selective binding for oxycodone. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 445.
In yet a further aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to oxycodone, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 446 or a fragment thereof which retains selective binding for oxycodone. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 447.
In yet a further aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to oxycodone, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 448 or a fragment thereof which retains selective binding for oxycodone. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 449.
In yet a further aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to oxycodone, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 450 or a fragment thereof which retains selective binding for oxycodone. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 451.
In yet a further aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to oxycodone, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 452 or a fragment thereof which retains selective binding for oxycodone. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 453.
In yet a further aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to oxycodone, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 454 or a fragment thereof which retains selective binding for oxycodone. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 455.
In yet a further aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to oxycodone, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 456 or a fragment thereof which retains selective binding for oxycodone. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 457. In yet a further aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to oxycodone, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 458 or a fragment thereof which retains selective binding for oxycodone. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 459.
In yet a further aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to oxycodone, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 460 or a fragment thereof which retains selective binding for oxycodone. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 461.
In yet a further aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to oxycodone, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 462 or a fragment thereof which retains selective binding for oxycodone. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 463.
In yet a further aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to oxycodone, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 464 or a fragment thereof which retains selective binding for oxycodone. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 465.
In yet a further aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to oxycodone, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 466 or a fragment thereof which retains selective binding for oxycodone. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 467.
Cluster analysis was also performed on the oxycodone aptamers disclosed herein using Clustal Omega. The results from these analyses is presented in Figure 21 where the level of structural (i.e. sequence) dis/similarity between the different aptamers in this drug class is pictorily illustrated. By way of exemplification only, the oxycodone aptamer designated OXY- 13 and OXY-15 share a higher degree of structural similarity compared to (e.g.) OXY-17. However, OXY-13, OXY-15 and OXY-17 collectively share a higher degree of structural similarity compared to (e.g.) OXY-7 and OXY-11. Geneious Prime pairwise alignments successfuly identified four discrete oxycodone aptamer consensus sequences defined in Tables 41-44 below and identified as SEQ ID NO: 555, SEQ ID NO: 556, SEQ ID NO: 557 and SEQ ID NO: 558.
Accordingly, in yet a further aspect of the present invention there is provided a polynucleotide or salt thereof which comprises or consists in a consensus sequence defined by SEQ ID NO: 555, which polynucleotide or salt thereof selectively binds oxycodone.
In an example according to this aspect of the present invention, the polynucleotide or salt thereof which selectively binds oxycodone comprises or consists in a sequence selected from SEQ ID Nos: 420, 462, 422, 424, 426, 428 and 440.
According to the information presented in Table 42, the range in Gibbs Free Energy associated with the Oxycodone Aptamer Cluster 1 ("OX1"; SEQ ID NO: 555) is DG = -3.52 to -9.28.
In yet a further aspect of the present invention there is provided a polynucleotide or salt thereof which comprises or consists in a consensus sequence defined by SEQ ID NO: 556, which polynucleotide or salt thereof selectively binds oxycodone.
In an example according to this aspect of the present invention, the polynucleotide or salt thereof which selectively binds oxycodone comprises or consists in a sequence selected from SEQ ID Nos: 466, 450, 452, 454, 456, 458 and 460.
According to the information presented in Table 43, the range in Gibbs Free Energy associated with the Oxycodone Aptamer Cluster 2 ("OX2"; SEQ ID NO: 556) is DG = -6.78 to -10.44.
In yet a further aspect of the present invention there is provided a polynucleotide or salt thereof which comprises or consists in a consensus sequence defined by SEQ ID NO: 557, which polynucleotide or salt thereof selectively binds oxycodone.
In an example according to this aspect of the present invention, the polynucleotide or salt thereof which selectively binds oxycodone comprises or consists in a sequence selected from SEQ ID Nos: 464, 442, 444, 446 and 448.
According to the information presented in Table 44, the range in Gibbs Free Energy associated with the Oxycodone Aptamer Cluster 3 ("OX3"; SEQ ID NO: 557) is DG = -7.79 to -12.17.
In yet a further aspect of the present invention there is provided a polynucleotide or salt thereof which comprises or consists in a consensus sequence defined by SEQ ID NO: 558, which polynucleotide or salt thereof selectively binds oxycodone.
In an example according to this aspect of the present invention, the polynucleotide or salt thereof which selectively binds oxycodone comprises or consists in a sequence selected from SEQ ID Nos: 430, 434 and 438.
Figure imgf000123_0001
Figure imgf000124_0001
According to the information presented in Table 45, the range in Gibbs Free Energy associated with the Oxycodone Aptamer Cluster 4 ("OX4"; SEQ ID NO: 558) is DG = -6.88 to -8.53.
Ketamine Aptamers
In reference to the Examples and Figures which follow, Applicant has further developed aptamer sequences which are highly selective for ketamine. Advantageously, these aptamers display highly selective binding affinity for ketamine, with minimal cross-reactivity to other drug targets.
By way of illustration only, in reference to Tables 45-51 below read in conjunction with Figures 22-23, Applicants have developed aptamer sequences which selectively bind to ketamine, the structure of which is depicted in Figure 22, as well as structual analogs of ketamine.
Accordingly, in yet a further aspect of the present invention there is provided a polynucleotide or salt thereof comprising the sequence ATACGAGCTTGTTCAATA[Y]TGATAGTAAGAGCAATC (SEQ ID NO: 593), where Y is any sequence comprising a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to any one of SEQ ID Nos: 469, 471, 473, 475, 477, 479, 481, 483, 485, 487, 489, 491, 493, 495, 497, 499, 501, 503, 505, 507, 509, 511, 513, 515, 517, 519, 521 and 523, which polynuceotide or salt thereof selectively binds to ketamine.
In yet a further aspect of the present invention there is provided a polynucleotide or salt thereof comprising the sequence ATACGAGCTTGTTCAATA[Y]TGATAGTAAGAGCAATC (SEQ ID NO: 594), where Y is any sequence comprising any one of SEQ ID Nos: 469, 471, 473, 475, 477, 479, 481, 483, 485, 487, 489, 491, 493, 495, 497, 499, 501, 503, 505, 507, 509, 511, 513, 515, 517, 519, 521 and 523, which polynuceotide or salt thereof selectively binds to ketamine.
In yet a further aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to ketamine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 468 or a fragment thereof which retains selective binding for ketamine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 469.
In yet a further aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to ketamine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 468 or a fragment thereof which retains selective binding for ketamine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 469.
Figure imgf000126_0001
Figure imgf000127_0001
In yet a further aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to ketamine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 470 or a fragment thereof which retains selective binding for ketamine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 471.
In yet a further aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to ketamine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 472 or a fragment thereof which retains selective binding for ketamine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 473.
In yet a further aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to ketamine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 474 or a fragment thereof which retains selective binding for ketamine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 475.
In yet a further aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to ketamine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 476 or a fragment thereof which retains selective binding for ketamine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 477.
In yet a further aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to ketamine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 478 or a fragment thereof which retains selective binding for ketamine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 479.
In yet a further aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to ketamine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 480 or a fragment thereof which retains selective binding for ketamine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 481.
In yet a further aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to ketamine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 482 or a fragment thereof which retains selective binding for ketamine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 483.
In yet a further aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to ketamine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 484 or a fragment thereof which retains selective binding for ketamine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 485.
In yet a further aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to ketamine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 486 or a fragment thereof which retains selective binding for ketamine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 487.
In yet a further aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to ketamine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 488 or a fragment thereof which retains selective binding for ketamine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 489.
In yet a further aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to ketamine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 490 or a fragment thereof which retains selective binding for ketamine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 491.
In yet a further aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to ketamine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 492 or a fragment thereof which retains selective binding for ketamine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 493.
In yet a further aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to ketamine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 494 or a fragment thereof which retains selective binding for ketamine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 495.
In yet a further aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to ketamine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 496 or a fragment thereof which retains selective binding for ketamine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 497.
In yet a further aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to ketamine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 498 or a fragment thereof which retains selective binding for ketamine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 499.
In yet a further aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to ketamine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 500 or a fragment thereof which retains selective binding for ketamine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 501.
In yet a further aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to ketamine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 502 or a fragment thereof which retains selective binding for ketamine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 503.
In yet a further aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to ketamine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 504 or a fragment thereof which retains selective binding for ketamine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 505.
In yet a further aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to ketamine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 506 or a fragment thereof which retains selective binding for ketamine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 507. In yet a further aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to ketamine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 508 or a fragment thereof which retains selective binding for ketamine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 509.
In yet a further aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to ketamine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 510 or a fragment thereof which retains selective binding for ketamine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 511.
In yet a further aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to ketamine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 512 or a fragment thereof which retains selective binding for ketamine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 513.
In yet a further aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to ketamine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 514 or a fragment thereof which retains selective binding for ketamine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 515.
In yet a further aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to ketamine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 516 or a fragment thereof which retains selective binding for ketamine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 517.
In yet a further aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to ketamine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 518 or a fragment thereof which retains selective binding for ketamine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 519.
In yet a further aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to ketamine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 520 or a fragment thereof which retains selective binding for ketamine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 521.
In yet a further aspect of the present invention there is provided a polynucleotide or salt thereof which selectively binds to ketamine, the polynucleotide or salt thereof comprising or consisting in a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 522 or a fragment thereof which retains selective binding for ketamine. In an example according to this aspect of the present invention, the fragment comprises or consists in a sequence defined by SEQ ID NO: 523.
Cluster analysis was also performed on the ketamine aptamers disclosed herein using Clustal Omega. The results from these analyses is presented in Figure 21 where the level of structural (i.e. sequence) dis/similarity between the different aptamers in this drug class is pictorily illustrated. By way of exemplification only, the ketamine aptamer designated 3KET6 and 3KET12 share a higher degree of structural similarity compared to (e.g.) 3KET2 and 3KET9. However, 3KET6, 3KET12, 3KET2 and 3KET9 collectively share a higher degree of structural similarity compared to (e.g.) 5KET50 and 5KET37.
Geneious Prime pairwise alignments successfuly identified six discrete ketamine aptamer consensus sequences defined in Tables 46-51 below and identified as SEQ ID NO: 559, SEQ ID NO: 560, SEQ ID NO: 561, SEQ ID NO: 562, SEQ ID NO: 563 and SEQ ID NO: 564.
Accordingly, in yet a further aspect of the present invention there is provided a polynucleotide or salt thereof which comprises or consists in a consensus sequence defined by SEQ ID NO: 559, which polynucleotide or salt thereof selectively binds ketamine.
In an example according to this aspect of the present invention, the polynucleotide or salt thereof which selectively binds ketamine comprises or consists in a sequence selected from SEQ ID Nos: 500, 506, 510, 512, 514 and 522.
According to the information presented in Table 47, the range in Gibbs Free Energy associated with the Ketamine Aptamer Cluster 1 ("KET1"; SEQ ID NO: 559) is DG = -3.61 to -8.21.
In yet a further aspect of the present invention there is provided a polynucleotide or salt thereof which comprises or consists in a consensus sequence defined by SEQ ID NO: 560, which polynucleotide or salt thereof selectively binds ketamine.
In an example according to this aspect of the present invention, the polynucleotide or salt thereof which selectively binds ketamine comprises or consists in a sequence selected from SEQ ID Nos: 486, 488, 492 and 508.
Figure imgf000133_0001
Figure imgf000134_0001
Figure imgf000135_0001
According to the information presented in Table 48, the range in Gibbs Free Energy associated with the Ketamine Aptamer Cluster 2 ("KET2"; SEQ ID NO: 560) is DG = -2.87 to -8.68.
In yet a further aspect of the present invention there is provided a polynucleotide or salt thereof which comprises or consists in a consensus sequence defined by SEQ ID NO: 561, which polynucleotide or salt thereof selectively binds ketamine.
In an example according to this aspect of the present invention, the polynucleotide or salt thereof which selectively binds ketamine comprises or consists in a sequence selected from SEQ ID Nos: 472, 480 and 496.
According to the information presented in Table 49, the range in Gibbs Free Energy associated with the Ketamine Aptamer Cluster 3 ("KET3"; SEQ ID NO: 561) is DG = -1.96 to -6.03.
In yet a further aspect of the present invention there is provided a polynucleotide or salt thereof which comprises or consists in a consensus sequence defined by SEQ ID NO: 562, which polynucleotide or salt thereof selectively binds ketamine.
In an example according to this aspect of the present invention, the polynucleotide or salt thereof which selectively binds ketamine comprises or consists in a sequence selected from SEQ ID Nos: 490, 498 and 516.
According to the information presented in Table 50, the range in Gibbs Free Energy associated with the Ketamine Aptamer Cluster 4 ("KET4"; SEQ ID NO: 562) is DG = -4.75 to -8.30.
In yet a further aspect of the present invention there is provided a polynucleotide or salt thereof which comprises or consists in a consensus sequence defined by SEQ ID NO: 563, which polynucleotide or salt thereof selectively binds ketamine.
In an example according to this aspect of the present invention, the polynucleotide or salt thereof which selectively binds ketamine comprises or consists in a sequence selected from SEQ ID Nos: 468, 474 and 476.
According to the information presented in Table 51, the range in Gibbs Free Energy associated with the Ketamine Aptamer Cluster 5 ("KET5"; SEQ ID NO: 563) is DG = -4.75 to -9.33.
In yet a further aspect of the present invention there is provided a polynucleotide or salt thereof which comprises or consists in a consensus sequence defined by SEQ ID NO: 564, which polynucleotide or salt thereof selectively binds ketamine.
In an example according to this aspect of the present invention, the polynucleotide or salt thereof which selectively binds ketamine comprises or consists in a sequence selected from SEQ ID Nos: 470, 478 and 482. According to the information presented in Table 52, the range in Gibbs Free Energy associated with the Ketamine Aptamer Cluster 6 ("KET6"; SEQ ID NO: 564) is DG = -3.55 to -4.65.
Methods for Detection of Illicit Drugs
The aptamer/polynucleotide sequences disclosed herein may be useful in performing assay methods for the detection of illicit drugs. This may include, for example, determining the drug status of an individual at a road-side testing station or in quality care governance for employees operating heavy machinary, (e.g.) in the mining industry.
Accordingly, in a further aspect of the present invention there is provided a method for determining the drug status of an individual, the method comprising combining a test sample obtained from the individual with a test kit or article of manufacture described herein, and using the results obtained from performance of the test kits to determine the drug status of an individual.
In certain examples according to the methods described herein, the test kit is configured to detect multiple drug targets from discrete drug classes to determine concurrent drug abuse. For example, detection of tetrahydrocannabinol in respect of cannabis use, tested alongside cocaine. Both are widely used recreational drugs, which are often abused by an individual at the same time.
In yet a further of the present invention there is provided a method for determining the drug status of an individual, the method comprising detecting an illicit drug from a test sample. In an example according to this aspect of the present invention, the illicit drug includes, but is not limited to, methamphetamine, tetrahydrocannabinol, cocaine, morphine, 3,4-methylenedioxymethamphetamine, benzodiazepine, AMB-FUBINACA, AM2201, oxycodone and ketamine, and includes structural analogs of methamphetamine, tetrahydrocannabinol, cocaine, morphine, 3,4-methylenedioxymethamphetamine, benzodiazepine, AMB-FUBINACA, AM2201, oxycodone and ketamine.
According to the present invention, detection of drugs of abuse is typically achieved by contacting a test sample with a polynucleotide (aptamer) selective for a drug target, and measuring a binding interaction between the aptamer and drug target, if present in the test sample.
Accordingly, in a further aspect of the present invention there is provided a method for detecting the presence of methamphetamine in a test sample, the method comprising the steps of:
(i) contacting a test sample with a polynucleotide or salt thereof comprising a sequence defined by any one of SEQ ID Nos: 1-74; and
(ii) measuring binding between the polynucleotide and methamphetamine from the test sample, wherein, a measured binding interaction between the polynucleotide and methamphetamine reflects the presence of methamphetamine in the test sample.
In a further aspect of the present invention there is provided a method for detecting the presence of tetrahydrocannabinol in a test sample, the method comprising the steps of:
(i) contacting a test sample with a polynucleotide or salt thereof comprising a sequence defined by any one of SEQ ID Nos: 75-136; and
(ii) measuring binding between the polynucleotide and tetrahydrocannabinol from the test sample, wherein, a measured binding interaction between the polynucleotide and tetrahydrocannabinol reflects the presence of tetrahydrocannabinol in the test sample.
In a further aspect of the present invention there is provided a method for detecting the presence of cocaine in a test sample, the method comprising the steps of:
(i) contacting a test sample with a polynucleotide or salt thereof comprising a sequence defined by any one of SEQ ID Nos: 137-172; and
(ii) measuring binding between the polynucleotide and cocaine from the test sample, wherein, a measured binding interaction between the polynucleotide and cocaine reflects the presence of cocaine in the test sample.
In a further aspect of the present invention there is provided a method for detecting the presence of 3,4-methylenedioxymethamphetamine in a test sample, the method comprising the steps of:
(i) contacting a test sample with a polynucleotide or salt thereof comprising a sequence defined by any one of SEQ ID Nos: 173-246; and
(ii) measuring binding between the polynucleotide and 3,4- methylenedioxymethamphetamine from the test sample, wherein, a measured binding interaction between the polynucleotide and 3,4- methylenedioxymethamphetamine reflects the presence of 3,4- methylenedioxymethamphetamine in the test sample.
In a further aspect of the present invention there is provided a method for detecting the presence of morphine in a test sample, the method comprising the steps of:
(i) contacting a test sample with a polynucleotide or salt thereof comprising a sequence defined by any one of SEQ ID Nos: 247-291; and
(ii) measuring binding between the polynucleotide and morphine from the test sample, wherein, a measured binding interaction between the polynucleotide and morphine reflects the presence of morphine in the test sample.
In a further aspect of the present invention there is provided a method for detecting the presence of benzodiazepine in a test sample, the method comprising the steps of: (i) contacting a test sample with a polynucleotide or salt thereof comprising a sequence defined by any one of SEQ ID Nos: 292-319; and
(ii) measuring binding between the polynucleotide and benzodiazepine from the test sample, wherein, a measured binding interaction between the polynucleotide and diazepam reflects the presence of diazepam in the test sample.
In a further aspect of the present invention there is provided a method for detecting the presence of AMB-FUBINACA in a test sample, the method comprising the steps of:
(i) contacting a test sample with a polynucleotide or salt thereof comprising a sequence defined by any one of SEQ ID Nos: 320-371; and
(ii) measuring binding between the polynucleotide and AMB-FUBINACA from the test sample, wherein, a measured binding interaction between the polynucleotide and AMB- FUBINACA reflects the presence of AMB-FUBINACA in the test sample.
In a further aspect of the present invention there is provided a method for detecting the presence of AM2201 in a test sample, the method comprising the steps of:
(i) contacting a test sample with a polynucleotide or salt thereof comprising a sequence defined by any one of SEQ ID Nos: 372-419; and
(ii) measuring binding between the polynucleotide and AM2201 from the test sample, wherein, a measured binding interaction between the polynucleotide and AM2201 reflects the presence of AM2201 in the test sample.
In a further aspect of the present invention there is provided a method for detecting the presence of oxycodone in a test sample, the method comprising the steps of:
(i) contacting a test sample with a polynucleotide or salt thereof comprising a sequence defined by any one of SEQ ID Nos: 420-467; and
(ii) measuring binding between the polynucleotide and oxycodone from the test sample, wherein, a measured binding interaction between the polynucleotide and oxycodone reflects the presence of oxycodone in the test sample.
In a further aspect of the present invention there is provided a method for detecting the presence of ketamine in a test sample, the method comprising the steps of:
(i) contacting a test sample with a polynucleotide or salt thereof comprising a sequence defined by any one of SEQ ID Nos: 468-523; and
(ii) measuring binding between the polynucleotide and oxycodone from the test sample, wherein, a measured binding interaction between the polynucleotide and ketamine reflects the presence of ketamine in the test sample. In certain examples according to the methods described herein, a binding interaction between the polynucleotide and drug target (or molecule associated with a drug target) is measured using a detection means. Examples of detection means include, but are not limited, optical methods, spectroscopy, visible spectroscopy, Raman spectroscopy, UV spectroscopy, surface plasmon resonance, electrochemical methods, impedance, resistance, capacitance, mechanical sensing by changes in mass, changes in mechanical resonance, electrophoresis, gel electrophoresis, gel retardation, imaging, fluorescence, fluorescence resonance energy transfer.
Test Kits & Articles of Manufacture
Further contemplated by the present invention is a test kit or article of manufacture suitable for performing a method or assay described herein, in particular for the detection of an illicit drug from a test sample.
In yet a further aspect of the present invention there is provided a test kit or article of manufacture for detecting the presence of methamphetamine in a test sample, the test kit or article of manufacture comprising a polynucleotide or salt thereof comprising a sequence defined by any one of SEQ ID Nos: 1-74, and optionally instructions for how to detect the presence of methamphetamine in the test sample.
In yet a further aspect of the present invention there is provided a test kit or article of manufacture for detecting the presence of tetrahydrocannabinol in a test sample, the test kit or article of manufacture comprising a polynucleotide or salt thereof comprising a sequence defined by any one of SEQ ID Nos: 75-136, and optionally instructions for how to detect the presence of tetrahydrocannabinol in the test sample.
In yet a further aspect of the present invention there is provided a test kit or article of manufacture for detecting the presence of cocaine in a test sample, the test kit or article of manufacture comprising a polynucleotide or salt thereof comprising a sequence defined by any one of SEQ ID Nos: 137-172, and optionally instructions for how to detect the presence of cocaine in the test sample.
In yet a further aspect of the present invention there is provided a test kit or article of manufacture for detecting the presence of 3,4-methylenedioxymethamphetamine in a test sample, the test kit or article of manufacture comprising a polynucleotide or salt thereof comprising a sequence defined by any one of SEQ ID Nos: 173-246, and optionally instructions for how to detect the presence of 3,4-methylenedioxymethamphetamine in the test sample.
In yet a further aspect of the present invention there is provided a test kit or article of manufacture for detecting the presence of morphine in a test sample, the test kit or article of manufacture comprising a polynucleotide or salt thereof comprising a sequence defined by any one of SEQ ID Nos: 247-291, and optionally instructions for how to detect the presence of morphine in the test sample.
In yet a further aspect of the present invention there is provided a test kit or article of manufacture for detecting the presence of benzodiazepine in a test sample, the test kit or article of manufacture comprising a polynucleotide or salt thereof comprising a sequence defined by any one of SEQ ID Nos: 292-319, and optionally instructions for how to detect the presence of benzodiazepine in the test sample.
In yet a further aspect of the present invention there is provided a test kit or article of manufacture for detecting the presence of AMB-FUBINACA in a test sample, the test kit or article of manufacture comprising a polynucleotide or salt thereof comprising a sequence defined by any one of SEQ ID Nos: 320-371, and optionally instructions for how to detect the presence of AMB-FUBINACA in the test sample.
In yet a further aspect of the present invention there is provided a test kit or article of manufacture for detecting the presence of AM2201 in a test sample, the test kit or article of manufacture comprising a polynucleotide or salt thereof comprising a sequence defined by any one of SEQ ID Nos: 372-419, and optionally instructions for how to detect the presence of AM2201 in the test sample.
In yet a further aspect of the present invention there is provided a test kit or article of manufacture for detecting the presence of oxycodone in a test sample, the test kit or article of manufacture comprising a polynucleotide or salt thereof comprising a sequence defined by any one of SEQ ID Nos: 420-467, and optionally instructions for how to detect the presence of oxycodone in the test sample.
In yet a further aspect of the present invention there is provided a test kit or article of manufacture for detecting the presence of oxycodone in a test sample, the test kit or article of manufacture comprising a polynucleotide or salt thereof comprising a sequence defined by any one of SEQ ID Nos: 420-467, and optionally instructions for how to detect the presence of oxycodone in the test sample.
In yet a further aspect of the present invention there is provided a test kit or article of manufacture for detecting the presence of ketamine in a test sample, the test kit or article of manufacture comprising a polynucleotide or salt thereof comprising a sequence defined by any one of SEQ ID Nos: 468-523, and optionally instructions for how to detect the presence of oxycodone in the test sample.
In certain examples according to the present invention, the test kits or articles of manufacture optionally include instructions for how to perform the detection of an illicit drug from the test sample interrogated. Exemplary Samples for Performance of Test Kits/Methods
The test kits and methods described herein may be performed on any test sample. In an example according to all aspects of the test kits, assays and methods described herein, the test sample is derived from biological material selected from the group consisting of urine, saliva, stool, hair, tissues including, but not limited to, blood (plasma and serum), muscle, tumors, semen, etc.
In yet a further example according to all aspects of the test kits, assays and methods described herein, the biological sample is derived from a human.
In yet a further example according to all aspects of the test kits, assays and methods described herein, the test sample is derived from a food selected from the group consisting of vegetable, meat, beverage including but not limited to sports drink and milk, supplements including, but not limited to, food supplements and sports supplements, nutritional supplements, herbal extracts, etc.
In yet a further example according to all aspects of the test kits, assays and methods described herein, the test sample is derived from a medication including, without limitation, a drug, a tonic, a syrup, a pill, a lozenge, a cream, a spray and a gel.
In yet a further example according to all aspects of the test kits, assays and methods described herein, the sample is derived from the environment including, without limitation, a liquid, water, plastics and a mineral.
Modified Aptamers/Polynucleotides
Various modifications can be made to the polynucleotide aptamers disclosed herein to reduce exonuclease degradation, increase half-life for diagnostic or therapeutic applications, and/or for other purposes. Modification of the 3'end of the aptamer with inverted thymidine, deoxythymidine nucleotide, and polyethylene glycol (PEG) can reduce degradation of the oligonucleotide aptamer and increases stability of the aptamer. In some examples, PEG has an average molecular weight from about 20 to 80 kDa.
Further, the phosphodiester linkages of the deoxyribose-phosphate backbone of the aptamer can also be modified to improve stability.
In some examples, the aptamer is a polynucleotide comprising repeating units of the structure shown in Formula 1. Wavy lines demarcate one nucleotide and/or repeat unit from a neighboring nucleotide and/or repeat unit.
Figure imgf000143_0001
In some examples, each repeat unit of Formula 1 has a deoxyribose moiety linked to one of the common nucleotide bases (B) , such as, adenosine, cytidine, guanosine, thymidine, and uridine. The base (B) for each repeating unit is independent from the other repeat units. The nucleotide sequences disclosed herein describe the order of appearance of bases (B) in an aptamer from the repeat unit on the 5'end of the aptamer to the 3'end of the aptamer.
In some examples, "L" is a linker group that links the deoxyribose moiety of adjacent repeat units. In the well-known structure of DNA, the L group is a phosphate group PO4H, which can exist as a salt or in a neutral protonated form. The deoxyribose moiety together with the linker group forms the backbone of the aptamer, where the nucleotide base "B" varies independently of bonds and linkers between repeat units. Typically, the majority of the linker groups (L) forming the repeat units of Formula 1 in the aptamer are phosphate groups. As such, a majority of the backbone of the aptamer can be referred to as a deoxyribose-phosphate backbone. Many nuclease enzymes exist that can degrade oligonucleotide molecules without specificity for the specific nucleotide base sequence of the oligonucleotide molecule. Without wishing to be bound by any one particular theory, linker groups "L" other than phosphate can be incorporated into an oligonucleotide or aptamer to reduce or prevent degradation by nucleases.
In some examples, L can be replaced with a group as shown in Formula 2, where X1-4 are independently O or S. X2 and X3 can be bonded to either the 3'carbon or the 5'carbon of a deoxyribose moiety. In some examples, X1 is O and X4 is O that can be either protonated or unprotonated. In some examples, one or more of X2 and/or X3 is S and X1 and X4 are O, where O can be either protonated or unprotonated. Where one of X2 and/or X3 are S, the aptamer can be referred to as having a thioester linkage in the deoxyribose-phosphate backbone.
Figure imgf000144_0001
In some examples, the linker group "L" is an amide-containing group as shown in Formula 3, where R can be selected from hydrogen substituted or unsubstituted C1-C10 hydrocarbyl group. A "hydrocarbon" or "hydrocarbyl" refers to organic compounds or radicals consisting exclusively of the elements carbon and hydrogen. Hydrocarbyl includes alkyl, alkenyl, alkynyl, and aryl moieties. Hydrocarbyl also includes alkyl, alkenyl, alkynyl, and aryl moieties substituted with other aliphatic, cyclic or aryl hydrocarbon groups, such as alkaryl, alkenaryl and alkynaryl. In some examples, the linker group "L" is a group having Formula 3, and the aptamer comprises amide linkage (s) in the deoxyribose-phosphate backbone. The "NR" group of Formula 3 can be bonded to either the 3'carbon or the 5'carbon of a deoxyribose moiety. In some examples, R is methoxymethyl or methoxyethyl.
Figure imgf000144_0002
In some examples, the aptamer has from about 14 to about 100 nucleotide bases and/or repeat units, or any number of nucleotide bases or repeating units between 14 to 100, such as between 20 to 50 nucleotide bases and/or repeating units. In some examples, the aptamer has from about 14 to about 50 nucleotide bases and/or repeat units. In some examples, the aptamer has from about 30 to about 35 nucleotide bases and/or repeat units. In some examples, the aptamer has from 14 to 100, 14 to 95, 14 to 90, 14 to 85, 14 to 80, 14 to 75, 14 to 70, 14 to 65, 14 to 60, 14 to 55, 14 to 50, 14 to 45, 14 to 40, 14 to 35, 14 to 30, 14 to 25, 14 to 20, 14 to 18, 14 to 16, 14 to 15 nucleotide bases and/or repeating units, inclusive.
In some examples, the aptamer has from 20 to 100, 20 to 95, 20 to 90, 20 to 85, 20 to 80, 20 to 75, 20 to 70, 20 to 65, 20 to 60, 20 to 55, 20 to 50, 20 to 45, 20 to 40, 20 to 35, 20 to 30, 20 to 25 nucleotide bases and/or repeating units, inclusive. In some examples, the aptamer has from 25 to 100, 25 to 95, 25 to 90, 25 to 85, 25 to 80, 25 to 75, 25 to 70,
25 to 65, 25 to 60, 25 to 55, 25 to 50, 25 to 45, 25 to 40, 25 to 35, 25 to 30 nucleotide bases and/or repeating units, inclusive. In some examples, the aptamer has from 30 to 100, 30 to
95, 30 to 90, 30 to 85, 30 to 80, 30 to 75, 30 to 70, 30 to 65, 30 to 60, 30 to 55, 30 to 50,
30 to 45, 30 to 40, 30 to 35 nucleotide bases and/or repeating units, inclusive. In some examples, the aptamer has from 35 to 100, 35 to 95, 35 to 90, 35 to 85, 35 to 80, 35 to 75, 35 to 70, 35 to 65, 35 to 60, 35 to 55, 35 to 50, 35 to 45, 35 to 40 nucleotide bases and/or repeating units, inclusive. In some examples, the aptamer has from 40 to 100, 40 to 95, 40 to 90, 40 to 85, 40 to 80, 40 to 75, 40 to 70, 40 to 65, 40 to 60, 40 to 55, 40 to 50, 40 to 45 nucleotide bases and/or repeating units, inclusive. In some examples, the aptamer has from 45 to 100, 45 to 95, 45 to 90, 45 to 85, 45 to 80, 45 to 75, 45 to 70, 45 to 65, 45 to 60, 45 to 55, 45 to 50 nucleotide bases and/or repeating units, inclusive. In some examples, the aptamer has from 50 to 100, 50 to 95, 50 to 90, 50 to 85, 50 to 80, 50 to 75, 50 to 70, 50 to 65, 50 to 60, 50 to 55 nucleotide bases and/or repeating units, inclusive. In some examples, the aptamer has from 55 to 100, 55 to 95, 55 to 90, 55 to 85, 55 to 80, 55 to 75, 55 to 70, 55 to 65, 55 to 60 nucleotide bases and/or repeating units, inclusive. In some examples, the aptamer has from 60 to 100, 60 to 95, 60 to 90, 60 to 85, 60 to 80, 60 to 75, 60 to 70, 60 to 65 nucleotide bases and/or repeating units, inclusive. In some examples, the aptamer has 14, 15, 16, 18, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 nucleotide bases and/or repeating units.
In some examples, the aptamer comprises one or more repeating units having the linker "L" selected independently from Formulae 2 and 3. In some examples of the aptamer, the number of repeating units having the linker "L" selected independently from Formulae 2 and 3 is between 1 to 15 (inclusive), or any number there between, such as, for example, 1 to 10, 2 to 8, and 3 to 5, inclusive. In some examples of the aptamer, the number of repeating units having the linker "L" selected independently from Formulae 2 and 3 is between 1 to 15, 1 to 14, 1 to 12, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, inclusive. In some examples of the aptamer, the number of repeating units having the linker "L" selected independently from Formulae 2 and 3 is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, or 15. Linker groups in repeat units not selected from Formulae 2 or 3 are phosphate.
In some examples of the aptamer, about 10% to about 100% (or any percentages there between, such as about 20%to about 90%, about 30%to about 50%) of the repeat units have the linker "L" selected independently from Formulae 2 and 3. In some examples of the aptamer, about 10% to about 70%of the repeat units have the linker "L" selected independently from Formulae 2 and 3. In some examples of the aptamer, about 10% to about 50% of the repeat units have the linker "L" selected independently from Formulae 2 and 3. In some examples of the aptamer, about 10% to about 30% of the repeat units have the linker "L" selected independently from Formulae 2 and 3. In some examples of the aptamer, about 10% to about 20% of the repeat units have the linker "L" selected independently from Formulae 2 and 3. Linker groups in repeat units not selected from Formulae 2 or 3 are phosphate.
Various nucleases are exonucleases that degrade oligonucleotides from the 5' or 3'end. As such, in some examples, a linker group L selected from Formulae 2 or 3 is located within about 5 repeat units from the 5' or the 3'end of the aptamer. In some examples, a linker group L selected from Formulae 2 or 3 is located within about 3 repeat units from the 5' or the 3'end of the aptamer. In some examples, a linker group L selected from Formulae 2 or 3 is located within 3 repeat units from the 5' or the 3'end of the aptamer. In some examples, a linker group L selected from Formulae 2 or 3 is located within 2 repeat units from the 5' or the 3' end of the aptamer. In some examples, a linker group L selected from Formulae 2 or 3 is part of the repeat unit on the 5' or the 3'end of the aptamer.
Degradation of the aptamers can also be reduced by the inclusion of modified nucleotide bases (B) . The pyrimidine nucleotide bases, cytosine, thymine and uracil can be replaced with alkylated pyrimidines. Examples of alkylated pyrimidines include pseudoisocytosine; N4, N4-ethanocytosine; 4-acetylcytosine, 5- (carboxyhydroxylmethyl) uracil; 5-fluorouracil; 5-bromouracil; 5-carboxymethylaminomethyl-2-thiouracil; 5- carboxymethylaminomethyl uracil; dihydrouracil; 1-methylpseudouracil; 3-methylcytosine; 5-methylcytosine; 5-methylaminomethyl uracil; 5-methoxy amino methyl-2-thiouracil; 5- methoxycarbonylmethyluracil; 5-methoxyuracil; uracil-5-oxyacetic acid methyl ester; pseudouracil; 2-thiocytosine; 5-methyl-2 thiouracil, 2-thiouracil; 4-thiouracil; 5-methyluracil; N-uracil-5-oxyacetic acid methylester; uracil 5-oxyacetic acid; 2-thiocytosine; 5-propyluracil; 5-propylcytosine; 5-ethyluracil; 5-ethylcytosine; 5-butyluracil; 5-pentyluracil; 5- pentylcytosine; methylpseudouracil; and 1-methylcytosine. The purine nucleotide bases, adenine and guanine, can be replaced by alkylated purines. Examples alkylated purines include 8-hydroxy-N6-methyladenine; inosine; N6-isopentyl-adenine; 1-methyladenine; 1- methylguanine; 2, 2-dimethylguanine; 2-methyladenine; 2-methylguanine; N6- methyladenine; 7-methylguanine; 2-methylthio-N6-isopentenyladenine; and 1- methylguanine.
In some examples, at least one deoxyribose or ribose of the nucleic acid aptamer is replaced with a morpholine ring. In one form, at least one phosphorothioate or phosphodiester linkage of the nucleic acid aptamer is replaced with phosphorodiamidate.
The nucleotides that can be substituted for natural nucleotides of DNA have a base moiety such as inosine, 5-fluorouracil, 5-bromouracil, hypoxanthine, 1-methylguanine, 5- methylcytosine, or tritylated bases. The sugar moiety of the nucleotide in a sequence can also be modified with a group such as arabinose, xylulose, or hexose. In addition, the adenine, cytosine, guanine, thymine, and uracil bases of the nucleotides can be modified with acetyl, methyl, hydroxyl, and thio groups. Sequences containing nucleotide substitutions, deletions, and/or insertions can be prepared and tested using standard techniques known in the art.
The disclosed polynucleotide aptamers may be modified to promote an association with a substrate (e.g. particle or solid surface). In certain examples, the disclosed polynucleotide aptamers may be coupled or conjugated to one or more chemical entities or moieties to aid functionalization (e.g.) in an illicit drug detection assay involving a substrate including but not limited to a particle or solid surface.
Detection Labels
To aid in detection of the illicit drug targets disclosed herein, detection labels may be directly associated with or coupled to a polynucleotide aptamer. As used herein, a detection label is any molecule that can be associated with an aptamer, directly or indirectly, and which results in a measurable, detectable signal, either directly or indirectly. Many such labels for are known to those of skill in the art. Examples of suitable detection labels include radioisotopes (e.g., 3H, 14C, 35S, 125I, 131I), fluorescent labels (e.g., methylene blue, FITC, rhodamine, lanthanide phosphors), luminescent labels such as luminol; enzymatic labels (e.g., horseradish peroxidase, beta-galactosidase, luciferase, alkaline phosphatase, acetylcholinestease), biotinyl groups (which can be detected by marked avidin, e.g., streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or calorimetric methods), predetermined polypeptide epitopes recognized by a secondary reporter (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags).
The detection labels associated with the aptamers contemplated by the present invention can be part of, and detectable with, enzyme-linked detection systems. Enzyme- linked detection generally involves an enzyme as a label or tag on a component where the presence of the enzyme (and thus of the analyte with which the enzyme is associated) is detected by having the enzyme convert an enzymatic substrate into a form that produces a detectable signal. For example, analytes labeled or associated with alkaline phosphatase can be detected by adding the chemiluminescent substrate CSPD (Tropix, Inc). The fluorescent reaction product can then be detected. Preferred forms of detection labels are enzymes, such as alkaline phosphatases and peroxidases, for use in an enzyme-linked detection system.
Examples of suitable fluorescent labels include fluorescein (FITC), 5, 6-carboxy methyl fluorescein, Texas red, nitrobenz-2-oxa-1, 3-diazol-4-yl (NBD), coumarin, dansyl chloride, rhodamine, 4'-6-diamidino-2-phenylinodole (DAPI), and the cyanine dyes Cy3, Cy3.5, Cy5, Cy5.5 and Cy7. Preferred fluorescent labels are fluorescein (5-carboxyfluorescein-N- hydroxysuccinimide ester) and rhodamine (5, 6-tetramethyl rhodamine) . Preferred fluorescent labels for combinatorial multicolor coding are FITC and the cyanine dyes Cy3, Cy3.5, Cy5, Cy5.5 and Cy7. The absorption and emission maxima, respectively, for these fluors are: FITC (490 nm; 520 nm), Cy3 (554 nm; 568 nm), Cy3.5 (581 nm; 588 nm), Cy5 (652 nm: 672 nm), Cy5.5 (682 nm; 703 nm) and Cy7 (755 nm; 778 nm), thus allowing their simultaneous detection. Detection labels such as biotin can be subsequently detected using sensitive methods well-known in the art. For example, biotin can be detected using streptavidin-alkaline phosphatase conjugate (Tropix, Inc), which is bound to the biotin and subsequently detected by chemiluminescence of suitable substrates (for example, chemiluminescent substrate CSPD: disodium, 3- (4-methoxyspiro- [1, 2, -dioxetane-3-2'- (5'-chloro) tricyclo [3.3.1.1 3, 7] decane] -4-yl) phenyl phosphate; Tropix, Inc. ).
Molecules that combine two or more of these detection labels are also contemplated by the present invention. Any of the known detection labels can be used with the disclosed detection agents. Methods for detecting and measuring signals generated by detection labels are also known to those of skill in the art. For example, radioactive isotopes can be detected by scintillation counting or direct visualization; fluorescent molecules can be detected with fluorescent spectrophotometers; phosphorescent molecules can be detected with a spectrophotometer or directly visualized with a camera; enzymes can be detected by detection or visualization of the product of a reaction catalyzed by the enzyme; antibodies can be detected by detecting a secondary detection element coupled to the antibody. Such methods can be used directly in the disclosed method of amplification and detection. As used herein, detection agents are molecules which interact with amplified nucleic acid and to which one or more detection labels are coupled.
Aptamers - General
Aptamers are nucleic acid species that have been engineered through repeated rounds of in vitro selection equivalently, SELEX (systematic evolution of ligands by exponential enrichment) to bind to various molecular targets such as small molecules, proteins, nucleic acids, and even cells, tissues and organisms. Aptamers offer molecular binding and recognition equivalent to antibodies. In addition to their discriminate recognition, aptamers offer advantages over antibodies as they can be engineered completely in vitro, are readily produced by chemical synthesis, possess desirable storage properties, and elicit little or no immunogenicity in therapeutic applications.
According to an example of the present invention, the aptamer is a monomer (one unit). According to another example of the invention, the aptamer is a multimeric aptamer. The multimeric aptamer may comprise a plurality of aptamer units (mers). Each of the plurality of units of the aptamer may be identical. In such a case the multimeric aptamer is a homomultimer having a single specificity but enhanced avidity (multivalent aptamer).
Alternatively, the multimeric aptamer may comprise two or more aptameric monomers, wherein at least two mers of the multimeric aptamer are non-identical in structure, nucleic acid sequence or both. Such a multimeric aptamer is referred to herein as a heteromultimer. The heteromultimer may be directed to a single binding site i.e., monospecific (such as to avoid steric hindrance). The heteromultimer may be directed to a plurality of binding sites i.e., multispecific. The heteromultimer may be directed to a plurality of binding sites on different analytes, including for different drugs of abuse Further description of the multimeric aptamer is provided below.
A plurality of multimeric aptamers may be conjugated to form a conjugate of multimeric aptamers. The multimeric aptamer may comprise, two (dimer), three (trimer), four (tetramer), five (pentamer), six (hexamer), and even more units.
Aptamers of the invention can be synthesized and screened by any suitable methods known in the art.
For example, aptamers can be screened and identified from a random aptamer library by SELEX (systematic evolution of ligands by exponential enrichment). Aptamers that bind to an antigen of interest can be suitably screened and selected by a modified selection method herein referred to as cell-SELEX or cellular-SELEX. In other examples, aptamers that bind to a cell surface target molecule can be screened by capillary electrophoresis and enriched by SELEX based on the observation that aptamer-target molecule complexes exhibited retarded migration rate in native polyacrylamide gel electrophoresis as compared to unbound aptamers.
A random aptamer library can be created that contains monomeric, dimeric, trimeric, tetrameric or other higher multimeric aptamers. A random aptamer library (either ssDNA or RNA) can be modified by including oligonucleotide linkers to link individual aptamer monomers to form multimeric aptamer fusion molecules. In other examples, a random oligonucleotide library is synthesized with randomized 45 nt sequences flanked by defined 20 nt sequences both upstream and downstream of the random sequence, i.e., known as 5'-arm and 3'-arm, which are used for the amplification of selected aptamers. A linking oligonucleotide (i.e., linker) is designed to contain sequences complementary to both 5'-arm and 3'-arm regions of random aptamers to form dimeric aptamers. For trimeric or tetrameric aptamers, a small trimeric or tetrameric (i.e., a Holiday junction-like) DNA nanostructure is engineered to include sequences complementary to the 3'-arm region of the random aptamers, therefore creating multimeric aptamer fusion through hybridization. In addition, 3-5 or 5-10 dT rich nucleotides can be engineered into the linker polynucleotides as a single stranded region between the aptamer-binding motifs, which offers flexibility and freedom of multiple aptamers to coordinate and synergize multivalent interactions with cellular ligands or receptors. Alternatively, multimeric aptamers can also be formed by mixing biotinylated aptamers with streptavidin.
A modified cellular SELEX procedure can be employed to select target-binding aptamers. Multimeric aptamers may be multivalent but be of single binding specificity (i.e., homomultimeric aptamers). Alternatively, the multimeric aptamer may be multivalent and multi- specific (i.e., heteromultimeric aptamers). Thus, each monomer of the homomultimeric aptamer binds the target protein (e.g., selected drug target) in an identical manner. Thus according to an example of the invention, all monomeric components of the homomultimeric aptamer are identical.
Conversely, a heteromultimeric aptamer comprises a plurality of monomeric aptamers at least two of which bind different sites on a single target protein or bind at least two different target proteins.
Selection of DNA or RNA-aptamers is well-established using protocols described in the scientific literature.
In certain examples, a suitable nucleotide length for an aptamer ranges from about 15 to about 100 nucleotide (nt), and in various other examples, 12-30, 14-30, 15-30 nt, 30-100 nt, 30-60 nt, 25-70 nt, 25-60 nt, 40-60 nt, or 40-70 nt in length.
In other examples, the aptamer has affinity at the range of 10-100 nM, which, after binding of the aptamer to a molecule, permits dissociation of the aptamer from the target molecule, which leads to the release and recycle of the aptamer nucleic acid nanostructure. The affinity of individual aptamers can be increased by 4-50 fold by constructing multimeric aptamers linked together by covalent or non-covalent linkages. Methods of multimerizing aptamers are further described below.
Thus, in certain examples, the desirable affinity of an aptamer to an analyte of interest can be fine-tuned by adjusting the multiplexity of the monomeric aptamer.
Multimerization can be done at the library level as follows.
In certain examples, a linker polynucleotide has a length between about 5 nucleotides (nt) and about 100 nt; in various examples, 10-30 nt, 20-30 nt, 25-35 nt, 30-50 nt, 40-50 nt, 50-60 nt, 55-65 nt, 50-80 nt, or 80-100 nt. It is within the ability of one of skill in the art to adjust the length of the linker polynucleotide to accommodate each monomeric aptamer in the multimeric structure.
In certain examples, the multimeric aptamers can be identified and screened from a random multimeric aptamer library as described herein. In other examples, the monomeric aptamers are linked to each other by one or a plurality of linker polynucleotides to form multimeric aptamers. Monomeric aptamers can be linked to form multimeric aptamers by any suitable means and in any configurations.
It will be appreciated that the monomeric structures of the invention can be further multimerized by post SELEX procedures.
Multimers can be linearly linked by continuous linear synthesis of DNA without spacers or with nucleic acid spacers. Aptamer synthesis usually relies on standard solid phase phosphoramitide chemistry.
Thus, dimers, trimers and tetramers or higher oligomeric structures (e.g., pentamers, hexamers, heptamers, octamers etc.) can be linked by a polymeric spacer. In certain examples, the aptamers are further modified to protect the aptamers from nuclease and other enzymatic activities. The aptamer sequence can be modified by any suitable methods known in the art. For example, phosphorothioate can be incorporated into the backbone, and 5'-modified pyrimidine can be included in 5' end of ssDNA for DNA aptamer. For RNA aptamers, modified nucleotides such as substitutions of the 2'-OH groups of the ribose backbone, e.g., with 2'-deoxy-NTP or - fluoro-NTP, can be incorporated into the RNA molecule using T7 RNA polymerase mutants. The resistance of these modified aptamers to nuclease can be tested by incubating them with either purified nucleases or nuclease from mouse serum, and the integrity of aptamers can be analyzed by gel electrophoresis.
The monomeric or multimeric aptamer of the invention can be further attached or conjugated to a detectable or therapeutic moiety (i.e., a pharmaceutical moiety).
In one example a diagnostic moiety such as a detectable moiety e.g., label (e.g., His tag, flag tag), fluorescent, radioactive, biotin/avidin etc., can be bound to the aptamer, and imaging, immunohistochemistry, for target identification.
The present invention provides polynucleotides and salts thereof. In some examples, a polynucleotide is an oligonucleotide or a single strand of RNA or DNA. In some examples, the polynucleotide or functional fragment thereof (e.g. including a target molecule/ligand binding domain), or salt of either is an aptamer. The term "aptamer" refers to a polynucleotide or functional fragment thereof, or salt of either that specifically binds a target molecule. The term "specifically binds" is used interchangeably herein with "selectively binds" and means the same thing. As used herein the terms "specifically binds" and "selectively binds" in reference to an aptamer, describe the binding of an aptamer to a target molecule and mean that aptamer binding to the target molecule does not involve the formation of nucleotide base pairs between the aptamer and the target molecule. The skilled person would recognize it is well-known in the art that the polynucleotide sequence of an aptamer may include base pairs that are not required for specific binding of the aptamer to a given target molecule, and that smaller fragments of an aptamer, even fragments having below 50% sequence identity may still be capable of effectively binding to a target molecule (Alsager, Omar A., et al. "Ultrasensitive Colorimetric Detection of 17b-Estradiol: The Effect of Shortening DNA Aptamer Sequences." Analytical chemistry 87.8 (2015): 4201-4209).
In some examples, an aptamer exerts an inhibitory effect on a target, e.g., by binding of the target, by catalytically altering the target, by reacting with the target in a way which modifies the target or the functional activity of the target, by ionically or covalently attaching to the target or by facilitating the reaction between the target and another molecule. The aptamer can comprise a ribonucleotide, deoxyribonucleotide, or other type of nucleic acid, or two or more different types of nucleic acids. An aptamer can also comprise one or more modified bases, sugars, polyethylene glycol spacers or phosphate backbone units. In some examples, the aptamer comprises one or more 2' sugar modifications, such as a 2'-0- alkyl (e.g., 2'-0-methyl or 2'-0-methoxyethyl) or a 2'-fluoro modification.
In some examples, an aptamer is a polynucleotide of about 80, about 75, about 70, about 65, about 60, about 55, about 50, about 45, about 40, about 35, about 30, about 25, about 20, about 15 nucleotides in length.
In some examples, an aptamer is a polynucleotide of less than 80, less than 75, less than 70, less than 65, less than 60, less than 55, less than 50, less than 45, less than 40, less than 35, less than 30, less than 25, less than 20, less than 15 nucleotides in length.
In some examples an aptamer is a polynucleotide of about 70 to 80, is about 60 to 70, is about 50 to 60, is about 40 to 50, is about 30 to 40, is about 20 to 30, is about 10 to 20 nucleotides in length.
In some examples an aptamer is a polynucleotide of about 75 to 85, is about 65 to 75, is about 55 to 65, is about 45 to 55, is about 35 to 45, is about 25 to 35, is about 15 to 25 nucleotides in length.
In some examples an aptamer is a polynucleotide of 70 to 80, is 60 to 70, is 50 to 60, is 40 to 50, is 30 to 40, is 20 to 30, is 10 to 20 nucleotides in length.
In some examples an aptamer is a polynucleotide of 75 to 85, is 65 to 75, is 55 to 65, is 45 to 55, is 35 to 45, is 25 to 35, is 15 to 25 nucleotides in length. In some examples the aptamer is about 73 to about 77 or about 74 to about 76 nucleotides in length.
A person of skill in the art will appreciate that an aptamer may be any length polynucleotide that falls within the size parameters set out herein. By way of non-limiting example an aptamer may be about 76, about 61, about 54, about 43, about 29 or about 27 nucleotides in length or may be 77, 62, 55, 44, 28 or 26 nucleotides in length. What is important is that the aptamer specifically binds the target molecule.
In some examples an aptamer of the invention is a polynucleotide of about 80, about 75, about 70, about 65, about 60, about 55, about 50, about 45, about 40, about 35, about 30, about 25, about 20, about 15 nucleotides in length that selectively binds a target molecule as described herein.
In some examples, an aptamer is a polynucleotide of less than 80, less than 75, less than 70, less than 65, less than 60, less than 55, less than 50, less than 45, less than 40, less than 35, less than 30, less than 25, less than 20, less than 15 nucleotides in length that selectively binds a target molecule as described herein.
In some examples an aptamer is a polynucleotide of about 70 to 80, is about 60 to 70, is about 50 to 60, is about 40 to 50, is about 30 to 40, is about 20 to 30, is about 10 to 20 nucleotides in length that selectively binds a target molecule as described herein.
In some examples an aptamer is a polynucleotide of about 75 to 85, is about 65 to 75, is about 55 to 65, is about 45 to 55, is about 35 to 45, is about 25 to 35, is about 15 to 25 nucleotides in length that selectively binds a target molecule as described herein. In some examples an aptamer is a polynucleotide of 70 to 80, is 60 to 70, is 50 to 60, is 40 to 50, is 30 to 40, is 20 to 30, is 10 to 20 nucleotides in length that selectively binds a target molecule as described herein.
In some examples an aptamer is a polynucleotide of 75 to 85, is 65 to 75, is 55 to 65, is 45 to 55, is 35 to 45, is 25 to 35, is 15 to 25 nucleotides in length that selectively binds a target molecule as described herein.
The term "salt" includes a non-toxic salt of an inorganic or organic acid, including, but not limited to, a halide (chloride, bromide, iodide, fluoride), acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptanoate, hydrochloride, hydrobromide, hydroiodide, 2- hydroxyethanesulfonate, lactate, maleate, malonate, methanesulfonate, nitrate, oxalate, persulfate, phosphate, picrate, pivalate, propionate, p-toluenesulfonate, salicylate, succinate, sulfate, tartrate, thiocyanate, and undecanoate.
The term "salt" also includes a non-toxic salt of an organic or inorganic base, including, but not limited to, sodium (Na+), potassium (K+), calcium (Ca2+), magnesium (Mg2+), and lithium (Li+) salts.
In some examples, the target molecule is a small molecule. In some examples, the small molecule has a molecular weight (MW) of from about 60 to about 2000 g mol-1. In other examples, the small molecule has a MW of from about 100 to about 500 g mol-1. In other examples, the small molecule has a MW of from about 150 to about 350 g mol-1. The molecular weight of such small molecules and the calculation of their molecular weight are well known to those of skill in the art.
In some examples, the illicit drug (or molecule) is a naturally occurring or synthetic opiate or opioid, or derivative or metabolite thereof (e.g., morphine, oxycodone, hydromorphone, propoxyphene, nicomorphine, dihydrocodeine, diamorphine, papavereturn, codeine, ethylmorphine, phenylpiperidine and derivates thereof, methadone, dextropropoxyphene, buprenorphine, pentazocine, tilidine, tramadol, hydrocodone, meperidine, oxymorphone, alphaprodine, anileridine, dextromoramide, metopone, levorphanol, phenazocine, etoheptazine, propiram, profadol, phenampromide, thiambuten, pholcodeine, codeine, dihydrocodeinon, fentanyl, 3-trans-dimethylamino-4-phenyl-4-trans- carbethoxy-A'-cyclohexen, 3-dimethylamino-O-(4-methoxyphenyl-carbamoyl)- propiophenone oxime, (-)b-2'-hydroxy-2,9-dimethyl-5-phenyl-6,7-benzomorphane, (-)2'- hydroxy-2-(3-methyl-2-butenyl)-9-methyl-5-phenyl-6,7-benzomorphane, pirinitramide, (- )a-5,9-diethyl-2'hydroxy-2-methyl-6,7-benzomorphane, ethyl 1-(2-dimethylaminoethyl)- 4,5,6,7-tetrahydro-3-methyl-4-oxo-6-phenyl-indol- -2-carboxylate, 1-benzoylmethyl-2,3- dimethyl-3-(m-hydroxy-phenyl)-piperidine, N-allyl-7a(1-R-hydroxy-1-methylbutyl)-6,14- endo-ethanotetrahydronororipavine, (-) 2'-hydroxy-2-methyl-6,7-benzomorphane, noracylmethadol, phenoperidine, a-d1-methadol, a-1-methadol, b-d1-acetylmethadol, a-1- acetylmethadol and b-1-acetylmethadol), or a naturally occurring or synthetic organohalide.
In some examples, the small molecule is cocaine, heroin, tetrahydrocannabinol (THC), or any derivative or metabolite thereof. This includes synthetic cannabis, for example, AM2201 and AMB-FUBINACA. In other examples, the small molecule is methamphetamine.
The invention is further described with reference to the following Examples. It will be appreciated that the invention as claimed is not intended to be limited in any way by these Examples.
EXAMPLES
EXAMPLE 1: APTAMER GENERATION/SELECTION
The aptamers described herein were generated/selected using affinity matrix-based systematic evolution of ligands by exponential enrichment (SELEX), capture SELEX, or in silico SELEX methodologies. SELEX is a process by which a random single stranded polynucleotide library is iteratively evolved to produce sequences that can bind to a specific target.
Briefly, affinity matrix-based SELEX methodology is where a target for selection is immobilised onto a solid phase (often, sepharose beads). The immobilisation steps usually include EDC/NHS or epoxy activation chemistry, depending on the structure of the target molecule. The beads are incubated with a single-stranded random polynucleotide library at room temperature in binding and washing buffer (BWB; 20 mM Tris-HCI, pH 7.5, 100 mM NaCI, 5 mM KCI, 2 mM MgCl2, 1 mM CaCl2, and between 0% and 0.1% IGEPAL) for up to 18 hours. The beads are washed with 5 mL BWB, and unbound aptamers are discarded in the supernatant while target binding aptamers are retained with the beads and processed for the next round of selection . The sequences that are bound to the beads are amplified by standard amplification techniques such as polymerase chain reaction (PCR). PCR is performed using the HotMaster™ Taq kit with 200 mM dNTPs, 220 nM of forward primer, 220 nM or biotin- labelled forward primer, and 1.5 U HotMaster Taq in a final volume of 50 pL The amplified sequences are incubated with 0.3 NaOH to separate the double stranded sequences into single stranded sequences, and the biotin-labelled strand is removed using streptavidin magnetic beads. The single stranded sequences are then incubated with the beads, as described above. This process can be repeated iteratively until the required target affinity is reached. The incubation, elution, and PCR conditions can be adjusted at each round of SELEX to ensure the sequences retain the desired binding characteristics. Capture SELEX methodology is where target for selection is free in solution and the ssDNA library is immobilised onto beads. Briefly, a polynucleotide probe is conjugated via NHS/EDC chemistry to magnetic beads functionalised with carboxylic acid groups on the surface. A random polynucleotide library is attached to the beads by hybridization with the complementary probe. The ssDNA-beads are added to the solution containing the target and incubated at room temperature in BWB for up to 18 hours. The target-bound sequences dissociate from the probe and remain in the supernatant, while the unbound sequences remain bound to the beads which are discarded. The target-bound sequences are amplified by standard amplification techniques such as polymerase chain reaction (PCR), as described above. The amplified sequences are incubated with 0.3 NaOH to separate the double stranded sequences into single stranded sequences, and the biotin-labelled strand is removed using streptavidin magnetic beads. The resulting single stranded sequences are then hybridised to the complementary probe attached to the beads, and the next round of SELEX is carried out. This process can be repeated iteratively until the required target affinity is reached. The incubation, elution, and PCR conditions can be adjusted at each round of SELEX to ensure the sequences retain the desired binding characteristics .Affinity matrix-based and capture SELEX methodologies would be well known to those skilled in the art. For example, refer to the methodologies described in Paniel et al. (2017) Talanta 162:232-240 (capture SELEX) and Stoltenburg et at. (2012) Journal of Analytical Methods in Chemistry doi: 10.1155/2012/415697 (affinity matrix SELEX), and both publications are incorporated by reference in their entirety.
According to the aptamers described herein, the following methodologies were used:
Figure imgf000155_0001
EXAMPLE 2: MEASUREMENT OF APTAMER BINDING AFFINITIES
To determine binding affinities for the aptamers described herein, either a fluorescent imaging assay a Sybr green assay, or a fluorescent microscale thermophoresis (MST) binding assay were used.
Briefly, the fluorescent imaging assay may be used to determine Kd when the affinity matrix SELEX method is used for aptamer generation and selection. A serial dilution of hexachloro-fluorescein (HEX)-labelled aptamer is prepared with BWB. The beads with the target immobilised on the surface are washed twice with BWB before being incubated for up to 2 hours at room temperature with the fluorescently labelled aptamer candidate. Beads are then washed twice with BWB to remove loosely bound aptamers and the beads are imaged with a fluorescent microscope at 535/556 nm excitation/emission. Average fluorescence values of at least 20 beads per concentration are computed and plotted against the aptamer concentration to determine Kd.
The Sybr Green I assay may be used to determine Kd when the capture SELEX method is used for aptamer generation and selection. Sybr Green I is a fluorescent nucleic acid dye that preferentially binds to double stranded DNA. Binding of an aptamer to its target may elicit a conformational change in the aptamer structure, thereby changing the amount of double stranded regions to which the Sybr Green I dye may bind. To perform the Sybr Green I assay, a serial dilution of the target is prepared in BWB and added to a constant concentration of the unlabelled aptamer. After incubation at room temperature for up to 2 hours, a constant concentration of Sybr Green I is added and incubated for up to 2 more hours. The final concentration of aptamer and Sybr Green I are optimised to maximise the difference between a high concentration of target and no target. The fluorescence is measured at excitation/emission 480 nm/524 nm and plotted against target concentration to determine Kd.
An alternative approach to the Sybr Green I assay may be used, whereby a complementary probe is hybridised to the aptamer sequence. Addition of the target induces the aptamer to preferentially bind to the target, thereby displacing the probe and reducing the number of double stranded DNA to which the Sybr Green I dye may bind. To perform the Sybr Green I strand displacement assay, the aptamer is hybridised with the complementary probe in BWB at room temperature for up to 2 hours. A serial dilution of the target is prepared and is added to the aptamer-probe mixture and incubated at room temperature for up to 2 hours. Sybr Green I is added and incubated for up to 2 hours at room temperature. The fluorescence is measured at excitation/emission 480 nm/524 nm and plotted against target concentration to determine Kd.
The fluorescent MST binding assay may be used to determine Kd regardless of the aptamer SELEX method used. Microscale thermophoresis is based on two principles. First, the temperature related intensity changes of a fluorophore, and second, the movement of molecules along a temperature gradient. Briefly, a serial dilution of the target is prepared in BWB before being added to a Cy5-labelled aptamer. The solution is loaded into the capillaries and analysed using a Monolith NT.115. The fluorescence was analysed using a 1: 1 binding model to determine the Kd.
Figure imgf000157_0001
Although the invention has been described by way of example, it should be appreciated that variations and modifications may be made without departing from the scope of the invention as defined in the claims. Furthermore, where known equivalents exist to specific features, such equivalents are incorporated as if specifically referred in this specification.
***
All patents, publications, scientific articles, web sites, and other documents and materials referenced or mentioned herein are indicative of the levels of skill of those skilled in the art to which the invention pertains, and each such referenced document and material is hereby incorporated by reference to the same extent as if it had been incorporated by reference in its entirety individually or set forth herein in its entirety. Applicants reserve the right to physically incorporate into this specification any and all materials and information from any such patents, publications, scientific articles, web sites, electronically available information, and other referenced materials or documents.
The terms and expressions that have been employed are used as terms of description and not of limitation, and there is no intent in the use of such terms and expressions to exclude any equivalent of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention as claimed. Thus, it will be understood that although the present invention has been specifically disclosed by preferred examples and optional features, modification and variation of the concepts disclosed herein may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as described herein, and as defined by the appended claims.
The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.
Other examples are within the following claims. In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.

Claims

1. A polynucleotide or salt thereof comprising or consisting in:
(i) a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to any one of SEQ ID Nos: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71 and 73, which polynucleotide of salt thereof selectively binds to methamphetamine;
(ii) a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to any one of SEQ ID Nos: 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 133 and 135, which polynucleotide of salt thereof selectively binds to tetrahydrocannabinol;
(iii) a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to any one of SEQ ID Nos: : 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169 and 171, which polynucleotide of salt thereof selectively binds to cocaine;
(iv) a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to any one of SEQ ID Nos: 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243 and 245, which polynucleotide of salt thereof selectively binds to 3,4- methylenedioxymethamphetamine;
(v) a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to any one of SEQ ID Nos: 247, 248, 250, 252, 254, 256, 258, 260, 262, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288 and 290, which polynucleotide of salt thereof selectively binds to morphine;
(vi) a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to any one of SEQ ID Nos: 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316 and 318, which polynucleotide of salt thereof selectively binds to benzodiazepine;
(vii) a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to any one of SEQ ID Nos: 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368 and 370, which polynucleotide of salt thereof selectively binds to AMB-FUBINACA;
(viii) a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to any one of SEQ ID Nos: 372, 374, 376, 378, 380, 382, 384, 386, 388, 390 ,392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416 and 418, which polynucleotide of salt thereof selectively binds to AM2201;
(ix) a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to any one of SEQ ID Nos: 420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440, 442, 444, 446, 448, 450, 452, 454, 456, 458, 460, 462, 464 and 466, which polynucleotide of salt thereof selectively binds to oxycodone; or
(x) a sequence that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to any one of SEQ ID Nos: 468, 470, 472, 474, 476, 478, 480, 482, 484, 486, 488, 490, 492, 494, 496, 498, 500, 502, 504, 506, 508, 510, 512, 514, 516, 518, 520 and 522, which polynucleotide of salt thereof selectively binds to ketamine.
2. A polynucleotide or salt thereof comprising the sequence:
ATACGAGCTTGTTCAATA[Y]TGATAGTAAGAGCAATC wherein:
(i) Y is a nucleic acid sequence comprising or consisting in any one of SEQ ID Nos: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72 and 74, and the polynuceotide or salt thereof selectively binds to methamphetamine; or
(ii) Y is a nucleic acid sequence comprising or consisting in any one of SEQ ID Nos: 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134 and 136and the polynuceotide or salt thereof selectively binds to tetrahydrocannabinol; or
(iii) Y is a nucleic acid sequence comprising or consisting in any one of SEQ ID Nos: 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170 and 172, and the polynuceotide or salt thereof selectively binds to cocaine; or
(iv) Y is a nucleic acid sequence comprising or consisting in any one of SEQ ID Nos: 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242 and 246, and the polynuceotide or salt thereof selective binds to 3,4 methylenedioxymethamphetamine; or
(v) Y is a nucleic acid sequence comprising or consisting in any one of SEQ ID Nos: 249, 251, 253, 255, 257, 259, 261, 263, 264, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289 and 291, and the polynuceotide or salt thereof selectively binds to morphine; or
(vi) Y is a nucleic acid sequence comprising or consisting in any one of SEQ ID Nos: 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317 and 319, and the polynuceotide or salt thereof selectively binds to diazepam; or
(vii) Y is a nucleic acid sequence comprising or consisting in any one of SEQ ID Nos: 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 355, 357, 359, 361, 363, 365, 367, 369 and 371, and the polynuceotide or salt thereof selectively binds to AMB-FUBINACA; or
(viii) Y is a nucleic acid sequence comprising or consisting in any one of SEQ ID Nos: 373, 375, 377, 379, 381, 383, 385, 387, 389, 391, 393, 395, 397, 399, 401, 403, 405, 407, 409, 411, 413, 415, 417 and 419, and the polynuceotide or salt thereof selectively binds to AM2201;
(ix) Y is a nucleic acid sequence comprising or consisting in any one of SEQ ID Nos: 421, 423, 425, 427, 429, 431, 433, 435, 437, 439, 441, 443, 445, 447, 449, 451, 453, 455, 457, 459, 461, 463, 465, and the polynuceotide or salt thereof selectively binds to oxycodone; or
(x) Y is a nucleic acid sequence comprising or consisting in any one of SEQ ID Nos: 469, 471, 473, 475, 477, 479, 481, 483, 485, 487, 489, 491, 493, 495, 497, 499, 501, 503, 505, 507, 509, 511, 513, 515, 517, 519, 521 and 523, and the polynuceotide or salt thereof selectively binds to ketamine.
3. A polynucleotide or salt thereof comprising or consisting in a consensus sequence defined by any one of SEQ ID Nos 524, 525, 526 and 527, which polynucleotide of salt thereof selectively binds to methamphetamine.
4. A polynucleotide or salt thereof according to claim 3, comprising or consisting in a sequence defined by any one of SEQ ID Nos: 1, 5, 13, 27, 29, 67 and 71.
5. A polynucleotide or salt thereof comprising or consisting in a consensus sequence defined by any one of SEQ ID Nos 528, 529, 530 and 531, which polynucleotide of salt thereof selectively binds to tetrahydrocannibinol.
6. A polynucleotide or salt thereof according to claim 5 comprising or consisting in a sequence defined by any one of SEQ ID Nos: 97, 133 and 135.
7. A polynucleotide or salt thereof comprising or consisting in a consensus sequence defined by any one of SEQ ID Nos 524, 525, 526 and 527, which polynucleotide of salt thereof selectively binds to morphine.
8. A polynucleotide or salt thereof according to claim 7, comprising or consisting in a sequence defined by any one of SEQ ID Nos: 268, 280, 282, 288 and 290.
9. The polynucleotide according to any one of claims 1 to 8, wherein the binding affinity
(Kd) of the polynucleotide for its target is between about 1 nM and about 100 uM.
10. The polynucleotide according to any one of claims 1 to 9, wherein the Gibbs Free
Energy of the polynucleotide or salt thereof is between about -1.0 and about -15.0.
11. The polynucleotide or salt thereof according to any one of claims 1 to 10, wherein the polynucleotide or salt thereof is modified with a detectable label.
12. The polynucleotide or salt thereof according to any one of claims 1 to 11, wherein the polynucleotide or salt thereof is modified to promote an association with a substrate.
13. A method for detecting the presence of an illicit drug in a test sample, the method comprising the steps of:
(i) for the detection of methamphetamine or a structural analog thereof, contacting a test sample with a polynucleotide or salt thereof comprising a sequence defined by any one of SEQ ID Nos: 1-74; and/or
(ii) for the detection of tetrahydrocannabinol or a structural analog thereof, contacting a test sample with a polynucleotide or salt thereof comprising a sequence defined by any one of SEQ ID Nos: 75-136; and/or
(iii) for the detection of cocaine or a structural analog thereof, contacting a test sample with a polynucleotide or salt thereof comprising a sequence defined by any one of SEQ ID Nos: 137-172; and/or
(iv) for the detection of 3,4 methylenedioxymethamphetamine or a structural analog thereof, contacting a test sample with a polynucleotide or salt thereof comprising a sequence defined by any one of SEQ ID Nos: 173-246; and/or
(v) for the detection of morphine or a structural analog thereof, contacting a test sample with a polynucleotide or salt thereof comprising a sequence defined by any one of SEQ ID Nos: 247-291; and/or
(vi) for the detection of benzodiazepine or a structural analog thereof, contacting a test sample with a polynucleotide or salt thereof comprising a sequence defined by any one of SEQ ID Nos: 292-319; and/or (vii) for the detection of AMB-FUBINACA or a structural analog thereof, contacting a test sample with a polynucleotide or salt thereof comprising a sequence defined by any one of SEQ ID Nos: 320-371; and/or
(viii) for the detection of AM2201 or a structural analog thereof, contacting a test sample with a polynucleotide or salt thereof comprising a sequence defined by any one of SEQ ID Nos: 372-419;
(ix) for the detection of oxycodone or a structural analog thereof, contacting a test sample with a polynucleotide or salt thereof comprising a sequence defined by any one of SEQ ID Nos: 420-467; and/or
(x) for the detection of ketamine or a structural analog thereof, contacting a test sample with a polynucleotide or salt thereof comprising a sequence defined by any one of SEQ ID Nos: 468-523; and
(xi) measuring binding between the polynucleotide and drug target from the test sample, wherein, a measured binding interaction between the polynucleotide and drug target reflects the presence of one or more illicit drugs in the test sample.
14. A test kit or article of manufacture for detecting the presence of an illicit drug in a test sample, the test kit or article of manufacture comprising:
(i) a polynucleotide or salt thereof comprising a sequence defined by any one of SEQ ID Nos: 1-74, which polynucleotide of salt thereof selectively binds methamphetamine; and/or
(ii) a polynucleotide or salt thereof comprising a sequence defined by any one of SEQ ID Nos: 75-136, which polynucleotide of salt thereof selectively binds tetrahydrocannabinol; and/or
(iii) a polynucleotide or salt thereof comprising a sequence defined by any one of SEQ ID Nos: 137-172, which polynucleotide of salt thereof selectively binds cocaine; and/or
(iv) a polynucleotide or salt thereof comprising a sequence defined by any one of SEQ ID Nos: 173-246, which polynucleotide or salt thereof selectively binds 3,4 methylenedioxymethamphetamine; and/or
(v) a polynucleotide or salt thereof comprising a sequence defined by any one of SEQ ID Nos: 247-291, which polynucleotide or salt thereof selectively binds morphine; and/or
(vi) a polynucleotide or salt thereof comprising a sequence defined by any one of SEQ ID Nos: 292-319, which polynucleotide or salt thereof selectively binds benzodiazepine; and/or (vii) a polynucleotide or salt thereof comprising a sequence defined by any one of SEQ ID Nos: 320-371, which polynucleotide or salt thereof selectively binds AMB-FUBINACA; and/or
(viii) a polynucleotide or salt thereof comprising a sequence defined by any one of SEQ ID Nos: 372-419, which polynucleotide or salt thereof selectively binds AM2201; and/or
(ix) a polynucleotide or salt thereof comprising a sequence defined by any one of SEQ ID Nos: 420-467, which polynucleotide or salt thereof selectively binds oxycodone;
(x) a polynucleotide or salt thereof comprising a sequence defined by any one of SEQ ID Nos: 468-523, which polynucleotide or salt thereof selectively binds ketamine; and optionally
(xi) instructions for how to detect the presence of methamphetamine, tetrahydrocannabinol, cocaine, 3,4-methylenedioxymethamphetamine, benzodiazepine, morphine, AMB-FUNIBACA, AM2201, oxycodone and/or ketamine in the test sample.
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WO2017025921A1 (en) * 2015-08-13 2017-02-16 Victoria Link Limited Aptamer biosensors useful for detecting hormones, hormone mimics, and metabolites thereof
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JP2010158238A (en) * 2008-12-09 2010-07-22 Tokyo Univ Of Agriculture & Technology C-reactive protein-binding aptamer and use of the same
KR20130032128A (en) * 2011-09-22 2013-04-01 고려대학교 산학협력단 Dna aptamers for porcine reproductive and respiratory syndrome(prrs) virus
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