Methods for identifying RNA segments bound by RNA-binding proteins or ribonucleoprotein complexes

US8841073B2 · kind B2 · utility

Assignee

• The Rockefeller University · US

Inventors

• Markus Landthaler · Berlin, DE
• Manuel Ascano, Jr. · New York, US
• Markus Hafner · New York, US
• Thomas Tuschl · Oettingen, DE
• Mohsen Khorshid · Basel, CH
• Lukas Burger · Basel, CH
• Mihaela Zavolan · Binningen, CH

Key dates

• Filing date: Jul 28, 2009
• Grant date: Sep 23, 2014
• Expiry date: Apr 8, 2031

Classification

• Technology area (CPC C): Chemistry; Metallurgy
• CPC primary: C12Q1/6869
• WIPO field: Biotechnology
• WIPO sector: Chemistry

Abstract

The present invention relates to a method for identifying a binding site on an RNA transcript, wherein the binding site binds to one or more binding moieties. The method includes, among other things, introducing a photoreactive nucleoside into living cells wherein the living cells incorporate the photoreactive nucleoside into RNA transcripts during transcription thereby producing modified RNA transcripts; reverse transcribing the RNA of isolated cross-linked segments thereby generating cDNA transcripts with one mutation wherein the photoreactive nucleoside is transcribed to a mismatched deoxynucleoside; amplifying the cDNA transcripts thereby generating amplicons; and analyzing the sequences of the amplicons aligned against the reference sequence so as to identify the binding site, wherein the sequences of each amplicon having a mutation resulting from the introduction of the photoreactive nucleoside is considered to be a valid amplicon comprising at least a portion of a binding site on the RNA transcript.