Nucleic acid probes and microarrays for analysis of polynucleotides

• US 7,754,475 B2
• Filed: 10/31/2006
• Issued: 07/13/2010
• Est. Priority Date: 01/25/2006
• Status: Active Grant

• Current Assignee: Agilent Technologies, Inc.
• Original Assignee: Agilent Technologies, Inc.
• Inventors: Wang, Hul
• Primary Examiner(s): Nguyen; Dave
• Assistant Examiner(s): BHAT, NARAYAN KAMESHWAR
• Application Number:
  US11/555,230
• Publication Number:
  US 20070172845A1
• Time in Patent Office: 1,351 Days
• Field of Search: 435/6, 435/287.2, 422/61, 536/23.1, 536/24.3
• US Class Current: 435/287.2
• CPC Class Codes: C12Q 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 2525/121 incorporating both deoxyribonucleotides and ribonucleotides
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides
• CPC Class Codes: C12Q 2549/126 using oligonucleotides as clamps

Abstract

The invention provides a probe for detecting a target polynucleotide. The probe contains a region that base-pairs with a target polynucleotide to form a duplex and a RNA hairpin extension domain that increases the stability of the duplex. The probe may further include a nucleotide clamp, a stem-complementary region and/or a linker moiety. Also provided is an array of subject probes bound to a surface of a solid support. Methods of using a subject probe to assess target polynucleotides, e.g., small RNAs, in a sample are provided, as are kits for use in practicing the subject methods.

Claims

1. A probe set for detecting a target polynucleotide, comprising:

a) a first oligonucleotide bound to a surface of a solid support by its 3′

end and comprises;

i) a surface-proximal target complementary region that base-pairs with said target polynucleotide to form a probe/target duplex; and

ii) a surface-distal stem-complementary region; and

b) a second oligonucleotide comprising a loop region and a stem region, wherein said stem region is complementary to said stem-complementary region,

wherein hybridization of said first oligonucleotide and said second oligonucleotide results in a stem-loop in which the 3′

terminal nucleotide of said second oligonucleotide is directly adjacent to the 5′

terminal nucleotide of said first oligonucleotide, wherein said stem-loop stabilizes said probe/target duplex.

2. The probe set of claim 1, wherein said target 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 probe set of claim 1, wherein said first oligonucleotide further comprises a nucleotide clamp region between the target complementary region and the stem-complementary region.

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

end of said target polynucleotide.

5. The probe set of claim 3, wherein the target complementary region is from 10 to 25 nucleotides long.

6. The probe set of claim 1, wherein said stem-loop provides for steno hindrance for nontarget polynucleotides.

7. The probe set of claim 1, wherein said second oligonucleotide is from 15 to 50 nucleotides long.

8. An array comprising:

a) a solid support having a surface; and

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

9. The array of claim 8, wherein said array comprises at least 100 different features, each feature having a different probe set according to claim 1.

10. The array of claim 8, wherein said at least one probe set comprises a linker between said target complementary region and said surface.

11. A kit for assessing target polynucleotides in a polynucleotide sample, the kit comprising:

a probe set according to claim 1; and

one or more components selected from the group consisting of instructions for using said probe set to assess target polynucleotides in a sample;

control polynucleotides;

reagents for isolating target polynucleotides from a cell; and

reagents for labeling said target polynucleotides.

12. A method, comprising:

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

contacting said labeled sample with a probe of claim 1; and

detecting any label bound to said probe.

13. The method 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 the test sample containing said small RNAs to produce a sample containing labeled small RNAs;

contacting said labeled sample with an array of claim 8; and

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

15. The method of claim 14, wherein said labeling comprises addition of a nucleotide label moiety to a terminus of said small 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 sample and said reference samples are obtained from test and control cells, respectively.