5 years ago

Base-Modified Nucleic Acids as a Powerful Tool for Synthetic Biology and Biotechnology

Base-Modified Nucleic Acids as a Powerful Tool for Synthetic Biology and Biotechnology
Michail Abramov, Piet Herdewijn, Elena Eremeeva, Lia Margamuljana
The ability of various nucleoside triphosphate analogues of deoxyguanosine and deoxycytidine with 7-deazadeoxyadenosine (A1) and 5-chlorodeoxyuridine (T1) to serve as substrates for Taq DNA polymerase was evaluated. The triphosphate set composed of A1, T1, and 7-deazadeoxyguanosine with either 5-methyldeoxycytidine or 5-fluorodeoxycytidine was successfully employed in the polymerase chain reaction (PCR) of 1.5 kb fragments as well as random oligonucleotide libraries. Another effective combination of triphosphates for the synthesis of a 1 kb PCR product was A1, T1, deoxyinosine, and 5-bromodeoxycytidine. In vivo experiments using an antibiotic-resistant gene containing the latter set demonstrated that the bacterial machinery accepts fully modified sequences as genetic templates. Moreover, the ability of the base-modified segments to selectively protect DNA from cleavage by restriction endonucleases was shown. This approach can be used to regulate the endonuclease cleavage pattern. A modified alphabet: Four letters of the genetic alphabet (A, T, G, and C) can be replaced by non-canonical nucleobases to build fully modified nucleic acid systems, denoted ‘DZA’ (see figure). Here, DZA segments are shown to be a versatile molecular tool that can be used to produce diverse DNA libraries and extended PCR fragments, as well as to control restriction enzymes cleavage or synthesize functional genetic templates.

Publisher URL: http://onlinelibrary.wiley.com/resolve/doi

DOI: 10.1002/chem.201700679

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