WO2002014476A2

WO2002014476A2 - Identifying and quantifying rnas and dnas associated with rna and dna binding proteins

Info

Publication number: WO2002014476A2
Application number: PCT/US2001/025804
Authority: WO
Inventors: James H. Eberwine
Original assignee: The Trustees Of The University Of Pennsylvania
Priority date: 2000-08-17
Filing date: 2001-08-17
Publication date: 2002-02-21
Also published as: AU2001288289A1; WO2002014476A3

Abstract

Methods and kits for identifying RNAs and DNAs associated with an RNA or DNA binding protein via a detector specific for the binding protein or protein bound thereto which is linked to an oligonucleotide capable of directing the synthesis of cDNA copies of RNA or DNA that are in close proximity to or in the vicinity of the bound detector are provided.

Claims

- 12 -What is claimed is:

1. A method for identifying RNAs or DNAs associated with an RNA or DNA binding protein, respectively, comprising:

(a) adding to live cells or a fixed tissue or cell sample a detector specific for an RNA or DNA binding protein or a protein bound thereto, wherein the detector is linked to an oligonucleotide which directs the synthesis of cDNA copies of mRNA or DNA that are in the vicinity of the bound detector;

(b) synthesizing cDNA copies of mRNAs or DNAs in the vicinity of the bound detector; and

(c) using the cDNA or aRNA amplified therefrom as a probe to identify RNAs or DNAs by hybridization to a set of RNAs or DNAs .

2. The method of claim 1 wherein the set of RNAs or DNAs is selected from the group consisting of RNA or DNA microarrays, RNA or DNA macroarrays, cDNA libraries, differential display libraries, and other clone or oligonucleotide sets.

3. The method of claim 1 wherein the oligonucleotide attached to the detector comprises from its 5' to 3 ' end an activatable primary amine group on a carbon linker, a short nonspecific sequence, a restriction enzyme site of an infrequent cutter, a T7 RNA polymerase promoter site in sense orientation, a long stretch of nonspecific sequence, and a totally degenerate sequence 8 to 20 bases in length.

4. A method for quantifying the relative abundance of RNA or DNA in close proximity or bound to a RNA or DNA binding protein, respectively, comprising:

(a) adding to live cells ^■ or a fixed tissue or cell sample a detector specific for an RNA or DNA binding protein or a protein bound thereto, wherein the detector is linked to an oligonucleotide which directs the synthesis of cDNA copies - 13 - of RNA or DNA that are in the vicinity of the bound detector, said oligonucleotide comprising a T7 RNA polymerase promoter site;

(b) synthesizing cDNA copies of RNAs or DNAs in the vicinity of the bound detector;

(c) using the cDNA or aRNA amplified therefrom as a probe to identify RNAs or DNAs by hybridization to a set of RNAs or DNAs; and

(d) quantifying the amount of probe hybridized to the set of RNAs or DNAs wherein, wherein the amount of hybridized probe is indicative of the relative abundance of RNAs or DNAs bound to the RNA or DNA binding protein.

5. A method for identifying a cis-acting element in an RNA responsible for binding of the RNA to an RNA binding protein comprising:

(a) adding to live cells or a fixed tissue or cell sample a detector specific for an RNA binding protein or a protein bound thereto, wherein the detector is linked to an oligonucleotide which directs the synthesis of cDNA copies of mRNA or DNA that are in the vicinity of the bound detector;

(b) synthesizing cDNA copies of mRNAs in the vicinity of the bound detector;

(c) amplifying the cDNA into aRNA;

(d) preparing a library from the aRNA; and (e) sequencing the library; and

(f) comparing the cDNA sequence to the full length sequence so that cis-acting elements in the RNA in the vicinity of the bound detector are identified.

6. A method for determining a translational state of a selected RNA or set of RNAs comprising:

(a) adding to live cells or a fixed tissue or cell sample a first detector specific for a protein associated with a first translational complex, wherein the first detector is - 14 - linked to an oligonucleotide which directs the synthesis of cDNA copies of mRNA that are in the vicinity of the first detector bound to the protein associated with the first translational complex; (b) synthesizing cDNA copies of mRNAs in the vicinity of the bound detector;

(c) amplifying the cDNA into aRNA;

(d) using the aRNA as a probe to identify RNAs by hybridization to a set of RNAs; (e) quantifying the amount of probe hybridized to the set of RNAs, wherein the amount of hybridized probe is indicative of the relative abundance of RNAs associated with the first translational complex;

(f) repeating steps (a) through (e) with a second detector specific for a protein associated with a second translational complex, wherein the second detector is linked to an oligonucleotide which directs the synthesis of cDNA copies of mRNA or DNA that are in the vicinity of the second detector bound to the protein associated with the second translational complex, so that the relative abundance of RNAs associated the second translational complex can be quantified; and

(g) comparing the relative abundances of the RNAs of the first and second translational complexes so that the translational state of a selected RNA or set of RNAs can be determined.

7. A kit for identifying RNAs associated with a selected RNA binding protein or DNAs associated with a selected DNA binding protein comprising a detector which specifically binds the selected RNA binding protein or DNA binding protein or proteins attached thereto, wherein the detector is linked to an oligonucleotide which directs the synthesis of cDNA copies of RNA or DNA that are in the vicinity of the bound detector.