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To explore the possibility of using restriction enzymes in a synthetic biology based on artificially expanded genetic information systems (AEGIS), 24 type-II restriction endonucleases (REases) were challenged to digest DNA duplexes containing recognition sites where individual Cs and Gs were replaced by the AEGIS nucleotides Z and P [respectively, 6-amino-5-nitro-3-(1'-β-D-2'-deoxyribofuranosyl)-2(1H)-pyridone and 2-amino-8-(1'-β-D-2'-deoxyribofuranosyl)-imidazo[1,2-a]-1,3,5-triazin-4(8H)-one]. These AEGIS nucleotides implement complementary hydrogen bond donor-donor-acceptor and acceptor-acceptor-donor patterns. Results allowed us to classify type-II REases into five groups based on their performance, and to infer some specifics of their interactions with functional groups in the major and minor grooves of the target DNA. For three enzymes among these 24 where crystal structures are available (BcnI, EcoO109I and NotI), these interactions were modeled. Further, we applied a type-II REase to quantitate the fidelity polymerases challenged to maintain in a DNA duplex C:G, T:A and Z:P pairs through repetitive PCR cycles. This work thus adds tools that are able to manipulate this expanded genetic alphabet in vitro, provides some structural insights into the working of restriction enzymes, and offers some preliminary data needed to take the next step in synthetic biology to use an artificial genetic system inside of living bacterial cells.

Recognition of an expanded genetic alphabet by type-II restriction endonucleases and their application to analyze polymerase fidelity