Capture probes immobilizable via L-nucleotide tail
The invention provides chimeric capture probes immobilizable via an L-nucleic acid tail that can bind to a complementary L-nucleic acid in an immobilized probe. The capture probes are useful for capturing a target nucleic acid from a sample. The L-nucleic acid in the tail of the capture probe bind to the complementary L-nucleic acid in the immobilized probe with similar affinity as would otherwise equivalent D-nucleic acids. However, the L-nucleic acid of the capture probe tail and immobilized probes do not form stable duplexes with D-nucleic acids present in the sample containing the target nucleic acid. Binding of nucleic acids in the sample directly to immobilized probe or to the tail of the capture probe is reduced or eliminated increasing the sensitivity and/or specificity of the assay.
1. A method of capturing a target nucleic acid, comprising:
contacting a target nucleic acid in a sample with a capture probe and an immobilized probe, the capture probe comprising a first segment that binds to the target nucleic acid and a second segment that binds to the immobilized probe, wherein the second segment of the capture probe and the immobilized probe comprise complementary homopolymeric L-nucleic acids that can hybridize to one another, wherein the homopolymeric L-nucleic acids are polyA and polyT, wherein the target nucleic acid binds to the first segment of the capture probe, and the second segment of the capture probe binds to the immobilized probe, thereby capturing the target nucleic acid, wherein the sample includes mRNA or a nucleic acid derived therefrom, wherein the copy number of the target nucleic acid in the sample is up to 33 copies per reaction.
2. The method of claim 1 , wherein the first segment includes a D nucleic acid of at least 10 D-nucleobase units complementary to the target nucleic acid.
3. The method of claim 2 , wherein the D-nucleic acid comprises any of adenine-D-deoxyribose, guanine D-deoxyribose, thymine D-deoxyribose and cytosine D-deoxyribose.
4. The method of claim 1 , wherein the first segment includes a D-nucleic acid of 10-30 D-nucleobase units complementary to the target nucleic acid.
5. The method of claim 1 , wherein the first segment binds non-specifically to the target nucleic acid.
6. The method of claim 5 , wherein the first segment includes a random sequence of D-nucleobase units that binds nonspecifically to the target nucleic acid.
7. The method of claim 1 , wherein the second segment includes an L-nucleic acid of at least six adenine L-deoxyribose units complementary to an L-nucleic acid of at least six thymine L-deoxyribose units in the immobilized probe.
8. The method of claim 1 , wherein the second segment includes an L-nucleic acid of 10-30 adenine L-deoxyribose units complementary to an L-nucleic acid of 10-30 contiguous thymine L-deoxyribose units in the immobilized probe.
9. The method of claim 8 , wherein the L-nucleic acid of the second segment is polyT and the L-nucleic acid of the immobilized probe constitutes polyA.
10. The method of claim 1 , wherein the target nucleic acid is contacted with the capture probe and immobilized probe simultaneously.
11. The method of claim 1 , wherein the target nucleic acid is contacted with the capture probe before the immobilized probe.
12. The method of claim 1 , wherein the binding of the target nucleic acid to the capture probe occurs under first hybridization conditions and the binding of the capture probe to the immobilized probe occurs under second hybridization conditions.
13. The method of claim 12 , wherein the first conditions are more stringent than the second conditions.
14. The method of claim 1 , wherein the binding of the target nucleic acid to the capture probe and the binding of the capture probe to the immobilized probe occur under the same hybridization conditions.
15. The method of claim 1 , wherein the immobilized probe is immobilized to a magnetic bead.
16. The method of claim 1 , wherein the target nucleic acid is provide as a component of a sample and the method further comprising separating the captured target nucleic acid from other components of the sample.
17. The method of claim 16 , further comprising dissociating the captured target nucleic acid from the immobilized probe.
18. The method of claim 17 , further comprising amplifying the target nucleic acid.
19. The method of claim 18 , wherein the amplifying is performed after dissociating the target nucleic acid from the capture probe.
20. The method of claim 17 , further comprising contacting the target nucleic acid with a detection probe and detecting the detection probe.
21. The method of claim 17 , wherein the target nucleic is contacted with the detection probe after dissociating the target nucleic acid from the capture probe.
22. The method of claim 1 , wherein the target nucleic acid is an HIV nucleic acid.
23. The method of claim 1 , wherein the second segment of the capture probe comprises (T) 0-5 /(A) 10-40 L-deoxyribose units and the immobilized probe comprises polyT.