Preparation of Monovalent Circular ssDNA Aptamer Using Rolling Circle Amplification Method

ISSN: 0973-7510

E-ISSN: 2581-690X

Huiyong Yang^1,2, Li Yang^1,2, Junsheng lin^{1, 2, 3} and Yong Diao^1,2

¹School of Biomedical sciences/Institute of Molecular Medicine, Huaqiao University, Quanzhou – 362 021, Fujian, China.

²Molecular Medicine Engineering Research Center of the Ministry of Education, Huaqiao University, Quanzhou – 362 021, China.

³Centre for Reproduction and Genomics, AgResearch, Invermay, Mosgiel 9053, New Zealand.

J Pure Appl Microbiol. 2013;7(Spl. Edn.: April):481-485

Received: 03/03/2013 | Accepted: 14/04/2013 | Published: 30/04/2013

Abstract

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.

Keywords

Aptamer, Stability, Circularization, Rolling circle amplification

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