Transcriptome in vivo analysis (TIVA) and transcriptome in situ analysis (TISA)

Patent metadata

Field	Value
Publication number	US-11873312-B2
Application number	US-202117333376-A
Country	US
Kind code	B2
Filing date	May 28, 2021
Priority date	Aug 4, 2014
Publication date	Jan 16, 2024
Grant date	Jan 16, 2024

Abstract

Compositions and methods of capturing one or more nucleic acid molecules of a cell or subcellular compartment are described. In certain aspects, the compositions comprise a caged molecule comprising one or more photolinkers and an antisense oligonucleotide, which when uncaged hybridizes to a target nucleic acid molecule.

First claim

What is claimed is:

1. A caged nucleic acid molecule comprising: a first, second, and third oligonucleotide, wherein the first oligonucleotide is linked to the second oligonucleotide through a first photocleavable linker and the second oligonucleotide is linked to the third oligonucleotide through a second photocleavable linker, wherein the first oligonucleotide comprises one or more terminal deoxynucleotides, wherein the first oligonucleotide comprises 2′-fluoro uridine, and wherein the one or more terminal deoxynucleotides comprises one or more thymidines.

2. The molecule of claim 1 , wherein the second oligonucleotide and third oligonucleotide each comprise a nucleotide sequence that is complementary to the first oligonucleotide, thereby forming a hairpin where the first oligonucleotide hybridizes to both the second oligonucleotide and third oligonucleotide.

3. The molecule of claim 2 , wherein the first photocleavable linker and the second photocleavable linker comprise nitrobenzyl.

4. The molecule of claim 1 , wherein the first oligonucleotide comprises an antisense oligonucleotide comprising a nucleic acid sequence that is complementary to the polyA tail of an mRNA.

5. The molecule of claim 4 , wherein irradiation of the photocleavable linkers exposes the antisense oligonucleotide thereby allowing the antisense oligonucleotide to hybridize with a target nucleic acid, wherein the antisense oligonucleotide is complementary to the target nucleic acid.

6. The molecule of claim 5 , wherein the first oligonucleotide and one or more terminal deoxynucleotides serve as a primer to synthesize cDNA from the target nucleic acid.

7. The molecule of claim 1 , wherein the molecule further comprises an index oligonucleotide.

8. The molecule of claim 1 , further comprising one or more fluorophores.

9. The molecule of claim 8 , wherein the one or more fluorophores is a FRET pair.

Assignees

• Univ Pennsylvania

Inventors

• Eberwine James
• Dmochowski Ivan J
• Yeldell Sean B
• Griepenburg Julianne C
• Rapp Teresa L
• Singh Jennifer M
• Sul Jai-Yoon
• Lee Jaehee

Classifications

• C07F15/0053 Primary

  without a metal-carbon linkage · CPC title

• B01J35/004

  Operations & Transport · mapped topic

• C07H23/00

  Compounds containing boron, silicon or a metal, e.g. chelates or vitamin B12 (esters with inorganic acids C07H11/00) · CPC title

• C12N13/00

  Treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves · CPC title

• C12N15/113

  Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; {Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing (when used in plants C12N15/8218)} · CPC title