METHODS, SYSTEMS AND KITS FOR DETECTING PROTEIN-NUCLEIC ACID INTERACTIONS

• US 20100323361A1
• Filed: 06/16/2010
• Published: 12/23/2010
• Est. Priority Date: 06/18/2009
• Status: Active Grant

• Current Assignee: Penn State Research Foundation
• Original Assignee: Penn State Research Foundation
• Inventors: Pugh, Benjamin Franklin, Rhee, Ho Sung
• Granted Patent: US 8,367,334 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6804 Nucleic acid analysis using immunogens immunoassay G01N33/53
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• 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 2521/319 Exonuclease
• CPC Class Codes: C12Q 2521/325 Single stranded exonuclease
• CPC Class Codes: C12Q 2522/101 Single or double stranded nucleic acid binding proteins
• CPC Class Codes: C12Q 2523/101 Crosslinking agents, e.g. psoralen
• CPC Class Codes: C12Q 2523/301 Sonication
• CPC Class Codes: C12Q 2565/531 characterised by the capture moiety being a protein for target oligonucleotides

Abstract

Methods, systems and kits for detecting protein-nucleic acid interactions, in particular, detecting the genomic location to near-base pair resolution at which a particular protein (e.g., transcription factor) binds includes combining steps of a convention chromatin immunoprecipitation (ChIP) assay with use of an exonuclease that digests DNA strands in the 5′-3′ or 3′-5′ direction until it reaches a bound protein including a protein crosslinked to DNA. A significant improvement of the resolution and dynamic range of the ChIP assay will increase one'"'"'s ability to determine with confidence where a particular protein is binding in the genome. Importantly, proteins that inefficiently crosslink to DNA (either intrinsically or due to indirect crosslinking via another protein) and thus are very difficult to detect, are expected to be significantly detected by the kits and methods described herein. Inasmuch as most aspects of infectious diseases, inborn diseases, and cancers are rooted in the mis-expression of genes, having a better measure of the binding of proteins to genes or promoter regions will greatly enhance the ability of investigators to understand the driving molecular mechanisms behind these human maladies.

Claims

1. A method of identifying a plurality of locations in a genome at which a protein of interest binds to the genome comprising the steps of:

(a) subjecting a plurality of cells or extract thereof to chromatin immunoprecipitation resulting in at least one nucleic acid fragment bound to the protein of interest;

(b) subjecting the at least one nucleic acid fragment bound to the protein of interest to exonuclease treatment resulting in a single-stranded nucleic acid fragment bound to the protein of interest having an exonuclease-treated end demarcating at least one location in the genome at which the protein of interest binds, wherein the exonuclease treatment comprises a double-stranded nucleic acid-specific exonuclease that degrades a nucleic acid either in a 5′

-to-3′

or a 3′

-to-5′

direction and a single-stranded nucleic acid-specific exonuclease that degrades a nucleic acid in the same direction as the double-stranded nucleic acid-specific exonuclease; and

(c) identifying the exonuclease-treated end of the single-stranded nucleic acid fragment bound to the protein of interest.

2. The method of claim 1, wherein identifying the exonuclease-treated end of the single-stranded nucleic acid fragment bound to the protein of interest identifies the plurality of locations in the genome at which the protein of interest binds.

3. The method of claim 1, wherein the protein of interest is any protein that binds directly or indirectly to a nucleic acid.

4. The method of claim 1, wherein step (c) of identifying the exonuclease-treated end comprises at least one selected from the group consisting of:

DNA sequencing, microarrays, and polymerase chain reaction (PCR).

5. The method of claim 1, wherein the genome is any nucleic acid.

6. The method of claim 1, wherein the single-stranded nucleic acid-specific exonuclease and the double-stranded nucleic acid-specific exonuclease degrade only one nucleic acid strand in either a 5′

-to-3′

direction or a 3′

-to-5′

direction.

7. The method of claim 1, wherein each of the plurality of locations in the genome at which the at least one protein of interest binds is identified within a five base pair or less limit of resolution.

8. The method of claim 1, wherein the genome is a human genome.

19. The method of claim 1, wherein the plurality of locations comprise a majority of locations at which the protein of interest binds to the genome.

9. A method for identifying the nucleotides of a nucleic acid sequence to which a peptide or a polypeptide binds, the method comprising the steps of:

(a) obtaining a sample comprising at least a first nucleic acid sequence and at least a first peptide or polypeptide;

(b) binding the at least first nucleic acid sequence to the at least first peptide or polypeptide;

(c) subjecting the at least first nucleic acid sequence bound to the at least first peptide or polypeptide to exonuclease treatment whereby nucleotides of the at least first nucleic acid that are not bound to the at least first peptide or polypeptide are degraded, resulting in a single-stranded fragment of the at least first nucleic acid, the fragment consisting of nucleotides of the at least first nucleic acid that bind to the at least first peptide or polypeptide, wherein the exonuclease treatment comprises a double-stranded nucleic acid-specific exonuclease that degrades a nucleic acid in a 5′

-to-3′

or a 3′

-to-5′

direction and a single-stranded nucleic acid-specific exonuclease that degrades a nucleic acid in the same direction as the double-stranded nucleic acid-specific exonuclease; and

(d) identifying the nucleotides of the at least first nucleic acid that bind to the at least first peptide or polypeptide.

10. The method of claim 9, wherein step (d) of identifying the nucleotides of the at least first nucleic acid that bind to the at least first peptide or polypeptide comprises at least one selected from the group consisting of:

DNA sequencing, microarrays, and polymerase chain reaction (PCR).

11. The method of claim 9, wherein the nucleic acid sequence is a human genome.

12. The method of claim 9, wherein the double-stranded nucleic acid-specific exonuclease is lambda exonuclease.

13. The method of claim 9, wherein the at least one location in the genome at which the protein of interest binds is identified with a resolution of five base pairs or less.

14. A kit for identifying at least one location in a genome at which a protein of interest binds, the kit comprising:

(a) an exonuclease having 5′

-3′

single-stranded-specific exonuclease activity;

(b) an exonuclease having 5′

-3′

double-stranded-specific exonuclease activity;

(c) at least one buffer;

(d) at least one wash solution; and

(e) instructions for use.

15. The kit of claim 14, further comprising:

(f) at least one elution solution;

(g) at least one primer;

(h) at least one adapter

(i) a least one DNA polymerase

(j) a polynucleotide kinase; and

(k) a DNA ligase.

16. The kit of claim 14, wherein the exonuclease having S′

-3′

double-stranded-DNA-specific exonuclease activity is lambda exonuclease, and the exonuclease having S′

-3′

single-stranded-DNA-specific exonuclease activity is RecJ_f exonuclease.

17. A kit for identifying at least one location in a genome at which a protein of interest binds, the kit comprising:

(a) an exonuclease having 3′

-5′

single-stranded-specific exonuclease activity;

(b) an exonuclease having 3′

-5′

double-stranded-specific exonuclease activity;

(c) at least one buffer;

(d) at least one wash solution; and

(e) instructions for use.

18. The kit of claim 17, further comprising:

(f) at least one elution solution;

(g) at least one primer;

(h) at least one adapter

(i) a DNA polymerase

(j) a polynucleotide kinase; and

(k) a DNA ligase.