Building better polymerases: Engineering the replication of expanded genetic alphabets

Abstract

DNA polymerases are today used throughout scientific research, biotechnology and medicine, in part for their ability to interact with unnatural forms of DNA created by synthetic biologists. Here especially, natural DNA polymerases often do not have the “performance specifications" needed for transformative technologies. This creates a need for science-guided rational (or semi-rational) engineering to identify variants that replicate unnatural base pairs (UBPs), unnatural backbones, tags, or other evolutionarily novel features of unnatural DNA. In this review, we provide a brief overview of the chemistry and properties of replicative DNA polymerases and their evolved variants, focusing on the Klenow fragment of Taq DNA polymerase (Klentaq). We describe comparative structural, enzymatic, and molecular dynamics studies of wild type and Klentaq variants, complexed with natural or non-canonical substrates. Combining these methods provides insight into how specific amino acid substitutions distant from the active site in a Klentaq DNA polymerase variant (ZP Klentaq) contribute to its ability to replicate unnatural base pairs (UBPs) with improved efficiency compared to Klentaq. This approach can therefore serve to guide any future rational engineering of replicative DNA polymerases.

Chemical Modifications for a Next Generation of Nucleic Acid Aptamers

“…In another genetic alphabet expansion approach developed by Benner (Figure 5c), the hydrogen bonding pattern of nucleobases is modified so that the resulting analogues specifically form specific hydrogen bonding interactions [49–51] . These unnatural base pairs are fully orthogonal to the canonical, Watson‐Crick base pairs.…”

“…In another genetic alphabet expansion approach developed by Benner (Figure 5c), the hydrogen bonding pattern of nucleobases is modified so that the resulting analogues specifically form specific hydrogen bonding interactions. [49][50][51] These unnatural base pairs are fully orthogonal to the canonical, Watson-Crick base pairs. This versatile method, coined artificially expanded genetic information systems (AEGIS), has been exploited to generate highly potent modified aptamers against cell cancer lines [22,52,53] as well as protein [54] targets.…”

From polymerase engineering to semi-synthetic life: artificial expansion of the central dogma

“…Unfortunately, for many of the exotic unnatural nucleic acids, especially those with modified sugar-phosphate backbones, natural DNA polymerases (DNAPs) and RNA polymerases (RNAPs) are unable to synthesize them efficiently. Approaches of protein engineering have thus been applied on tailoring polymerases for the efficient synthesis of various unnatural nucleic acids, and a number of strategies specifically effective for screening or selecting polymerase mutants have also been developed to facilitate these efforts [2][3][4][5] .…”

Transcription, Reverse Transcription, and Amplification of Backbone‐Modified Nucleic Acids with Laboratory‐Evolved Thermophilic DNA Polymerases

“…Many efforts have been made to identify efficient polymerases for unnatural nucleic acids through the development and use of novel directed evolution methods. The most successful of these have employed methods based on emulsion techniques, such as compartmentalized self-replication (CSR) and compartmentalized self-tagging (CST), or on phage display techniques (Chen & Romesberg, 2014;Houlihan, Arangundy-Franklin, & Holliger, 2017;Morrison, Podracky, & Liu, 2020;Ouaray, Benner, Georgiadis, & Richards, 2020). method based on an optimized phage display system (Chen et al, 2016).…”