Synthetic DNA Aptamers to Detect Protein Molecular Variants in a High‐Throughput Fluorescence Quenching Assay

Abstract

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.

Using photons to manipulate enzyme inhibition by an azobenzene-modified nucleic acid probe

“…Each probe contained a FRET pair (fluorescein and dabcyl) as a signaling element to monitor the hybridization and dehybridization between the regulatory and inhibitory domains (36). The working principle is that The working principle is that dissociation and association of the 2 domains report high and quenched fluorescence signal, respectively.…”

Real-time PCR detection of protein analytes with conformation-switching aptamers

“…Low nanomolar detection limits have previously been observed with an aptamer beacon developed by Tan's group [43], with an aptamer-based fluorescence anisotropy assay [44], with a colorimetric determination technique using aptamer-modified gold nanoparticles [45], with a luminescence detection strategy [46], and with an exciton detection strategy [47]. It should be noted, though, that proximity ligation coupled with real time PCR could detect zeptomole amounts of PDGF [12].…”

Aptamers evolved from live cells as effective molecular probes for cancer study

“…Aptamers are obtained through an in vitro selection process known as SELEX (systematic evolution of ligands by exponential enrichment) (7,8), in which aptamers are selected from a library of random sequences of synthetic DNA or RNA by repetitive binding of the oligonucleotides to target molecules. Aptamers have had many important applications in bioanalysis, biomedicine, and biotechnology (9)(10)(11)(12). Most aptamers reported so far have been selected by using simple targets, such as a purified protein.…”