Assessing protein-RNA interactions using microfluidic capillary mobility shift assays.
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.
Department of Antiviral Research, Merck Research Laboratories, Montreal, Quebec, Canada H9H 3L1.
Jimmy
J
Fourtounis
Jean-Pierre
JP
Falgueyret
Camil Elie
CE
Sayegh
D019075:Electrophoresis, Capillary; D024202:Electrophoretic Mobility Shift Assay / Q000379:methods*; D016325:HIV Long Terminal Repeat / Q000235:genetics*; D006801:Humans; D044085:Microfluidics / Q000379:methods*; D011485:Protein Binding; D012367:RNA, Viral / Q000378:metabolism*; D054322:tat Gene Products, Human Immunodeficiency Virus / Q000378:metabolism*
D012367:RNA, Viral; D054322:tat Gene Products, Human Immunodeficiency Virus