Nicking enzymes are a class of restriction enzymes which cut one strand of the DNA duplex to produce shorter DNA fragments. Their major application is in the Nicking Enzyme Amplification Reaction (NEAR), which has been reported to rapidly synthetize short oligonucleotides (ONs) under mild isothermal conditions.(1) The amplification is based on the continuous repetition of two steps: polymerase-mediated primer extension and nicking enzyme-mediated cleavage. The cleaved ON is too short to form a stable duplex with the template and leaves the duplex, thus regenerating the initial dimer primer-template.NEAR has so far been used on an analytical scale for the detection and quantification of DNA. Here, we present a method based on NEAR developed for the preparation of base-modified ONs.(2) Modified cytidines came out as the most efficient substrates, although the reaction proceeded well also with modified adenosines and uridines. The reaction was scaled-up to obtain nanomolar amounts of modified ONs.The modified ONs were successfully used as primers in primer extension and PCR.(3) Two simple and efficient methods for fluorescent labeling of the PCR products were developed, either based on direct fluorescent labeling of primers or on NEAR synthesis of ethynylated primers, PCR and final click labeling with fluorescent azides.This work was supported by GA AV CR (IAA400040901), by the Czech Science Foundation (203/09/0317, P206/12/G151) and by Gilead Sciences, Inc. (Foster City, CA, USA).1. Van Ness, J.; Van Ness, L.K.; Galas, D.J. Proc. Nat. Ac. Sci. 2003 , 100, 4504.2. M�nov�, P.; Hocek, M. Chem. Commun. 2012 , 48, 6921.3. M�nov�, P.; Raindlov�, V.; Hocek, M. Bioconjugate Chem. 2013 , 24, 1081.

Preparation of modified oligonucleotides by nicking enzyme amplification reaction

248th National Meeting (2014)

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