A microfluidic-FCS platform for investigation on the dissociation of Sp1-DNA complex by doxorubicin.
Nucleic Acids Res
; 34(21): e144, 2006.
Article
em En
| MEDLINE
| ID: mdl-17108358
ABSTRACT
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.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Espectrometria de Fluorescência
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Doxorrubicina
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Fator de Transcrição Sp1
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Técnicas Analíticas Microfluídicas
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Avaliação Pré-Clínica de Medicamentos
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Substâncias Intercalantes
Tipo de estudo:
Evaluation_studies
Idioma:
En
Revista:
Nucleic Acids Res
Ano de publicação:
2006
Tipo de documento:
Article
País de afiliação:
Estados Unidos