Double-stranded probes for detecting nucleic acids by fluorescence.
2006
A double-stranded probe, intended for the detection of a target nucleic acid, single-stranded or double-stranded, by fluorescence, which comprises: - a first strand of formula X1- the (L1) a-S1-S ' '1- (L'1) b-Y1, intended for a first strand of the target nucleic acid which comprises a sequence of formula T' 1-T 1; - a second strand of formula X2- the (L2) c-S2-S''2- (L'2) dy2, intended for a second strand of the target nucleic acid which comprises a sequence of formula T '' 2-T2; where, - two of X1, X2, Y1, and Y2 represent a fluorescent donor, while the two others represent a fluorescent acceptor, and X 1 and X 2 can not represent both a fluorescent donor; - a, b, c and d are 0 or 1 with the proviso that a, b, c or d represent 1 when respectively X1, X2, Y1, or Y2, represent a fluorescent donor; - T 1 and T 2 represent oligonucleotide sequences having from 10 to 35 nucleotides, more preferably 12 to 20 nucleotides, which are complementary to one relative to the other; - independently one with respect to the other, T''1 and T''2, they represent oligonucleotide sequences having from 2 to 8 nucleotides; - S1 and S2 represent oligonucleotide sequences having from 10 to 35 nucleotides, which are complementary to one each other, with S1, at least 85% complementary to T1, and being, S2, so least 85% complementary to T2; - independently one with respect to the other, and S''2 S''1, represent oligonucleotide sequences having from 2 to 8 nucleotides, S''1 being at least 65% complementary to T''1, and being, S''2, at least 65% complementary to T''2; - L 1 and L 2 are spacer moieties, such that the respective turning radius of X 1 and X 2 with respect to the binding sites of S 1 to L 1 and S 2 to L 2 is of at least 3.4 '' A; - L'1 and L'2 are spacer moieties, such that the respective turning radius of Y1 and Y2 with respect to the binding sites S''1 L' '1 and S '' 2 to L '2 is of at least 3.4' 'a; - being the melting temperature of the double-stranded probe, below the melting temperature of the complex formed between the first strand and the second strand of the double-stranded probe target nucleic acid; and lower than the melting temperature of the complex formed between the second strand of the double stranded-probe and the second strand of the target nucleic acid, if present in.
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