WO2011087456A1 - Reconnaissance d'acides nucléiques sélective vis-à-vis d'une séquence utilisant des sondes nanoparticulaires - Google Patents

Reconnaissance d'acides nucléiques sélective vis-à-vis d'une séquence utilisant des sondes nanoparticulaires Download PDF

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WO2011087456A1
WO2011087456A1 PCT/SG2011/000016 SG2011000016W WO2011087456A1 WO 2011087456 A1 WO2011087456 A1 WO 2011087456A1 SG 2011000016 W SG2011000016 W SG 2011000016W WO 2011087456 A1 WO2011087456 A1 WO 2011087456A1
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conjugate
nucleic acid
target nucleic
acid molecule
target
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PCT/SG2011/000016
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English (en)
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Yanbing Zu
Zhiqiang Gao
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Agency For Science, Technology And Research
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Priority to CN201180013638.3A priority Critical patent/CN102884204B/zh
Publication of WO2011087456A1 publication Critical patent/WO2011087456A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
    • C07D487/04—Ortho-condensed systems
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y15/00—Nanotechnology for interacting, sensing or actuating, e.g. quantum dots as markers in protein assays or molecular motors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D413/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07D413/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings
    • C07D413/04—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings directly linked by a ring-member-to-ring-member bond
    • 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
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism

Definitions

  • the present invention relates to a conjugate for nucleic acid detection comprising a nanoparticle and at least one oligonucleotide analog, methods of using the same, uses thereof as well as a kit comprising at least one of the conjugates of the invention.
  • SNPs single nucleotide polymorphisms
  • detection of single-nucleotide variants is critical in many research fields, such as genotyping of single nucleotide polymorphisms (SNPs) and detection of acquired point mutations.
  • SNPs represent the most abundant type of genetic variations and SNP genotyping plays an important role in associating sequence variants to phenotypic changes.
  • the demand for mutation detection arises because a large number of human diseases originate from mutations in one or more genes.
  • Point mutations are emerging as important molecular markers of cancer, and the detection of low-level somatic point mutations within a large excess (> 100-fold) of wild-type alleles is essential for early diagnostic and risk assessment.
  • Hybridization-based strategies are commonly used for SNP discrimination.
  • the stability differences between perfectly-matched and single-base- mismatched duplexes can be distinguished by measuring their different melting curves.
  • Stringent hybridization conditions have to be employed to reduce cross-binding of the allele-specific probes and achieve selective detection.
  • fluorescence measurements still dominate in these studies, a variety of gold nanoparticle (NP)-based methods have been proposed during the last decade.
  • Gold NPs exhibit strongly distance-dependent optical properties and large surface areas. Their extinction coefficients could be ⁇ 3 orders of magnitude larger than those of organic dyes.
  • the analyte-related aggregation of gold NPs shifts the surface plasmon resonance (SPR) absorption peak toward longer wavelength, leading to colouiimetric sensing with easy readout.
  • SPR surface plasmon resonance
  • a colourimetric DNA detection method based on the formation of a polymeric network of ssDNA- modified gold nanoparticles was developed (Elghanian, R. et al, Science 1997, 277, 1078-1081). By controlling the temperature stringently, single base mismatches could be discriminated.
  • the DNA detection can also be conducted on the basis of nanoparticle stability change induced by hybridization.
  • low stringency conditions i.e., high salt or low temperature
  • high stringency conditions i.e., low salt or high temperature
  • PNA peptide nucleic acid
  • the invention provides for a conjugate including a nanoparticle and at least one oligonucleotide analog.
  • the at least one oligonucleotide analog is a phosphorodiamidate morpholino oligo (PMO) or a derivative thereof that is covalently coupled to the nanoparticle.
  • PMO phosphorodiamidate morpholino oligo
  • the invention provides for a method for detecting a target nucleic acid.
  • the method includes contacting at least one conjugate according to the invention with a sample containing the target nucleic acid, under conditions which allow the at least one conjugate and the target nucleic acid to hybridize to each other, wherein the phosphorodiamidate morpholino oligo or derivative thereof comprises a nucleotide sequence that is complementary to a nucleotide sequence comprised in the target nucleic acid, and detecting the formed complex.
  • the invention provides for a method for detecting at least one single nucleotide polymorphism (SNP) in a target nucleic acid molecule.
  • the method includes i) contacting the target nucleic acid molecule with a conjugate of the invention to form a complex of conjugate and target nucleic acid molecule; ii) measuring the melting transition temperature of the complex; and iii) comparing the melting transition temperature of the complex measured in ii) with the melting transition temperature of a control complex, wherein, if the complex of conjugate and target nucleic acid molecule has a lower melting transition temperature compared to the control complex, this indicates that the target nucleic acid molecule comprises at least one single nucleotide polymorphism.
  • the invention provides for a use of at least one conjugate of the invention for the detection of a target nucleic acid molecule.
  • the invention provides for a kit for the detection of a target nucleic acid molecule.
  • the kit includes at least one conjugate of the invention.
  • Figure 1 shows a graph that illustrates the surface density of the thiolated morpholino oligos on 40 nm gold nanoparticles (number of strands/NP) as a function of incubation time. The error bars represent the standard deviation from three measurements.
  • the mixture solution contained 10 mM phosphate, ⁇ 2 nM fluorosurfactant (FSN)-capped gold NPs, and ⁇ 1 ⁇ of the thiolated morpholino oligos.
  • the loading density of the thiolated morpholino (MO) on 40 nm gold NPs is -1200 strands/particle, which is much higher than the largest surface coverage that thiolated ssDNA could be loaded on the same size gold NPs. Without wishing to be bound to any theory, this could be attributed to the absence of electrostatic repulsion force between the immobilized MOs. Fast attachment kinetics was observed in which saturated loading density could be achieved within 1 hour.
  • Figure 2 shows the time courses of the extinction (absorbance at 540 nm) of the conjugates solutions upon the addition of NaCl at concentrations of 20 mM, 80 mM and 200 mM. The measurements were conducted using a 384- well microplate. The colour change of the conjugate solution occurred upon the addition of NaCl.
  • FIG 3 shows the UV-Vis spectra of the MOl/gold NP conjugates prior and after a 0.5 h incubation with 200 mM NaCl.
  • MOl indicates the morpholino oligonucleotide as set forth as SEQ ID NO: 1.
  • Line 1 (“dispersed") of the graph represents the UV-Vis spectrum of the MOl/gold NP conjugate before aggregation, i.e. before incubation with NaCl.
  • Line 2 (“aggregated”) of the graph represents the UV-Vis spectrum of the MOl/gold NP conjugate after aggregation, i.e., after incubation with NaCl.
  • Figure 4 shows a schematic representation of the hybridization of DNA targets with the morpholino (MO)/NP conjugates.
  • Figure 5 shows a graph that measures the absorbance (at 540 nm) of the conjugate solutions as a function of the concentration of the DNA target (PM1) after incubation for 1 h in the presence of 50 mM NaCl.
  • the DNA target PM1 is obtained in Table 1 and as set forth ;as SEQ ID NO: 4.
  • the measurements were conducted using a 384- well microplate. The detection limit of this method was found to be -0.5 nM.
  • Figure 6 shows the melting behaviour of the hybridized PMl-MOl/NP conjugates.
  • the absorbance values were obtained after a 0.5 h incubation of the gold NP solution at each temperature.
  • the solutions contain 0.06 nM MOl/NP conjugates, 10 nM DNA targets (PM1; SEQ ID NO: 4), 200 mM NaCl, and 0.01% Triton X-100.
  • the inset graph shows the thermal dissociation curves for the free PM1-MO duplexes in solution (500 nM oligos, 10 mM phosphate buffer, pH7.5).
  • Figure 7 shows a schematic representation of the melting transition of the DNA- MO/NP conjugates.
  • the states of "a” to “e” correspond to that shown in Figure 6.
  • the stability of the DNA-MO/NP conjugates depends on the amount of DNA strands attached on the NPs.
  • Figure 8 shows the melting transitions of the hybridized PM and SNP DNA- MOl/NPs conjugates.
  • the absorbance values were obtained after a 0.5 h incubation of the NP solution at each temperature.
  • Solutions contain 0.06 nM MO/NP conjugates, 100 nM DNA targets PM1 (SEQ ED NO: 4) or SNP1 (SEQ ID NO: 5), 200 mM NaCl, and 0.01% Triton X- 100.
  • the unique melting behaviour of the hybridized NP conjugates resulted in distinct separation of the melting curves for the PM1 and the SNP1 targets (100 nM).
  • the NP conjugates hybridized with the perfectly complementary DNA remained stable pink colour in the presence of 200 mM NaCl, while the mutant DNA (SNP) (single mismatch) could not stabilize the conjugates and aggregation of the NPs proceeded rapidly.
  • SNP single mismatch
  • Figure 9 shows the melting transition temperature of the hybridized PM1 and SNP l DNA-MOl/NP conjugates as a function of the respective target DNA (PMl or SNPl) concentration.
  • Figure 10 shows the absorbance values at 540 ran after 0.5hr incubation of the MO/NP conjugate solutions containing 10 ⁇ of SNPl (SEQ ID NO: 5) in the presence or absence of 50 nM of PM1 (SEQ ID NO: 4). The background absorbance signal of the conjugate solution (no target) after incubation under the identical condition has been subtracted.
  • Figure 11 shows the melting transitions of the hybridized PM-10 (SEQ ID NO: 8) and SNP-10 (SEQ ID NO: 9) MO/NPs conjugates. Solutions contain 200 mM NaCl, 0.01% Triton X-100. The 10-base specific sequence, the T m values for the PM-10 and the single-base mismatched targets SNP-10 were 19 °C and 32.5 °C, respectively. The AT m> PM-SNP increased to 13.5 °C. In addition, the employment of the short probe sequence allowed convenient discrimination of SNP at room temperature. In this context, without being bound by any theory, the recognition selectivity of an oligo probe towards a single base mismatch increases as its length decreases.
  • the percentage of the mismatched base pair increases from 7% to 10%, which may have a substantially higher impact on duplex stability.
  • the melting transitions of the NP conjugate systems may shift to a lower temperature region.
  • Figure 12 shows the UV-Vis spectra of the MO/silver NP conjugates prior and after a 0.5 h-incubation with 200 mM NaCl.
  • Line 1 (“dispersed") of the graph represents the UV-Vis spectrum of the MOl/Ag NP conjugate before aggregation, i.e. before incubation with NaCl.
  • Line 2 (“aggregated”) represents the UV-Vis spectrum of the MOl/Ag NP conjugate after aggregation, i.e., after incubation with NaCl.
  • Figure 13 shows the UV-Vis spectra for testing heterozygous and homozygous types.
  • Assay solutions contain MOl-modified gold NPs, MOI SNP (SEQ ID NO: 2)-modified silver NPs, 200 mM NaCl, and 0.01% Triton X-100.
  • Sample 1 10 nM PM 1 and 10 nM SNPl.
  • Sample 2 10 nM PM 1.
  • Sample 3 10 nM SNP 1.
  • Figure 14(a) shows a schematic presentation of the colourimetric detection of nucleic acids using a pair of conjugates (MOl; SEQ ID NO: 1 and M02; SEQ ID NO: 3) in 5mM of NaCl and 2mM of Tris buffer solution.
  • the DNA target acts as a linker to align the pair of conjugates through hybridization with the NP -bound morpholino sequences.
  • Figure 14(b) shows the UV-Vis spectra of MO/Au NP conjugates before and after aggregation induced by target DNA.
  • the "dispersed NPs” represents the UV-Vis spectrum of the MO/Au NP conjugates dispersed in a 2 mM Tris buffer solution.
  • the "DNA-linked NPs” represents the UV-Vis spectrum of at least one pair of MO/Au NP conjugates linked by the DNA target.
  • Figure 14(c) shows the colourimetric change of the MO/Au NP conjugates solution (absorbance at 532 nm) as a function of the DNA target concentration.
  • the 13-nm NP conjugates respond to the DNA target with concentrations >1 nM.
  • Figure 16 shows the first derivative curves of the melting profiles shown in Figure
  • the present invention is based on the surprising finding that the melting transition of the conjugates of the present invention hybridized to a target nucleic acid occurs within a narrow temperature range of ⁇ 1°C, allowing single-mismatch detection. This can result in a selectivity factor greater than 200: 1 for single nucleotide polymorphism (SNP) discrimination. Furthermore, the cross-linking of two conjugates of the invention by way of hybridization to a target nucleic acid enables the target nucleic acid to be detected under extremely low salt conditions, for example, at ⁇ 5mM NaCl.
  • the low salt conditions used in the method of the present invention can also result in improved selectivity for detecting a target nucleic acid and SNPs for example. This also eliminates the requirement of the stringent control over the assay conditions and may significantly reduce the generation of false positives or negatives when detecting a target nucleic acid.
  • the conjugates of the present invention dispersed well in 5 mM phosphate buffer solution, due to the much higher solubility of the PMO as compared to that of PNA. Therefore, the preparation of the conjugates of the present invention is much easier than previously known methods. It was further found that the conjugates of the present invention can achieve stability by way of cross-linking of two conjugates hybridized to a target nucleic acid.
  • the conjugates of the present invention hybridized to a target nucleic acid display sharp melting transitions, allowing unambiguous discrimination of the perfectly-matched target from the sequences with single-base substitution, deletion, or insertion.
  • the sequence-selective method may provide a new tool for the study of gene variation in a more effective way.
  • the invention thus provides for a conjugate for the detection of a target nucleic acid molecule.
  • the conjugate includes a nanoparticle and at least one oligonucleotide analog.
  • the at least one oligonucleotide analog is a phosphorodiamidate morpholino oligo (PMO) or a derivative thereof that is covalently coupled to the nanoparticle.
  • PMO phosphorodiamidate morpholino oligo
  • nanoparticle refers to any particle having a size from about 1 to about 250 nm and has the capacity to be covalently coupled to at least one oligonucleotide analog as described herein.
  • the nanoparticle is a metal nanoparticle. In other embodiments, the nanoparticle is a colloidal metal.
  • the metal is a noble metal.
  • a noble metal that can be used can include silver, gold, platinum, palladium, ruthenium, osmium, iridium or mixtures thereof, not to mention a few.
  • Other metals that can also be used in the formation of the nanoparticle can include but are not limited to aluminium, copper, cobalt, indium, nickel, or any other metal amenable to nanoparticle formation).
  • the nanoparticle as described herein can also comprise a semiconductor (including for example and without limitation, CdSe, CdS, and CdS or CdSe coated with ZnS) or magnetic (for example, ferromagnetite) colloidal materials.
  • nanoparticles useful in the practice of the invention include, also without limitation, ZnS, ZnO, Ti, Ti0 2 , Sn, Sn0 2 , Si, Si0 2 , Fe, Ag, Cu, Ni, Al, steel, cobalt-chrome alloys, Cd, titanium alloys, Agl, AgBr, Hgl 2 , PbS, PbSe, ZnTe, CdTe, In 2 S 3 , In 2 Se 3 , Cd 3 P 2 , Cd 3 As 2 , InAs, and GaAs.
  • nanoparticles are also commercially available from, for example, Ted Pella, Inc. (gold), Amersham Corporation (gold) and Nanoprobes Inc (gold).
  • the nanoparticles comprising materials described herein are available commercially or they can be produced from progressive nucleation in solution (e.g., by colloid reaction), or by various physical and chemical vapour deposition process, such as sputter disposition.
  • the nanoparticles as described herein can also be produced using HAuCl 4 and a citrate-reducing agent, using methods known in the art (see for example, Grabar, K. C. et al, Anal. Chem., 1995, 67, 735-743).
  • the size of the nanoparticle used in the conjugate of the present invention can vary in any size when desired, as long as the nanoparticle is capable of providing optical properties; for example, generate optical signals sensitive to hybridization reactions.
  • the diameter of the nanoparticle as described herein can range in the size from about 1 nm to about 250 nm; about 1 nm to about 200 nm; about 1 nm to about 160 nm; about 1 nm to about 140 nm; about 1 nm to about 120 nm; about 1 nm to about 80 nm; about 1 nm to about 60 nm; about 1 nm to about 50 nm; about 5 nm to about 250 nm; about 8 nm to about 250 nm; about 10 nm to about 250 nm; about 20 nm to about 250 nm; about 30 nm to about 250 nm; about 40 nm to about 250 nm; about 85 nm to about 250 nm; about 100 n
  • the nanoparticle comprises a surfactant.
  • surfactant refers to a surface active agent which has both hydrophilic and hydrophobic parts in the molecule.
  • the surfactant can for example be used to stabilize the nanoparticles.
  • the surfactant can also be used to prevent non-specific adsorption of the oligonucleotide analog on the surface of the nanoparticles.
  • the surfactant is a non-ionic surfactant.
  • Other types of surfactants that can be used can include but are not limited to cationic, anionic, or zwitterionic surfactants. A particular surfactant may be used alone or in combination with other surfactants.
  • One class of surfactants comprises a hydrophilic head group and a hydrophobic tail.
  • Hydrophilic head groups associated with anionic surfactants include carboxylate, sulfonate, sulfate, phosphate, and phosphonate.
  • Hydrophilic head groups associated with cationic surfactants include quaternary amine, sulfonium, and phosphonium. Quaternary amines include quaternary ammonium, pyridinium, bipyridinium, and imidazolium.
  • Hydrophilic head groups associated with non-ionic surfactants include alcohol and amide.
  • Hydrophilic head groups associated with zwitterionic surfactants include betaine.
  • the hydrophobic tail typically comprises a hydrocarbon chain. The hydrocarbon chain typically comprises between about six and about 24 carbon atoms, more typically between about eight to about 16 carbon atoms.
  • Exemplary anionic surfactants include alkyl phosphonates, alkyl ether phosphates, alkyl sulfates, alkyl ether sulfates, alkyl sulfonates, alkyl ether sulfonates, carboxylic acid ethers, carboxylic acid esters, alkyl aryl sulfonates, and sulfosuccinates.
  • Anionic surfactants include any sulfate ester, such as those sold under the trade name ULTRAFAX, including, sodium lauryl sulfate, sodium laureth sulfate (2 EO), sodium laureth, sodium laureth sulfate (3 EO), ammonium lauryl sulfate, ammonium laureth sulfate, TEA-lauryl sulfate, TEA-laureth sulfate, MEA-lauryl sulfate, MEA-laureth sulfate, potassium lauryl sulfate, potassium laureth sulfate, sodium decyl sulfate, sodium octyl/decyl sulfate, sodium 2-ethylhexyl sulfate, sodium octyl sulfate, sodium nonoxynol-4 sulfate, sodium nonoxynol-6 sulfate, sodium cumen
  • Exemplary cationic surfactants include quaternary ammonium salts such as dodecyl trimethyl ammonium chloride, cetyl trimethyl ammonium salts of bromide and chloride, hexadecyl trimethyl ammonium salts of bromide and chloride, alkyl dimethyl benzyl ammonium salts of chloride and bromide, and the like.
  • a class of non-ionic surfactants can include those comprising polyether groups, based on, for example, ethylene oxide (EO) repeat units and/or propylene oxide (PO) repeat units. These surfactants are typically non-ionic. Surfactants having a polyether chain may comprise between about 1 and about 36 EO repeat units, between about 1 and about 36 PO repeat units, or a combination of between about 1 and about 36 EO repeat units and PO repeat units. More typically, the polyether chain can comprise between about 2 and about 24 EO repeat units, between about 2 and about 24 PO repeat units, or a combination of between about 2 and about 24 EO repeat units and PO repeat units.
  • EO ethylene oxide
  • PO propylene oxide
  • the polyether chain can comprise between about 6 and about 15 EO repeat units, between about 6 and about 15 PO repeat units, or a combination of between about 6 and about 15 EO repeat units and PO repeat units.
  • These surfactants may comprise blocks of EO repeat units and PO repeat units, for example, a block of EO repeat units encompassed by two blocks of PO repeat units or a block of PO repeat units encompassed by two blocks of EO repeat units.
  • Another class of polyether surfactants cam comprise alternating PO and EO repeat units. Within these classes of surfactants are the polyethylene glycols, polypropylene glycols, and the polypropylene glycol/polyethylene glycols.
  • non-ionic surfactants can comprise EO, PO, or EO/PO repeat units built upon an alcohol or phenol base group, such as glycerol ethers, butanol ethers, pentanol ethers, hexanol ethers, heptanol ethers, octanol ethers, nonanol ethers, decanol ethers, dodecanol ethers, tetradecanol ethers, phenol ethers, alkyl substituted phenol ethers, o>naphthol ethers, and j8-naphthol ethers.
  • glycerol ethers such as glycerol ethers, butanol ethers, pentanol ethers, hexanol ethers, heptanol ethers, octanol ethers, nonanol ethers, decanol ether
  • the phenol group is substituted with a hydrocarbon chain having between about 1 and about 10 carbon atoms, such as about 8 (octylphenol) or about 9 carbon atoms (nonylphenol).
  • the polyether chain may comprise between about 1 and about 24 EO repeat units, between about 1 and about 24 PO repeat units, or a combination of between about 1 and about 24 EO and PO repeat units. More typically, the polyether chain comprises between about 8 and about 16 EO repeat units, between about 8 and about 16 PO repeat units, or a combination of between about 8 and about 16 EO and PO repeat units. Even more typically, the polyether chain comprises about 9, about 10, about 11, or about 12 EO repeat units; about 9, about 10, about 11, or about 12 PO repeat units; or a combination of about 9, about 10, about 11, or about 12 EO repeat units and PO repeat units.
  • An exemplary j3-naphthol derivative non-ionic surfactant is Lugalvan BN012 which is a /8-naphtholethoxylate having 12 ethylene oxide monomer units bonded to the naphthol hydroxyl group.
  • a similar surfactant is Polymax NPA-15, which is a polyethoxylated nonylphenol.
  • Another surfactant is Triton®-X100 nonionic surfactant, which is an octylphenol ethoxylate, typically having around 9 or 10 EO repeat units. Additional commercially available non-ionic surfactants include the Pluronic® series of surfactants, available from BASF.
  • Pluronic® surfactants include the P series of EO/PO block copolymers, including P65, P84, P85, P103, P104, P105, and P123, available from BASF; the F series of EO/PO block copolymers, including F108, F127, F38, F68, F77, F87, F88, F98, available from BASF; and the L series of EO/PO block copolymers, including L10, L101, L121, L31, L35, L44, L61, L62, L64, L81, and L92, available from BASF.
  • non-ionic surfactants include water soluble, ethoxylated nonionic fluorosurfactants available from DuPont and sold under the trade name Zonyl®, including Zonyl® FSN (Telomar B Monoether with Polyethylene Glycol nonionic surfactant), Zonyl® FSN-100, Zonyl® FS-300, Zonyl® FS-500, Zonyl® FS-510, Zonyl® FS- 610, Zonyl® FSP, and Zonyl® UR.
  • Zonyl® FSN (Telomar B Monoether with Polyethylene Glycol nonionic surfactant) is particularly preferred.
  • non-ionic surfactants include the amine condensates, such as cocoamide DEA and cocoamide MEA, sold under the trade name ULTRAFAX.
  • Other classes of non-ionic surfactants include acid ethoxylated fatty acids (polyethoxy-esters) comprising a fatty acid esterified with a polyether group typically comprising between about 1 and about 36 EO repeat units.
  • Glycerol esters comprise one, two, or three fatty acid groups on a glycerol base.
  • oligonucleotide analog refers to an oligonucleotide having (i) a modified backbone structure, e.g., a backbone other than the standard phosphodiester linkage found in natural oligo- and polynucleotides, and (ii) optionally, modified sugar moieties, e.g., morpholino moieties rather than ribose or deoxyribose moieties.
  • the analog supports bases capable of hydrogen bonding by Watson-Crick base pairing to standard polynucleotide bases, where the analog backbone presents the bases in a manner to permit such hydrogen bonding in a sequence- specific fashion between the oligonucleotide analog molecule and bases in a standard polynucleotide (e.g., single-stranded RNA or single-stranded DNA).
  • the analogs can for example, include those having a substantially uncharged, phosphorus containing backbone.
  • a substantially uncharged, phosphorus containing backbone in an oligonucleotide analog can for example be one in which a majority of the subunit linkages, e.g., between 60- 100%, are uncharged at physiological pH, and contain a single phosphorous atom.
  • the oligonucleotide analog can comprise a nucleotide sequence complementary to a target nucleic acid sequence as defined below.
  • oligonucleotide analogs of the present invention are phosphorodiamidate morpholino oligos, wherein the sugar and phosphate backbone is replaced by morpholine groups linked by phosphoramidates and the nucleobases, such as cytosine, guanine, adenine, thymine and uracil, are coupled to the morpholine ring or derivatives thereof.
  • the term "complementary” or “complementarity” relates to the relationship of nucleotides/bases on two different strands of DNA or RNA, or the relationship of nucleotides/bases of the nucleotide sequence of the oligonucleotide analog and a DNA/RNA strand, where the bases are paired (for example by Watson-Crick base pairing: guanine with cytosine, adenine with thymine (DNA) or uracil (RNA)).
  • the at least one oligonucleotide analog as described herein can comprise a nucleotide sequence that can form hydrogen bond(s) with another nucleotide sequence, for example a DNA or RNA sequence, by either conventional Watson-Crick base pairing or other non-traditional types of pairing such as Hoogsteen or reversed Hoogsteen hydrogen bonding between complementary nucleosides or nucleotides.
  • hybridize refers to an interaction between two different strands of DNA or RNA or between nucleotides/bases of the nucleotide sequence of the oligonucleotide analog and a DNA/RNA sequence by hydrogen bonds in accordance with the rules of Watson-Crick DNA complementarity, Hoogsteen binding, or other sequence-specific binding known in the art.
  • a nucleotide sequence of an oligonucleotide analog described herein need not be 100% complementary to a target nucleic acid sequence to be specifically or selectively hybridizable.
  • the oligonucleotide analog may hybridize with each other over one or more segments such that intervening or adjacent segments are not involved in the hybridization event (e.g., a loop structure or hairpin structure).
  • Complementarity is indicated by a percentage of contiguous residues in a nucleic acid molecule that can form hydrogen bonds with a second nucleic acid molecule. For example, if a first nucleic acid molecule has 10 nucleotides and a second nucleic acid molecule has 10 nucleotides, then base pairing of 5, 6, 7, 8, 9, or 10 nucleotides between the first and second nucleic acid molecules represents 50%, 60%, 70%, 80%, 90%, or 100% complementarity, respectively, not to mention a few.
  • the oligonucleotide analog used herein can be 100% complementary to a target nucleic acid (i.e., a perfect match). In other embodiments, the oligonucleotide analog can be at least about 95% complementary, at least about 85% complementary, at least about 70% complementary, at least about 65% complementary, at least about 55% complementary, at least about 45 % complementary, or at least about 30% complementary to the target nucleic acid. In one embodiment, "complementarity" relates to hydrogen bonding by Watson-Crick base pairing only.
  • the at least one oligonucleotide analog as described herein can be a phosphorodiamidate morpholino oligo (PMO) or a derivative thereof.
  • PMO phosphorodiamidate morpholino oligo
  • the monomelic unit of PMO or derivative thereof can be represented by Formula (I):
  • Pi is a purine or pyrimidine base-pairing moiety
  • X is NH 2 , NHR, or NR 2
  • R is Ci-C 6 alkyl.
  • Ci-Ce alkyl refers to a fully saturated aliphatic hydrocarbon comprising 1 to 6 carbon atoms. The alkyls can for example be optionally substituted. In some embodiments, "Ci-Ce alkyl” refers to an alkyl group comprising only 1 carbon atom, 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, 5 carbon atoms, or 6 carbon atoms.
  • alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec- butyl, tert-butyl, tert-amyl, pentyl, hexyl and the like.
  • the term "optionally substituted” refers to a group in which none, one, or more than one of the hydrogen atoms has been replaced with one or more group(s) are independently selected from: alkyl, heteroalkyl, haloalkyl, heterohaloalkyl, cycloalkyl, aryl, arylalkyl, heteroaryl, non-aromatic heterocycle, hydroxy, alkoxy, aryloxy, mercapto, alkylthio, arylthio, cyano, halo, carbonyl, thiocarbonyl, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, S-sulfonamido, N-sulfonamido, C-carboxy, O-carboxy, isocyanato, thiocyanato, isothiocyanato, nitro, silyl,
  • cycloalkyl refers to a completely saturated hydrocarbon ring.
  • the cycloalkyl group used in this invention may range from C 3 to Cg.
  • a cycloalkyl group of this invention can for example be optionally substituted.
  • Examples of cycloalkyl groups include, but are not limited to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and the like.
  • aryl refers to an aromatic ring wherein each of the atoms forming the ring is a carbon atom.
  • Aryl rings may be formed by five, six, seven, eight, nine, or more than nine carbon atoms.
  • Aryl groups may be optionally substituted.
  • aromatic refers to a group comprising a covalently closed planar ring having a delocalized [pi]-electron system comprising 4n+2 [pi] electrons, where n is an integer.
  • Aromatic rings may be formed by five, six, seven, eight, nine, or more than nine atoms.
  • Aromatics may be optionally substituted. Examples of aromatic groups include, but are not limited to phenyl, naphthalenyl, phenanthrenyl, anthracenyl, tetralinyl, fluorenyl, indenyl, and indanyl.
  • aromatic includes, for example, benzenoid groups, connected via one of the ring-forming carbon, atoms, and optionally carrying one or more substituents selected from an aryl, a heteroaryl, a. cycloalkyl, a non-aromatic heterocycle, a halo, a hydroxy, an amino, a cyano, a nitro, an alkylamido, an acyl, a Ci-Ce alkoxy, a Ci-C 6 alkyl, a Ci-C 6 aminoalkyl, alkylamino, an alkylsulfenyl, an alkylsulfinyl, an alkylsulfonyl, an sulfamoyl, or a trifluoromethyl.
  • an aromatic group is substituted at one or more of the para, meta, and/or ortho positions.
  • aromatic groups comprising substitutions include, but are not limited to, phenyl, 3-halophenyl, 4-halophenyl, 3-hydroxyphenyl, 4- hydroxyphenyl, 3-aminophenyl, 4-aminophenyl, 3-methylphenyl, 4-methylphenyl, 3- methoxyphenyl, 4-methoxyphenyl, 4-trifluoromethoxyphenyl, 3-cyanophenyl, 4-cyanophenyl, dimethylphenyl, naphthyl, hydroxynaphthyl, hydroxymethylphenyl, (trifluoromethyl)phenyl, alkoxyphenyl, 4-mo holin-4-ylphe yl, 4-pyrrolidin-l-ylphenyl, 4-pyrazolylphenyl, 4- triazolylphenyl, and 4-(2-oxopyrrolidin-l- yl
  • arylalkyl refers to a group comprising an aryl group bound to an alkyl group.
  • heteroaryl refers to an aromatic heterocycle. Heteroaryl rings may be formed by three, four, five, six, seven, eight, nine, or more than nine atoms. Heteroaryls may be optionally substituted. Examples of heteroaryl groups include, but are not limited to, aromatic C3-8 heterocyclic groups comprising one oxygen or sulfur atom or up to four nitrogen atoms, or a combination of one oxygen or sulfur atom and up to two nitrogen atoms, and their substituted as well as benzo- and pyrido-fused derivatives, for example, connected via one of the ring-forming carbon atoms.
  • non-aromatic heterocycle refers to a group comprising a non-aromatic ring wherein one or more atoms forming the ring is a heteroatom.
  • Non-aromatic heterocyclic rings may be formed by three, four, five, six, seven, eight, nine, or more than nine atoms.
  • Non- aromatic heterocycles may be optionally substituted.
  • non-aromatic heterocycles comprise one or more carbonyl or thiocarbonyl groups such as, for example, oxo- and thio-containing groups.
  • non-aromatic heterocycles include, but are not limited to, lactams, lactones, cyclic imides, cyclic thioimides, cyclic carbamates, tetrahydrothiopyran, 4H-pyran, tetrahydropyran, 1,3-dioxane, 1,4-dioxin, 1,4-dioxane, piperazine, 1,3-oxathiane, 1 ,4- oxathiane, tetrahydro-l,4-thiazine, 2H- 1,2- oxazine, maleimide, succinimide, barbituric acid, thiobarbituric acid, dioxopiperazine, hydantom, dihydrouracil, morphinone, trioxane, hexahydro- 1,3,5-triazine, tetrahydrothiophene, tetrahydrofuran, pyrroline, pyrrol
  • heteroatom refers to an atom other than carbon or hydrogen. Heteroatoms are typically independently selected from oxygen, sulphur, nitrogen, and phosphorus, but are not limited to those atoms. In embodiments in which two or more heteroatoms are present, the two or more heteroatoms may all be the same as one another, or some or all of the two or more heteroatoms may each be different from the others. [0058]
  • cyano refers to a group of formula -CN.
  • isocyanato refers to a group of formula -NCO
  • thiocyanato refers to a group of formula -CNS.
  • isothiocyanato refers to a group of formula -NCS.
  • R appearing by itself and without a number designation refers to a substituent selected from alkyl, cycloalkyl, aryl, heteroaryl (bonded through a ring carbon) and non-aromatic heterocycle (bonded through a ring carbon).
  • X is NR 2 and R is methyl.
  • the purine or pyrimidine base-pairing moiety (P is selected from the group consisting of cytosine, guanine, adenosine, uracil or thymine.
  • the purine or pyrimidine base pairing moiety can vary between the different monomeric units of the PMO.
  • the purine or pyrimidine base-pairing moiety is selected from nucleobase analogs, such as hypoxanthine or xanthine.
  • a "monomeric unit" of an oligonucleotide analog refers to one nucleotide unit of the oligonucleotide analog.
  • the length of the oligonucleotide analog described herein can comprise about 5 monomelic units to about 40 monomelic units; about 10 monomelic units to about 35 monomelic units; or about 15 monomelic units to about 35 monomelic units.
  • the conjugate of the invention is for the detection of at least one target nucleic acid molecule and wherein the at least one oligonucleotide analog can include a target complementary sequence. In other embodiments, the at least one oligonucleotide analog can include a target non-complementary sequence.
  • the "target non-complementary sequence” may have any sequence which does not interfere with the ability of the target complementary sequence to hybridize to a target nucleic acid molecule.
  • the target non-complementary sequence is not complementary to the rest of the oligonucleotide analog, i.e. the target-complementary region, or to that of a target nucleic acid molecule.
  • the target non-complementary sequence can also be selected such that it does not form an intramolecular secondary structure.
  • the target non-complementary sequence of the oligonucleotide analog may be designed such that it is bound to the nanoparticle, so that the target complementary sequence of the oligonucleotide analog is spaced away from the surface of the nanoparticle and is more accessible for hybridization with the target nucleic acid molecule.
  • the length and sequence of the target non-complementary sequence providing good spacing of the target complementary sequence away from the nanoparticle can be determined empirically known to persons of average skill in the art.
  • the target non-complementary sequence can comprise about 10 to 30 monomelic units, or can have about 6, 7, 8, 9 or 10 monomelic units.
  • the "target complementary sequence” relates to a sequence stretch that can hybridize to a sequence comprised in the target nucleic acid molecule.
  • the degree of complementarity is selected such that the target complementary sequence hybridizes to the target under hybridization conditions, such as high stringency hybridization conditions. Hybridization occurs preferably via Watson-Crick base pairs.
  • oligonucleotide analog in relation to an PMO as used herein relates to chemical derivatives of the PMOs, wherein, for example, the morpholine ring bears further substituents or the phosphoramidate group is substituted or modified, for example by replacement of one or both oxygen atoms with other atoms or groups or by substitution of the nitrogen atom by suitable substituents.
  • the oligonucleotide analog as described herein can comprise any of the following base sequences:
  • MOl 5'-CGG ACT ATG GAC ACC TTT TTT TTT TTT T-3' -Disulfide amide (SEQ ID NO: 1)
  • MOl SNP 5'-CGG ACT AGG GAC ACC TTT TTT TTT TTT T-3 '-Disulfide amide (SEQ ID NO: 2)
  • the oligonucleotide analog is covalently coupled to the nanoparticle via a functional group.
  • the functional group is typically included in the spacer portion of the oligonucleotide analog for covalently binding to the nanoparticle.
  • the functional group can include a thiol (SH) group, which can for example be used to covalently attach to the surface of the nanoparticle.
  • SH thiol
  • Oligonucleotides functionalized with thiols at their 3 '-end or 5 '-end can readily attach to gold nanoparticles. See for example, Mucic et al. Chem. Commun.
  • 555-557 (1996) which describes a method of attaching 3' thiol DNA to flat gold surfaces.
  • the thiol moiety also can be used to attach oligonucleotides to other metal, semiconductor, and magnetic colloids and to the other types of nanoparticles described herein.
  • Other functional groups for attaching oligonucleotides to solid surfaces include phosphorothioate groups (see, for example, U.S. Pat. No. 5,472,881 for the binding of oligonucleotide-phosphorothioates to gold surfaces), substituted alkylsiloxanes (see, for example Grabar et al., Anal. Ghent., 67, 735-743).
  • Oligonucleotides having a 5' thionucleoside or a 3 ' thionucleoside may also be used for attaching oligonucleotides to solid surfaces.
  • Other functional groups that are known to persons skilled in the art that can be used to attach the oligonucleotide analog to nanoparticles can include but are not limited to disulfides such as disulfide amides; carboxylic acids; aromatic ring compounds; sulfolanes; sulfoxides; silanes, not to mention a few.
  • the at least one oligonucleotide analog is coupled via its 3' or 5' end to the nanoparticle.
  • the conjugate of the invention comprises more than one oligonucleotide analogs, wherein the more than one oligonucleotide analogs have different base sequences.
  • the conjugate of the invention is for the detection of at least one target nucleic acid molecule, wherein more than one oligonucleotide analogs have different target complementary sequences.
  • the invention also provides a method for detecting a target nucleic acid molecule.
  • the method includes i) contacting at least one conjugate with a sample containing the target nucleic acid molecule, under conditions which allow the at least one conjugate and the target nucleic acid molecule to hybridize to each other, to form a conjugate:target nucleic acid molecule complex, and ii) detecting the formed complex.
  • the conjugate:target nucleic acid molecule complex can for example be detected by observing by the naked eye the colourimetric changes in an assay or by measuring the absorption of light in the visible, ultraviolet or infrared regions, for example by a spectrometer, such as an UV/Vis spectrometer or an IR spectrometer, or by any other suitable methods known in the art.
  • the detection of the conjugate:target nucleic acid molecule complex by observing the colorimetric changes in the assay is based on the optical properties (plasmon resonance) of the nanoparticles.
  • Such nanoparticles for example, metal nanoparticles are sensitive to changes in the surrounding medium.
  • the complex when detecting the conjugate:target nucleic acid molecule complex by the naked eye, the complex can be detected by a colour change.
  • the colour variation can for example be the result of the aggregation of the nanoparticles in the presence of a target nucleic acid molecule.
  • the observation of the colour change with the naked eye is* for example, made against a background of a contrasting colour.
  • the observation of a colour change can be facilitated by spotting a sample of solution containing the conjugate:target nucleic acid molecule complex on a solid white surface (such as, without limitation, silica or alumina TLC plates, filter paper, cellulose nitrate membranes, nylon membranes, or a C- 18 silica TLC plate) and allowing the spot to dry.
  • a solid white surface such as, without limitation, silica or alumina TLC plates, filter paper, cellulose nitrate membranes, nylon membranes, or a C- 18 silica TLC plate
  • the colour of the solution containing the conjugate:target nucleic acid molecule complex typically ranges from pink/red to purplish-red/purple, depending on the degree of hybridization between the target nucleic acid molecule and the conjugate.
  • one conjugate can be used for complex formation and detection.
  • the conjugate:target nucleic acid molecule complex can, for example be detected in the presence of an electrolyte in a concentration of about 50 mM to about 500 mM.
  • the method for detecting a target nucleic acid using one conjugate can result in high selectivity in recognizing specific nucleic acids in samples containing mixed sequences.
  • the high selectivity of detecting a nucleic acid using the method of the present invention is found to be much higher than that of other hybridization probes known in the art. It has been found that target concentrations as low as 0.5 nM could be detected. Therefore, the method of the present invention can be employed for the detection of rare somatic point mutations.
  • the two different conjugates can be used for complex formation and detection.
  • the two different conjugates may comprise oligonucleotide analogs that differ in their target-complementary region.
  • the target-complementary regions of the two conjugates bind to different sequence stretches of the same target nucleic acid. This leads to cross-linking of the conjugates via the target nucleic acid molecule. It has been found that by such a use of two different conjugates for complex formation and detection, the target nucleic acid can be detected under extremely low salt conditions, for example in the range of 0 to 5 mM of an electrolyte.
  • the method may further comprise the step of freezing the sample to accelerate cross-linking.
  • nucleic acid molecule used herein is a term of art that refers to a sequence of at least two base-sugar-phosphate combinations. Nucleotides are the monomeric units of nucleic acid polymers. Nucleic acid molecules can include deoxyribonucleic acid (DNA), ribonucleic acid (RNA) in the form of an oligonucleotide messenger RNA, anti-sense, plasmid DNA, parts of a plasmid DNA or genetic material derived from a virus for example.
  • DNA deoxyribonucleic acid
  • RNA ribonucleic acid
  • anti-sense plasmid DNA
  • parts of a plasmid DNA parts of a plasmid DNA or genetic material derived from a virus for example.
  • RNA may be in the form of a tRNA (transfer RNA), snRNA (small nuclear RNA), rRNA (ribosomal RNA), mRNA (messenger RNA), anti-sense RNA, and ribozymes.
  • DNA may be in the form of plasmid DNA, viral DNA, linear DNA, or chromosomal DNA or derivatives of these groups. In addition, these forms of DNA and RNA may be single, double, triple, or quadruple stranded. Therefore, the nucleic acid molecule can also refer to any of the following structures including primary, secondary, tertiary or quaternary structure(s).
  • the nucleic acid molecule also includes PNAs (peptide nucleic acids), phosphorothioates, and other variants of the phosphate backbone of native nucleic acids.
  • the nucleic acid molecule used herein can be derived from the genetic material of any living organisms, containing the specific information that allows a complete characterization of that organism.
  • the nucleic acid molecule used herein can for example be derived from the genome of an individual.
  • Such a nucleic acid molecule can contain single nucleotide variation in a specific nucleotide sequence, for example, single nucleotide polymorphisms (SNPs).
  • SNPs single nucleotide polymorphisms
  • Other variations in the specific nucleotide sequence can include, but are not limited to, insertions, deletions or duplications of one or more nucleotides.
  • SNPs are spread all over the entire human genome, and can for example be present at coding regions for proteins, in which the properties and/or expression of that protein can be altered. SNPs can also be present at non-coding regions where they can alter expression parameters and modify gene expression such as exon-excision or splicing. Other SNPs which do not modulate the properties and/or expression in proteins can for example, change the stability, maturation and location of messenger RNA.
  • target nucleic acid molecule refers to a nucleic acid molecule that comprises a nucleotide sequence capable of hybridizing to at least one conjugate of the present invention. Therefore, the target nucleic acid molecule can for example hybridize to a target complementary sequence of the phosphorodiamidate morpholino oligo or derivative thereof covalently coupled to the nanoparticle.
  • the target nucleic acid molecule can for example contain at least one single nucleotide polymorphism.
  • At least two different conjugates can be used for forming and detecting the conjugate:target nucleic acid molecule complex.
  • different regions of a target nucleic acid molecule can for example hybridize to the at least two different conjugates.
  • concentration of the target nucleic acid molecule can be used as long as a sufficient amount of target nucleic acid molecule can be detected using the method of the present invention.
  • the concentration of the target nucleic acid molecule can be in the range of about 0.05 nM to about 2000 nM; about 0.05 nM to about 1500 nM; about 0.05 nM to about 1200 nM; about 0.05 nM to about 1000 nM; about 0.05 nM to about 800 nM; about 0.05 nM to about 500 nM; about 0.05 nM to about 200 nM; about 0.05 nM to about 100 nM; about 0.05 nM to about 50 nM; about 0.05 nM to about 20 nM; about 0.1 nM to about 10 nM; about 0.1 nM to about 100 nM; about 5 nM to about 100 nM; about 5 nM to about 50 nM; about 10 nM to about 50 nM; or
  • Non-limiting examples of the target nucleic acid molecules used in the present invention are provided in Table 1 as follows.
  • Table 1 The specific sequences are underlined. The SNP positions are highlighted in bold.
  • the target nucleic acid molecule is detected in the presence of an electrolyte.
  • electrolyte refers to any material or substance containing electrolyte solids, for example free ions.
  • the substance or material can for example be in liquid form, containing the target nucleic acid(s), at least one conjugate and the ions in solution.
  • Typical ions can include but are not limited to sodium, potassium, calcium, magnesium, chloride, phosphate and bicarbonate.
  • Exemplary electrolytes that can be used include but are not limited to NaCl, MgCl 2 , NiCl 2 , NaBr, ZnCl 2 , MnCl 2 , BrCl, CdCl 2 , CaCl 2 , CoCl 2 , CoCl 3 , CuCl 2 , CuCl, PbCl 2 , PtCl 2 , PtCL,, KC1, RbCl, AgCl, SnCl 2 , BrF, LiBr, KBr, AgBr, NaN0 2 , Na 3 P0 4 , Na 2 HP0 4 , NaH 2 P0 4 , KH 2 P0 4 or K 2 HP0 4 .
  • the concentration of the electrolyte used can be in the range of about 0 mM to about 700 mM; about 0 mM to about 650 mM; about 0 mM to about 500 mM; about 10 mM to about 700 mM; about 20 mM to about 600 mM; about 50 mM to about 500 mM; about 1 mM to about 250 mM; about 1 mM to about 200 mM; about 1 mM to about 150 mM; about 1 mM to about 80 mM; about 1 mM to about 50 mM; about 5 mM to about 250 mM; about 10 mM to about 250 mM; about 20 mM to about 250 mM; about 50 mM to about 250 mM; about 80 mM to about 250 mM; or about 100 mM to about 250 mM.
  • the invention also provides a method for detecting at least one single nucleotide polymorphism in a target nucleic acid molecule.
  • the method includes i) contacting the target nucleic acid molecule with at least one conjugate to form a conjugate:target nucleic acid molecule complex; ii) measuring the melting transition temperature of the conjugate:target nucleic acid molecule complex; and iii) comparing the melting transition temperature of the complex in ii) with a control complex, wherein, if the conjugate complex has a lower melting transition temperature compared to the control complex, this indicates that the target nucleic acid molecule comprises at least one single nucleotide polymorphism.
  • control complex comprises a target nucleic acid molecule that does not contain a single nucleotide polymorphism.
  • conjugate:target nucleic acid molecule complex can comprise at least one conjugate in which at least one phosphorodiamidate morpholino oligonucleotide or a derivative thereof covalently coupled to the nanoparticle is hybridized to a target nucleic acid molecule.
  • the conjugate:target nucleic acid molecule complex can also comprise at least two different conjugates to a target nucleic acid molecule.
  • the conjugate:target nucleic acid molecule complex can for example include at least one single nucleotide polymorphism in the target nucleic acid molecule.
  • melting transition temperature refers to the temperature at which the half-maximum absorbance is measured.
  • the melting profiles of the conjugate:target nucleic acid molecule complexes can for example be obtained by measuring the solution absorbance after half hour incubation at a given temperature when a relatively stable value is reached. The melting profiles can be carried out using a spectrophometer with a temperature controller. In this context, without wishing to be bound by theory, the stability of the conjugate:target nucleic acid molecule complexes described herein depends on the amount of target DNA strands attached to the nanoparticles.
  • the initial dissociation of the DNA and PMO duplexes may not result in the aggregation of the nanoparticles. Only when the number of the binding nucleic acid decreased below a certain threshold value, aggregation of the nanoparticles derived by the London- van der Waals attractive force could start to proceed. Therefore, the initial release of a small amount of DNA (states a to b) exerts little effect on the optical signal. Once the nanoparticles start to aggregate, large-sized particles can be formed (states c to d). The increase of mass and decrease of surface charge density made the larger particles even less stable and tend to aggregate further until precipitating from the solution (state e).
  • the melting transition of the conjugate:target nucleic acid molecule complexes is irreversible and the aggregation can proceed rapidly. As a result, extremely steep melting curves occurring within 1°C for example, can be obtained.
  • the invention also provides use of at least one conjugate described herein for the detection of a target nucleic acid molecule.
  • the target nucleic acid molecule can include at least one single nucleotide polymorphism.
  • the invention further provides a kit for the detection of a target nucleic acid molecule.
  • the kit can contain at least one conjugate as described herein.
  • the kit can comprise one or more control nucleic acid molecules that are perfectly complementary to the target-complementary sequence of the at least one oligonucleotide analog of the conjugate.
  • the kit can comprise at least one target non- complementary sequence of the at least one oligonucleotide analog of the conjugate.
  • the kit can also comprise one or more solutions, for example a hybridization buffer.
  • the hybridization buffer can for example include but is not limited to phosphate, citrate, Tris, Hepes (4- (2- hydroxyethyl)-l-piperazineethanesulfonic acid), TAPS (N-Tris(hydroxymethyl)methyl-3- aminopropanesulfonic acid), MOPS (3-( ⁇ ⁇ 1 ⁇ ) ⁇ 3 ⁇ 8 ⁇ 1 ⁇ ⁇ acid), PIPES (Piperazine-l,4-bis(2-ethanesulfonic acid) buffers, hypersolutes (for example, mannosylglycerate) or any other buffer solution, optionally containing, denaturing agents, salts, inert polymers, surfactants, among others.
  • the one or more solutions in the kit can also include any one of the electrolytes as described herein.
  • the one or more solutions of the kit can be supplied in wells of one or more microplate(s), for example a 384-well microplate, or in containers for later application in the wells of the microplate(s).
  • the capping of the gold and silver (-40 nm) NPs with FSN was carried out by adding -0.05 wt% FSN to the colloid solutions.
  • the capping of -13 nm gold NPs with FSN was carried out by adding 0.6 mL 2 wt% FSN- 100.
  • the MO modified with disulfide amide at the 3' terminal was treated with 0.1 M DTT in 0.2 M phosphate buffered saline (PBS, pH 8.0) for 1 h, by activating the thiol groups; and then the thiolated MOs were purified using an NAP-5 column (GE Healthcare).
  • the purified thiolated MO samples were stored at 4 °C until further use.
  • the purified MOs were incubated with 5 mM tris(2- carboxyethyl)phosphine (TCEP) (pH 7.5) for 10 min. before mixing with the gold or silver NPs.
  • TCEP tris(2- carboxyethyl)phosphine
  • the final concentration of the NP conjugates was determined by measuring the SPR absorbance at 532 nm with a standard cuvette.
  • A sic
  • An ⁇ value of 1.5 x 10 8 L/(mol e cm) at 524 nm was used.
  • Example 4 Quantification of Morpholino Loaded on the Gold NPs.
  • the concentration of the NP conjugates was determined by measuring the absorbance of the conjugate solution with the UV- visible spectrophotometer.
  • the melting studies were carried out using a spectrophotometer with a temperature controller (Agilent G1 103A). The temperature accuracy is ⁇ 0.3°C.
  • the temperature was increased from 25 to 80 °C at 1 °C intervals with a holding time of 1 min at each point prior to each measurement.
  • the temperature was changed at 0.5 °C intervals in a narrow range ( ⁇ 5 °C) where the melting transition occurred. At each point, a fresh sample was used and the equilibrium time was 30 min prior to each measurement. Because the melting transition occurred within ⁇ 1 °C, the T m values were determined as the middle point of the transition.
  • Example 6 Study of Average Zeta Potential of Morpholino Loaded Gold NPs in Increasing
  • Table 2 shows the increase of negative charge of the NPs upon hybridization with the target DNA in increasing concentrations. No obvious change of NP surface charge has been measured when the DNA sequence is non-complementary to the MO probe. The stability of the NPs increased obviously as more target ssDNA strands were attached. In the presence of 5 nM DNA targets, the conjugates were stable upon the addition of 50 mM NaCl, indicating a sufficient amount of DNA strands were hybridized to the NP conjugates. When the concentration of the DNA target was lower, the amount of hybridized DNA decreased and the conjugates were less stable.
  • ni/PM 1.11 Ln[PM] + 49.86 (2)
  • Table 3 shows the predicted SNP discrimination ability of the current method at different concentration of PM targets/Because of the level-off of the SNP curve at higher concentrations (Figure 9), the detection selectivity became extremely high (a > 200: 1) when the concentration of the target PM was close or higher than 50 nM.
  • Example 8 Competitive Assay [00123] To validate the unusually high selectivity based on Example 7, a competitive assay was conducted at 48.5 °C. One aliquot of the conjugates solution contained 10 ⁇ of SNP target, while another aliquot contained 10 ⁇ of SNP target and 50 nM of PM target. After a 0.5 h-incubation, a distinct colour difference was observed ( Figure 10). No colour change was observed in the presence of the PM targets, while the solution became nearly colourless in the absence of the PM targets. The selectivity factor in this experiment is ⁇ 200: 1.
  • T m values of the conjugates hybridized with PM and different SNPs are shown in Table 4.
  • the order of stability of the systems i.e., T-A> T-G > T-T ⁇ T-C, is in agreement with that predicted for the Watson-Crick base pairs.
  • the differences of T m between the perfectly matched and SNP systems are generally ⁇ 10 °C, which enable accurate detection of a single-base substitution.
  • Conjugates of morpholino and 40 nm silver rianoparticles based on Example 3 are prepared to detect both alleles (i.e., a wild-type allele and a mutant of the allele) of a SNP at a specific locus.
  • the silver NP conjugates shows an absorption peak at -420 nm, well separated from that of the 40 nm gold NP conjugates.
  • Figure 12 shows the UV-Vis spectral features of the dispersed and aggregate MOl/Ag NP conjugates (the aggregation was obtained after 0.5-h incubation with 200 mM NaCl). Similar features of the silver NP system in responding to the DNA targets were observed.
  • the method can be used to identify the genotype of an individual.
  • the absorption peak of the gold NPs at 538 nm maintained while that of the silver NPs at 410 nm disappeared (trace 2).
  • the absorption peak of the silver NPs was observed in the case of the SNP sample (trace 3).
  • both absorption peaks were detected (trace 1).
  • the method can be used to identify the homozygous and heterozygous genotypes.
  • Example 11 Preparation of Thiolated Morpholino-modified metal NPs and Hybridization of two MO/Au NP Conjugates with a DNA Target
  • the NP conjugates were prepared by immobilizing thiolated 25-base MOs on Au NPs (average diameter 13 nm). Each MO strand used here contains both of a 10-base (Tio) spacer and a 15-base specific sequence for hybridization. The loading of MOs was carried out by incubating a mixing solution of thiolated MO and Au NPs for 2h at room temperature. To minimize the nonspecific adsorption of MOs on NP surfaces, the Au NPs were capped with non- ionic fluorosurfactants (e.g., Zonyl FSN) before mixing with the thiolated MOs. The resulting NP conjugates were dispersed in a 2 mM Tris buffer solution ( Figure 14b).
  • non- ionic fluorosurfactants e.g., Zonyl FSN
  • the surface charge of the NPs is around -20 mV.
  • the conjugate solution was stable for at least 3 months when stored at 5°C.
  • Two sets of NP conjugates (probes 1 and 2) were designed so that the DNA targets could act as linkers to align a pair of NP probes through hybridization with the NP-bound MO sequences ( Figure. 14a).
  • a solution of DNA target and the probes 1 and 2 ( ⁇ 2 nM each in 2 mM Tris buffer) containing 5 mM NaCl was prepared.
  • the DNA targets used in this method are obtained from Table 1. After incubation at room temperature for 24 h, no solution colour change was observed. However, after a freezing-thaw process, i.e., freezing the solution in a bath of dry ice (10 min) and then thawing it at room temperature, the solution colour changed from red to purple or gray, indicating the formation of target-linked larger particles ( Figure 14b). Without wishing to be bound by theory, the cross-linking of the MO/NP conjugates by DNA targets was accelerated by the freezing step.
  • the perfectly-matched sequence can be differentiated unambiguously from the single-base-mismatched strands (Fig. 15).
  • Table 5 shows the T m and FWHM values for different sequences.
  • the single-base substitutions generally reduced T m by ⁇ 12-14°C.
  • the order of the basepair stability of the MO-target duplexes was C:G > C:T > C:A > C:C, which is in good agreement with the destabilizing effects of mismatched basepairs observed for DNA duplexes known in the art. While one base deletion led to a decrease of 14.3°C in T m , the effect of one base insertion on the stability of the hybrid was much less significant (with a decrease of 3.3°C in T m ).
  • Table 5 The values of melting temperature and full width at half maximum of the first derivative for the melting profiles.

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Abstract

La présente invention concerne un conjugué comprenant une nanoparticule et au moins un analogue oligonucléotidique, ledit analogue oligonucléotidique étant un phosphorodiamidate morpholino oligo (PMO) ou un dérivé de celui-ci qui est couplé de manière covalente à la nanoparticule, des procédés de détection d'un acide nucléique cible utilisant le conjugué et une trousse comprenant le conjugué.
PCT/SG2011/000016 2010-01-12 2011-01-12 Reconnaissance d'acides nucléiques sélective vis-à-vis d'une séquence utilisant des sondes nanoparticulaires WO2011087456A1 (fr)

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CN102778492A (zh) * 2012-07-13 2012-11-14 首都师范大学 一种用于汞离子检测的电化学传感器及其制作方法和检测方法
CN102778492B (zh) * 2012-07-13 2014-07-02 首都师范大学 一种用于汞离子检测的电化学传感器及其制作方法和检测方法
WO2014168582A1 (fr) 2013-04-11 2014-10-16 Agency For Science, Technology And Research Analyse génétique à base de nanosondes
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WO2015041603A1 (fr) * 2013-09-17 2015-03-26 Agency For Science, Technology And Research Procédé de détection d'un variant génétique
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US10704101B2 (en) 2013-09-17 2020-07-07 Agency For Science, Technology And Research Method for detection of a genetic variant
WO2017091156A1 (fr) 2015-11-27 2017-06-01 Agency For Science, Technology And Research Détermination de méthylation d'acides nucléiques
US11021743B2 (en) 2015-11-27 2021-06-01 Agency For Science, Technology And Research Determination of nucleic acid methylation

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