Surface plasmon resonance-based sensors to identify cis-regulatory elements.
In eukaryotes, transcription is regulated by multiprotein complexes binding to specific regions of genomic DNA, called cis-regulatory elements. Comprehensive identification of these elements is an important goal of functional genomics. Hence, it is of practical interest to develop a high-throughput assay to identify cis-regulatory elements. Toward that goal, we demonstrate that a surface plasmon resonance-based assay can identify whether a specific region of DNA binds to proteins present in raw nuclear lysate. Specifically, we immobilized a 16-basepair double-stranded DNA region of the SQSTM1 promoter to the Texas Instruments Spreeta, a surface plasmon resonance sensor. As a control, in a separate experiment, we immobilized a similar piece of DNA that differed by only a single base pair. We observed a significant difference in surface plasmon resonance signal when these two probes were exposed to raw nuclear lysate from NIH/3T3 cells. Using a luciferase-reporter vector transfected into live NIH/3T3 cells, we measured a significant difference in transcriptional activity between the two pieces of DNA. We conclude that a surface plasmon resonance-based sensor is capable of identifying physiologically significant cis-regulatory elements.
Lin
Limin
L
Department of Biomedical Engineering, University of California-Irvine, Irvine, California 92697-2715, USA.
Harris
Joseph W
JW
Thompson
H Garrett R
HG
Brody
James P
JP
D048868:Adaptor Proteins, Signal Transducing; D000327:Adsorption; D000818:Animals; D051379:Mice; D041681:NIH 3T3 Cells; D011401:Promoter Regions, Genetic; D011506:Proteins / Q000235:genetics; D012045:Regulatory Sequences, Nucleic Acid; D000071456:Sequestosome-1 Protein; D020349:Surface Plasmon Resonance / Q000295:instrumentation*
D048868:Adaptor Proteins, Signal Transducing; D011506:Proteins; C099718:SQSTM1 protein, human; D000071456:Sequestosome-1 Protein