A microfluidic-FCS platform for investigation on the dissociation of Sp1-DNA complex by doxorubicin.
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 microM, 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.
Yeh
Hsin-Chih
HC
Department of Mechanical Engineering, The Johns Hopkins University, Baltimore, MD 21218, USA.
Puleo
Christopher M
CM
Lim
Teck Chuan
TC
Ho
Yi-Ping
YP
Giza
Paul E
PE
Huang
Ru Chih C
RC
Wang
Tza-Huei
TH
D004247:DNA / Q000187:drug effects / Q000378:metabolism; D004317:Doxorubicin / Q000494:pharmacology*; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D024202:Electrophoretic Mobility Shift Assay; D007364:Intercalating Agents / Q000494:pharmacology*; D046210:Microfluidic Analytical Techniques; D016329:Sp1 Transcription Factor / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
D007364:Intercalating Agents; D016329:Sp1 Transcription Factor; D004317:Doxorubicin; D004247:DNA