Functional nucleic acids are of great interest due to potential applications in chemical biology, bioanalysis and nanotechnology. Apart from traditional chemical synthesis, nucleobase-functionalized DNA can be prepared enzymatically by the polymerase incorporation of modified deoxyribonucleoside triphosphates. PCR is used for the synthesis of long dsDNA with high density of modifications, whereas primer extension (PEX) is the most common method for the synthesis of medium-sized oligonucleotides (ONs) bearing several modifications.(1)A general methodology for enzymatic construction of ONs bearing one single modification at a specific site within the sequence was developed by a single-nucleotide incorporation (SNI) followed by primer extension with natural dNTPs.(2) The process of SNI was monitored using a fluorescent label.(3) The methodology was applied in the synthesis of diverse labeled sequences for the study of their electrochemical properties, including sequence-dependence of the redox potential.Nicking enzyme amplification reaction (NEAR) was optimized for the use of modified dNTPs and afforded nanomolar amounts of short modified oligonucleotides.(4) These were successfully used as primers in PEX and PCR.(5) Direct incorporation of fluorescent labels into primer in NEAR afforded fluorescently labeled primers. Indirect fluorescent labeling of PCR products was achieved by NEAR synthesis of ethynylated primers, subsequent 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. Hocek, M.; Fojta, M. Org. Biomol. Chem. 2008 , 6, 2233.2. M�nov�, P.; Cahov�, H.; Pluncara, M.; Havran, L.; Fojta, M.; Hocek, M. Chem. Commun. 2012 , 49, 4652.3. Riedl, J.; M�nov�, P.; Pohl, R.; Ors�g, P.; Fojta, M.; Hocek, M. J. Org. Chem. 2012 , 77, 8287.4. M�nov�, P.; Hocek, M. Chem. Commun. 2012 , 48, 6921.5. M�nov�, P.; Raindlov�, V.; Hocek, M. Bioconjugate Chem. 2013 , 24, 1081.

New enzymatic methods for the preparation of modified oligonucleotides

248th National Meeting (2014)

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