Inversion probes
View Patent ↗Unitary hybridization probes having stem-and-loop structures, wherein the stem portion of the structure comprises a pair of interactive arms that are substantially prevented from interacting with target polynucleotides. One arm of the invented parallel-stem hybridization probe has a backbone polarity opposite that of the target-complementary loop sequence of the probe. Rather than interacting in an antiparallel fashion, the arms of parallel-stem hybridization probes interact in a parallel fashion. The arms of the invented dual inversion probes interact in a conventional antiparallel fashion, but have backbone polarities opposite that of the target-complementary loop portion of the probe. Arm portions of the inversion probes do not substantially contribute to sequence-dependent stabilization of probe:target hybrids. Incorporating inversion linkages into the structures of these probes dramatically simplifies the process of designing stem-and-loop hybridization probes.
1. A hybridization probe for detecting a target polynucleotide, comprising:
(a) a loop comprising a target-complementary sequence of bases joined to a loop backbone, said target-complementary sequence of bases extending from a first boundary to a second boundary;
(b) a first arm joined to said target-complementary sequence of bases at said first boundary through a first arm linkage, said first arm comprising a first arm sequence of bases joined to a first arm backbone;
(c) a second arm joined to said target-complementary sequence of bases at said second boundary through a second arm linkage, said second arm comprising a second arm sequence of bases joined to a second arm backbone;
wherein both of said first arm linkage and said second arm linkage are inversion linkages different from each other, said hybridization probe being a dual inversion probe, each of said inversion linkages optionally including a non-nucleotide linker, and
(d) at least one detectable label joined to said loop, said first arm, said second arm or, said non-nucleotide linker,
wherein said first arm and said second arm interact with each other in the absence of said target polynucleotide to form a stem duplex.
2. The hybridization probe of claim 1 , wherein said at least one detectable label comprises a pair of interactive labels comprising a first label and a second label, said first label being joined to said first arm and said second label being joined to said second arm.
3. The hybridization probe of claim 1 , wherein said first arm linkage is a 3′-3′ inversion linkage, and wherein said second arm linkage is a 5′-5′ inversion linkage.
4. The hybridization probe of claim 1 , wherein said first arm linkage is a 5′-5′ inversion linkage, and wherein said second arm linkage is a 3′-3′ inversion linkage.
5. The hybridization probe of claim 2 , wherein at least one of said loop, said first arm or said second arm comprise at least one nucleotide analog.
6. The hybridization probe of claim 5 , wherein said loop comprises 2′-methoxy nucleotide analogs.
7. The hybridization probe of claim 2 , wherein the target-complementary sequence of bases has a length in the range of 10–25 bases.
8. The hybridization probe of claim 7 , wherein the target-complementary sequence of bases has a length in the range of 16–22 bases.
9. The hybridization probe of claim 7 , wherein the first arm has a length of 5–12 bases.
10. The hybridization probe of claim 9 , wherein the second arm has a length of 5–12 bases.
11. The hybridization probe of claim 7 , wherein both the first arm and the second arm have lengths in the range of 6–8 bases.
12. The hybridization probe of claim 2 , wherein said pair of interactive labels is a pair of FRET interactive labels.
13. The hybridization probe of claim 2 , wherein said pair of interactive labels is a pair of non-FRET interactive labels.
14. The hybridization probe of claim 13 , wherein one member of said pair of non-FRET interactive labels comprises fluorescein.
15. The hybridization probe of claim 10 , wherein said pair of interactive labels is a pair of FRET interactive labels.
16. The hybridization probe of claim 10 , wherein said pair of interactive labels is a pair of non-FRET interactive labels.
17. A method of determining whether a test sample contains a target polynucleotide, comprising the steps of:
(a) providing a hybridization probe of claim 1 ,
(b) contacting said hybridization probe with said target polynucleotide in the test sample under hybridization-promoting conditions; and
(c) detecting the formation of hybrid duplexes comprising said hybridization probe and said target polynucleotide as an indication of the presence of said target polynucleotide sequence in said test sample.
18. A kit for detecting a target polynucleotide sequence using a hybridization assay, comprising:
(a) a hybridization probe of claim 1 ; and
(b) a positive-control target polynucleotide having a sequence complementary to said target-complementary sequence of bases of said loop.
19. The kit of claim 18 , further comprising
(c) a hybridization solution.