The RNA-catalyzed replication of RNA has long been hypothesized as the basis of RNA life. RNA bridges the gap between genotype (DNA) and phenotype (protein) because RNA acts as both the information carrier and the enzyme. We aim to achieve sustained autocatalytic replication and evolution of RNA through two avenues: (i) a general cross-chiral RNA polymerase that catalyzes the template-directed polymerization of activated mononucleotides (NTPs) of the opposite handedness, currently enabling amplification of nuclease-resistant L-RNA and ultimately enabling cross-chiral replication; (ii) non-covalent assembly of component fragments of an existing homochiral RNA polymerase ribozyme.<br/>(i) Our laboratory developed the first example of an enzyme with cross-chiral activity, which can catalyze the RNA-templated assembly of its own enantiomer from oligonucleotide building blocks, but has only a limited ability to polymerize mononucleotides. Beginning with a library of ~10<sup>15</sup> polymerase variants, we selected molecules composed of D-RNA that catalyze the template-directed polymerization of multiple monomers of L-RNA. After 26 rounds of selection, the resulting optimized ribozyme is capable of cross-chiral polymerization of all four L-NTPs.<br/>(ii) A recent version of the homochiral polymerase ribozyme is the most robust RNA polymerase ribozyme published to date. However, it is unable to synthesize itself in its entirety. The ribozyme has been split into fragments that assemble non-covalently through Watson-Crick base pairing to form a functional polymerase that is capable of template-directed synthesis of RNA. Synthesis of component fragments, rather than the full-length ribozyme, lowers the barrier to sustained autocatalytic replication and evolution.

Advancing polymerase ribozymes towards self-replication

255th National Meeting (2018)

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