Encoding bacterial resistance using modified nucleobases

In present work, in order to extend and express a new third type of nucleic acids in living organisms, we have been evaluating the replication ability of alternative nucleic acids with substituted natural nucleobases both in vitro and in vivo. As an alternative to natural pyrimidines, we chose 5-substituted nucleotides such as 5-chloro-2’-deoxyuridine, 5-fluoro-, 5-methyl-, 5-bromo-2’-deoxycytidine paired with 7-deaza-, 8-aza-, 7-deaza-8aza-2’-deoxyadenosine or 2’-deoxyguanosine. Successful in vitro polymerization of short DNA templates (regular 57- or randomized 67-base templates) was achieved using thermophilic DNA polymerases, Taq and Vent exo -,with different combinations of nucleoside triphosphate analogues. We also tested PCR amplification of long plasmid template with these modified nucleobase derivatives and the best result, full-length 525 bp PCR product, was achieved using 7-deaza-dA paring with 5-Cl-dU and 7-deaza-dG paring with either 5-Me-dC or 5-F-dC and Taq DNA polymerase as catalyst. To further examine the possibility of using the artificial nucleic acid containing these alternative base pairs within the replication system of bacterial cells, we employed gene encoding trimethoprim-resistant dihydrofolate reductase (DHFR) as a template for synthesis modified sequences by PCR. All possible modified 234 bp long genes produced colonies after cloning and transformation into E.coli cells. These results illustrate that modified genes were successfully accepted, transcribed and translated into functional DHFR protein by bacterial expression machinery. As the first example in which a natural biological system was capable to recognize the fully modified gene with all components of an unnatural genetic set, our results may find applications in the next generation of gene engineering and synthetics biology.status: publishe