Fluorescent Branched RNAs for High-Throughput Analysis of Dbr1 Enzyme Kinetics and Inhibition

Adam Katolik, Nathaniel E. Clark, Nobuhiro Tago, Eric J. Montemayor, P. John Hart, Masad J. Damha

Research output: Contribution to journalArticlepeer-review

3 Scopus citations

Abstract

We have developed fluorescent 2′,5′ branched RNAs (bRNA) that permit real time monitoring of RNA lariat (intron) debranching enzyme (Dbr1) kinetics. These compounds contain fluorescein (FAM) on the 5′ arm of the bRNA that is quenched by a dabcyl moiety on the 2′ arm. Dbr1-mediated hydrolysis of the 2′,5′ linkage induces a large increase in fluorescence, providing a convenient assay for Dbr1 hydrolysis. We show that unlabeled bRNAs with non-native 2′,5′-phosphodiester linkages, such as phosphoramidate or phosphorothioate, can inhibit Dbr1-mediated debranching with IC50 values in the low nanomolar range. In addition to measuring kinetic parameters of the debranching enzyme, these probes can be used for high throughput screening (HTS) of chemical libraries with the aim of identifying Dbr1 inhibitors, compounds that may be useful in treating neurodegenerative diseases and retroviral infections.

Original languageEnglish (US)
Pages (from-to)622-627
Number of pages6
JournalACS Chemical Biology
Volume12
Issue number3
DOIs
StatePublished - Mar 17 2017

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Medicine

Fingerprint Dive into the research topics of 'Fluorescent Branched RNAs for High-Throughput Analysis of Dbr1 Enzyme Kinetics and Inhibition'. Together they form a unique fingerprint.

Cite this