Unnatural base pair systems toward the expansion of the genetic alphabet in the central dogma

Abstract

Toward the expansion of the genetic alphabet of DNA, several artificial third base pairs (unnatural base pairs) have been created. Synthetic DNAs containing the unnatural base pairs can be amplified faithfully by PCR, along with the natural A–T and G–C pairs, and transcribed into RNA. The unnatural base pair systems now have high potential to open the door to next generation biotechnology. The creation of unnatural base pairs is a consequence of repeating “proof of concept” experiments. In the process, initially designed base pairs were modified to address their weak points. Some of them were artificially evolved to ones with higher efficiency and selectivity in polymerase reactions, while others were eliminated from the analysis. Here, we describe the process of unnatural base pair development, as well as the tests of their applications.

Faithful PCR Amplification of an Unnatural Base‐Pair Analogue with Four Hydrogen Bonds

“…We concluded that the proofreading activity recognizes A:NaO O and/or G:NaO O mismatches. It has been reported that this proofreading activity of the DNA polymerases improves the accuracy of incorporating unnatural base‐pair analogues,7e, 8b and the benefits of this activity are also apparent in our case.…”

Unnatural Base Pairs for Synthetic Biology

“…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.…”

Overcoming Challenges in Engineering the Genetic Code

“…Additionally, stability is crucial to prevent loss of information. Replication error rates below 10 −3 per bp per replication have been recommended for PCR [80], but replication fidelity better than 10 −8 per bp per replication may be necessary in vivo in the absence of a strong selective pressure to maintain the unnatural base pair (as has been demonstrated for nsAAs [34,35]). The fidelities of transcription and translation are more flexible as long as they do not interfere with normal cell function, but net translational fidelity should be comparable with that of current suppression systems [81,82].…”