title: Uncovering the thermodynamics of monomer binding for RNA replication.
abstract: The nonenzymatic replication of primordial RNA is thought to have been a critical step in the origin of life. However, despite decades of effort, the poor rate and fidelity of model template copying reactions have thus far prevented an experimental demonstration of nonenzymatic RNA replication. The overall rate and fidelity of template copying depend, in part, on the affinity of free ribonucleotides to the RNA primer-template complex. We have now used (1)H NMR spectroscopy to directly measure the thermodynamic association constants, Kas, of the standard ribonucleotide monophosphates (rNMPs) to native RNA primer-template complexes. The binding affinities of rNMPs to duplexes with a complementary single-nucleotide overhang follow the order C > G > A > U. Notably, these monomers bind more strongly to RNA primer-template complexes than to the analogous DNA complexes. The relative binding affinities of the rNMPs for complementary RNA primer-template complexes are in good quantitative agreement with the predictions of a nearest-neighbor analysis. With respect to G:U wobble base-pairing, we find that the binding of rGMP to a primer-template complex with a 5'-U overhang is approximately 10-fold weaker than to the complementary 5'-C overhang. We also find that the binding of rGMP is only about 2-fold weaker than the binding of rAMP to 5'-U, consistent with the poor fidelity observed in the nonenzymatic copying of U residues in RNA templates. The accurate Ka measurements for ribonucleotides obtained in this study will be useful for designing higher fidelity, more effective RNA replication systems.
authors.affiliation: †Howard Hughes Medical Institute, Department of Molecular Biology and Center for Computational and Integrative Biology, Massachusetts General Hospital, 185 Cambridge Street, Boston, Massachusetts 02114, United States.|‡Department of Genetics, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, Massachusetts 02115, United States.
authors.forename: Enver Cagri
authors.initials: EC
authors.lastname: Izgu
authors.affiliation: †Howard Hughes Medical Institute, Department of Molecular Biology and Center for Computational and Integrative Biology, Massachusetts General Hospital, 185 Cambridge Street, Boston, Massachusetts 02114, United States.|‡Department of Genetics, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, Massachusetts 02115, United States.|§Earth-Life Science Institute, Tokyo Institute of Technology, 2-12-1-IE-1 Ookayama, Meguro-ku, Tokyo 152-8550, Japan.
authors.forename: Albert C
authors.initials: AC
authors.lastname: Fahrenbach
authors.affiliation: †Howard Hughes Medical Institute, Department of Molecular Biology and Center for Computational and Integrative Biology, Massachusetts General Hospital, 185 Cambridge Street, Boston, Massachusetts 02114, United States.
authors.forename: Na
authors.initials: N
authors.lastname: Zhang
authors.affiliation: †Howard Hughes Medical Institute, Department of Molecular Biology and Center for Computational and Integrative Biology, Massachusetts General Hospital, 185 Cambridge Street, Boston, Massachusetts 02114, United States.|‡Department of Genetics, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, Massachusetts 02115, United States.
authors.forename: Li
authors.initials: L
authors.lastname: Li
authors.affiliation: †Howard Hughes Medical Institute, Department of Molecular Biology and Center for Computational and Integrative Biology, Massachusetts General Hospital, 185 Cambridge Street, Boston, Massachusetts 02114, United States.|‡Department of Genetics, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, Massachusetts 02115, United States.
authors.forename: Wen
authors.initials: W
authors.lastname: Zhang
authors.affiliation: †Howard Hughes Medical Institute, Department of Molecular Biology and Center for Computational and Integrative Biology, Massachusetts General Hospital, 185 Cambridge Street, Boston, Massachusetts 02114, United States.|‡Department of Genetics, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, Massachusetts 02115, United States.
authors.forename: Aaron T
authors.initials: AT
authors.lastname: Larsen
authors.affiliation: †Howard Hughes Medical Institute, Department of Molecular Biology and Center for Computational and Integrative Biology, Massachusetts General Hospital, 185 Cambridge Street, Boston, Massachusetts 02114, United States.
authors.forename: J Craig
authors.initials: JC
authors.lastname: Blain
authors.affiliation: †Howard Hughes Medical Institute, Department of Molecular Biology and Center for Computational and Integrative Biology, Massachusetts General Hospital, 185 Cambridge Street, Boston, Massachusetts 02114, United States.|‡Department of Genetics, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, Massachusetts 02115, United States.|§Earth-Life Science Institute, Tokyo Institute of Technology, 2-12-1-IE-1 Ookayama, Meguro-ku, Tokyo 152-8550, Japan.|#Department of Chemistry and Chemical Biology, Harvard University, 12 Oxford St., Cambridge, Massachusetts 02138, United States.
authors.forename: Jack W
authors.initials: JW
authors.lastname: Szostak
mesh_terms: D001483:Base Sequence; D004247:DNA / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D000069497:Origin of Life; D012313:RNA / Q000235:genetics / Q000378:metabolism*; D012265:Ribonucleotides / Q000235:genetics / Q000378:metabolism; D013698:Templates, Genetic; D013816:Thermodynamics; D014158:Transcription, Genetic
chemical_list: D012265:Ribonucleotides; D012313:RNA; D004247:DNA