GENOME-WIDE METHOD FOR MAPPING OF ENGAGED RNA POLYMERASES QUANTITATIVELY AND AT HIGH RESOLUTION

• US 20100062946A1
• Filed: 09/04/2009
• Published: 03/11/2010
• Est. Priority Date: 09/08/2008
• Status: Abandoned Application

• Current Assignee: Cornell University
• Original Assignee: Cornell University
• Inventors: Core, Leighton J., Lis, John T.
• Application Number:
  US12/554,472
• Publication Number:
  US 20100062946A1
• Time in Patent Office: Days
• US Class Current: 506/7
• CPC Class Codes: C12Q 1/6809 Methods for determination or identification of nucleic acids involving differential detection
• CPC Class Codes: C12Q 2521/119 RNA polymerase
• CPC Class Codes: C12Q 2565/501 being an array of oligonucleotides

Abstract

A method is provided for detecting genome-wide transcriptionally-engaged RNA polymerases. The method can also be used to assess status and regulation of gene promoters. The method comprises permeabilizing a cell of interest or isolating the nucleus from a cell of interest; performing a nuclear run-on (NRO) reaction with the permeabilized cell or isolated nucleus, wherein a purifiable nucleotide analog is added to the NRO reaction; optimizing the number of bases traveled by engaged polymerases for high resolution and low bias for nucleotide content of transcribed sequences by limiting a second nucleotide concentration or duration of the NRO reaction; isolating NRO-RNA from the NRO reaction; hydrolyzing the NRO-RNA isolated from the NRO reaction to optimize resolution of polymerase location; selecting hydrolyzed NRO-RNA with a solid support to obtain an enriched, purified fraction of the hydrolyzed NRO-RNA; enzymatically repairing the hydrolyzed NRO-RNA; and ligating the hydrolyzed NRO-RNA to compatible adapter oligos.

Claims

1. A method for performing a genome-wide nuclear run-on assay in a cell of interest comprising:

1) permeabilizing the cell of interest or isolating the nucleus from the cell of interest;

2) performing a nuclear run-on (NRO) reaction with the permeabilized cell or the isolated nucleus, wherein a purifiable nucleotide analog is added to the NRO reaction;

3) optimizing the number of bases traveled by engaged polymerases for high resolution and low bias for nucleotide content of transcribed sequences by limiting a second nucleotide concentration or duration of the NRO reaction;

4) isolating NRO-RNA from the NRO reaction;

5) hydrolyzing the NRO-RNA isolated from the NRO reaction to optimize resolution of polymerase location;

6) selecting hydrolyzed NRO-RNA with a solid support to obtain a highly enriched and purified fraction of the hydrolyzed NRO-RNA;

7) enzymatically repairing the hydrolyzed NRO-RNA; and

8) ligating the hydrolyzed NRO-RNA to compatible adapter oligos.

2. The method of claim 1 wherein the cell of interest is a plurality of cells of interest and the step of permeabilizing comprises permeabilizing the plurality.

3. The method of claim 1 wherein the cell of interest is a plurality of cells of interest and the step of isolating the nucleus comprises isolating nuclei from the plurality.

4. The method of claim 1 wherein the step of isolating the nucleus comprises chemical or mechanical disruption of the outer cell membrane.

5. The method of claim 1 wherein the solid support is a bead support, column matrix, membrane support, biochip, microtiter plate or microfluidic device.

6. The method of claim 1 wherein the purifiable nucleotide analog comprises a purifiable affinity tag.

7. The method of claim 6 wherein the purifiable nucleotide analog is 5-Bromo-UTP (BrU) and the second nucleotide is not U or an analog thereof.

8. The method of claim 1 wherein the step of isolating the NRO-RNA comprises using a moiety that binds BrU contained within the NRO-RNA.

9. The method of claim 8 wherein the moiety is an antibody, an aptamer or a protein that reversibly binds BrU contained within the NRO-RNA.

10. The method of claim 1 wherein the step of enzymatically repairing the hydrolyzed NRO-RNA comprises removing the 5′

cap.

11. The method of claim 10 wherein removing the 5′

cap is accomplished through tobacco acid pyrophosphatase (TAP) treatment.

12. The method of claim 1 wherein the step of enzymatically repairing the hydrolyzed NRO-RNA comprises adding a 5′

-phosphate (5′

-P).

13. The method of claim 12 wherein adding the 5′

-P is accomplished through neutral pH T4 polynucleotide kinase (T4 PNK) treatment.

14. The method of claim 1 wherein the step of enzymatically repairing the hydrolyzed NRO-RNA comprises removing a 3′

-phosphate (3′

-P).

15. The method of claim 14 wherein removing the 3′

-P is accomplished through low pH T4 PNK treatment.

16. The method of claim 1 comprising reverse transcribing the NRO-RNA ligated to the compatible adapter oligos.

17. The method of claim 16 comprising producing a NRO-cDNA second strand by DNA extension.

18. The method of claim 17 comprising amplifying the double-stranded NRO-cDNA thereby producing a NRO-library.

19. The method of claim 18 comprising sequencing the amplified NRO-library.

20. The method of claim 19 comprising mapping one or more sequence reads to a reference genome.

21. The method of claim 20 comprising determining position, orientation or number of hits for the sequence read.

22. The method of claim 1 wherein the hydrolyzing step comprises base hydrolyzing.

23. The method of claim 1 wherein the hydrolyzing step comprises RNase hydrolyzing.

24. The method of claim 1 wherein the step of selecting hydrolyzed NRO-RNA comprises triple-selecting the hydrolyzed NRO-RNA.

25. The method of claim 1 comprising analyzing the hydrolyzed NRO-RNA ligated to compatible adapter oligos using sequencing analysis or microarray analysis.

26. The method of claim 25 wherein the sequencing analysis is massively parallel sequencing analysis.

27. The method of claim 25 wherein the analysis is microarray analysis and the NRO-RNA is ligated to an oligo containing a promoter for an RNA polymerase.

28. The method of claim 1 comprising analyzing production of nascent RNA.

29. The method of claim 28 comprising determining transcriptionally-engaged polymerase density.

30. The method of claim 28 wherein the production of nascent RNA is compared to accumulated mRNA levels to identify genes regulated by mRNA turnover.

35. The method of claim 1 comprising, after the step of ligating the hydrolyzed NRO-RNA to compatible adapter oligos, the step of amplifying the NRO-RNA.

36. The method of claim 35 comprising the step of:

performing reverse transcription after the step of ligating the hydrolyzed NRO-RNA to compatible adapter oligos;

wherein the ligating step comprises addition of a RNA oligomer to the 5′

-end of the NRO-RNA and addition of an RNA oligomer to the 3′

-end of the NRO-RNA.

37. The method of claim 1 comprising, after the step of amplifying the NRO-RNA, the step of purifying the amplified NRO-RNA by PAGE purification.

38. The method of claim 1 comprising:

treating the isolated nucleus with RNase prior to the step of running the NRO reaction; and

identifying polymerase active sites after the step of ligating the hydrolyzed NRO-RNA to compatible adapter oligos.

39. The method of claim 1, wherein the purifiable nucleotide analog does not allow further elongation.

31. A method for identifying a transcription start site in the genome of a cell of interest comprising the steps of:

1) permeabilizing the cell of interest or isolating the nucleus from the cell of interest;

2) performing a nuclear run-on (NRO) reaction with the permeabilized cell or the isolated nucleus, wherein a purifiable nucleotide analog is added to the NRO reaction;

3) optimizing the number of bases traveled by engaged polymerases for high resolution and low bias for nucleotide content of transcribed sequences by limiting a second nucleotide concentration or duration of the NRO reaction;

4) isolating NRO-RNA from the NRO reaction;

5) hydrolyzing the NRO-RNA isolated from the NRO reaction to optimize resolution of polymerase location;

6) selecting hydrolyzed NRO-RNA with a solid support to obtain a highly enriched and purified fraction of the hydrolyzed NRO-RNA;

7) enzymatically repairing the hydrolyzed NRO-RNA;

8) selecting capped NRO-RNAs through enzymatic enrichment by the oligo-capping method; and

9) ligating the hydrolyzed NRO-RNA to compatible adapter oligos.

32. A method for identifying the position of an active site of an engaged RNA polymerase in the genome of a cell of interest comprising the steps of:

1) permeabilizing the cell of interest or isolating the nucleus from the cell of interest;

2) hydrolyzing RNA in the permeabilized cell or the isolated nucleus with an RNase;

3) performing a nuclear run-on (NRO) reaction with the permeabilized cell or the isolated nucleus, wherein a purifiable nucleotide analog is added to the NRO reaction;

4) optimizing the number of bases traveled by engaged polymerases for high resolution and low bias for nucleotide content of transcribed sequences by limiting a second nucleotide concentration or duration of the NRO reaction;

5) isolating NRO-RNA from the NRO reaction;

6) selecting hydrolyzed NRO-RNA with a solid support to obtain a highly enriched and purified fraction of the hydrolyzed NRO-RNA;

7) enzymatically repairing the hydrolyzed NRO-RNA by removing a 5′

cap from the NRO-RNA and adding a 5′

-P to the NRO-RNA; and

8) ligating the hydrolyzed NRO-RNA to compatible adapter oligos.

33. The method of claim 32 wherein the step of enzymatically repairing the hydrolyzed NRO-RNA by removing the 5′

cap from the NRO-RNA and adding the 5′

-P to the NRO-RNA comprises TAP treatment and neutral pH PINK treatment.

40. The method of claim 32, wherein the purifiable nucleotide analog does not allow further elongation.

41. The method of claim 32 comprising analyzing production of nascent RNA.

42. The method of claim 41 comprising determining transcriptionally-engaged polymerase density.

34. A method for mapping a site of co-transcriptional cleavage that delineates the 3′

end of an mRNA comprising the steps of;

1) permeabilizing the cell of interest or isolating the nucleus from the cell of interest;

2) performing a nuclear run-on (NRO) reaction with the permeabilized cell or isolated nucleus, wherein a purifiable nucleotide analog is added to the NRO reaction;

3) optimizing the number of bases traveled by engaged polymerases for high resolution and low bias for nucleotide content of transcribed sequences by limiting a second nucleotide concentration or duration of the NRO reaction;

4) isolating NRO-RNA from the NRO reaction;

5) optionally hydrolyzing the NRO-RNA isolated from the NRO reaction to optimize resolution of polymerase location;

6) selecting hydrolyzed NRO-RNA with a solid support to obtain a highly enriched and purified fraction of the hydrolyzed NRO-RNA;

7) enzymatically repairing the hydrolyzed NRO-RNA removing a 3′

-P from the hydrolyzed NRO-RNA; and

8) ligating the hydrolyzed NRO-RNA to compatible adapter oligos.