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Albany 2013: Book of Abstracts

category image Albany 2013
Conversation 18
June 11-15 2013
©Adenine Press (2012)

Efficient and High-Fidelity Copying of an RNA-like Model Prebiotic System

Non-enzymatic RNA replication would provide an important bridge to the RNA world. However, the demonstration of efficient and high-fidelity copying chemistry remains a great experimental challenge. It requires an efficient mechanism that can lead to both a high rate of polymerization and a high degree of fidelity in the copying chemistry. Previous experiments concerning non-enzymatic template-directed syn-thesis of RNA with activated monomers have led to the copying of short RNA templates, but these reac-tions are generally slow (taking days to weeks) and highly error-prone. Therefore, the ability to efficiently and accurately copy arbitrary template sequences remains frustratingly out of reach. N3′-P5′-linked phos-phoramidate DNA is a highly reactive model for self-replicating genetic materials and has been used for studies of non-enzymatic RNA self-replication. It is also an excellent RNA mimic, due to its similar over-all duplex structure, rigidity, and level of hydration (Tereshko, Gryaznov & Egli, 1998). Our experiments show that the high reactivity imparted by the presence of an amino nucleophile allows rapid and efficient copying of all four nucleobases on both homopolymeric and mixed templates. On the other hand, G:T wobble pairing leads to a high error rate. We have therefore investigated the use of the modified nu-cleobase, 2-thio T (Ts) (Sintim & Kool, 2006), to suppress formation of the G:T wobble base-pair. Our results illustrate that the 2-thio modification can both increase polymerization rate and enhance fidelity in this self-replicating N3′-P5′-DNA system. These results suggest that this simple nucleobase modification may have played a role in primordial RNA (or proto-RNA) replication. In addition to sup-pressing the G:T mismatch, an additional benefit gained from its stronger base-pairing with A is that it also reduces A:C mismatch formation. Thus simple modifications of nucleobases might provide a means of suppressing mismatches to yield better fidelity. Taken together, our results show that a high rate of po-lymerization and a high degree of fidelity are not mutually exclusive, but can be achieved simultaneously in non-enzymatic copying of N3′-P5′-linked phosphoramidate DNA. The structural similarity of NP-DNA to RNA suggests that these results could be translated to an RNA-only system.

References

    V. Tereshko, S. Gryaznov, & M. Egli. (1998). Consequences of Replacing the DNA 3' -Oxygen by an Amino Group:  High-Resolution Crystal Structure of a Fully Modified N3' → P5' Phosphoramidate DNA Dodecamer Duplex. J. Am. Chem. Soc., 120, 269-283.

    H. O. Sintim, & E. T. Kool. (2006) Enhanced Base Pairing and Replication Efficiency of Thio-thymidines, Expanded-size Variants of Thymidine. J. Am. Chem. Soc., 128, 396-397.


Shenglong Zhang1,2,3
Sergei M. Gryaznov4
Jack W. Szostak*1,2,3

1Howard Hughes Medical Institute
2Harvard Medical School
3Department of Molecular Biology
Center for Computational and Integrative Biology
Massachusetts General Hospital
185 Cambridge Street
Boston, MA 02114, USA
4Geron Corporation
230 Constitution Drive
Menlo Park, CA 94025, USA.

Ph: (617) 726-5980
Fx: (617) 643-3328
szostak@molbio.mgh.harvard.edu
slzhang@molbio.mgh.harvard.edu