WO2019094968A1 - Detection of tenofovir - Google Patents

Detection of tenofovir Download PDF

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Publication number
WO2019094968A1
WO2019094968A1 PCT/US2018/060825 US2018060825W WO2019094968A1 WO 2019094968 A1 WO2019094968 A1 WO 2019094968A1 US 2018060825 W US2018060825 W US 2018060825W WO 2019094968 A1 WO2019094968 A1 WO 2019094968A1
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Prior art keywords
nucleic acid
signal
oligonucleotide
tenofovir
composition
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PCT/US2018/060825
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French (fr)
Inventor
Alexander CHIU
Robert Batchelor
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Base Pair Biotechnologies, Inc.
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Publication of WO2019094968A1 publication Critical patent/WO2019094968A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/6561—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing systems of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring or ring system, with or without other non-condensed hetero rings
    • C07F9/65616—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing systems of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring or ring system, with or without other non-condensed hetero rings containing the ring system having three or more than three double bonds between ring members or between ring members and non-ring members, e.g. purine or analogs
    • 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
    • G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
    • G01N33/542—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with steric inhibition or signal modification, e.g. fluorescent quenching

Definitions

  • This invention relates to methods and materials for functional ligands, particularly to methods and materials for utilizing structure-switching functional ligands which bind to a target, more particularly to methods and materials for utilizing structure-switching functional ligands which modulate the activity of a signal-interacting pair by binding to a target and further particularly to methods and materials for utilizing structure-switching functional ligands to release signal-interacting molecules to produce a detectable signal indicative of the presence and/or amount of a target in a sample.
  • DNA sequences which are disclosed in the ASCII text file entitled "PTENOUS00_ST25.txt", created on November 13, 2017 and of 689 bytes in size, which is incorporated by reference in its entirety, herein are intended to include other aptamers incorporating modifications, truncations (e.g. trivial truncations, such as 1-5 nucleotides removed at an end, which consist essentially of the same sequence and retains binding to the target molecule), incorporations into larger molecules or complexes (e.g.
  • aptamer sequence within a longer nucleic acid strand may also bind to homologous proteins or molecules from organisms other than the organisms listed herein, to recombinant or non-recombinant versions of the proteins or molecules, to modified versions of the proteins or molecules, to proteins or molecules from sources other than the source listed herein.
  • the aptamers are artificial, non-naturally occurring sequences designed and/or selected for specific and/or high affinity binding to a target molecule, such as, without limitation, SEQ ID 1 may bind to tenofovir.
  • Non-naturally occurring sequences of aptamers may also not be present in naturally occurring systems or situations, such as by, for example, not being already present or having a pre-existing function in a naturally occurring setting.
  • SEQ ID 2 is a complementary sequence to SEQ ID 1.
  • Aptamers which are nucleic acid ligands capable of binding to molecular targets, have recently attracted increased attention for their potential application in many areas of biology and biotechnology. They may be used as sensors, therapeutic tools, to regulate cellular processes, as well as to guide drugs to their specific cellular target(s). Contrary to the actual genetic material, their specificity and characteristics are not directly determined by their primary sequence, but instead by their secondary and/or tertiary structure. Aptamers have been recently investigated as immobilized capture elements in a microarray format. Others have recently selected aptamers against whole cells and complex biological mixtures. Aptamers are typically characterized by binding to their target molecules via non-Watson-Crick (i.e.
  • non- hybridization such as by intermolecular forces resulting from the secondary or tertiary structure of the aptamer. This is especially true of non-nucleic acid target molecules where Watson-Crick mechanisms typically do not apply. Aptamers may also, for example, exhibit changes in their secondary and/or tertiary structure depending on whether it is complexed or uncomplexed with a target molecule.
  • Aptamers are commonly identified by an in vitro method of selection sometimes referred to as Systematic Evolution of Ligands by Exponential enrichment or "SELEX".
  • SELEX typically begins with a very large pool of randomized polynucleotides which is generally narrowed to one aptamer ligand per molecular target. Once multiple rounds (typically 10-15) of SELEX are completed, the nucleic acid sequences are identified by conventional cloning and sequencing. Aptamers have most famously been developed as ligands to important proteins, rivaling antibodies in both affinity and specificity, and the first aptamer-based therapeutics are now emerging. More recently, however, aptamers have been also developed to bind small organic molecules and cellular toxins, viruses, and even targets as small as heavy metal ions.
  • Tenofovir disoproxil sold under the trade name Viread among others, is a medication used to treat chronic hepatitis B and to prevent and treat HIV/AIDS. It is generally recommended for use with other antiretrovirals. It may be used for prevention of HIV/ AIDS among those at high risk before exposure, and after a needlestick injury or other potential exposure. It is sold both by itself and together as emtricitabine/tenofovir and efavirenz/emtricitabine/tenofovir. It does not cure HIV/AIDS or hepatitis B. Tenofovir may be measured in plasma which may be useful for monitoring therapy and to prevent drug accumulation and toxicity in people with kidney or liver problems.
  • This invention relates to methods and materials for functional ligands, particularly to methods and materials for utilizing structure-switching functional ligands which bind to a target (e.g. tenofovir), more particularly to methods and materials for utilizing structure-switching functional ligands which modulate the activity of a signal-interacting pair by binding to a target (e.g. tenofovir) and further particularly to methods and materials for utilizing structure-switching functional ligands and a hybridized oligonucleotide to produce a detectable signal indicative of the presence and/or amount of a target (e.g. tenofovir) in a sample.
  • a target e.g. tenofovir
  • functional ligands may be selected for and/or utilized for their ability to bind or complex to particular target molecules, such as tenofovir, and/or for the manner in which they bind or complex to particular target molecules, such as by exhibiting a detectable structural change.
  • target molecules such as tenofovir
  • biomolecules such as nucleic acids and peptides, take on varied secondary and tertiary structures in response to different environments or by associating with other molecules.
  • Functional ligands may generally include biomolecules such as nucleic acids, such as single-stranded nucleic acids and double-stranded nucleic acids or combinations or regions of both, peptides, other biopolymers and/or combinations or modifications thereof, such as artificially modified nucleic acids, synthetic analogs and the like.
  • a method for utilizing functional ligands may include providing a functional ligand to a target molecule, such as a nucleic acid aptamer that binds to tenofovir, and an oligonucleotide which is complementary to at least a portion of the nucleic acid aptamer, such that the oligonucleotide is hybridized to the nucleic acid aptamer, and the binding of the target to the nucleic acid aptamer, such as tenofovir, may generate a detectable change in the interaction between the nucleic acid aptamer and the oligonucleotide.
  • the binding of the target to the nucleic acid aptamer may generally cause the oligonucleotide to dehybridize from the nucleic acid aptamer.
  • the nucleic acid aptamer and the oligonucleotide may each be attached to a signal interacting moiety, such as an interacting pair of chromophores where the proximity of the pair results in a decreased amount of detectable signal and increasing the distance between them results in an increase in detectable signal.
  • the binding of the target to the nucleic acid aptamer may generally cause an increase in the distance of the pair, such as, for example and without being bound to any particular theory, by causing the oligonucleotide to dehybridized and move away, such as by a structural or conformational change of the nucleic acid aptamer upon binding.
  • the oligonucleotide may be displaced and/or occluded from hybridizing upon binding of a target molecule to the nucleic acid aptamer.
  • the interaction of the substrate and the signal-interacting molecules may include, but are not limited to, colorimetric interactions, refractive index, fluorescence interactions (e.g. Fluorescence Energy Transfer (FRET)), fluorescence enhancement upon moving to a different solvent environment, redox interactions, enzymatic interactions, pH reporting mechanisms, surface enhanced Raman scattering (SERS), isothermal DNA amplification, thermal or temperature changes, and/or any other appropriate interaction to produce a detectable signal or change.
  • FRET Fluorescence Energy Transfer
  • SERS surface enhanced Raman scattering
  • FIG. 1 illustrates an embodiment of a composition comprising a nucleic acid aptamer, an oligonucleotide and an attached pair of signal interacting moieties;
  • FIG. 2 illustrates the interaction of the composition in FIG. 1 with a target molecule (e.g. tenofovir) to generate a signal;
  • a target molecule e.g. tenofovir
  • FIG. 3 illustrates an attachment embodiment between a nucleic acid aptamer with a signal interacting moiety
  • FIG. 4 shows the efficiency of the quenching of the signal interacting moiety attached to the nucleic acid aptamer by the signal interacting moiety attached to the oligonucleotide
  • FIG. 5 shows the fluorescence emissions of the signal interacting moiety attached to the nucleic acid aptamer due to binding of the target molecule (e.g. tenofovir).
  • This invention relates to methods and materials for functional ligands, particularly to methods and materials for utilizing structure-switching functional ligands which bind to a target (e.g. tenofovir), more particularly to methods and materials for utilizing structure-switching functional ligands which modulate the activity of a signal-interacting pair by binding to a target (e.g. tenofovir) and further particularly to methods and materials for utilizing structure-switching functional ligands and a hybridized oligonucleotide to produce a detectable signal indicative of the presence and/or amount of a target (e.g. tenofovir) in a sample.
  • a target e.g. tenofovir
  • FIG. 1 illustrates a composition 100 with a functional ligand, shown as nucleic acid aptamer 102, hybridized to an oligonucleotide 104 with 5 '-end 104b at a hybridization region 102b.
  • the nucleic acid aptamer 102 may, for example and without being bound to any particular theory, include a stem-loop structure, with regions 102c, 102d forming a stem hybridization in the absence of the oligonucleotide 104, and a loop portion 102f, which may, for example, include the binding region for the target, such as tenofovir.
  • the stem may be two nucleotides in length.
  • the nucleic acid aptamer 102 may also, for example, be modified, such as with terminal modifications (e.g. a 3 '-end amine at 3 '-end 102e).
  • the oligonucleotide 104 may be a truncated version of the complementary sequence to the nucleic acid aptamer 102, as illustrated in FIG. 1, such as, for example, a 3'-end truncation (i.e. complementary to the 3 '-end of the nucleic acid aptamer 102) of about 6 to 14 nucleotides in length, more particularly 8-10 nucleotides in length, such as 8 or 9 nucleotides in length.
  • a 3'-end truncation i.e. complementary to the 3 '-end of the nucleic acid aptamer 102
  • functional ligands may be selected for and/or utilized for their ability to bind or complex to particular target molecules, such as tenofovir, and/or for the manner in which they bind or complex to particular target molecules, such as by exhibiting a detectable structural change.
  • target molecules such as tenofovir
  • biomolecules such as nucleic acids and peptides, take on varied secondary and tertiary structures in response to different environments or by associating with other molecules.
  • Functional ligands may generally include biomolecules such as nucleic acids, such as single-stranded nucleic acids and double-stranded nucleic acids or combinations or regions of both, peptides, other biopolymers and/or combinations or modifications thereof, such as artificially modified nucleic acids, synthetic analogs and the like.
  • Non-naturally occurring sequences of functional ligands such as nucleic acids and nucleic acid analogs, such as aptamers, may also be useful by interacting with a target molecule in a manner not present in naturally occurring systems or situations, such as by, for example, not being already present or having a pre-existing function in a naturally occurring setting.
  • functional ligands may generally include nucleic acids, particularly single-stranded nucleic acids, peptides, other biopolymers and/or combinations or modifications thereof.
  • Nucleic acid sequences may include naturally-occurring biomolecules such as ribonucleic acid (RNA), deoxyribonucleic acid (DNA), artificially modified nucleic acids, and/or combinations thereof.
  • modified nucleic acid bases may be utilized and may include, but are not limited to, 2'-Deoxy-P- nucleoside-5'-Triphosphate, 2'-Deoxyinosine-5'-Triphosphate, 2'-Deoxypseudouridine-5'-Triphosphate, 2'-Deoxyuridine-5'-Triphosphate, 2'-Deoxyzebularine-5'-Triphosphate, 2-Amino-2'-deoxyadenosine-5'- Triphosphate, 2-Amino-6-chloropurine-2'-deoxyriboside-5'-Triphosphate, 2-Aminopurine-2'- deoxyribose-5'-Triphosphate, 2-Thio-2'-deoxycytidine-5'-Triphosphate, 2-Thiothymidine
  • Triphosphate 5-Aminoallyl-2'-deoxycytidine-5'-Triphosphate, 5-Aminoallyl-2'-deoxyuridine-5'- Triphosphate, 5-Bromo-2'-deoxycytidine-5'-Triphosphate, 5-Bromo-2'-deoxyuridine-5'-Triphosphate, 5- Fluoro-2'-deoxyuridine-5'-Triphosphate, 5-Trifluoromethyl-2-deoxyuridine-5'-Triphosphate, and/or any other appropriate modified nucleic acid base.
  • nucleoside triphosphates listed above may generally refer to any appropriate phosphate of the modified base, such as additionally, for example, monophosphates (NMPs) or diphosphates (NDPs) of the base.
  • NMPs monophosphates
  • NDPs diphosphates
  • Embodiments of the SELEX method may generally be utilized to select or preselect for aptamers to be used in a collection.
  • the basic SELEX protocol and aptamers are described in U.S. Patent No. 5,270,163, entitled "Methods for identifying nucleic acid ligands," the entire contents of which are hereby incorporated by reference.
  • Structural changes may be directly observable, such as through microscopy, microscale thermophoresi s (MST), backscattering interferometry (BSI), circular dichroism, Nuclear Magnetic Resonance (NMR) and/or any other appropriate observation method. Structural changes may also be detectable through secondary events, such as by detecting changes in fluorescence, spectral emission/color or other radiation emissions/signals due to alterations in the structure of a functional ligand.
  • MST microscale thermophoresi s
  • BSI backscattering interferometry
  • NMR Nuclear Magnetic Resonance
  • a method for utilizing functional ligands may include providing a functional ligand to a target molecule, such as a nucleic acid aptamer that binds to tenofovir, and an oligonucleotide which is complementary to at least a portion of the nucleic acid aptamer, such that the oligonucleotide is hybridized to the nucleic acid aptamer, and the binding of the target to the nucleic acid aptamer, such as tenofovir, may generate a detectable change in the interaction between the nucleic acid aptamer and the oligonucleotide.
  • a target molecule such as a nucleic acid aptamer that binds to tenofovir
  • the binding of the target to the nucleic acid aptamer may generally cause the oligonucleotide to dehybridize from the nucleic acid aptamer.
  • FIG. 2 illustrates an example of a nucleic acid aptamer 102 binding to a target molecule 90 (e.g. tenofovir), which may generally cause a hybridized oligonucleotide 104 to dehybridize.
  • a target molecule 90 e.g. tenofovir
  • the nucleic acid aptamer and the oligonucleotide may each be attached to a signal interacting moiety (e.g. at their 5'- or 3'- ends), such as an interacting pair of chromophores where the proximity of the pair results in a decreased amount of detectable signal and increasing the distance between them results in an increase in detectable signal.
  • a signal interacting moiety e.g. at their 5'- or 3'- ends
  • FIG. 1 illustrates a nucleic acid aptamer
  • the binding of the target to the nucleic acid aptamer may generally cause an increase in the distance of the pair, such as, for example and without being bound to any particular theory, by causing the oligonucleotide to dehybridized and move away, such as by a structural or conformational change of the nucleic acid aptamer upon binding.
  • the oligonucleotide may be displaced and/or occluded from hybridizing upon binding of a target molecule to the nucleic acid aptamer.
  • composition 100 illustrates the dehybndization and increased distance between the nucleic acid aptamer 102 and the oligonucleotide 104 due to the target 90 binding, causing an increased signal emission A from the signal interacting moiety 102a.
  • the composition 100 may be used, without being bound to any particular theory, in any appropriate solution or fluid/gel system, such as to detect and/or quantify tenofovir in a blood or other body fluid sample, such as to monitor the concentration of the drug (e.g. for aiding in maintaining the drug in a therapeutic range or detect accumulation/toxicity).
  • the signal interacting moieties may generally be attached to the nucleic acid aptamer and/or the oligonucleotide by any appropriate means, such as, for example, by covalent attachment, attachment through biotin-streptavidin/avidin coupling.
  • FIG. 3 illustrates an example of a 5 '-end of a nucleic acid aptamer 102 to a coupling moiety 103, which may be biotin, streptavidin, avidin, etc., and a signal interacting moiety 102a (e.g. a fluorophore emitting a signal A), attached to a coupling moiety 103a, which may be the corresponding agent to the coupling moiety 103 (i.e. streptavidin/avidin if the coupling moiety
  • streptavidin/avidin are multivalent to up to four biotins, but modified versions may be utilized with difference valency.
  • the interaction of the signal interacting moieties may include, but are not limited to, colorimetric interactions, fluorescence interactions (e.g. Fluorescence Energy Transfer (FRET)), fluorescence enhancement upon moving to a different solvent environment, redox interactions, enzymatic interactions, pH reporting mechanisms, surface enhanced Raman scattering (SERS), isothermal DNA amplification, and/or any other appropriate interaction to produce a detectable signal or change.
  • fluorescence interactions e.g. Fluorescence Energy Transfer (FRET)
  • FRET Fluorescence Energy Transfer
  • SERS surface enhanced Raman scattering
  • isothermal DNA amplification e.g.
  • Pairs of corresponding chromophores may be utilized for their ability of altering the conversion efficiency of the radiation converting chromophore in order to create a detectable signal or change.
  • converting and absorbing chromophore pairs in the radiation portion of interest may include, but are not limited to: ALEXA633TM/QSY21TM, CY5TM/QSY21TM, ALEXA647TM/QSY21TM, ALEXA647TM/ALEXA680TM, ALEXA680TM/allophycocyanin (APC), ALEXA700TM/APC, and/or ALEXA750TM/APC (Molecular Probes, Inc.), and/or any other appropriate fluorophore/chromophore pair. It is contemplated, however, that any suitable pair of first and/or second radiation converting chromophore and radiation absorbing chromophore, whether now known or later developed, is within the scope of the present invention.
  • a chromophore-quencher pair may also include a metallic quenching element, such as a gold or other metallic substrate, which may include, for example, gold nanoparticles, nanotubes, nanorods, and/or any other appropriate metallic substrate. Interactions between fluorophores and metallic quenchers may be desirable, for example, where utilization of plasmonic energy transfer or quenching, which may generally have a longer range than other forms of transfer such as FRET, may be useful.
  • a metallic quenching element such as a gold or other metallic substrate, which may include, for example, gold nanoparticles, nanotubes, nanorods, and/or any other appropriate metallic substrate.
  • a signal-interacting molecule may be labeled with a metallic nanoparticle/structure, such that when the signal-interacting molecule is combined with another metallic nanoparticle/structure (which may be labeled with an appropriate Raman reporter molecule), the combined metallic nanoparticles/structures may generate a specific and/or distinctive Raman signature under Raman spectroscopy.
  • chromophores or fluorophores such as quantum dots, may also be utilized.
  • nucleic acid aptamer 102 has the sequence of SEQ ID 1 and the oligonucleotide 104 has a truncated sequence of SEQ ID 2 (e.g. 3'-end truncated to 9 nucleotides in length)
  • the nucleic acid aptamer 102 is biotinylated at its 5 '-end and coupled to a streptavidin coupled to the fluorophore ALEXA647
  • the oligonucleotide 104 is coupled at its 3 '-end to a quencher QSY-21.
  • FIG. 1 The composition of a nucleic acid aptamer 102 and oligonucleotide 104 shown in FIG. 1, where the nucleic acid aptamer 102 has the sequence of SEQ ID 1 and the oligonucleotide 104 has a truncated sequence of SEQ ID 2 (e.g. 3'-end truncated to 9 nucleotides
  • FIG. 4 shows the relative quenching efficiency by showing the relative fluorescence difference of the nucleic acid aptamer 102 alone in the first bar and with the hybridized oligonucleotide 104 in the second bar.
  • FIG. 5 shows the concentration- dependent fluorescence emission for concentrations of tenofovir from 0 to 4 mM. As shown the increasing concentrations of tenofovir results in higher fluorescence emissions.
  • the term "or” as used herein is generally intended to mean “and/or” unless otherwise indicated. For example, a condition A or B is satisfied by any one of the following: A is true (or present) and B is false (or not present), A is false (or not present) and B is true (or present), and both A and B are true (or present).
  • a term preceded by “a” or “an” includes both singular and plural of such term, unless clearly indicated within the claim otherwise (i.e., that the reference “a” or “an” clearly indicates only the singular or only the plural).
  • the meaning of "in” includes “in” and “on” unless the context clearly dictates otherwise.

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Abstract

This invention relates to methods and materials for functional ligands, particularly to methods and materials for utilizing structure-switching functional ligands which bind to a target, more particularly to methods and materials for utilizing structure-switching functional ligands which modulate the activity of a signal-interacting pair by binding to a target and further particularly to methods and materials for utilizing structure-switching functional ligands to release signal-interacting molecules to produce a detectable signal indicative of the presence and/or amount of a target in a sample.

Description

DETECTION OF TENOFOVIR CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This Patent Cooperation Treaty international patent application claims the benefit and priority of U.S. provisional patent application Ser. No. 62/585,527, filed November 13, 2017, entitled, "DETECTION OF TENOFOVIR", the contents of which is hereby incorporated by reference in its entirety.
COPYRIGHT NOTICE
[0002] A portion of the disclosure of this patent document contains material which is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or the patent disclosure, as it appears in the U.S. Patent and Trademark Office patent file or records, but otherwise reserves all copyright rights whatsoever.
FIELD OF THE INVENTION
[0003] This invention relates to methods and materials for functional ligands, particularly to methods and materials for utilizing structure-switching functional ligands which bind to a target, more particularly to methods and materials for utilizing structure-switching functional ligands which modulate the activity of a signal-interacting pair by binding to a target and further particularly to methods and materials for utilizing structure-switching functional ligands to release signal-interacting molecules to produce a detectable signal indicative of the presence and/or amount of a target in a sample.
SEQUENCE LISTING
[0004] Deoxyribonucleic acid (DNA) sequences, which are disclosed in the ASCII text file entitled "PTENOUS00_ST25.txt", created on November 13, 2017 and of 689 bytes in size, which is incorporated by reference in its entirety, herein are intended to include other aptamers incorporating modifications, truncations (e.g. trivial truncations, such as 1-5 nucleotides removed at an end, which consist essentially of the same sequence and retains binding to the target molecule), incorporations into larger molecules or complexes (e.g. the aptamer sequence within a longer nucleic acid strand), and/or other aptamers having substantial structural or sequence homology, for example, greater than 75% sequence homology within a similar length of nucleic acid (e.g. similar to within 5-10 nucleotides in length with significant sequence homology within that length, such as greater than 75%), as well as RNA and/or other non-DNA/RNA aptamers. The disclosed aptamers may also bind to homologous proteins or molecules from organisms other than the organisms listed herein, to recombinant or non-recombinant versions of the proteins or molecules, to modified versions of the proteins or molecules, to proteins or molecules from sources other than the source listed herein. The aptamers are artificial, non-naturally occurring sequences designed and/or selected for specific and/or high affinity binding to a target molecule, such as, without limitation, SEQ ID 1 may bind to tenofovir. Non-naturally occurring sequences of aptamers, may also not be present in naturally occurring systems or situations, such as by, for example, not being already present or having a pre-existing function in a naturally occurring setting. SEQ ID 2 is a complementary sequence to SEQ ID 1.
BACKGROUND OF THE INVENTION
[0005] Aptamers, which are nucleic acid ligands capable of binding to molecular targets, have recently attracted increased attention for their potential application in many areas of biology and biotechnology. They may be used as sensors, therapeutic tools, to regulate cellular processes, as well as to guide drugs to their specific cellular target(s). Contrary to the actual genetic material, their specificity and characteristics are not directly determined by their primary sequence, but instead by their secondary and/or tertiary structure. Aptamers have been recently investigated as immobilized capture elements in a microarray format. Others have recently selected aptamers against whole cells and complex biological mixtures. Aptamers are typically characterized by binding to their target molecules via non-Watson-Crick (i.e. non- hybridization) mechanisms, such as by intermolecular forces resulting from the secondary or tertiary structure of the aptamer. This is especially true of non-nucleic acid target molecules where Watson-Crick mechanisms typically do not apply. Aptamers may also, for example, exhibit changes in their secondary and/or tertiary structure depending on whether it is complexed or uncomplexed with a target molecule.
[0006] Aptamers are commonly identified by an in vitro method of selection sometimes referred to as Systematic Evolution of Ligands by Exponential enrichment or "SELEX". SELEX typically begins with a very large pool of randomized polynucleotides which is generally narrowed to one aptamer ligand per molecular target. Once multiple rounds (typically 10-15) of SELEX are completed, the nucleic acid sequences are identified by conventional cloning and sequencing. Aptamers have most famously been developed as ligands to important proteins, rivaling antibodies in both affinity and specificity, and the first aptamer-based therapeutics are now emerging. More recently, however, aptamers have been also developed to bind small organic molecules and cellular toxins, viruses, and even targets as small as heavy metal ions.
[0007] Tenofovir disoproxil (Tenofovir), sold under the trade name Viread among others, is a medication used to treat chronic hepatitis B and to prevent and treat HIV/AIDS. It is generally recommended for use with other antiretrovirals. It may be used for prevention of HIV/ AIDS among those at high risk before exposure, and after a needlestick injury or other potential exposure. It is sold both by itself and together as emtricitabine/tenofovir and efavirenz/emtricitabine/tenofovir. It does not cure HIV/AIDS or hepatitis B. Tenofovir may be measured in plasma which may be useful for monitoring therapy and to prevent drug accumulation and toxicity in people with kidney or liver problems.
SUMMARY OF THE INVENTION
[0008] This invention relates to methods and materials for functional ligands, particularly to methods and materials for utilizing structure-switching functional ligands which bind to a target (e.g. tenofovir), more particularly to methods and materials for utilizing structure-switching functional ligands which modulate the activity of a signal-interacting pair by binding to a target (e.g. tenofovir) and further particularly to methods and materials for utilizing structure-switching functional ligands and a hybridized oligonucleotide to produce a detectable signal indicative of the presence and/or amount of a target (e.g. tenofovir) in a sample.
[0009] In general, functional ligands may be selected for and/or utilized for their ability to bind or complex to particular target molecules, such as tenofovir, and/or for the manner in which they bind or complex to particular target molecules, such as by exhibiting a detectable structural change. For example, numerous biomolecules, such as nucleic acids and peptides, take on varied secondary and tertiary structures in response to different environments or by associating with other molecules. Functional ligands may generally include biomolecules such as nucleic acids, such as single-stranded nucleic acids and double-stranded nucleic acids or combinations or regions of both, peptides, other biopolymers and/or combinations or modifications thereof, such as artificially modified nucleic acids, synthetic analogs and the like.
[0010] In one aspect of the present invention, a method for utilizing functional ligands may include providing a functional ligand to a target molecule, such as a nucleic acid aptamer that binds to tenofovir, and an oligonucleotide which is complementary to at least a portion of the nucleic acid aptamer, such that the oligonucleotide is hybridized to the nucleic acid aptamer, and the binding of the target to the nucleic acid aptamer, such as tenofovir, may generate a detectable change in the interaction between the nucleic acid aptamer and the oligonucleotide. In general, and without being bound to any particular theory, the binding of the target to the nucleic acid aptamer may generally cause the oligonucleotide to dehybridize from the nucleic acid aptamer.
[0011] In some embodiments, the nucleic acid aptamer and the oligonucleotide may each be attached to a signal interacting moiety, such as an interacting pair of chromophores where the proximity of the pair results in a decreased amount of detectable signal and increasing the distance between them results in an increase in detectable signal. The binding of the target to the nucleic acid aptamer may generally cause an increase in the distance of the pair, such as, for example and without being bound to any particular theory, by causing the oligonucleotide to dehybridized and move away, such as by a structural or conformational change of the nucleic acid aptamer upon binding. In some embodiments, the oligonucleotide may be displaced and/or occluded from hybridizing upon binding of a target molecule to the nucleic acid aptamer.
[0012] In some exemplary embodiments, the interaction of the substrate and the signal-interacting molecules may include, but are not limited to, colorimetric interactions, refractive index, fluorescence interactions (e.g. Fluorescence Energy Transfer (FRET)), fluorescence enhancement upon moving to a different solvent environment, redox interactions, enzymatic interactions, pH reporting mechanisms, surface enhanced Raman scattering (SERS), isothermal DNA amplification, thermal or temperature changes, and/or any other appropriate interaction to produce a detectable signal or change.
[0013] The present invention together with the above and other advantages may best be understood from the following detailed description of the embodiments of the invention and as illustrated in the drawings. The following description, while indicating various embodiments of the invention and numerous specific details thereof, is given by way of illustration and not of limitation. Many substitutions, modifications, additions or rearrangements may be made within the scope of the invention, and the invention includes all such substitutions, modifications, additions or rearrangements.
BRIEF DESCRIPTION OF THE FIGURES
[0014] FIG. 1 illustrates an embodiment of a composition comprising a nucleic acid aptamer, an oligonucleotide and an attached pair of signal interacting moieties;
[0015] FIG. 2 illustrates the interaction of the composition in FIG. 1 with a target molecule (e.g. tenofovir) to generate a signal;
[0016] FIG. 3 illustrates an attachment embodiment between a nucleic acid aptamer with a signal interacting moiety;
[0017] FIG. 4 shows the efficiency of the quenching of the signal interacting moiety attached to the nucleic acid aptamer by the signal interacting moiety attached to the oligonucleotide; and
[0018] FIG. 5 shows the fluorescence emissions of the signal interacting moiety attached to the nucleic acid aptamer due to binding of the target molecule (e.g. tenofovir).
DETAILED DESCRIPTION OF THE INVENTION
[0019] The detailed description set forth below is intended as a description of the presently exemplified methods, devices, and compositions provided in accordance with aspects of the present invention and is not intended to represent the only forms in which the present invention may be practiced or utilized. It is to be understood, however, that the same or equivalent functions and components may be accomplished by different embodiments that are also intended to be encompassed within the spirit and scope of the invention. [0020] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this invention belongs. Although any methods, devices and materials similar or equivalent to those described herein can be used in the practice or testing of the invention, the exemplified methods, devices and materials are now described.
[0021] This invention relates to methods and materials for functional ligands, particularly to methods and materials for utilizing structure-switching functional ligands which bind to a target (e.g. tenofovir), more particularly to methods and materials for utilizing structure-switching functional ligands which modulate the activity of a signal-interacting pair by binding to a target (e.g. tenofovir) and further particularly to methods and materials for utilizing structure-switching functional ligands and a hybridized oligonucleotide to produce a detectable signal indicative of the presence and/or amount of a target (e.g. tenofovir) in a sample.
[0022] FIG. 1 illustrates a composition 100 with a functional ligand, shown as nucleic acid aptamer 102, hybridized to an oligonucleotide 104 with 5 '-end 104b at a hybridization region 102b. The nucleic acid aptamer 102 may, for example and without being bound to any particular theory, include a stem-loop structure, with regions 102c, 102d forming a stem hybridization in the absence of the oligonucleotide 104, and a loop portion 102f, which may, for example, include the binding region for the target, such as tenofovir. For example, the stem may be two nucleotides in length. The nucleic acid aptamer 102 may also, for example, be modified, such as with terminal modifications (e.g. a 3 '-end amine at 3 '-end 102e).
[0023] The oligonucleotide 104 may be a truncated version of the complementary sequence to the nucleic acid aptamer 102, as illustrated in FIG. 1, such as, for example, a 3'-end truncation (i.e. complementary to the 3 '-end of the nucleic acid aptamer 102) of about 6 to 14 nucleotides in length, more particularly 8-10 nucleotides in length, such as 8 or 9 nucleotides in length.
[0024] In general, functional ligands may be selected for and/or utilized for their ability to bind or complex to particular target molecules, such as tenofovir, and/or for the manner in which they bind or complex to particular target molecules, such as by exhibiting a detectable structural change. For example, numerous biomolecules, such as nucleic acids and peptides, take on varied secondary and tertiary structures in response to different environments or by associating with other molecules. Functional ligands may generally include biomolecules such as nucleic acids, such as single-stranded nucleic acids and double-stranded nucleic acids or combinations or regions of both, peptides, other biopolymers and/or combinations or modifications thereof, such as artificially modified nucleic acids, synthetic analogs and the like.
[0025] Non-naturally occurring sequences of functional ligands, such as nucleic acids and nucleic acid analogs, such as aptamers, may also be useful by interacting with a target molecule in a manner not present in naturally occurring systems or situations, such as by, for example, not being already present or having a pre-existing function in a naturally occurring setting. [0026] In general, functional ligands may generally include nucleic acids, particularly single-stranded nucleic acids, peptides, other biopolymers and/or combinations or modifications thereof. Nucleic acid sequences may include naturally-occurring biomolecules such as ribonucleic acid (RNA), deoxyribonucleic acid (DNA), artificially modified nucleic acids, and/or combinations thereof. In general, modified nucleic acid bases may be utilized and may include, but are not limited to, 2'-Deoxy-P- nucleoside-5'-Triphosphate, 2'-Deoxyinosine-5'-Triphosphate, 2'-Deoxypseudouridine-5'-Triphosphate, 2'-Deoxyuridine-5'-Triphosphate, 2'-Deoxyzebularine-5'-Triphosphate, 2-Amino-2'-deoxyadenosine-5'- Triphosphate, 2-Amino-6-chloropurine-2'-deoxyriboside-5'-Triphosphate, 2-Aminopurine-2'- deoxyribose-5'-Triphosphate, 2-Thio-2'-deoxycytidine-5'-Triphosphate, 2-Thiothymidine-5'- Triphosphate, 2'-Deoxy-L-adenosine-5'-Triphosphate, 2'-Deoxy-L-cytidine-5'-Triphosphate, 2'-Deoxy- L-guanosine-5' -Triphosphate, 2' -Deoxy-L-thymidine-5' -Triphosphate, 4-Thiothymidine-5'-
Triphosphate, 5-Aminoallyl-2'-deoxycytidine-5'-Triphosphate, 5-Aminoallyl-2'-deoxyuridine-5'- Triphosphate, 5-Bromo-2'-deoxycytidine-5'-Triphosphate, 5-Bromo-2'-deoxyuridine-5'-Triphosphate, 5- Fluoro-2'-deoxyuridine-5'-Triphosphate, 5-Trifluoromethyl-2-deoxyuridine-5'-Triphosphate, and/or any other appropriate modified nucleic acid base. It may generally be understood that the nucleoside triphosphates (NTPs) listed above may generally refer to any appropriate phosphate of the modified base, such as additionally, for example, monophosphates (NMPs) or diphosphates (NDPs) of the base. Embodiments of the SELEX method may generally be utilized to select or preselect for aptamers to be used in a collection. The basic SELEX protocol and aptamers are described in U.S. Patent No. 5,270,163, entitled "Methods for identifying nucleic acid ligands," the entire contents of which are hereby incorporated by reference.
[0027] Structural changes may be directly observable, such as through microscopy, microscale thermophoresi s (MST), backscattering interferometry (BSI), circular dichroism, Nuclear Magnetic Resonance (NMR) and/or any other appropriate observation method. Structural changes may also be detectable through secondary events, such as by detecting changes in fluorescence, spectral emission/color or other radiation emissions/signals due to alterations in the structure of a functional ligand.
[0028] In one aspect of the present invention, a method for utilizing functional ligands may include providing a functional ligand to a target molecule, such as a nucleic acid aptamer that binds to tenofovir, and an oligonucleotide which is complementary to at least a portion of the nucleic acid aptamer, such that the oligonucleotide is hybridized to the nucleic acid aptamer, and the binding of the target to the nucleic acid aptamer, such as tenofovir, may generate a detectable change in the interaction between the nucleic acid aptamer and the oligonucleotide. In general, and without being bound to any particular theory, the binding of the target to the nucleic acid aptamer may generally cause the oligonucleotide to dehybridize from the nucleic acid aptamer. [0029] FIG. 2 illustrates an example of a nucleic acid aptamer 102 binding to a target molecule 90 (e.g. tenofovir), which may generally cause a hybridized oligonucleotide 104 to dehybridize.
[0030] In some embodiments, the nucleic acid aptamer and the oligonucleotide may each be attached to a signal interacting moiety (e.g. at their 5'- or 3'- ends), such as an interacting pair of chromophores where the proximity of the pair results in a decreased amount of detectable signal and increasing the distance between them results in an increase in detectable signal. FIG. 1 illustrates a nucleic acid aptamer
102 with a signal interacting moiety 102a attached at the 5 '-end and an oligonucleotide 104 with a signal interacting moiety 104a attached at its 3 '-end. The binding of the target to the nucleic acid aptamer may generally cause an increase in the distance of the pair, such as, for example and without being bound to any particular theory, by causing the oligonucleotide to dehybridized and move away, such as by a structural or conformational change of the nucleic acid aptamer upon binding. In some embodiments, the oligonucleotide may be displaced and/or occluded from hybridizing upon binding of a target molecule to the nucleic acid aptamer. FIG. 2 illustrates the dehybndization and increased distance between the nucleic acid aptamer 102 and the oligonucleotide 104 due to the target 90 binding, causing an increased signal emission A from the signal interacting moiety 102a. The composition 100 may be used, without being bound to any particular theory, in any appropriate solution or fluid/gel system, such as to detect and/or quantify tenofovir in a blood or other body fluid sample, such as to monitor the concentration of the drug (e.g. for aiding in maintaining the drug in a therapeutic range or detect accumulation/toxicity).
[0031] The signal interacting moieties may generally be attached to the nucleic acid aptamer and/or the oligonucleotide by any appropriate means, such as, for example, by covalent attachment, attachment through biotin-streptavidin/avidin coupling. FIG. 3 illustrates an example of a 5 '-end of a nucleic acid aptamer 102 to a coupling moiety 103, which may be biotin, streptavidin, avidin, etc., and a signal interacting moiety 102a (e.g. a fluorophore emitting a signal A), attached to a coupling moiety 103a, which may be the corresponding agent to the coupling moiety 103 (i.e. streptavidin/avidin if the coupling moiety
103 is biotin and vice versa). In general, streptavidin/avidin are multivalent to up to four biotins, but modified versions may be utilized with difference valency.
[0032] In some exemplary embodiments, the interaction of the signal interacting moieties may include, but are not limited to, colorimetric interactions, fluorescence interactions (e.g. Fluorescence Energy Transfer (FRET)), fluorescence enhancement upon moving to a different solvent environment, redox interactions, enzymatic interactions, pH reporting mechanisms, surface enhanced Raman scattering (SERS), isothermal DNA amplification, and/or any other appropriate interaction to produce a detectable signal or change. Examples of interacting systems include, but are not limited to, horseradish peroxidase and a chromogenic substrate (e.g. 3,3',5,5'-Tetramethylbenzidine, 3,3'-Diaminobenzidine, 2,2'-azino- bis(3-ethylbenzothiazoline-6-sulphonic acid), etc.) or fluorogenic substrates such as 10-acetyl-3,7- dihydroxyphenoxazine, alkaline phosphatase with substrates such as Nitroblue Tetrazolium and 5-Bromo- 4-Chloro-3-Indolyl Phosphate, p-nitrophenyl phosphate, and TR phosphate, diaphorase (NADPH: (acceptor) oxidoreductase) and resazurin, FRET or other fluorescence-quencher pairs as discussed above, and/or any other appropriate system.
[0033] Pairs of corresponding chromophores may be utilized for their ability of altering the conversion efficiency of the radiation converting chromophore in order to create a detectable signal or change. For example, converting and absorbing chromophore pairs in the radiation portion of interest may include, but are not limited to: ALEXA633™/QSY21™, CY5™/QSY21™, ALEXA647™/QSY21™, ALEXA647™/ALEXA680™, ALEXA680™/allophycocyanin (APC), ALEXA700™/APC, and/or ALEXA750™/APC (Molecular Probes, Inc.), and/or any other appropriate fluorophore/chromophore pair. It is contemplated, however, that any suitable pair of first and/or second radiation converting chromophore and radiation absorbing chromophore, whether now known or later developed, is within the scope of the present invention.
[0034] A chromophore-quencher pair may also include a metallic quenching element, such as a gold or other metallic substrate, which may include, for example, gold nanoparticles, nanotubes, nanorods, and/or any other appropriate metallic substrate. Interactions between fluorophores and metallic quenchers may be desirable, for example, where utilization of plasmonic energy transfer or quenching, which may generally have a longer range than other forms of transfer such as FRET, may be useful. In some embodiments, a signal-interacting molecule may be labeled with a metallic nanoparticle/structure, such that when the signal-interacting molecule is combined with another metallic nanoparticle/structure (which may be labeled with an appropriate Raman reporter molecule), the combined metallic nanoparticles/structures may generate a specific and/or distinctive Raman signature under Raman spectroscopy.
[0035] Other chromophores or fluorophores, such as quantum dots, may also be utilized.
EXAMPLE OF FLUORESCENCE EMISSION FROM TENOFOVIR DETECTION
[0036] The composition of a nucleic acid aptamer 102 and oligonucleotide 104 shown in FIG. 1, where the nucleic acid aptamer 102 has the sequence of SEQ ID 1 and the oligonucleotide 104 has a truncated sequence of SEQ ID 2 (e.g. 3'-end truncated to 9 nucleotides in length), the nucleic acid aptamer 102 is biotinylated at its 5 '-end and coupled to a streptavidin coupled to the fluorophore ALEXA647, and the oligonucleotide 104 is coupled at its 3 '-end to a quencher QSY-21. FIG. 4 shows the relative quenching efficiency by showing the relative fluorescence difference of the nucleic acid aptamer 102 alone in the first bar and with the hybridized oligonucleotide 104 in the second bar. FIG. 5 shows the concentration- dependent fluorescence emission for concentrations of tenofovir from 0 to 4 mM. As shown the increasing concentrations of tenofovir results in higher fluorescence emissions. [0037] Although the invention has been described with respect to specific embodiments thereof, these embodiments are merely illustrative, and not restrictive of the invention. The description herein of illustrated embodiments of the invention, including the description in the Abstract and Summary, is not intended to be exhaustive or to limit the invention to the precise forms disclosed herein (and in particular, the inclusion of any particular embodiment, feature or function within the Abstract or Summary is not intended to limit the scope of the invention to such embodiment, feature or function). Rather, the description is intended to describe illustrative embodiments, features and functions in order to provide a person of ordinary skill in the art context to understand the invention without limiting the invention to any particularly described embodiment, feature or function, including any such embodiment feature or function described in the Abstract or Summary. While specific embodiments of, and examples for, the invention are described herein for illustrative purposes only, various equivalent modifications are possible within the spirit and scope of the invention, as those skilled in the relevant art will recognize and appreciate. As indicated, these modifications may be made to the invention in light of the foregoing description of illustrated embodiments of the invention and are to be included within the spirit and scope of the invention. Thus, while the invention has been described herein with reference to particular embodiments thereof, a latitude of modification, various changes and substitutions are intended in the foregoing disclosures, and it will be appreciated that in some instances some features of embodiments of the invention will be employed without a corresponding use of other features without departing from the scope and spirit of the invention as set forth. Therefore, many modifications may be made to adapt a particular situation or material to the essential scope and spirit of the invention.
[0038] Reference throughout this specification to "one embodiment", "an embodiment", or "a specific embodiment" or similar terminology means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment and may not necessarily be present in all embodiments. Thus, respective appearances of the phrases "in one embodiment", "in an embodiment", or "in a specific embodiment" or similar terminology in various places throughout this specification are not necessarily referring to the same embodiment. Furthermore, the particular features, structures, or characteristics of any particular embodiment may be combined in any suitable manner with one or more other embodiments. It is to be understood that other variations and modifications of the embodiments described and illustrated herein are possible in light of the teachings herein and are to be considered as part of the spirit and scope of the invention.
[0039] In the description herein, numerous specific details are provided, such as examples of components and/or methods, to provide a thorough understanding of embodiments of the invention. One skilled in the relevant art will recognize, however, that an embodiment may be able to be practiced without one or more of the specific details, or with other apparatus, systems, assemblies, methods, components, materials, parts, and/or the like. In other instances, well-known structures, components, systems, materials, or operations are not specifically shown or described in detail to avoid obscuring aspects of embodiments of the invention. While the invention may be illustrated by using a particular embodiment, this is not and does not limit the invention to any particular embodiment and a person of ordinary skill in the art will recognize that additional embodiments are readily understandable and are a part of this invention.
[0040] As used herein, the terms "comprises," "comprising," "includes," "including," "has," "having," or any other variation thereof, are intended to cover a non-exclusive inclusion. For example, a process, product, article, or apparatus that comprises a list of elements is not necessarily limited only those elements but may include other elements not expressly listed or inherent to such process, process, article, or apparatus.
[0041] Furthermore, the term "or" as used herein is generally intended to mean "and/or" unless otherwise indicated. For example, a condition A or B is satisfied by any one of the following: A is true (or present) and B is false (or not present), A is false (or not present) and B is true (or present), and both A and B are true (or present). As used herein, including the claims that follow, a term preceded by "a" or "an" (and "the" when antecedent basis is "a" or "an") includes both singular and plural of such term, unless clearly indicated within the claim otherwise (i.e., that the reference "a" or "an" clearly indicates only the singular or only the plural). Also, as used in the description herein, the meaning of "in" includes "in" and "on" unless the context clearly dictates otherwise.

Claims

1. A method for detecting tenofovir in a sample comprising:
providing a composition comprising:
a ligand comprising a non-naturally occurring artificial nucleic acid sequence of SEQ ID i ;
a first signal interacting moiety attached to said ligand at a 5 '-end;
an oligonucleotide comprising a truncation of a non-naturally occurring artificial nucleic acid sequence of SEQ ID 2; and
a second signal interacting moiety attach to said oligonucleotide at a 3 '-end;
contacting said composition with a sample which may contain tenofovir;
detecting a signal emitted from said composition due to a change in the interaction between said first and second signal interacting moieties.
2. A composition for detecting tenofovir comprising:
a ligand comprising a non-naturally occurring artificial nucleic acid sequence of SEQ ID 1; a first signal interacting moiety attached to said ligand at a 5 '-end;
an oligonucleotide comprising a truncation of a non-naturally occurring artificial nucleic acid sequence of SEQ ID 2;
a second signal interacting moiety attach to said oligonucleotide at a 3 '-end;
3. The composition or method of claims 1-2, wherein said truncation is a 3 '-truncation to a length of between 6 and 14 nucleotides.
4. The composition or method of claims 1-2, wherein said truncation is a 3 '-truncation to a length of between 8 and 10 nucleotides.
5. The composition or method of claims 1-2, wherein said first signal interacting moiety and said second signal interacting moiety comprise a chromophore pair which decrease in signal when in proximity.
6. The composition or method of claims 1-2, wherein said first signal interacting moiety and said second signal interacting moiety comprise a pair selected from the group consisting of
ALEXA633/QSY21, CY5/QSY21, ALEXA647/QS Y21 , ALEXA647/ALEXA680,
ALEXA680/allophycocyanin (APC), ALEXA700/APC, ALEXA750/APC and a pair of nanoparticles that emit a distinctive Raman signature under Raman spectroscopy when said pair interacts.
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