Transcriptome in vivo analysis

Patent metadata

Field	Value
Publication number	US-9540634-B2
Application number	US-201113702415-A
Country	US
Kind code	B2
Filing date	Jun 7, 2011
Priority date	Jun 7, 2010
Publication date	Jan 10, 2017
Grant date	Jan 10, 2017

Abstract

Provided are compositions and methods that permit a hybrid nucleic acid-peptide molecule to enter a cell and when specifically activated within the cell, the molecule anneals to endogenous cellular RNA and permits the isolation of the RNA.

First claim

What is claimed is:

1. A hybrid nucleic acid-peptide molecule comprising:
a) a first, second, and third oligomer, wherein the first oligomer is linked to the second oligomer through a first photocleavable linker and the second oligomer is linked to the third oligomer through a second photocleavable linker, wherein the first, second, and third oligomers are arranged in a caged hairpin conformation
wherein the second oligomer comprises a poly-A nucleic acid sequence, comprising at least 7 consecutive adenosines, that is complementary to a first nucleic acid sequence of the first oligomer, such that the first oligomer is hybridized to the second oligomer, and
wherein the third oligomer comprises a poly-A nucleic acid sequence, comprising at least 7 consecutive adenosines, that is complementary to a second nucleic acid sequence of the first oligomer, such that the first oligomer is hybridized to the third oligomer;
b) a label for isolating nucleic acids, wherein the label is linked to the first oligomer;
c) a peptide moiety comprising a cell penetrating peptide (CPP).

2. The molecule of claim 1 , wherein the first oligomer comprises a nucleic acid sequence comprising at least 18 consecutive 2′-fluoro nucleotides, wherein the second oligomer comprises a poly-A nucleic acid sequence comprising at least 7 consecutive adenosines, and the third oligomer comprises a poly-A nucleic acid sequence comprising at least 7 consecutive adenosines.

3. The molecule of claim 1 , wherein the first oligomer comprises a nucleic acid sequence comprising at least 18 consecutive 2′-fluoro nucleotides, wherein the second oligomer comprises a poly-A nucleic acid sequence, comprising at least 14 consecutive adenosines, and the third oligomer comprises a poly-A nucleic acid sequence comprising at least 14 consecutive adenosines.

4. The molecule of claim 1 , wherein photocleavage of the first and second photocleavable linkers alters the conformation of the molecule from the caged conformation to an uncaged conformation wherein the at least a portion of first oligomer disassociates from at least a portion of at least one of the second oligomer and the third oligomer.

5. The molecule of claim 1 , wherein the label is biotin.

6. The molecule of claim 1 , wherein the CPP is linked to the third oligomer.

7. A method of isolating cellular RNA in a live cell, the method comprising the steps of: introducing the molecule of claim 1 into a cell, activating the cell under conditions suitable for binding of the nucleic acid portion of the hybrid molecule to cellular RNA wherein the activation cleaves the photo cleavable linkers in the molecule, allowing the formation of a hybridized nucleic acid molecule/cellular RNA complex, and isolating the hybridized nucleic acid molecule/cellular RNA complex.

Assignees

• Eberwine James
• Lovatt Ditte
• Richards Julia
• Dmochowski Ivan
• Ruble Brittani
• Univ Pennsylvania

Inventors

• Eberwine James
• Lovatt Ditte
• Richards Julia
• Dmochowski Ivan
• Ruble Brittani

Classifications

• C12Q1/6806 Primary

  Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence) · CPC title

• C12N15/1003 Primary

  Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor · CPC title

• C12Q2525/205

  Aptamer · CPC title