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...

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Bibliographic Details
Published in:Analytical biochemistry 2011-04, Vol.411 (1), p.161-163
Main Authors: Fourtounis, Jimmy, Falgueyret, Jean-Pierre, Elie Sayegh, Camil
Format: Article
Language:English
Subjects:
capillary electrophoresis
dose response
Electrophoresis, Capillary
Electrophoretic Mobility Shift Assay - methods
HIV Long Terminal Repeat - genetics
Human immunodeficiency virus
Human immunodeficiency virus 1
Humans
Microfluidics - methods
neomycin
Protein Binding
RNA
RNA, Viral - metabolism
screening
tat Gene Products, Human Immunodeficiency Virus - metabolism
transactivators
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Summary: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 = 1.6–3.0 μM). This microfluidic-based method is high-throughput screening compatible and may be applicable to targeting other nucleic acid–protein interactions.
ISSN:0003-2697
1096-0309
DOI:10.1016/j.ab.2010.11.042