Chemical Replication of Nucleic Acids

I. Regiospecificity of Chemical RNA Copying

During the nonenzymatic copying of an RNA template by primer extension, either the 2′ or the 3′ hydroxyl of the primer can attack the phosphate of the incoming activated monomer, generating either a 2′-5′- or a 3′-5′-linkage. The resulting complementary strand will therefore contain a mixture of 2′-5′- and 3′-5′-linkages. Such backbone heterogeneity was thought to disrupt the folding—and hence the function—of RNA molecules. Our studies challenged this view and showed that even in the presence of substantial backbone heterogeneity, RNA aptamers and ribozymes can retain their molecular recognition and catalytic functions. Our high-resolution crystal structures rationalized this observation at the atomic level: RNA duplexes can buffer local structural changes caused by 2′-5′-linkages, resulting in a minimally altered global structure. Furthermore, 2′-5′-linkages reduce the energetic barriers of the ribose pseudorotation cycle, allowing RNA molecules to sample a wider range of conformations.

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Giurgiu, C., Li, L., O’Flaherty, D.K., Tam, C.P., Szostak, J.W.*, 2017. A Mechanistic Explanation for the Regioselectivity of Nonenzymatic RNA Primer Extension. J. Am. Chem. Soc. 139, 16741–16747. PDF

Zhang, W., Tam, C.P., Wang, J., Szostak, J.W.*, 2016. Unusual Base-Pairing Interactions in Monomer–Template Complexes. ACS Cent. Sci. 2, 916–926. PDF

Engelhart, A.E., Powner, M.W., Szostak, J.W., 2013. Functional RNAs exhibit tolerance for non-heritable 2′–5′ versus 3′–5′ backbone heterogeneity. Nature Chem 5, 390–394. PDF

Sheng, J., Li, L., Engelhart, A.E., Gan, J., Wang, J., Szostak, J.W., 2014. Structural insights into the effects of 2′-5′ linkages on the RNA duplex. Proc. Natl. Acad. Sci. U.S.A. 111, 3050–3055. PDF

Li, L., Szostak, J.W., 2014. The Free Energy Landscape of Pseudorotation in 3′–5′ and 2′–5′ Linked Nucleic Acids. J. Am. Chem. Soc. 136, 2858–2865. PDF

II. Fidelity

In order for protocells to have evolved in a Darwinian manner, the process of RNA replication must have been accurate enough to allow for the transmission of genetic information. This requires an error rate less than the reciprocal of the effective genome size, or about 1-2% for an RNA genome encoding 1 or 2 small ribozymes. Unfortunately, chemical copying using the four standard nucleobases is error-prone, and a major source of error is the G:U mismatch. Our recent studies showed that this problem can be alleviated by using s²U, a modified nucleotide that stabilizes canonical A:U pairs and destabilizes G:U wobble pairs. This single atom substitution improves both the kinetics and the fidelity of chemical copying of mixed-sequence RNA templates. We are actively exploring the potential roles of such noncanonical nucleotides during the infancy of the RNA world.

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Zhang, S., Blain, J.C., Zielinska, D., Gryaznov, S.M., Szostak, J.W., 2013. Fast and accurate nonenzymatic copying of an RNA-like synthetic genetic polymer. Proc. Natl. Acad. Sci. U.S.A. 110, 17732–17737. PDF

Sheng, J., Larsen, A., Heuberger, B.D., Blain, J.C., Szostak, J.W.*, 2014. Crystal Structure Studies of RNA Duplexes Containing s ² U:A and s ² U:U Base Pairs. J. Am. Chem. Soc. 136, 13916–13924. PDF

Heuberger, B.D., Pal, A., Del Frate, F., Topkar, V.V., Szostak, J.W.*, 2015. Replacing Uridine with 2-Thiouridine Enhances the Rate and Fidelity of Nonenzymatic RNA Primer Extension. J. Am. Chem. Soc. 137, 2769–2775. PDF

Larsen, A.T., Fahrenbach, A.C., Sheng, J., Pian, J., Szostak, J.W.*, 2015. Thermodynamic insights into 2-thiouridine-enhanced RNA hybridization. Nucleic Acids Res 43, 7675–7687. PDF

Izgu, E.C., Oh, S.S., Szostak, J.W.*, 2016. Synthesis of activated 3′-amino-3′-deoxy-2-thio-thymidine, a superior substrate for the nonenzymatic copying of nucleic acid templates. Chem. Commun. 52, 3684–3686. PDF

III. Activated Helper Oligos

It has long been possible to copy C-rich templates by primer extension with 2-methylimidazole (2MeIm)-activated nucleotides. In contrast, the copying of mixed sequence templates containing all four nucleotides has been essentially impossible. We have recently found that by using an activated trimer helper oligo, e.g. with 2-MeIm on the 5′-phosphate, there is a dramatic acceleration of the reaction between the primer and the adjacent monomer. By using a set of activated helper trinucleotides, along with all four activated monomers, we can now copy short mixed sequence templates containing all four nucleotides, in good yield.

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Duzdevich, D., Carr, C.E., Ding, D., Zhang, S.J., Walton, T.S., Szostak, J.W.*, 2021. Competition between bridged dinucleotides and activated mononucleotides determines the error frequency of nonenzymatic RNA primer extension. Nucleic Acids Research 49, 3681–3691. PDF

Zhang, W., Tam, C.P., Zhou, L., Oh, S.S., Wang, J., Szostak, J.W., 2018. Structural Rationale for the Enhanced Catalysis of Nonenzymatic RNA Primer Extension by a Downstream Oligonucleotide. J. Am. Chem. Soc. 140, 2829–2840. PDF

Prywes, N., Blain, J.C., Del Frate, F., Szostak, J.W.*, 2016. Nonenzymatic copying of RNA templates containing all four letters is catalyzed by activated oligonucleotides. eLife 5, e17756. PDF

IV. Alternative Genetic Polymers

In addition to RNA, many alternative genetic polymers have been proposed and some may have played a role in the early evolution of life. Our lab has characterized an extensive array of such genetic polymers, including threose nucleic acids, glycerol nucleic acids, as well as ribonucleotides with 2′-amino or 3′-amino modifications. Both 2′-amino-modified and 3′-amino-modified nucleotides are more reactive than their hydroxyl counterparts, enabling rapid and efficient copying of all four nucleobases on homopolymeric RNA and DNA templates. Future investigations will focus on copying mixed templates with all four nucleobases, within in fatty acid vesicles.

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Jia, X., Zhang, S.J., Zhou, L., Szostak, J.W.*, 2024. Constraints on the emergence of RNA through non-templated primer extension with mixtures of potentially prebiotic nucleotides. Nucleic Acids Research 52, 5451–5464. PDF

Jia, X., Fang, Z., Kim, S.C., Ding, D., Zhou, L., Szostak, J.W.*, 2024. Diaminopurine in Nonenzymatic RNA Template Copying. J. Am. Chem. Soc. 146, 15897–15907. PDF

O’Flaherty, D.K., Zhou, L., Szostak, J.W.*, 2019. Nonenzymatic Template-Directed Synthesis of Mixed-Sequence 3′-NP-DNA up to 25 Nucleotides Long Inside Model Protocells. J. Am. Chem. Soc. 141, 10481–10488. PDF

Ding, D., Fang, Z., Kim, S.C., O’Flaherty, D.K., Jia, X., Stone, T.B., Zhou, L.*, Szostak, J.W.*, 2024. Unusual Base Pair between Two 2-Thiouridines and Its Implication for Nonenzymatic RNA Copying. J. Am. Chem. Soc. 146, 3861–3871. PDF

Pal, A., Das, R.S., Zhang, W., Lang, M., McLaughlin, L.W., Szostak, J.W.*, 2016. Effect of terminal 3′-hydroxymethyl modification of an RNA primer on nonenzymatic primer extension. Chem. Commun. 52, 11905–11907. PDF

Blain, J.C., Ricardo, A., Szostak, J.W., 2014. Synthesis and Nonenzymatic Template-Directed Polymerization of 2′-Amino-2′-deoxythreose Nucleotides. J. Am. Chem. Soc. 136, 2033–2039. PDF