Electrophilic activity-based RNA probes reveal a self-alkylating RNA for RNA labeling

Abstract

Probes that form covalent bonds with RNA molecules based on their chemical reactivity would advance our ability to study the transcriptome. We developed a set of electrophilic activity-based RNA probes designed to react with unusually nucleophilic RNAs. We used these probes to identify reactive genome-encoded RNAs, resulting in the discovery of a 42-nt catalytic RNA from an archaebacterium that reacts with a 2,3-disubstituted epoxide at N7 of a specific guanosine. Detailed characterization of the catalytic RNA revealed the structural requirements for reactivity. We developed this catalytic RNA into a general tool to selectively conjugate a small molecule to an RNA of interest. This strategy enabled up to 500-fold enrichment of target RNA from total mammalian RNA or from cell lysate. We demonstrated the utility of this approach by selectively capturing proteins in yeast cell lysate that bind to the ASH1 mRNA.

Chemo‐enzymatic treatment of RNA to facilitate analyses

“…In an alternative approach, a 41 nt long RNA was selected, capable of transferring 2,3‐disubstituted epoxides specifically onto a guanosine, forming a C ‐ N ‐bond at the N 7‐position. The range of epoxide substrates included biotinylated, azide‐ and alkyne‐modified substrates and one directly equipped with a tetramethylrhodamine fluorescent dye (TAMRA) (McDonald et al, ). The ability to specifically modify a ROI was demonstrated by transfecting HEK cells with plasmid DNA coding for 5S‐rRNA fused to the ribozyme.…”

Making the Message Clear: Concepts for mRNA Imaging

“…Using an azide-epoxide probe (2-(3-(3-methyloxiran-2-yl)propoxy)ethyl 4-azidobutanoate, 6 ) enables a subsequent click reaction, as shown for the fluorophore tetramethylrhodamine (TAMRA) ( Figure 2 B). 44 The approach is appealing; however, both self-alkylating appendages were only used in cell lysate until now. Similar to the aptamer approaches, a turn-on effect or the appendage of multiple fluorophores will be necessary to obtain the signal intensity and signal-to-noise ratio required for in vivo applications.…”

A new strategy for site-specific alkylation of DNA using oligonucleotides containing an abasic site and alkylating probes

“…13 The combination of catalytic RNA and electrophilic 35 probes was developed as a tool for selective, irreversible RNA modification. 14 Here, we propose a strategy for the site-specific alkylation of DNA using oligonucleotides containing an abasic (AP) site and the alkylating probes. We previously reported that a 2'-OMe 40 oligonucleotide containing 2-amino-6-vinylpurine (1: AVP) efficiently forms a covalent linkage with the complementary sequence of mRNA at the uridine residue across from the AVP.…”

“…Interestingly, the alkylated product was nearly absent in the 15 duplex DNA containing the Hoechst binding site (AATT) at the 3' side of the target base in the fluorescein-labelled strand (Rev.AP). For the non-reactive Hoechst-AVP derivative (14) that was prepared by a route described in the supporting information, the binding constants to duplex DNA targets were determined 20 by fluorescence titrations. Fig.…”