Aptamer-Based Regionally Protected PCR for Protein Detection

Abstract

AbstractBackground: DNA aptamers are single-stranded nucleotide sequences that bind specifically to target molecules. By combining the advantages of PCR for amplifying specific DNA sequences and aptamer technology, we have developed a new strategy to detect target molecules such as proteins.Methods: Ovine follicle-stimulating hormone α subunit (oFSHα) was used as the model protein to generate a specific DNA aptamer via an in vitro evolutionary process. A targeted regional-mapping approach and a target-capturing assay were used to identify the binding region on the aptamer molecule. In the detection assay, referred to as “aptamer-based regionally protected PCR” (ARP-PCR), the aptamer was allowed to bind to the target protein in solution before digestion with DNase I. The region of the aptamer bound to the target was protected from DNase I cleavage. The target-binding region of the aptamer protected from the enzymatic treatment was then amplified by the PCR.Results: Aptamers against oFSHα were generated. Six sequences of 20 selected aptamer clones were identical. This aptamer sequence was divided into 4 regions according to the aptamer’s secondary structure. From examination of the target-binding ability of each region, we determined the specific binding region, for which primers were designed. With the aptamer and primers to detect oFSHα by means of the ARP-PCR method, we were able to detect the target protein at concentrations as low as 10−14 mol/L.Conclusions: Combining the use of a DNA aptamer with the PCR is a potentially useful analytic tool for detection of proteins at low concentrations. .