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Among wide applications of nucleotide analogs, their roles in enzyme catalytic reactions are significant in both fundamental and medical researches. By introducing analogs into circular templates, we succeeded in determining effects of four analogs on RCA efficiency for three different DNA polymerases. Results showed an obvious suppression effect for 2′-OMeRNA modification, which might be due to the size of the C2′-modified moieties. 2′-F RNA, LNA and PS had little interference, suggesting good analog candidates for application in RCA. Different polymerases and nucleobases made a little difference according to analogs we used. These results are useful for understanding polymerase catalytic mechanism and analogs applications in RCA reaction. Schematic of RCA affected by nucleotide analogs. (A) The structure comparison of DNA and nucleotide analogs used. (B) Analogs were substituted in circular template for RCA.