Functional Comparison of Laboratory-Evolved XNA Polymerases for Synthetic Biology

Abstract

Artificial genetic polymers (XNAs) have enormous potential as new materials for synthetic biology, biotechnology, and molecular medicine; yet, very little is known about the biochemical properties of XNA polymerases that have been developed to synthesize and reverse-transcribe XNA polymers. Here, we compare the substrate specificity, thermal stability, reverse transcriptase activity, and fidelity of laboratory-evolved polymerases that were established to synthesize RNA, 2′-fluoroarabino nucleic acid (FANA), arabino nucleic acid (ANA), hexitol nucleic acid (HNA), threose nucleic acid (TNA), and phosphonomethylthreosyl nucleic acid (PMT). We find that the mutations acquired to facilitate XNA synthesis increase the tolerance of the enzymes for sugar-modified substrates with some sacrifice to protein-folding stability. Bst DNA polymerase was found to have weak reverse transcriptase activity on ANA and uncontrolled reverse transcriptase activity on HNA, differing from its known recognition of FANA and TNA templates. These data benchmark the activity of current XNA polymerases and provide opportunities for generating new polymerase variants that function with greater activity and substrate specificity.

Synthesis and Polymerase Recognition of Threose Nucleic Acid Triphosphates Equipped with Diverse Chemical Functionalities

“…The stereochemistry of the modified nucleosides was unambiguously determined by solving the small-molecule X-ray crystal structures of two representative members. We further show that the set of tUTP substrates is universally recognized by the engineered polymerase Kod-RSGA, which is currently the best example of a DNA-dependent TNA polymerase . We obtained insight into the mechanism of TNA synthesis by solving a high-resolution X-ray crystal structure of the postcatalytic complex of Kod-RSGA bound to the primer–template duplex.…”

“…We further show that the set of tUTP substrates is universally recognized by the engineered polymerase Kod-RSGA, which is currently the best example of a DNA-dependent TNA polymerase. 33 We obtained insight into the mechanism of TNA synthesis by solving a high-resolution X-ray crystal structure of the postcatalytic complex of Kod-RSGA bound to the primer− template duplex. The crystal structure reveals a large cavity in the major groove region of the primer−template binding site that can accommodate the side chain of C-5-modified tUTP substrates.…”

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

“…Evolved mutants 'RGFA', ('RGVG', E593G, V685A), 'RGFH' and 'RGLH' showed good activity when 2'-OMe UTP was used as a substrate, and mutant ('RGVG', E593G, V685A) showed the best activity when more kinds of Tgo-EPFLH was demonstrated to be a tPhoNA synthase, while Tgo RT521 and KOD RT521K showed efficient ability to reverse transcribe tPhoNA into DNA 69 . Bst DNAP displayed good activities for the reverse transcriptions of FANA and TNA, but much lower activity for the reverse transcription of ANA [177][178][179] . Other than the extensively explored polymerases described above, some other natural or mutated polymerases have also been investigated for the activities towards unnatural substrates.…”

Towards polymerase-mediated synthesis of artificial RNA–DNA metal base pairs

“…This might be ascribed to the fact that DNA polymerases are finely tuned biological machineries that have the capacity of strongly distinguishing dNTPs from sugar modified nucleotides including NTPs. 25,26 The strong discrimination of NTPs (by a factor of up to 10 5 ) stems from a steric exclusion caused by a clash between the 2 0 -hydroxyl moiety of the incoming rNTP and an active site residue, usually equipped with a bulky side chain such as tryptophan or phenylalanine. [26][27][28][29] Hence, most naturally occurring DNA polymerases predominantly incorporate deoxynucleoside monophosphates (dNMPs) and depending on the polymerase, insert only one ribonucleoside monophosphate every hundred thousand correct nucleotides.…”