Assessing protein-RNA interactions using microfluidic capillary mobility shift assays

Jimmy Fourtounis; Jean-Pierre Falgueyret; Camil Elie Sayegh

doi:10.1016/j.ab.2010.11.042

Assessing protein-RNA interactions using microfluidic capillary mobility shift assays

Anal Biochem. 2011 Apr 1;411(1):161-3. doi: 10.1016/j.ab.2010.11.042. Epub 2010 Dec 2.

Authors

Jimmy Fourtounis¹, Jean-Pierre Falgueyret, Camil Elie Sayegh

Affiliation

¹ Department of Antiviral Research, Merck Research Laboratories, Montreal, Quebec, Canada H9H 3L1.

PMID: 21130065
DOI: 10.1016/j.ab.2010.11.042

Abstract

We describe a novel method of characterizing protein-RNA interactions using a fluorescence-based multiwell capillary electrophoresis platform based on microfluidic technology. As a proof of concept, we studied the binding of human immunodeficiency virus 1 (HIV-1) transactivator of transcription (Tat) to the transactivation-responsive RNA (TAR). We established conditions to quantify the binding of recombinant HIV-1 Tat to TAR RNA and validated the assay by demonstrating the dependence of this interaction on the presence of the UCU bulge in TAR. In addition, we showed that neomycin inhibited Tat-TAR binding in a dose-dependent manner (IC(50)=1.6-3.0μM). This microfluidic-based method is high-throughput screening compatible and may be applicable to targeting other nucleic acid-protein interactions.

MeSH terms

Electrophoresis, Capillary
Electrophoretic Mobility Shift Assay / methods*
HIV Long Terminal Repeat / genetics*
Humans
Microfluidics / methods*
Protein Binding
RNA, Viral / metabolism*
tat Gene Products, Human Immunodeficiency Virus / metabolism*

Substances

RNA, Viral
tat Gene Products, Human Immunodeficiency Virus