Structural basis for TNA synthesis by an engineered TNA polymerase

Abstract

Darwinian evolution experiments carried out on xeno-nucleic acid (XNA) polymers require engineered polymerases that can faithfully and efficiently copy genetic information back and forth between DNA and XNA. However, current XNA polymerases function with inferior activity relative to their natural counterparts. Here, we report five X-ray crystal structures that illustrate the pathway by which α-(l)-threofuranosyl nucleic acid (TNA) triphosphates are selected and extended in a template-dependent manner using a laboratory-evolved polymerase known as Kod-RI. Structural comparison of the apo, binary, open and closed ternary, and translocated product detail an ensemble of interactions and conformational changes required to promote TNA synthesis. Close inspection of the active site in the closed ternary structure reveals a sub-optimal binding geometry that explains the slow rate of catalysis. This key piece of information, which is missing for all naturally occurring archaeal DNA polymerases, provides a framework for engineering new TNA polymerase variants.

The Structure of an Archaeal B‐Family DNA Polymerase in Complex with a Chemically Modified Nucleotide

“…Recently, crystal structures of closed ternary complexes of KOD DNA pol [6] and a mutant [7] were published, giving first insights into the coordination of the triphosphate and the metal ions in the active site. However, structural data for DNA pols in complex with the above-mentioned nucleobasemodified nucleotides is so far only limited to the A-family KlenTaq DNA pol.…”

Engineering-driven biological insights into DNA polymerase mechanism

“…This dynamic process represents a key bottleneck in the engineering of XNA polymerases, where the unnatural triphosphate substrates (or XNA templates, or both) are likely to have suboptimal fit in the active site, as recently reported for a KOD-based TNA synthase crystallised as a ternary complex with a nascent DNA duplex and TNA nucleotide triphosphate in its active site [34].…”

Modified nucleic acids: replication, evolution, and next-generation therapeutics

“…Chim et al recently reported ternary structures of a hyperthermophilic B-family polymerase, an engineered KOD mutant, polymerizing TNA [152]. Given the prevalent use of B-family hyperthermophiles in polymerase engineering, the structures of the apo, binary, and ternary polymerase complexes will prove useful in developing hypotheses for further work in the field.…”