Investigation of molecular beacon aptamer-based bioassay for platelet-derived growth factor detection.
This report describes studies on the use of a molecular-beacon aptamer (MBA) as a synthetic high-affinity DNA probe that exhibits fluorescence resonance energy transfer (FRET) in response to a specific protein biomarker, platelet-derived growth factor (PDGF). As a step toward the application of the MBA in a fluorescence-based assay for biological specimens, we examined the influence of certain physical and chemical parameters of incubation that would affect DNA conformation and DNA-backbone modification, and thus improve nuclease resistance. This bioassay is compatible with pH, temperature, and monovalent cation levels typically encountered in biological samples, and phosphorothioate backbone-modified MBA is able to exhibit specific FRET. With minimal sample processing and without assay optimization, the MBA is able to detect as little as 10 ng PDGF per mug of serum proteins from cell-culture media. We also show that different sets of known fluorophore-quencher pairs can be successfully used in the MBA for sensitive detection of the PDGF target. It should, therefore, be possible to develop multiplex bioassays that monitor either quenching or enhancement for the simultaneous detection of several biomarkers by using MBAs created from high-affinity DNA ligands for the desired protein targets. Interestingly, we observed that, with a DNA ligand with multiple binding sites for a standard multimeric protein target, the FRET bioassay could be accomplished by using a mixture of two individually labeled DNAs-one carrying the fluorophore and the other with the matching quencher. This observation has significant implications in the future design of more selective DNA-based FRET bioassays that use more than one ligand for the same protein target.
Vicens
Marie C
MC
Center for Research at Bio/nano Interface, Department of Chemistry and Shands Cancer Center, McKnight Brain Institute, University of Florida, Gainesville, FL 32611-7200, USA.
Sen
Arup
A
Vanderlaan
Andrew
A
Drake
Timothy J
TJ
Tan
Weihong
W
D001681:Biological Assay / Q000379:methods*; D017077:Culture Media, Conditioned / Q000737:chemistry; D004247:DNA / Q000378:metabolism; D005453:Fluorescence; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D006863:Hydrogen-Ion Concentration; D015335:Molecular Probes / Q000032:analysis* / Q000235:genetics; D010982:Platelet-Derived Growth Factor / Q000032:analysis*; D012680:Sensitivity and Specificity; D013696:Temperature
D017077:Culture Media, Conditioned; D015335:Molecular Probes; D010982:Platelet-Derived Growth Factor; D004247:DNA