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.
Highlights
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
Previous analyses indicate that Kod-RI functions with a modest rate of ~1 nucleotide per minute[16], which is ~10,000-fold slower than the rate of DNA synthesis by wild-type Kod DNA polymerase[27]
These values, which are within the range of natural archaeal B-family DNA polymerases[28], led us to speculate that the slow rate of threofuranosyl nucleic acid (TNA) synthesis is due to an imperfect active site that positions tNTP substrates in a geometry that is suboptimal for phosphodiester bond formation
Summary
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. 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. By engineering polymerases to synthesize and recover genetic information encoded in XNA, researchers are developing complex molecular systems that are capable of undergoing Darwinian evolution in response to imposed selection constraints[4]. These studies, which expand our ability to store, propagate, and evolve genetic information, have profound implications for biotechnology, molecular medicine, and the origin of life[5]. Structural information about the ternary complex must be derived from distantly related viral (RB69 Pol and Phi[29] Pol) and eukaryotic polymerases (Pols α, δ, and ε), which share only ~20% identity with archaeal B-family polymerases[21,22,23,24,25]
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