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

Expanding the chemical space of evolvable non-natural genetic polymers (XNAs) to include functional groups that enhance protein target binding affinity offers a promising route to therapeutic aptamers with high biological stability. Here we describe the chemical synthesis and polymerase recognition of 10 chemically diverse functional groups introduced at the C-5 position of alpha-L-threofuranosyl uridine nucleoside triphosphate (tUTP). We show that the set of tUTP substrates is universally recognized by the laboratory- evolved polymerase Kod-RSGA. Insights into the mechanism of TNA synthesis were obtained from a high-resolution X-ray crystal structure of the postcatalytic complex bound to the primer-template duplex. A structural analysis reveals a large cavity in the enzyme active site that can accommodate the side chain of C-5-modified tUTP substrates. Our findings expand the chemical space of evolvable nucleic acid systems by providing a synthetic route to artificial genetic polymers that are uniformly modified with diversity-enhancing functional groups.

Automated summary

This study expands the chemical space of evolvable nucleic acid systems by introducing diverse functional groups at the C-5 position of alpha-L-threofuranosyl uridine nucleoside triphosphate (tUTP), providing a synthetic route to therapeutic aptamers with high biological stability. Structural insights into TNA synthesis were obtained from a high-resolution X-ray crystal structure analysis, revealing a large cavity in the enzyme active site that accommodates the modification of tUTP substrates. The findings offer a promising route to artificially modified genetic polymers uniformly enhanced with diversity-enhancing functional groups.