Patent ID: 8916345

Claim:
A method of detecting a target oligonucleotide in a sample, comprising: contacting the sample with a composition comprising a first oligonucleotide having the structure 5′-Y 1 -L 1 -X-L 2 -Y 2 -3′, wherein Y 1 comprises a sequence of DNA or RNA nucleotides, including a first nucleotide N 1 having a 3′ phosphate covalently linked to L 1 ; Y 2 comprises a sequence of DNA or RNA nucleotides, including a second nucleotide N 2 having a 5′ phosphate covalently linked to L 2 ; L 1 and L 2 each independently are a direct bond or a C 1 -C 7 alkyl, alkynyl, alkenyl, heteroalkyl, substituted alkyl, aryl, heteroaryl, substituted aryl, cycloalkyl, alkylaryl, or alkoxyl group; X is R 1 is a hydrogen or a C 1 -C 8 alkyl; M is L 3 is a direct bond or a C 1 -C 8 alkyl, alkenyl, alkenyl, heteroalkyl, substituted alkyl, cycloalkyl, or alkoxyl; R 2 -R 6 each independently are a hydrogen, an alkenyl, a heteroalkyl, a substituted alkyl, an aryl, a heteroaryl, a substituted aryl, a cycloalkyl, an alkylaryl, an alkoxyl, an electron withdrawing group, or an electron donating group, and one of R 2 -R 6 comprises wherein P is R 7 -R 9 each independently are a hydrogen, an alkoxyl, an alkyl, an alkylamino, an arylamino, a cycloalkyl, a heteroalkoxyl, a heteroalkyl, or an amino; and R 10 -R 13 each independently are a hydrogen, a nitro, a cyano, a carboxylate, a sulfonyl, a sulfamoyl, an alkenyl, an alkynyl, an amino, an aryl, a heteroaryl, a biaryl, a bialkenyl, a bialkenyl, an alkoxycarbonyl or a carbamoyl; wherein the first oligonucleotide is adapted to hybridize to a second oligonucleotide having the structure 3′-Y 3 -Y 4 -5′; wherein Y 3 comprises a sequence of DNA or RNA nucleotides including a third nucleotide N 3 ; and wherein Y 4 comprises a sequence of DNA or RNA nucleotides including a fourth nucleotide N 4 that is directly attached to nucleotide N 3 ; wherein if the first oligonucleotide hybridizes to the second oligonucleotide, N 1 base pairs with N 3 and N 2 base pairs with N 4 to form a duplex having a T m that is greater than the T m of a duplex formed between the second oligonucleotide and a third oligonucleotide having the structure 5′-Y 1 -Y 2 -3′; wherein the first oligonucleotide is also adapted to hybridize to the target oligonucleotide; wherein the first oligonucleotide is labeled with a fluorophore; and wherein fluorescence of the fluorophore is reduced by fluorescence resonance energy transfer to the quencher or by ground state quenching by the quencher when the first oligonucleotide is not hybridized to the target oligonucleotide; and detecting an increase in fluorescence indicating the presence of the target oligonucleotide in the sample.