Method for detecting a structured target
View Patent ↗The present invention concerns oligonucleotides containing one or more modified nucleotides which increase the binding affinity of the oligonucleotides to target nucleic acids having a complementary nucleotide base sequence. These modified oligonucleotides hybridize to the target sequence at a faster rate than unmodified oligonucleotides having an identical nucleotide base sequence. Such modified oligonucleotides include oligonucleotides containing at least one 2′-O-methylribofuranosyl moiety joined to a nitrogenous base. Oligonucleotides can be modified in accordance with the present invention to preferentially bind RNA targets. The present invention also concerns methods of using these modified oligonucleotides and kits containing the same.
1. A method for detecting an RNA analyte, the method comprising:
(a) contacting a sample with a probe molecule under conditions permitting the probe to form a detectable probe:analyte hybrid containing at least one 2′-O-methyl ribonucleotide, wherein the probe binds to a double-stranded region of the analyte, and wherein the probe does not form a detectable probe:non-analyte hybrid with non-target nucleic acid present in the sample; and
(b) determining whether the probe:analyte hybrid has formed as an indication of the presence or absence of a target nucleic acid in the sample.
2. The method of claim 1 , wherein the probe:analyte hybrid includes a cluster of at least 4 contiguous 2′-O-methyl ribonucleotides.
3. The method of claim 1 , wherein at least one nucleotide of the probe is not a 2′-O-methyl ribonucleotide.
4. The method of claim 1 , wherein each nucleotide of the probe is a 2′-O-methyl ribonucleotide.
5. The method of claim 1 , wherein the probe includes a conjugate molecule.
6. The method of claim 2 , wherein the probe includes a conjugate molecule joined to the probe at a site located within the cluster.
7. The method of claim 1 , wherein the probe is between 10 and 100 bases in length.
8. The method of claim 1 , wherein the probe is between 10 and 16 bases in length.
9. The method of claim 1 , wherein the probe includes a detectable label.
10. The method of claim 9 , wherein the label is a chemiluminescent or a fluorescent agent.
11. The method of claim 1 , wherein the probe comprises first and second base regions that are hybridized to each other and include at least one 2′-O-methyl ribonucleotide prior to the formation of the probe:analyte hybrid.
12. The method of claim 1 , wherein the analyte is the target nucleic acid.
13. The method of claim 1 , wherein the analyte is a product of an amplification procedure.
14. The method of claim 13 , wherein the amplification procedure is a transcription-based method of amplification.
15. The method of claim 13 further comprising quantifying the amount of the target nucleic acid determined to be present in the sample.
16. The method of claim 1 , wherein at least one of the probe and the target nucleic acid is directly or indirectly immobilized by a solid support.
17. The method of claim 1 , wherein the sample contains DNA.
18. The method of claim 17 , wherein the DNA is chromosomal DNA.
19. The method of claim 1 , wherein the target nucleic acid is mRNA.
20. The method of claim 1 , wherein the target nucleic acid is ribosomal RNA.
21. The method of claim 1 , wherein the sample is obtained from a human.
22. The method of claim 1 , wherein the determining step provides an indication of the presence or absence of a virus or microorganism in the sample.
23. The method of claim 1 , wherein the probe comprises the at least one 2′-O-methyl ribonucleotide, and wherein the rate of hybridization of the probe to the analyte is greater than the rate of hybridization of an unmodified form of the probe to the analyte.
24. The method of claim 23 , wherein the nucleotide sequence of the unmodified form of the probe consists of deoxynucleotides.
25. The method of claim 1 , wherein the probe:analyte hybrid forms in solution.