Double-stranded probes for the fluorescent detection of nucleic acids

1. A double-stranded probe intended for the fluorescent detection of a single-stranded or double-stranded target nucleic acid, comprising:

a first strand of formula X 1 -L 1 -S 1 -S′ 1 -Y 1 , intended for the detection of a first strand of the target nucleic acid which comprises a sequence of formula T′ 1 -T 1 ; and

a second strand of formula X 2 -L 2 -S 2 -S′ 2 -Y 2 , intended for the detection of a second strand of the target nucleic acid, if present, said second strand of the target nucleic acid comprising a sequence of formula T′ 2 -T 2 ;

wherein:

X 1 and X 2 represent fluorescent donors and Y 1 and Y 2 represent a fluorescent acceptors;

T 1 and T 2 represent oligonucleotide sequences having from 10 to 35 nucleotides which are complementary to one another;

independently from one another, T′ 1 and T′ 2 represent oligonucleotide sequences having from 2 to 8 nucleotides;

S 1 and S 2 represent oligonucleotide sequences having from 10 to 35 nucleotides which are complementary to one another, S 1 being at least 85% complementary to T 1 , and S 2 being at least 85% complementary to T 2 ;

independently from one another, S′ 1 and S′ 2 , represent oligonucleotide sequences having from 3 to 8 nucleotides, S′ 1 being at least 65% complementary to T′ 1 , and S′ 2 being at least 65% complementary to T′ 2 ;

L 1 and L 2 are spacer moieties such that the respective gyration radius of X 1 and X 2 with respect to the attachment sites of S 1 to L 1 and S 2 to L 2 is at least 3.4 Å, wherein L 1 and L 2 are polynucleotides, and

wherein L 1 and L 2 have less than 35% complementarity with S′ 2 and S′ 1 respectively,

wherein L 1 and L 2 are independently selected from the group consisting of a polynucleotide having from 2 to 10 nucleotides, and the length of one or both of L 1 and L 2 is shorter than that of one or both of S′ 2 and S′ 1 respectively; and

a melting temperature of the double-stranded probe is lower than a melting temperature of a complex formed between the first strand of the double stranded-probe and the first strand of the target nucleic acid; and lower than a melting temperature of a complex formed between the second strand of the double stranded-probe and the second strand of the target nucleic acid, if present.

2. The double-stranded probe according to claim 1 , wherein the fluorescent donor is selected from the group consisting of xanthene dyes, rhodamine dyes, carbopyronine dyes and carboxamide dyes.

3. The double-stranded probe according to claim 1 , wherein the fluorescent acceptor is a dark quencher.

4. The double-stranded probe according to claim 1 , wherein L 1 and L 2 respectively quench less than 25% of the fluorescence of X 1 and X 2 .

5. The double-stranded probe according to claim 1 , wherein at least one of L 1 and L 2 comprise at least one positive charge.

6. The double-stranded probe according to claim 1 , wherein one or more of L 1 and L 2 is a polyT polynucleotide.

7. The double-stranded probe according to claim 1 , wherein a melting temperature of the first strand of the probe with respect to the second strand of the probe is at least 10% lower than the melting temperature of either the first strand or the second strand with respect to their respective target nucleic acid strands.

8. The double-stranded probe according to claim 1 , wherein said double-stranded probe comprises:

a first nucleic acid strand of formula X 1 -L 1 -S 1 -S′ 1 -Y 1 , and

a second nucleic acid strand of formula X 2 -L 2 -S 2 -S′ 2 -Y 2 ;

and wherein:

X 1 and X 2 are selected from the group consisting of FAM, Atto532, NK141, NK230 and Atto647N;

Y 1 and Y 2 represent Dabcyl;

L 1 and L 2 represent a polyT polynucleotide having from 3 to 6 nucleotides;

S 1 and S 2 represent an oligonucleotide sequence having from 14 to 18 nucleotides; and

S′ 1 and S′ 2 represent an oligonucleotide sequence having from 4 to 6 nucleotides.

9. The double-stranded probe according to claim 8 intended for the detection of Salmonella typhi , wherein said double-stranded probe comprises:

a first nucleic acid strand of formula X 1 -L 1 -S 1 -S′ 1 -Y 1 , in the 5′ to 3′ orientation, and

a second nucleic acid strand of formula X 2 -L 2 -S 2 -S′ 2 -Y 2 , in the 5′ to 3′ orientation;

and wherein:

L 1 and L 2 represent 5′-TTT-3′ (SEQ ID NO: 86);

S 1 represents 5′-GAATAGTGAGCGTGCCT-3′ (SEQ ID NO: 87);

S′ 1 represents 5′-TACCG-3′ (SEQ ID NO: 88);

S 2 represents 5′-AGGCACGCTCACTATTC-3′ (SEQ ID NO: 89); and

S′ 2 represents 5′-TGACA-3′ (SEQ ID NO: 90).

10. The double-stranded probe according to claim 1 , wherein the fluorescent acceptor is 4-(4-dimethylaminophenyl)diazenyl benzoic acid ; 4-N,N -dimethylaminoazobenzene-4′ sulfonic acid; Xanthylium, 9-[2-[[4-[[(2,5-dioxo-1-pyrrolidinyl)oxy]carbonyl]-1-piperidinyl]sulfonyl]phenyl]-3,6-bis(methylphenylamino)-, chloride; N-(9-{2-[(4-{[(2,5-dioxopyrrolidin-1-yl)oxy]carbonyl}piperidin-1-yl)sulfonyl]phenyl}-6-[methyl(4-sulfophenyl)amino]-3H-xanthen-3-ylidene)-N-methyl-4-sulfoanilinium; 2-[6-(1,3-dihydro-2H-isoindol-2-yl)-9-{2-[(4-{[(2,5-dioxopyrrolidin-1-yl)oxy]carbonyl}piperidin-1-yl)sulfonyl]phenyl}-3H-xanthen-3-ylidene]-2,3-dihydro-1H-isoindolium chloride; 4-N-methyl-N-(4′nitro-2′-chloroazobenzen-4-yl)-aminobutanamido-1-(2-O-dimethoxytrityloxymethyl)-pyrrolidin -4-yl-succinoyl long chain alkylamino-CPG; 4-dimethylaminophenylazophenyl-4′-maleimid; 4-[(4-dimethylaminophenyl)phenyl-methyl]-N,N-dimethylaniline; or 2H-chromen-2-one.

11. A kit intended for the fluorescent detection of a single-stranded or double-stranded target nucleic acid comprising:

a first nucleic acid strand of formula X 1 -L 1 -S 1 -S′ 1 -Y 1 as defined in claim 1 ; and

a second nucleic acid strand of formula X 2 -L 2 -S 2 -S′ 2 -Y 2 as defined in claim 1 .

12. A kit for the fluorescent detection of at least one single-stranded or double-stranded target nucleic acid in an enzyme-based nucleic acid amplification method, comprising:

at least one double-stranded probe according to claim 1 ;

an enzyme for enzyme-based nucleic acid amplification; and

a reagent mixture adapted for enzyme-based nucleic acid amplification.

13. A kit according to claim 12 , further comprising nucleotide primers adapted for enzyme-based amplification of the target nucleic acid.

14. A kit according to claim 12 for the detection of more than one single-stranded or double-stranded target nucleic acid in a multiplex enzyme-based nucleic acid amplification method, said kit comprising more than one double-stranded probe according to claim 1 , wherein each of said target nucleic acids is able to be detected by at least one of said double-stranded probes.