Amplification, Mutation, and Sequencing of a Six-Letter Synthetic Genetic System

Abstract

The next goals in the development of a synthetic biology that use artificial genetic systems will require chemistry-biology combinations that allow the amplification of DNA containing any number of sequential and non-sequential non-standard nucleotides. This amplification must ensure that the non-standard nucleotides are not unidirectionally lost during PCR amplification (unidirectional loss would cause the artificial system to revert to an all-natural genetic system). Further, technology is needed to sequence artificial genetic DNA molecules. The work reported here meets all three of these goals for a six-letter artificially expanded genetic information system (AEGIS) that comprises four standard nucleotides (G, A, C, and T) and two additional non-standard nucleotides (Z and P). We report polymerases and PCR conditions that amplify a wide range of GACTZP DNA sequences having multiple consecutive unnatural synthetic genetic components with low (0.2% per theoretical cycle) levels of mutation. We demonstrate that residual mutation processes both introduce and remove unnatural nucleotides, allowing the artificial genetic system to evolve as such, rather than revert to a wholly natural system. We then show that mechanisms for these residual mutation processes can be exploited in a strategy to sequence “six-letter” GACTZP DNA. These are all not yet reported for any other synthetic genetic system.

Unnatural Base Pairs for Synthetic Biology

“…9) Recently, they applied the six-letter genetic system with the P : Z pair to the cell-systematic evolution of ligands by exponential enrichment (SELEX) system and succeeded in obtaining highly active aptamers against HepG2 liver cancer cells. and 2,4-difluorotoluene (F) as steric isosteres of the natural A : T pair 12,13) (Fig.…”

“…7d). Although the replication fidelity of the ImN N : NaO O pair is slightly inferior to those of other unnatural base-pair analogs, 8,9,26,28,34,46,47) it is strongly indicated that the ImN N : NaO O pair acts as an orthogonal base pair for WC base pairs during PCR amplification.…”

Polymerize it all! A critical review

“…In this context, the dZ:dP pair consisting of two non-standard nucleoside analogues (dZ 7 and dP 8, Figure 2) with a different hydrogen bond pattern, has advanced as a potent surrogate of a natural base pair and is thus a potential candidate for a six-letter (ACGTZP) synthetic genetic system 40 . While polymerases such as…”

Chemical biology: DNA's new alphabet

“…In 2011, Hirao's team reported that DNA containing an unnatural hydrophobic base pair, called Ds and Diol1-Px, could be copied with 99.77-99.92% fidelity per replication 9 . The same year, Benner and his colleagues showed that another unnatural base pair -P and Z, which join using hydrogen bonds -achieved fidelity of 99.8% per replication 10 . And in July, Romesberg's team reported rates of 99.66-99.99% for optimized versions of his bases, called NaM and 5SICS (ref.…”

“…Researchers haven't shown that polymerases can copy more than four of the paired bases in a row 10 . The polymerase is "the hard nut to crack", says Benner.…”