Witnessing the structural evolution of an RNA enzyme

Abstract

An RNA polymerase ribozyme that has been the subject of extensive directed evolution efforts has attained the ability to synthesize complex functional RNAs, including a full-length copy of its own evolutionary ancestor. During the course of evolution, the catalytic core of the ribozyme has undergone a major structural rearrangement, resulting in a novel tertiary structural element that lies in close proximity to the active site. Through a combination of site-directed mutagenesis, structural probing, and deep sequencing analysis, the trajectory of evolution was seen to involve the progressive stabilization of the new structure, which provides the basis for improved catalytic activity of the ribozyme. Multiple paths to the new structure were explored by the evolving population, converging upon a common solution. Tertiary structural remodeling of RNA is known to occur in nature, as evidenced by the phylogenetic analysis of extant organisms, but this type of structural innovation had not previously been observed in an experimental setting. Despite prior speculation that the catalytic core of the ribozyme had become trapped in a narrow local fitness optimum, the evolving population has broken through to a new fitness locale, raising the possibility that further improvement of polymerase activity may be achievable.

Cross-Chiral, RNA-Catalyzed Exponential Amplification of RNA

“…The ability to carry out cross-chiral exponential amplification of functional RNAs opens the door to directed evolution experiments where the cross-chiral polymerase is selected based on the function of the RNA it produces. This approach has proven to be powerful in optimizing the activity of a homochiral RNA polymerase ribozyme. ,, By comparison, the cross-chiral polymerase is a relatively young enzyme that has not had yet the benefit of extensive evolutionary optimization.…”

Trinucleotide building blocks enable exponential ribozyme-catalysed RNA replication and open-ended growth of diverse RNA sequence pools

“…Surprisingly, a similar (if fainter) pattern was also seen in an unseeded control, implying emergence of products even in the absence of a seed template. Upon continued cycling (with serial dilution), levels of synthesised RNA in both seeded and unseeded reaction pools grew substantially and product lengths increased: after 73 cycles (and over 2.5 × 10 5 -fold dilution of the initial reaction), a robust ladder of products up to 36 nt in length was present in both seeded and unseeded reactions (Fig. 5b), consuming a substantial fraction of the ppp NNN triplet substrates supplied upon each dilution step (Fig.…”

“…All life on Earth relies upon the faithful copying of its genetic material -replication -to enable heredity and evolution. This critical behaviour is widely thought to have begun with the templated polymerisation of activated mono-or oligo-nucleotide building blocks by chemical replication processes [1][2][3] and later RNA-catalyzed RNA replication [4][5][6][7] (Fig. 1a).…”

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“…But a longer strand may be able to replicate itself. If they achieve this they will show an RNA world cell can in theory replicate its own RNA without using proteins (Portillo et al, 2021).…”