Nucleic-acid aptamer is a promising therapeutic reagent. The poor stability of aptamer in vivo limits its application in the field of clinical therapy. Chemical modification can improve the stability of aptamer in serum; however it will bring a risk by the toxic nucleic acid metabolites. Circular aptamer is an attractive choice to improve the stability of aptamer. Here we developed a novelty rolling circle amplification (RCA) method to produce the monovalent circular ssDNA aptamer. In this RCA assay, the digestion reaction mediated by EcoR I was applied to ensure that the monovalent ssDNA aptamer molecules were obtained, the ligation reaction mediated by heat-resisted Taq DNA ligase was also applied to constructed circular aptamer template (caApt), and the digestion reaction mediated by Exonuclease I and III was used to detect and purify the circular ssDNA aptamer. A real-rime RCA also was designed to estimate the efficiency of the cyclization. The result showed that our RCA method can efficiently produced monovalent circular ssDNA aptamer with exonuclease-resistant properties. The RCA method is a simple, fast, low-cost, and easy-to-automated method to prepare monovalent circular ssDNA aptamers.
Aptamer, Stability, Circularization, Rolling circle amplification
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