Ultrasensitive detection of protein using an aptamer-based exonuclease protection assay

Currently, methods for protein detection are not as sensitive and specific as methods for detection of specific nucleic acid sequences. Here, we present an analogous technique for detection of proteins using aptamers as ligands for target binding. We have named this method the aptamer-based exonuclease protection assay. We applied a special oligonucleotide probe containing a thrombin aptamer, which has the capacity to recognize thrombin with high affinity and specificity. The aptamer probe is a 22-base-long single-strand oligonucleotide with the thrombin aptamer sequence at the 3'-terminus and 7 additional nucleotides at the 5'-terminus, which is able to bind thrombin with high affinity and specificity. In the exonuclease protection assay, thrombin binds the aptamer and thereby protects it from degradation by exonuclease I, whereas any unbound aptamer probe is degraded by exonuclease I. Subsequently, the aptamer probes that were protected from exonuclease I by thrombin act as linkers to join two free connectors, which contain sequences matching the probe. The joined products, which reflect the identity and amount of the target protein, are amplified by PCR. The exonuclease protection assay is extremely sensitive, since it is based on PCR amplification. This method can detect as few as several hundred molecules of target protein without using washes or separations. In addition, this new method for protein detection is simple and inherits all the advantages of aptamers. The mechanism, moreover, may be generalized and used for other forms of protein analysis.