Nucleic Acids Research Advance Access published online on November 15, 2006
Nucleic Acids Research, doi:10.1093/nar/gkl787
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Methods Online |
A microfluidic-FCS platform for investigation on the dissociation of Sp1-DNA complex by doxorubicin
1 Department of Mechanical Engineering Baltimore, MD 21218, USA 2 Department of Biomedical Engineering Baltimore, MD 21218, USA 3 Department of Biology Baltimore, MD 21218, USA 4 Whitaker Biomedical Engineering Institute, The Johns Hopkins University Baltimore, MD 21218, USA
*To whom correspondence should be addressed. Tel: +1 410 516 7086; Fax: +1 410 516 7254; Email: thwang{at}jhu.edu
Received August 24, 2006. Revised September 19, 2006. Accepted September 28, 2006.
The transcription factor (TF) Sp1 is a well-known RNA polymerase II transcription activator that binds to GC-rich recognition sites in a number of essential cellular and viral promoters. In addition, direct interference of Sp1 binding to DNA cognate sites using DNA-interacting compounds may provide promising therapies for suppression of cancer progression and viral replication. In this study, we present a rapid, sensitive and cost-effective evaluation of a GC intercalative drug, doxorubicin (DOX), in dissociating the Sp1–DNA complex using fluorescence correlation spectroscopy (FCS) in a microfluidic system. FCS allows assay miniaturization without compromising sensitivity, making it an ideal analytical method for integration of binding assays into high-throughput, microfluidic platforms. A polydimethylsiloxane (PDMS)-based microfluidic chip with a mixing network is used to achieve specific drug concentrations for drug titration experiments. Using FCS measurements, the IC50 of DOX on the dissociation of Sp1–DNA complex is estimated to be 0.55 µM, which is comparable to that measured by the electrophoretic mobility shift assay (EMSA). However, completion of one drug titration experiment on the proposed microfluidic-FCS platform is accomplished using only picograms of protein and DNA samples and less than 1 h total assay time, demonstrating vast improvements over traditional ensemble techniques.