SENSOR

Abstract

The present invention relates to a genetic probe for the detection of a single nucleotide polymorphism (SNP) or a single nucleotide modification of a target nucleic acid, the genetic probe comprising: a nanoparticle; an oligonucleotide probe anchored to the surface of the nanoparticle, comprising an oligonucleotide backbone with a tag incorporated therein via a linker group; and a reference probe anchored to the surface of the nanoparticle, wherein the reference probe comprises a marker; wherein either (a) the tag is an organic fluorescent tag and the marker is a transition metal-based fluorescent marker; or (b) the tag is a redox-active tag and the marker is a transition metal-based redox-active marker. The invention also relates to a composition or kit containing a probe of the invention, or to the use of a probe of the invention. The invention also relates to a method of determining the percentage of single nucleotide polymorphisms (SNPs) or single nucleotide modifications of a target nucleic acid in a pool of the target nucleic acid, or to a method of determining the status of a condition associated with a known SNP in a subject

Claims

1.-46. (canceled)

47. A genetic probe for the detection of a single nucleotide polymorphism (SNP) or a single nucleotide modification of a target nucleic acid, the genetic probe comprising: a nanoparticle; an oligonucleotide probe anchored to the surface of the nanoparticle, comprising an oligonucleotide backbone with a tag incorporated therein via a linker group; and a reference probe anchored to the surface of the nanoparticle, wherein the reference probe comprises a marker; wherein either (a) the tag is an organic fluorescent tag and the marker is a transition metal-based fluorescent marker; or (b) the tag is a redox-active tag and the marker is a transition metal-based redox-active marker.

48. The genetic probe of claim 47, wherein the nanoparticle is formed from a material selected from: metals, metal oxides, silica, graphene, and quantum dots.

49. The genetic probe of claim 47, wherein the tag is a planar aromatic or heteroaromatic moiety or a planar macrocyclic transition metal complex.

50. The genetic probe of claim 47, wherein the tag is a fluorescent tag based on any of the following chemical families: thiazine or cyanine or pyrene or xanthene or acridine or anthracene or anthraquinone.

51. The genetic probe of claim 47, wherein the tag is a redox-active tag based on any of the following chemical families: phenanthridines, phenothiazines, phenazines, acridines, anthraquinones; or is based on metal complexes containing intercalating ligands; or is based on a planar macrocyclic transition metal complex.

52. The genetic probe of claim 51, wherein the redox-active tag is a transition metal complex with a cyclidene [14] ligand as shown below: ##STR00019## where M is Ni(II) or Cu(II) and where there are optionally one or more pendant groups extending from the ring which are selected from hydroxyl, carboxyl, C1-4 alkyl, amino (NR2, where each R is independently selected from H and C1-4 alkyl), C1-C4 ether, and C1-C4 ester.

53. The genetic probe of claim 47, wherein the linker group is of formula (I): ##STR00020## wherein L is connected to the tag and is selected from C3-16 alkyl (e.g. C3-14 or C3-12 or C3-10 alkyl), C3-16 alkenyl (e.g. C3-14 or C3-12 or C3-10 alkenyl), and C3-16 alkynyl (e.g. C3-14 or C3-12 or C3-10 alkynyl), wherein one, two or three carbon atoms may optionally be substituted with a heteroatom independently selected from O, S and N, and wherein one, two, three or four hydrogen atoms may optionally be substituted with a group independently selected from hydroxyl, carboxyl, amino (NR2, where each R is independently selected from H and C1-4 alkyl), C1-C4 alkoxy, C1-C4 ether, C1-C4 thioether, nitro, nitrile, C1-C4 ester, phenyl, pyridinyl, pyrimidinyl, furanyl, pyrrolyl, thiophenyl, imidazolyl, and thiazolyl; A, B and Z are each independently selected from hydrogen, C1-4 alkyl, amino (NR.sub.2, where each R is independently selected from H and C1-4 alkyl), and C1-4 alkoxy.

54. The genetic probe of claim 47, wherein the marker is a transition metal-based fluorescent marker which is a complex of a transition metal with an aromatic ligand or a chelating carboxylate-based ligand.

55. The genetic probe of claim 47, wherein the marker is a transition metal-based redox-active marker that is selected from ferricyanide/ferrocyanide, ferrocene and derivatives thereof, hexacyanoruthenate, and hexacyanoosmate.

56. A composition that comprises a plurality of genetic probes as defined in claim 47.

57. A method of determining the percentage of single nucleotide polymorphisms (SNPs) or single nucleotide modifications of a target nucleic acid in a pool of the target nucleic acid, the method comprising: contacting the pool of target nucleic acid with an oligonucleotide probe capable of detecting the SNP or single nucleotide modification, wherein the oligonucleotide probe is substantially complimentary to the target polynucleotide, and wherein the oligonucleotide probe comprises an oligonucleotide backbone with a tag incorporated therein via a linker group, wherein either the tag is an organic fluorescent tag or a redox-active tag, and wherein the tag is in a position that is arranged to be paired with a nucleotide of the target nucleic acid to be interrogated, whereby (i) the light emission of the organic fluorescent tag differs in intensity depending on the nucleotide's identity or modified structure; or (ii) the electrical charge of the redox-active tag differs in intensity depending on the nucleotide's identity or modified structure; detecting the percentage change in the fluorescent emission intensity of the organic fluorescent tag when the pool of target nucleic acid is contacted by the oligonucleotide probe comprising the organic fluorescent tag, or detecting the percentage change in electrical charge intensity of the redox-active tag when the pool of target nucleic acid is contacted by the oligonucleotide probe comprising the redox-active tag; and determining the percentage of single nucleotide polymorphisms (SNPs) or single nucleotide modifications by comparing the percentage change in intensity of the tag to a calibration value that has been determined by linear regression of the percentage change in intensity of known standards.

58. The method according to claim 57, wherein the pool of target nucleic acid is in a sample or in situ in a single cell or a population of cells.

59. The method according to claim 57, wherein the sample comprises a cell lysate, a bodily fluid sample, or a nucleic acid sample.

60. The method according to claim 57, wherein the target nucleic acid comprises circulating DNA, optionally circulating tumour DNA (ctDNA), mRNA, or cDNA.

61. The method according to claim 57, wherein the target nucleic acid is amplified nucleic acid.

62. The method according to claim 57, wherein the target nucleic is from a subject who has, or is suspected to have, or is at risk of having, a condition associated with an SNP or single nucleotide modification.

63. The method according to claim 57, wherein the method further comprises the use of a second genetic probe.

64. A method of determining the status of a condition associated with a known SNP in a subject, the method comprising: providing a sample from the subject comprising a target nucleic acid, wherein the target nucleic acid may comprise the SNP; determining the percentage of the SNP in the sample relative to target nucleic acid not having the SNP in accordance with the method according to claim 57, wherein the percentage of the SNP is indicative of the status of the condition associated with the SNP in the subject.

65. A method determining the epigenetic status of a target nucleic acid of a subject, the method comprising determining the percentage of single nucleotide modifications of the target nucleic acid in accordance with the method according to claim 57, wherein the percentage of the single nucleotide modifications of the target nucleic acid is indicative of the epigenetic status of the target nucleic acid in the subject.

66. A kit for the detection and analysis of the ratio of a SNP and/or a single nucleotide modification of a target nucleic acid in a pool of the target nucleic acid, wherein the kit comprises: the genetic probe according to claim 47, or an oligonucleotide probe comprising an oligonucleotide backbone with a tag incorporated therein via a linker group, wherein either the tag is an organic fluorescent tag or a redox-active tag, and wherein the tag is in a position that is arranged to be paired with a nucleotide of the target nucleic acid to be interrogated; and a first standard target nucleic acid for use as a standard in a calibration, wherein the first target nucleic acid comprises the SNP or single nucleotide modification to be analysed; and a second standard target nucleic acid for use as a standard in calibration, wherein the second target nucleic acid does not comprise the SNP or single nucleotide modification to be analysed.

Description

[0296] Embodiments of the invention will now be described in more detail, by way of example only, with reference to the accompanying drawings.

[0297] FIG. 1Schematic of the SNP sensing mechanism, whereby the signal response from an attached tag (normally a fluorophore) to duplex formation with a target strand depends upon the identity of the nucleobase opposite (or opposite and adjacent to) the tag.

[0298] FIG. 2Schematic of the structure of an anthracene-tagged fluorescent probe. The stereochemistry of the tag is L (R,R). The linker group can be varied, for example n=5.

[0299] FIG. 3Calibration of % A variant present versus % change in emission intensity, performed on 1 mL samples with 1 M probe and 1.5 M target in 10 mM sodium phosphate buffer pH 7 and 100 mM NaCl, .sub.ex=350 nm, 293 K. ex=350 nm, em=426 nm, rt.

[0300] FIG. 4Calibration of % C variant present versus % change in emission intensity, performed on 1 mL samples with 1 M probe and 1.5 M target in 10 mM sodium phosphate buffer pH 7 and 100 mM NaCl, .sub.ex=350 nm, 293 K. ex=350 nm, em=426 nm, rt.

[0301] FIG. 5Calibration of % C variant present versus % change in emission intensity, performed on 1 mL samples with 1 M probe and 1 M target in 10 mM sodium phosphate buffer pH 7 and 100 mM NaCl, .sub.ex=350 nm, 293 K. ex=350 nm, em=426 nm, rt.

[0302] FIG. 6Anthacene-tagged DNA strands were functionalised with a tether consisting of a 1,2-dithiolane end group (left) by reacting a strand of DNA containing an aminoalkyl group with thiooctic acid under standard peptide coupling conditions.

[0303] FIG. 7Shows the structure of the RubpySS probe.

[0304] FIG. 8AuNPs containing (left) DNA-dithiolane strands, AuNP-A, and (right) both DNA-dithiolane strands and Ru(bipy).sub.3-tagged bis-dithiolane molecules, AuNPB, for ratiometric sensing.

[0305] FIG. 9Calibration of % A variant present versus % change in emission intensity using AuNP-A particles. Error bars are standard deviation of three repeats with three separate AuNP syntheses at RT. .sub.ex=350 nm, .sub.em=426 nm.

[0306] FIG. 10Calibration of % A present versus ratio of anthracene signal (426 nm)/ruthenium signal (630 nm) for AuNPB. Error bars are standard deviation of three repeats at RT.

[0307] FIG. 11Percentage change in fluorescence of 173 base synthetic oligonucleotide strands once fully hybridised with the 5 L-anthracene tagged oligonucleotide probe. The error bars are the standard deviation from 3 repeats. 1 M of target with 1 M of probe and that is able to detect the A/T SNP difference in a BRAF gene.

[0308] FIG. 123% agarose gels to show the 150 base pair PCR product. The gels represent two different PCRs. Gel A: Lanes 1-4 show the PCR product of the genomic DNA from tumour tissue and lanes 5-8 show the PCR product of the genomic DNA from healthy tissue.

[0309] FIG. 13Sequencing data of the PCR product from the genomic DNA from healthy tissue (wild type).

[0310] FIG. 14Sequencing data of the PCR product from the genomic DNA from tumour tissue.

[0311] FIG. 15Graph (A) shows the full fluorescence emission intensity at 426 nm of the unpurified PCR product samples when added to the probe 5-AGATTTCXCTGTAGC-3 (BRAF, X=anthracene 5 L, 1 M); Graph (B) shows the data focused between 3000 and 5000 units to demonstrate the difference between each sample more clearly. The error bars show the standard deviation from 3 experimental repeats.

EXAMPLES

Example 1Preparation of Genetic Probe

Anthracene Probe Synthesis

Synthesis of 2-(anthracen-9-yloxy) Acetate

[0312] ##STR00010##

[0313] In a round bottomed flask anthrone (5.83 g) and K.sub.2CO.sub.3 (4.15 g) were dissolved in degassed acetone (200 mL) and stirred for 15 minutes in the dark. Ethyl Bromoacetae (3.3 mL) was then syringed into the flask and the reaction refluxed under N.sub.2 overnight in the dark. The solution was filtered to remove the K.sub.2CO.sub.3 and the solvent removed in vacuo. The solid was re-dissolved in DCM (100 mL) and washed with water (50 mL) before drying with MgSO.sub.4. The solvent was removed by reduced pressure and the solid (compound 2) purified by column chromatography (10% hexane: DCM) giving a yellow solid (1.91 g, 23%).

[0314] Rf=0.54 in 10% hexane: DCM; .sup.1HNMR (300 MHz, CDCl.sub.3) 8.23 (2H, d, J 8.5, H.sub.7), 8.09 (1H, s, H.sub.12), 7.84 (2H, d, J 8.1, H.sub.10), 7.28-7.44 (4H, m, H.sub.8H.sub.9), 4.66 (2H, s, H.sub.4), 4.24 (2H, q, J 7.1, H.sub.2), 1.23 (3H, t, J 7.1, H.sub.1);

[0315] TOF-MS-ES.sup.+ [M+H].sup.+ 281.12 [M+Na].sup.+ 303.09

Synthesis of 2-(anthracen-9-ylox) acetic Acid

[0316] ##STR00011##

[0317] In a round bottomed flask, 1 g of 2-(anthracen-9-yloxy) acetate was dissolved in 10% NaOH in EtOH 1:1 (200 mL), this was refluxed overnight in the absence of light under N.sub.2. The EtOH was removed in vacuo and water was added (400 mL), conc HCl was then added dropwise whilst swirling the solution until a creamy precipitate appears and does not disappear upon mixing, to give a cream solid (800 mg, 89%).

[0318] .sup.1HNMR (300 MHz, CDCl.sub.3) 8.34 (1H, s, H.sub.10), 8.31 (2H, d, J 8.3, 2H.sub.5), 8.06 (2H, d, J 8.0, 2H.sub.8), 7.49-7.60 (4H, m, 2H.sub.6, H.sub.7), 4.91 (2H, s, H.sub.2); .sup.13CNMR (100 MHz, CDCl.sub.2) 172.1 (C.sub.1), 149.5 (C.sub.3), 132.6 (2C.sub.9), 128.9 (2C.sub.8), 127.2 (2C.sub.6), 126.1 (2C.sub.7), 125.7 (2C.sub.6), 123.6 (C.sub.10), 121.6 (2C.sub.5), 71.2 (C.sub.2); TOF-MS-ES.sup. [MH].sup.+ 251.06

Synthesis of 2-(anthracen-9-yloxy)-N-((2S,3S)-1,3-dihydroxybutan-2-yl)acetamide

[0319] ##STR00012##

[0320] In a round bottomed flask the 2-(anthracen-9-ylox) acetic acid (300 mg) was dissolved in dry DMF (20 mL) under inert conditions. 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) (0.455 g) was added and the solution stirred for 15 minutes in the dark. L-threoninol (0.126 g) and DIPEA (0.21 mL) were added and the solution stirred at 40 C. for 40 hours. The solvent was removed in vacuo the product was then purified by column chromatography (5% MeOH: DCM) giving a pale yellow solid (203 mg, 49%).

[0321] Rf=0.27 in DCM: 5% MeOH; .sup.1HNMR (300 MHz, CDCl.sub.3) 8.38 (1H, s, H.sub.15), 8.30-8.35 (2H, m, H.sub.10), 8.06-8.11 (2H, dd J 8.4 & 2.0, H.sub.13), 7.49-7.60 (4H, m, H.sub.11, H.sub.12), 4.74 (2H, d, J 5.8, H.sub.7), 4.15-4.23 (1H, m, H.sub.2), 4.07-4.14 (1H, m, H.sub.3), 3.83 (2H, m, H.sub.4), 1.35 (3H, d, J 6.4, H.sub.1); .sup.13CNMR (100 MHz, CDCl.sub.3) 132.1 (C.sub.14), 128.3 (C.sub.13), 125.7 (C.sub.11), 125.3 (C.sub.12), 124.0 (C.sub.9), 122.9 (C.sub.15), 121.1 (C.sub.10), 73.2 (C.sub.7), 65.6 (C.sub.2), 61.6 (C.sub.4), 55.8 (C.sub.3), 19.3 (C.sub.1);

[0322] TOF-MS-ES.sup.+ [M+Na].sup.+ 362.1

Synthesis of Anthracene-Tagged Fluorescent Probes

[0323] The probes were synthesised using standard phosphoramidite automated DNA synthesis, purified by MS and characterised by mass spectrometry (ESMS).

[0324] The stereochemistry of the tag in each case was L (R,R), with the linker group as n=5 (see below). Each probe was a 15-mer, with the eighth position occupied by the anthracene tag.

##STR00013##

[0325] The anthracene probe used for this work is a randomly designed 15-mer which will not display any secondary structure. P.UM is probe sequence; P.AF is probe sequence with thioctic acid modification. The probe strands (P.UM, P.AF, P.1L and P.1D) are the strands that are bound to the gold nanoparticles and used to detect the target strands (DNA-T.1, DNA-T.2).

[0326] Each strand was synthesised on an Applied Biosystems 9394 DNA/RNA Synthesizer within the group. They were purified by reverse phase HPLC and characterised by mass spectrometry.

DNA Synthesis with Amine-Termination

[0327] The DNA was first modified with an amine group to which the activated ester could be coupled. A 5 amine C6 modification was added as the final base on the DNA synthesiser, giving an amine-terminated probe strand.

[0328] Sequences were synthesised on an Applied Biosystems 394 DNA/RNA synthesizer as per normal oligo synthesis, using standard Syn Base CPG 1 M (Link Technologies).

[0329] The 2-(anthracen-9-yloxy)-N-((2S,3S)-1,3-dihydroxybutan-2-yl)acetamide was integrated into the oligonucleotide backbone via the threoninol unit.

[0330] Base de-protection was performed off column, in 1M ammonia heated to 55 C. for 6 hours using a heating block (Grant Instruments). The ammonia is removed under reduced pressure.

[0331] The sequences were then purified by semi-prep HPLC (Dionex UVD 1705) using a Clarity 5 Oligo-RP 1504.6 mm (Phenomenex) column. After collecting the purified peak, the solvent is removed in vacuo (Thermo Scientific SAVANT SPD 131 DDA) before dissolving in water and passing through a NAP-10 column (GE Healthcare). The samples were then characterised by Mass Spectrometry and analytical HPLC (Shimadzu UFLC) on a Clarity 5 Oligo-RP 1504.6 mm 5 micron (Phenomenex) column.

RNA Synthesis with Amine-Termination

[0332] Sequences were synthesised on an Applied Biosystems 394 DNA/RNA synthesizer as per standard RNA oligo synthesis, using standard RNA bases (Link Technologies). The sequences were left on column after the synthesis; columns were then removed from the synthesiser and washed with 2.5 mL ammonia: ethanol (3:1) using syringes, washing through every 30 minutes for 2 hours. The mixture was then poured into a vial, washing the column once more with 1 mL ammonia: ethanol (3:1). The solution was then heated for 6 hours at 55 C. Once cooled, the solution was dried on a rotor evaporator. The dry sample was vortexed in 0.5 mL of TBAF (1 M in THF) and left overnight.

[0333] The samples were then passed through a NAP-10 column (GE Healthcare) using water as eluent. The solution was then concentrated, 375 mL of sample was placed into a 2 mL Eppendorf, to this 0.2 mL of Sodium acetate (0.1 M) and 1.4 mL of iso-propanol were added. The mixture was centrifuged in a refrigerated centrifuge at 15000 rpm at 4 C. for 15 minutes. The supernatant was removed in a speed vac, the pellets were recombined in 1 mL water, vortexing to re-dissolve them. The samples were then purified by semi-prep HPLC using a Clarity 5 Oligo-RP 1504.6 mm column. The solvent was then removed and the samples passed through a NAP-10 column to desalt. The samples were then characterised by Mass Spectrometry and analytical HPLC on a Clarity 5 Oligo-RP 1504.6 mm 5 micron column.

Synthesis of 2,5-dioxopyrrolidin-1-yl 5-(1,2-dithiolan-3-yl)pentanoate=NHS ester of Thioctic Acid

[0334] ##STR00014##

[0335] In a round bottomed flask 1.84 g N-(3-Dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride was dissolved in anhydrous DCM (20 mL). 1.7 mL of N,N-Diisopropylethlyamine (DIPEA) was added the reaction and the reaction was allowed to stir for 10 minutes. The reaction vessel was placed in an ice bath before 1.29 g of N-Hydroxy succinimide (NHS) was added to the mixture and allowed to stir. 1.643 g of thioctic acid was firstly dissolved in 10 mL of anhydrous DCM, then added to the reaction over 5 minutes. The reaction was then left to stir overnight at room temperature. The reaction was washed with HCl (aq) (5% [v/v], 50 mL) twice and then with 50 mL water. The organic layer was dried over Na.sub.2SO.sub.4 and then solvents concentrated in vacuo. The residue was dry loaded onto Na.sub.2SO.sub.4 and purified by column chromatography (50% EtAc: 50% Hexane) giving the purified product (compound 1) as a yellow powder (0.451 g, 1.49 mmol, 19%).

[0336] Rf=0.57 in EtAc: Hexane 50:50; .sup.1HNMR (300 MHz, DMSO) 3.56-3.65 (1H, m, H.sub.3), 3.12-3.21 (2H, m, H.sub.1), 2.81 (4H, s, H.sub.10), 2.69 (2H, t, J 7.1, H.sub.7), 2.38-2.46 (1H, m, H.sub.2), 1.84-1.93 (1H, m, H.sub.2), 1.4-1.7 (6H, m, H.sub.4H.sub.5H.sub.6); .sup.13CNMR (100 MHz, DMSO) 170.2 (2C.sub.9), 168.9 (C.sub.8), 55.9 (C.sub.3), 40.1 (C.sub.2), 38.1 (C.sub.1), 33.8 (C.sub.4), 30.0 (C.sub.2), 27.6 (C.sub.5), 25.4 (2C.sub.10), 24.0 (C.sub.6),

[0337] TOF MS EI.sup.+ [M].sup.+ 303.09

Coupling Oligonucleotide to Thioctic Acid Binding Agent

[0338] Amine-modified oligonucleotide was dissolved in sodium carbonate buffer (100 L, 0.1 M). A 5 molar excess of the NHS ester of thioctic acid was added dissolved in DMSO (5 L).

[0339] The reaction was vortexed and heated overnight at 37 C. The reaction was then passed through a NAP-5 column (GE Healthcare) using TEAA buffer (0.1 M) as eluent. The modified oligonucleotide was then purified by HPLC and characterised by Mass Spectrometry.

##STR00015##

Thioctic Acid Phosphoramidite Synthesis

Synthesis of 5-(1,2-dithiolan-3-yl)-N-(2-hydroxyethyl)pentanamide (Compound 7)

[0340] ##STR00016##

[0341] In a round bottomed flask, thioctic acid (0.71 g) and HOBt (0.46 g) were dissolved in dry DMF (10 mL) and stirred over ice. To this, EDC.HCl (0.66 g) was added and the mixture was stirred for 1 hour, still over ice. The solution was allowed to warm to room temperature for 1 hour. In a separate flask, 4-ethylmorpholine (0.39 g) and 2-aminoethan-1-ol (0.15 g) was dissolved in dry DMF (5 mL), this was then added to the thioctic acid containing solution and then allowed to stir overnight. The solution was washed with DCM: H.sub.2O, the DCM was then removed under reduced pressure. The product was purified by column chromatography on silica (5% MeOH, CHCl.sub.3) giving the product as a yellow solid (455 mg, 73%).

[0342] Rf=0.41 in CHCl.sub.3: 5% MeOH; .sup.1HNMR (300 MHz, CDCl.sub.3) 6.57 (1H, s, H.sub.9), 3.58-3.77 (2H, m, H.sub.11), 3.45-3.56 (1H, m, H.sub.3), 3.28-3.40 (2H, m, H.sub.10), 3.20-2.97 (2H, m, H.sub.1), 2.40 (1H, dq, J 12.6, 6.1, H.sub.2a), 2.16 (2H, t, J 7.3, H.sub.7), 1.85 (1H, dq, J 13.2, 6.7, H.sub.8b), 1.52-1.72 (4H, m, H.sub.4H.sub.6), 1.33-1.45 (2H, m, H.sub.5); .sup.13CNMR (100 MHz, CDCl.sub.3) 174.2 (C.sub.8), 61.9 (C.sub.11), 56.4 (C.sub.3), 42.4 (C.sub.10), 40.3 (C.sub.2), 38.5 (C.sub.1), 36.3 (C.sub.2), 34.6 (C.sub.4), 28.9 (C.sub.5), 25.4 (C.sub.6); TOF-MS-ES.sup.+ [249.1].sup.+

Synthesis of 2-(5-(1,2-dithiolan-3-yl)pentanamido)ethyl (2-cyanoethyl) diisopropylphosphoramidite (Compound 8)

[0343] ##STR00017##

[0344] In a round bottomed flask, the 5-(1,2-dithiolan-3-yl)-N-(2-hydroxyethyl)pentanamide (125 mg) was dissolved in dry DCM (5 mL) under an inert atmosphere. To this DIPEA (0.716 mL) was added. (i-Pr.sub.2N)PClO(CH.sub.2).sub.2CN (0.314 mL) was added dropwise to the reaction mixture, the reaction was then left to stir for 2 hours. Once the reaction was complete, degassed EtAc (5 mL) was added, the solution was then washed with degassed Na.sub.2CO.sub.3 (2 M, 250 mL) then degassed brine (50 mL) before being dried over Na.sub.2SO.sub.4. The solution was filtered and dried under reduced pressure before being purified by column chromatography on activated alumina (EtAc: Hexane: TEA, 50:49:1) giving a yield of 75%.

[0345] Rf; 0.9; .sup.1HNMR (300 MHz, CD.sub.3CN) 3.44-3.90 (7H, m, H.sub.3H.sub.11H.sub.12H.sub.14) 3.34-3.44 (2H, m, H.sub.10) 2.99-3.17 (2H, m, H.sub.1) 2.60 (2H, t, J 6.2, H.sub.15) 2.40 (1H, dtd, J 12.9, 6.6, 5.5, H.sub.2) 2.13 (2H, t, J 7.5, H.sub.7) 1.84 (1H, dq, J 12.7, 7.0, H.sub.2) 1.5-1.73 (4H, m, H.sub.4H.sub.6) 1.33-1.47 (2H, m, H.sub.5) 1.12 (12H, dd, J 6.9, 4.6, H.sub.13); .sup.13CNMR (100 MHz, CD.sub.3CN) 173.0 (C.sub.8), 117.8 (C.sub.16), 62.5 (C.sub.11), 58.9 (C.sub.14), 56.9 (C.sub.3), 54.6 (C.sub.7), 43.3 (C.sub.12), 40.6 (C.sub.10), 38.8 (C.sub.2), 36.1 (C.sub.1), 34.8 (C.sub.4), 29.0 (C.sub.5), 25.6 (C.sub.6), 24.6 (C.sub.13), 20.4 (C.sub.15); .sup.31PNMR (120 MHz, CD.sub.3CN) 148.0;

[0346] TOF-MS-ES.sup.+ [M+H].sup.+ 450, [M+Na].sup.+ 472.

Synthesis of ruthenium-4,4-Di(5-lipoamido-1-pentoxy)-2,2-bi-pyridine (RubpySS)

[0347] ##STR00018##

[0348] The ruthenium based probe, ruthenium-4,4-di(5-lipoamido-1-pentoxy)-2,2-bi-pyridine (RubpySS) was synthesised in accordance with Adams, S. J., et al, ACS Appl. Mater. Interfaces 6, 11598-11608 (2014).

Coupled Oligonucleotides

[0349]

TABLE-US-00001 MassSpec Probe Sequence ES+ P.UM TGGACTCTCTCAATG 4696[M+H].sup.+ P.AF W-TGGACTCTCTCAATG 4911[M].sup.+ P.1L W-TGGACTCLCTCAATG 5008[M].sup.+ P.1D W-TGGACTCDCTCAATG 5008[M].sup.+ P.5L W-CAUUGAGXGAGUCCA 5065[M].sup.+ m.P.1L W-AAAAATGGACTCLCTCA 6573[MH].sup. ATG l.P.1L W-AAAAAAAAAATGGACTCLC 8139[MH].sup. TCAATG DNA-T.1 CATTGAGAGAGTCCA 4601[M+H].sup.+ DNA-T.2 CATTGAGAAAGTCCA 4585[M] RNA-T.1 CAUUGAGAGAGUCCA 4798[MH].sup. RNA-T.2 CAUUGAGAAAGUCCA 4782[MH].sup. DNA-T.3 AGCTGAGACGCGACT 4602[M] DNA-T.4 AGCTGAGCCGCGACT 4577[MH].sup. RNA.1 Q-CAAUCAGGGUCGACGAGA 6458[M] A RNA.2 UUCUCGUCGACCCUGAUUG 5952[M] W =thioctic acid modification L =1L anthracene probe D =1D anthracene probe X =5L anthracene probe Q =Compound 8 modification

[0350] The numbers 1 or 5 refer to the carbon linker length between the anthracene molecule and the threoninol unit. The L and D indicate the isomer of threoninol used in the synthesis.

Gold Nanoparticle (AuNP) Synthesis

[0351] Particles are grown by the particle seeding method starting with 13 nm particles, which are synthesised as described below. Initially stock solutions of the reactants were made up as: 5 mM HAuCl.sub.4.H.sub.2O (100 mg in 50 mL deionised H.sub.2O), 57 mM ascorbic acid (500 mg in 50 mL deionised H.sub.2O) and 34 mM trisodium citrate dehydrate (500 mg in 50 mL deionised H.sub.2O).

13 nm Gold Nanoparticles

[0352] All glassware was soaked in aqua regia for at least 30 minutes prior to reaction, the glassware was then rinsed 10 times with deionised water and placed in an oven prior to use. In a 250 mL round bottomed flask fitted with a condenser, 100 mL of 2.75 mM citrate buffer (75:25 sodium citrate: citric acid) was heated until boiling, ensuring a vortex is formed by the vigorous stirring. After 15 minutes of boiling, 1.6 mg of Ethylenediaminetetraacetic acid (EDTA) was added to the solution. In a separate flask, 25 mL of HAuCl.sub.4.3H.sub.2O (Sigma Aldrich) (8.5 mg) is placed in an oven to heat until 90 C. The solution of gold was then added rapidly to the centre of the vortex of the citrate buffer solution and allowed to boil for 20 minutes. After 20 minutes the heat is turned off, and the solution is allowed to cool to room temperature still with vigorous stirring.

[0353] SPR=519 nm, number distribution=12 nm (3 nm), intensity distribution=21 nm (6 nm)

25 nm Gold Nanoparticles

[0354] A solution of 30 mL 13 nm AuNP (2 nM) was diluted to 40 mL with deionised water in a 250 mL three necked round bottomed flask. The solution was vigorously stirred. Two solutions were then made up using the stock solutions, solution A=1 mM HAuCl.sub.4.H.sub.2O (20 mL) solution B=2.85 mM ascorbic acid and 1.7 mM trisodium citrate dehydrate (20 mL). Solution A and B were then added to the AuNP solution using a peristaltic pump over 45 mins. Once added, the AuNP solution was refluxed for 30 minutes and allowed to cool to room temperature.

[0355] Number distribution=22 nm (5 nm), intensity distribution=31 nm (8 nm)

50 nm Gold Nanoparticles

[0356] A solution of 9 mL 25 nm AuNP (0.7 nM) was diluted to 40 mL with deionised water in a 250 mL three necked round bottomed flask. The solution was vigorously stirred. Two solutions were then made up using the stock solutions, solution A=1 mM HAuCl.sub.4.H.sub.2O (20 mL) solution B=2.85 mM ascorbic acid and 1.7 mM trisodium citrate dehydrate (20 mL). Solution A and B were then added to the AuNP solution using a peristaltic pump over 45 mins. Once added, the AuNP solution was refluxed for 30 minutes and allowed to cool to room temperature, the solution was neutralised with 0.01 M NaOH.

[0357] Number distribution=38 nm (9 nm), intensity distribution=56 nm (16 nm)

100 nm Gold Nanoparticles

[0358] A solution of 40 mL 50 nm AuNP (80 pM) was placed in a 250 mL three necked round bottomed flask. The solution was vigorously stirred. Two solutions were then made up using the stock solutions, solution A=4 mM HAuCl.sub.4.H.sub.2O (20 mL) solution B=11.4 mM ascorbic acid and 3.4 mM trisodium citrate dehydrate (20 mL). Solution A and B were then added to the AuNP solution using a peristaltic pump over 45 mins. Once added, the AuNP solution was refluxed for 30 minutes and allowed to cool to room temperature.

[0359] SPR=561 nm, number distribution=68 nm (20 nm), intensity distribution=105 nm (32 nm) (100%)

Characterising AuNP

Sizing

[0360] 1 mL of 2 nM AuNP sample is placed into a disposable DT50012 cuvette (SARSTEDT) before sizing the particles on a Zetasizer NANO (Malvern). Each sample was run 12 times, repeating this 3 times and taking an average of the value.

Transmission Electron Microscopy

[0361] Using tweezers to hold the copper grid (3 mm, FORMVAR) in a clean and safe area, 20 L of 2 nM sample of AuNP is pippeted onto the grid. The sample is left to settle for 20 minutes, after 20 minutes wick away excess using filter paper. The samples were then imaged using a JEOL 1200 Transmission Electron Microscope. Coated AuNP procedure

Coating AuNP Procedures

[0362] Coating 13 nm AuNP with DNA and RubpySS

[0363] In an Eppendorf containing a magnetic stirrer, citrate coated AuNPs (13 nm, 3 nM) are made up in 10 mM pH 7.0 phosphate buffer. Thioctic acid modified DNA (0.33 M) is added to the AuNPs and stirred for 2 minutes before the solution is sonicated for 20 seconds. This process is repeated a further two times giving a final DNA concentration of 0.98 RubpySS (1.5 M) probe is added to the particles and allowed to stir for 20 minutes. This process is repeated, giving a final RubpySS concentration of 3 The particles are passed through a Sephadex G-50 column (stored in 20% ethanol) with deionised water as eluent. A final UV-vis spectrum is taken to confirm concentration.

TABLE-US-00002 SPR Size: number Size: intensity Particle (nm) distribution (nm) distribution (nm) P.AF-AuNP-Ru 524 14 (4) 40 (19) 97% P.1L-AuNP-Ru 524 14 (4) 34 (17) 96% P.1D-AuNP-Ru 524 14 (4) 23 (9) 87% 286 (97) 10% l.P.1L-AuNP-Ru 524 16 (5) 36 (6) 96% P.5L-AuNP-Ru 524 15 (4) 35 (16) 96%
Coating 100 nm AuNP with P.1L and RubpySS

[0364] In an Eppendorf containing a magnetic stirrer, citrate coated AuNPs (100 nm, 40 pM) are made up in 10 mM pH 7.0 phosphate buffer. P.1L (0.25 M) is added to the AuNPs and stirred for 2 minutes before the solution is sonicated for 20 seconds. This process is repeated a further two times giving a final DNA concentration of 0.75 M. RubpySS (1.13 M) probe is added to the particles and allowed to stir for 20 minutes. This process is repeated, giving a final RubpySS concentration of 2.3 M. The particles are then spun down to form a pellet at 13000 rpm for 90 seconds. The supernatant is removed, with care taken to not disturb the pellet. The particles are re-dispersed in deionised water.

[0365] SPR=565 nm, number distribution=73 nm26 nm, intensity distribution=10638 nm.

Coating 13 nm AuNP with siRNA and RubpySS

[0366] In an Eppendorf containing a magnetic stirrer, citrate coated AuNPs (13 nm, 3 nM) are made up in 10 mM pH 7.0 phosphate buffer. RNA.1 (0.33 M) is added to the AuNPs and stirred for 2 minutes before the solution is sonicated for 20 seconds. The UV-vis spectrum of the particles is taken prior to purification. This process is repeated a further two times giving a final RNA.1 concentration of 0.98 M. RubpySS (1.5 M) probe is added to the particles and allowed to stir for 20 minutes. This process is repeated, giving a final RubpySS concentration of 3 M. The particles are passed through a Sephadex G-50 column (stored in 20% ethanol) with deionised water as eluent. A final UV-vis spectrum is taken to confirm concentration. The particles are made up in 10 mM pH 7.0 phosphate buffer and 100 mM NaCl. To this solution RNA.2 (0.4 M) is added.

[0367] SPR=524 nm, number distribution=15 nm3 nm, intensity distribution=30 nm 15 nm.

Fluorescence Testing

[0368] A 1 M solution of the duplex DNA was made up with 10 mM phosphate buffer (pH 7.0) and 100 mM NaCl, the solution is thoroughly mixed using a pipette. For DNA-AuNP samples, 2.5 nM AuNP sample was made up with 10 mM phosphate buffer (pH 7.0) and 100 mM NaCl, the solution is thoroughly mixed using a pipette.

[0369] For samples run on Shimadzu RF-5301 PC Spectrofluorophotometer, anthracene .sub.ex=350 nm .sub.em=370-550 nm, slit widths for excitation were 5 nm and emission 10 nm. Each run dwell time 1.0 second, 1 nm bandwidth 1 accumulation.

[0370] For samples run on FLSP920 Times Resolved Spectrometer (Edinburgh), anthracene .sub.ex=350 nm .sub.em=390-550 nm, slit widths for excitation 5 nm and emission 10 nm. Each run dwell time 1.0 second, 1 nm bandwidth 1 accumulation. For the ruthenium probe, .sub.ex=465 nm .sub.em=580-800 nm, slit widths for excitation 15 nm and emission 15 nm. Each run dwell time 1.0 second, 3 nm bandwidth 3 accumulations.

Example 2

Read-Out of the Allelic Ratio of Nucleobase Variants in Target Strands of DNA and RNA Using Fluorophore-Tagged Probes

1. INTRODUCTION

[0371] Single Nucleotide Polymorphisms (SNPs), variations in one nucleobase at one site in a particular sequence of genomic DNA, play an important role in the development and prognosis of diseases with a genetic component, including cancer. In clinical research, surgery and diagnostics, there is a need for a method that gives a rapid, cheap and reliable read-out out of the allelic (i.e. SNP) ratio to inform clinical decision making.

[0372] Over the past few years, an SNP sensing methodology has developed in which SNP identities can be read-out routinely from target samples of DNA. This approach uses duplex formation (hybridisation), involving a tagged DNA probe to generate a fluorescent signal. However a crucial difference in the approach used in the invention herein is that analysis is based on the strength of the signal generated upon duplex formation, not on how well the duplex forms to give a signal. This means the assay can be done at room temperature and obviates the need to use narrow temperature windows to ensure only one transcript (or transcript product) binds. Published work has so far revealed the results of studies on samples of target strands containing either one nucleobase at a locus (homozygous) or a 50/50 mixture (heterozygous). In all these reports, the sensing signal comes from the fluorescence emission from an anthracene tag on the probe strand either increasing or decreasing at a particular monitoring wavelength (e.g. 426 nm) upon duplex formation, with the intensity of the signal directly depending on the identity of the base opposite (FIG. 1).

[0373] The invention herein demonstrates on a series of DNA and RNA sequences that there is a linear dependence in the emission signal as a function of the SNP/ratio in the target, thus allowing the SNP ratio (i.e. allelic ratio) to be calibrated and then read-out for unknown mixtures through a simple measure of the emission intensity at a given wavelength.

[0374] The results of sensing studies on short (<20-mer) synthetic DNA and RNA targets are described below in Sections 2 (free probes) and 3 (AuNP-bound probes). Section 4 details studies carried out on longer (>100-mer) targets, including PCR-amplified strands derived from patient samples.

2. STUDIES ON SHORT (<20-MER) SYNTHETIC DNA AND RNA TARGETS

[0375] This section describes work on sensing two different SNPs, using anthracene-tagged fluorescent probes. The stereochemistry of the tag in each case was L (R,R), with the linker group as n=5 (see FIG. 2). Each probe was a 15-mer, with the eighth position occupied by the anthracene tag.

[0376] The probes were synthesised as set out above, using standard phosphoramidite automated DNA synthesis, purified by MS and characterised by mass spectrometry (ESMS).

2.1 DNA Sensing (BRAF Gene Mutation)

[0377]

TABLE-US-00003 Probe:5-AGATTTCXCTGTAGC-3(BRAF,X=anthracene 5L) Target:3-TCTAAAGXGACATCG-5 SNP:BRAFgenetransversion(V600E;X=TtoA); associatedwithcancer T.sub.minvalues:45.5(A);46(T)(5Mduplexin 10mMsodiumphosphatebufferpH7;100mMNaCl).

[0378] The resulting calibration graph is shown in FIG. 3.

2.2 DNA Sensing (P21 Gene SNP)

[0379]

TABLE-US-00004 Probe:5-AGTCGCGXCTCAGCT-3(Zsuzsa,X= anthracene5L) Target:3-TCAGCGCXGAGTCGA-5 SNP:P21genetransversion(rs1801270;CtoA); associatedwithAlzheimer'sDisease T.sub.mvalues:60C.(A),63C.(C)(5Mduplexin 10mMsodiumphosphatebufferpH7;100mMNaCl)

[0380] The resulting calibration graph is shown in FIG. 4.

[0381] Using the calibration, unknown heterozygous samples of C and A target were analysed to determine the percentage of C/A base present. The results were calculated using the equation of the linear correlation shown in FIG. 4. The y value was obtained by calculating the percentage change in fluorescence at 426 nm in relation to the probe alone fluorescence. The results of this trial (see Table 1) are shown to be very close to the actual values of the unknown samples.

TABLE-US-00005 TABLE 1 Percentage Change in Calculated Percentage Actual Percentage Fluorescence at 426 nm of C base present of C base present (%) (%) (%) +28 62 60 37 99 100 +34 59 55 0 78 No target

[0382] The results obtained when no target is present highlights one issue: at the point at which the calibration crosses the x-axis, it would not be clear in a test whether (i) there is no target present in solution or (ii) 80% of target strands have the C nucleobase variant and 20% have the A. However this is addressed by: [0383] Use of a second probe: An identical assay can be run with a separate probe containing a different linker length to the anthracene tag, a different linker stereochemistry or a different fluorophore. In each case, the intercept with the x-axis would occur at a different C/A ratio value. A dual-probe approach also gives a further verification of the results obtained; and/or [0384] Use of a second fluorophore tag on one probe: a second fluorophore simply reads out duplex formation through a change in emission intensity; a separate tag allows a ratiometric method for reading out the SNP variant ratio (see Section 3).

2.3 RNA Sensing (P21 Gene SNP)

[0385]

TABLE-US-00006 Probe:5-AGTCGCGXCTCAGCT-3(Zsuzsa,X= anthracene5L) Target:3-UCAGCGCXGAGUCGA-5 SNP:P21genetransversion(rs1801270;CtoA); associatedwithAlzheimer'sDisease T.sub.mvalues:55.5C.(A),57C.(C)(5Mduplexin 10mMsodiumphosphatebufferpH7;100mMNaCl)

[0386] The resulting calibration graph is shown in FIG. 5.

3. STUDIES ON SHORT (<20-MER) SYNTHETIC DNA TARGETS USING PROBES ATTACHED TO GOLD NANOPARTICLES (AU-NPS)

[0387] It is known that DNA can be attached to gold nanoparticles (AuNPs) by modification with sulfur-containing groups allow to surface immobilisation through Au-thiolate bonds. It was decided to immobilise the fluorophore-tagged SNP-sensing strands used in the method of the invention onto AuNPs. This approach provides the advantage that in biological media and upon entering cells, AuNP-immobilised DNA strands tend to be more resistant to nucleases that would otherwise quickly degrade the DNA. This approach would therefore be useful for probing target species (e.g. mRNA) in biological environments. In addition AuNPs can be tailored with additional luminescent groups for ratiometric sensing. Ratiometric sensing consists of analysing the sensing signal from two separate fluorophores at two distinct wavelengths. Dividing one signal intensity by another obviates the need to determine the initial probe concentration; this both simplifies and facilitates the sensing process, in particular for analysis in cellular environments where probe concentrations would be difficult to determine. Further luminescent groups could be used to facilitate tracking in cells as well.

[0388] Anthacene-tagged DNA strands were functionalised with a tether consisting of a 1,2-dithiolane end group (FIG. 6) by reacting a strand of DNA containing an aminoalkyl group with thiooctic acid under standard peptide coupling conditions. AuNPs (ca. 13 nm in diameter) were then functionalised with these strands (AuNP-A, FIG. 8A), with strand immobilisation checked by monitoring changes to the SPR band on the UV/vis spectrum in aqueous media. For the mixed nanoparticles designed to explore ratiometric sensing (AuNPB, FIG. 8B), a Ru(bipy).sub.3 complex (FIG. 7), with well characterised luminescent properties and containing two 1,2-dithiolane end groups, was used in addition to the DNA strands.

TABLE-US-00007 Probe:5-AGTCGCGXCTCAGCT-3(Zsuzsa,X= anthracene5L) Target:3-TCAGCGCXGAGTCGA-5 SNP:P21genetransversion(rs1801270;CtoA); associatedwithAlzheimer'sDisease T.sub.mvalues(AuNP-A):57.5C.(A),59C.(C) (2nMAuNPsin10mMsodiumphosphatebufferpH7; 100mMNaCl)

[0389] The resulting calibration graph for AuNP-A is shown in FIG. 9. The resulting calibration graph for AuNPB is shown in FIG. 10.

[0390] The data in FIG. 9 surprisingly show that a linear correlation is also possible for the surface-immobilised probes, with the percentage changes similar to the data for the corresponding free probe (FIG. 4). The graph in FIG. 10 also shows that ratiometric sensing is possible using this methodology.

4. STUDIES ON LONGER DNA STRAND TARGETS, INCLUDING THOSE FROM PATIENT SAMPLES

4.1 173-Mer Synthetic Targets

[0391]

TABLE-US-00008 Probe:5-AGATTTCXCTGTAGC-3(BRAF,X=anthracene 5L) Target:173-mercontaininginmiddle: 3-TCTAAAGXGACATCG-5 SNP:BRAFgenetransversion(V600E;X=TtoA); associatedwithcancer

[0392] The fluorescence studies undertaken with varying percentages of the BRAF V600E T-to-A cancer mutation within samples of 15-mer synthetic targets demonstrated a linear dependence (Section 2.1). However it was decided to repeat the experiment on a longer synthetic 173-mer oligonucleotide target since such a length would be a more realistic example of the size of a strand that would be generated through amplification by PCR of genomic DNA from actual patient samples (Section 4.2). Due to the BRAF V600E cancer mutation being normally heterozygous rather than homozygous (i.e. cancerous tissue would not be expected to show more than 50% T-A conversion), more data points were chosen between 0% and 50% A in the sample than between 50% and 100% A. The results are shown below in FIG. 11 and again reveal a linear dependence.

[0393] This plot could be used to distinguish the amount of A vs T alleles in a mixture of cells by using the percentage fluorescence change upon binding of the anthracene tagged oligonucleotide probe. For example, if the sample gave a 150% change in fluorescence the amount of the A allele present in the sample would be approximately 47%. As with the 15-mer product (FIG. 3), the A target gave a large increase in emission compared to probe alone. However the percentage increase in emission (ca. 300%) was significantly less than that found for the shorter 15-mer (ca. 700% increase), presumably because unbound (single-stranded) sequences in the 173-mer can fold and interact with the fluorophore tag, which would change its immediate environment to some extent.

4.2. PCR of Genomic DNA

[0394] PCR cycles were then performed on patient samples containing either wt DNA (100% T, healthy tissue) or mutated DNA (cancerous tissue). The samples were run on an agarose gel and the results can be seen in FIG. 12. Strong bands demonstrate a good yield, which only came once the conditions of the PCR reaction had been optimised by varying different components.

[0395] Once the double stranded PCR products were digested to single stranded DNA (the target strand contained phosphorothioate primers to prevent digestion), the samples were submitted for sequencing. The results can be seen in FIGS. 13 and 14. The SNP position that changes malignant melanomas has been circled within the figure. In wt DNA, the position is occupied by a T base (FIG. 13), whereas the DNA from the tumour tissue shows both A and T (FIG. 14) due to the heterozygous nature of the BRAF V600E mutation (vide supra). These results therefore confirmed the correct sequences of single stranded DNA in the PCR products, being either 100% T for DNA from healthy tissue or (presumably) 50% T and 50% A for DNA from fully cancerous tissue.

4.3 Fluorescence Studies of PCR Product

[0396] The next task was to determine whether the SNP sensing technique was viable on the samples obtained by PCR amplification of genomic DNA derived from patient samples. The fluorescence studies required at least equimolar concentrations of the BRAF probe and the target PCR product, with ideally an excess of product to ensure full binding of the probe. However it was found that after the PCR cycles, followed by digestion of the strands to ssDNA and then finally purification, the yield had dropped by approximately 85%. Therefore preliminary fluorescence studies were undertaken after the digestion step but without any further purification steps.

[0397] Gratifyingly, the data show that whereas binding to wtDNA gives a slight decrease in anthracene emission intensity compared to the probe alone, the tumour tissue DNA gives the expected increase (FIG. 15). The data therefore indicates that the SNP sensing protocol is indeed reproducible on DNA derived from patient samples. However, one unexpected finding was the higher than expected emission intensity of the sample containing the probe alone. Control experiments were undertaken to determine whether the fluorescence increase was due to the PCR by-product or another component of the unpurified PCR mixture; these revealed that the formamide used to deactivate the T7 exonuclease had caused the high fluorescence intensity of the mixture of the probe and the reaction mix (3600 units) compared to the probe alone in buffer (1600 units). However, as the results in FIG. 15 show, this issue did not preclude the observation of expected increases and decreases in emission intensity for the samples from cancerous and healthy tissue respectively.

[0398] There could be several reasons as to why the increase in fluorescence using the PCR products was not as high as expected from the results with the synthetic sequences, which predicted a ca. 150% increase for the heterozygous (50/50) mixture (FIG. 12). The most likely reason is that given the concentration issues encountered, the PCR target may have not being fully in excess, resulting in some unbound probe in the mixture, reducing the signal intensity. Alternatively, the formamide may have had more of an adverse effect on the anthracene emission intensity in the single stranded probe than the corresponding duplexes. Finally the sample from the cancerous tissue may not have been fully heterozygous (i.e. 50% A and 50% T).

5. CONCLUSIONS

[0399] These examples have proven that it is possible to quantify SNP (allelic) ratios in target samples of DNA or RNA using a novel hybridization assay that operates through monitoring changes in the emission intensity of a fluorophore-tagged DNA probe upon duplex formation. Quantification comes from a demonstration of a linear dependence of the emission intensity on the SNP ratio in the target strand mixture.

[0400] The assay works at room temperature and could be adapted for use on a standard plate reader.

[0401] The methodology has been demonstrated for two different medical conditions, Alzheimer's disease and cancer. However, the methodology is, in principle, universal for any SNP combination associated with a medical condition (here the targets studied are both transversions, i.e. C/A and T/A), so long as the two duplexes have significantly different emission profiles and similar stabilities (T.sub.m values).

[0402] The findings have also shown that the methodology works for (i) RNA targets as well as DNA targets; (ii) probes immobilized on gold nanoparticle surfaces that may be applied for ratiometric sensing in cellular environments or in blood plasma, for example to target circulating tumour DNA (ctDNA) and (iii) different sequence lengths in the target, from 15-mers to >100-mers.

[0403] PCR products derived from actual patient samples have been shown to bring about the same trends.

[0404] This methodology could also be used for epigenetic screening purposes (i.e. to establish the Me-C/C ratio within a sample), given that these strands can also discern base modifications (i.e. methylation of cytosine) as well as base changes (Duprey et al., ACS Chem. Biol., 2016, 11, 717-721).

[0405] An advantage of this method over other known sequencing or sensing approaches (e.g. RNA-seq, TaqMan,) concerns the assessment of heterozygous nucleic acid samples; currently there is no one method that is quick, cheap and quantitative for accurately determining allelic ratios, either from DNA samples (for example in those arising from regions of cancerous tissue) or from mRNA transcripts. The examples provide evidence that all three of these issues can be addressed by the claimed invention.