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.
J Am Chem Soc. 2020 Sep 16;142(37):15961-15965. doi: 10.1021/jacs.0c06722. Epub 2020 Sep 1.
Our current understanding of the chemistry of the primordial genetic material is fragmentary at best. The chemical replication of oligonucleotides long enough to perform catalytic functions is particularly problematic because of the low efficiency of nonenzymatic template copying. Here we show that this problem can be circumvented by assembling a functional ribozyme by the templated ligation of short oligonucleotides. However, this approach creates a new problem because the splint oligonucleotides used to drive ribozyme assembly strongly inhibit the resulting ribozyme. We explored three approaches to the design of splint oligonucleotides that enable efficient ligation but which allow the assembled ribozyme to remain active. DNA splints, splints with G:U wobble pairs, and splints with G to I (Inosine) substitutions all allowed for the efficient assembly of an active ribozyme ligase. Our work demonstrates the possibility of a transition from nonenzymatic ligation to enzymatic ligation and reveals the importance of avoiding ribozyme inhibition by complementary oligonucleotides.
我们目前对原始遗传物质化学性质的了解充其量只能说是零散的。由于非酶模板复制的效率很低，因此，对足够长的寡核苷酸进行化学复制以实现催化功能是特别成问题的。在这里，我们表明可以通过短寡核苷酸的模板连接来组装功能性核酶来解决这个问题。但是，这种方法产生了一个新的问题，因为用于驱动核酶组装的衔接子寡核苷酸强烈抑制了所得核酶。我们探索了三种设计衔接子寡核苷酸的方法，这些方法可以实现有效的连接，但同时使组装的核酶保持活性。DNA 衔接子、具有 G:U 摆动对的衔接子和具有 G 到 I（肌苷）取代的衔接子都允许有效组装具有活性的核酶连接酶。我们的工作证明了从非酶连接到酶连接的转变的可能性，并揭示了避免互补寡核苷酸抑制核酶的重要性。
