Synthetic DNA aptamers to detect protein molecular variants in a high-throughput fluorescence quenching assay.
Real-time protein detection in homogeneous solutions is necessary in many biotechnology and biomedical studies. The recent development of molecular aptamers, combined with fluorescence techniques, may provide an easy and efficient approach to protein elucidation. This report describes the development of a fluorescence-based assay with synthetic DNA aptamers that can detect and distinguish molecular variants of proteins in biological samples in a high-throughput process. We used an aptamer with high affinity for the B chain of platelet-derived growth factor (PDGF), labeled it with a fluorophore and a quencher at the two termini, and measured fluorescence quenching by PDGF. The specific quenching can be used to detect PDGF at picomolar concentrations even in the presence of serum and other cell-derived proteins in cell culture media. This is the first successful application of a synthetic aptamer for the detection of tumor-related proteins directly from the tumor cells. We also show that three highly related molecular variants of PDGF (AA, AB, and BB dimers) can be distinguished from one another in this single-step assay, which can be readily adapted to a microtiter plate assay for high-throughput analysis. The use of fluorescence quenching as a measure of binding between the DNA probe and the target protein eliminates potential false signals that may arise in traditional fluorescence enhancement assays as a result of degradation of the DNA aptamer by contaminating nucleases in biological specimens. This assay is applicable to proteins that are not naturally DNA binding. The excellent specificity, ultrahigh sensitivity, and simplicity of this one-step assay addresses a growing need for high-throughput methods that detect changes in the expression of gene products and their variants in cell cultures and biological specimens.
Fang
Xiaohong
X
Department of Chemistry and McKnight Brain Institute, Shands Cancer Center, College of Medicine, University of Florida, Gainesville, FL 32611, USA.
Sen
Arup
A
Vicens
Marie
M
Tan
Weihong
W
D000818:Animals; D041702:BALB 3T3 Cells; D001667:Binding, Competitive; D004247:DNA / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism; D005456:Fluorescent Dyes; D006801:Humans; D051379:Mice; D010982:Platelet-Derived Growth Factor / Q000032:analysis* / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D014407:Tumor Cells, Cultured
D005456:Fluorescent Dyes; D010982:Platelet-Derived Growth Factor; D004247:DNA