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

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.