Nucleic acid probes for analysis of small RNAs and other polynucleotides

• US 20070003940A1
• Filed: 07/01/2005
• Published: 01/04/2007
• Est. Priority Date: 07/01/2005
• Status: Active Grant

• Current Assignee: Agilent Technologies, Inc.
• Original Assignee: Agilent Technologies, Inc.
• Inventors: Wang, Hui
• Granted Patent: US 9,297,036 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides

Abstract

The invention provides a nucleic acid probe for detecting a polynucleotide. The probe contains a region that base-pairs with a polynucleotide to form a duplex and a T_m enhancement domain that increases the stability of the duplex. The T_m enhancement domain may contain a nucleotide clamp and/or a hairpin structure, for example. Also provided is an array of subject nucleic acid probes bound to a surface of the solid support. Methods of using a subject probe to assess polynucleotides, e.g., small RNAs in a sample are provided, as are kits for use in practicing the subject methods.

Claims

1. A nucleic acid probe for detecting a polynucleotide, comprising:

a region that base-pairs with said polynucleotide to form a duplex; and

a T_m enhancement domain that increases stability of said duplex.

2. The nucleic acid probe of claim 1, wherein said polynucleotide is a small RNA selected from the group consisting of a short interfering RNA (siRNA), microRNA (miRNA), tiny non-coding RNA (tncRNA) and a small modulatory RNA (smRNA).

3. The nucleic acid probe of claim 1, wherein said T_m enhancement domain comprises a nucleotide clamp.

4. The nucleic acid probe of claim 1, wherein said nucleotide clamp base-pairs with a nucleotide label moiety present at a 3′

end of said polynucleotide.

5. The nucleic acid probe of claim 1, wherein said T_m enhancement domain comprises a hairpin structure.

6. The nucleic acid probe of claim 5, wherein said hairpin structure provides for stacking in said duplex.

7. The nucleic acid probe of claim 5, wherein said hairpin structure provides for steric hindrance for non-target polynucleotides.

8. The nucleic acid probe of claim 1, wherein said nucleic acid probe comprises a T_m enhancement domain that contains a nucleotide clamp and a hairpin region.

9. An array comprising:

a) a solid support having a surface; and

b) at least one nucleic acid probe of claim 1 bound to said surface.

10. The array of claim 9, wherein said at Ieast one nucleic acid probe is present on said surface as a discrete feature.

11. The array of claim 9, wherein said at least one nucleic acid probe comprises a linker between said region and said surface.

12. A method, comprising:

labeling a sample containing a polynucleotide to produce a labeled sample;

contacting said labeled sample with a nucleic acid probe of claim 1;

and

detecting any label bound to said nucleic acid probe.

13. The assay of claim 12, wherein said labeling comprises addition of a nucleotide label moiety to a terminus of said polynucleotide.

14. A method for evaluating small RNAs in a test sample, comprising:

labeling a test sample containing said small RNAs to produce a sample containing labeled small RNAs;

contacting said labeled sample with an array of claim 9;

and

detecting any label bound to said array to evaluate binding of said labeled small RNAs to said at least one nucleic acid probe.

15. The method of claim 14, wherein said labeling comprises addition of a nucleotide label moiety to a terminus of said RNAs.

16. The method of claim 14, wherein said binding is evaluated relative to binding of small RNAs in a reference sample.

17. The method of claim 16, wherein said test and reference samples are made from test and controls cells, respectively.

18. A kit for assessing polynucleotides in a polynucleotide sample, comprising:

a nucleic acid probe according to claim 1;

and

instructions for using said nucleic acid probe to assess polynucleotides in said polynucleotide sample.

19. The kit of claim 18, wherein said kit contains control polynucleotides.

20. The kit of claim 18, further comprising reagents for isolating polynucleotides from a cell.

21. The kit of claim 18, further comprising reagents for labeling said polynucleotide.

22. The kit of claim 18, wherein said labeling reagents distinguishably label polynucleotides in two samples.

23. The kit of claim 18, wherein said nucleic acid probe is bound to a surface of a solid support and is part of an array of nucleic acid probes.

24. A method of producing an array comprising:

designing a nucleic acid probe for detecting a polynucleotide, said nucleic acid probe comprising;

a region that base-pairs with said polynucleotide to form a duplex; and

a T_m enhancement domain that increases stability of said duplex; and

fabricating an array comprising said nucleic acid probe.

25. A computer readable medium comprising:

programming for designing a nucleic acid probe for detecting a polynucleotide, said nucleic acid probe comprising;

a region that base-pairs with said polynucleotide to form a duplex; and

a T_m enhancement domain that increases stability of said duplex.