Zhang, WenKim, Seohyun ChrisTam, Chun PongLelyveld, Victor S.Bala, SaikatChaput, John C.Szostak, Jack W.2022-02-242022-02-242021-01-25Zhang, W., Kim, S. C., Tam, C. P., Lelyveld, V. S., Bala, S., Chaput, J. C., & Szostak, J. W. (2021). Structural interpretation of the effects of threo-nucleotides on nonenzymatic template-directed polymerization. Nucleic Acids Research, 49(2), 646–656. https://doi.org/10.1093/nar/gkaa12151362-4962https://hdl.handle.net/1805/27930The prebiotic synthesis of ribonucleotides is likely to have been accompanied by the synthesis of noncanonical nucleotides including the threo-nucleotide building blocks of TNA. Here, we examine the ability of activated threo-nucleotides to participate in nonenzymatic template-directed polymerization. We find that primer extension by multiple sequential threo-nucleotide monomers is strongly disfavored relative to ribo-nucleotides. Kinetic, NMR and crystallographic studies suggest that this is due in part to the slow formation of the imidazolium-bridged TNA dinucleotide intermediate in primer extension, and in part because of the greater distance between the attacking RNA primer 3'-hydroxyl and the phosphate of the incoming threo-nucleotide intermediate. Even a single activated threo-nucleotide in the presence of an activated downstream RNA oligonucleotide is added to the primer 10-fold more slowly than an activated ribonucleotide. In contrast, a single activated threo-nucleotide at the end of an RNA primer or in an RNA template results in only a modest decrease in the rate of primer extension, consistent with the minor and local structural distortions revealed by crystal structures. Our results are consistent with a model in which heterogeneous primordial oligonucleotides would, through cycles of replication, have given rise to increasingly homogeneous RNA strands.enAttribution 4.0 United StatesMolecular ConformationMolecular StructurePolymerizationArticle