Engineered regulation of an RNA ligase ribozyme

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Robertson, Michael Paul

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Catalytic RNA has been implicated as a critical component of the origin of life on Earth. Compelling arguments have been made for an ‘RNA world’ era during the early evolution of life in which all living systems consisted solely of RNA functioning as both genetic storage media and catalytic agents. Since the discovery of catalytic RNA in the early 1980s, the types of chemical reactions that RNA has been shown to catalyze has steadily increased and with it the feasibility of an all-RNA metabolism. However, a key aspect of modern biocatalysts is their ability to attenuate their level of activity in response to changing environmental conditions. Presumably, a similar regulation strategy would also be necessary to sustain a reasonably complex RNA-based metabolism.

We have isolated and designed several RNA ligase ribozymes whose activities are regulated by the presence of a variety of different effectors. An oligonucleotidedependent ligase (L1) was isolated from a randomized pool of RNA using in vitro selection. Other allosterically regulated ribozymes were engineered by replacing a non-essential stem loop of L1 with various small molecule binding RNA aptamers to create aptamer-ribozyme hybrids (aptazymes). Alternatively, we vi have employed a two stage in vitro selection procedure with a partially randomized pool based on the L1 ligase to isolate protein-activated aptazymes that are extremely dependent on their cognate effector for activity.

In addition to the regulation of RNA catalysis, we have been investigating ways to increase the proficiency and versatility of ribozymes by augmenting the chemical functional groups of RNA by the incorporation of modified nucleosides. These modified nucleotides can be efficiently transcribed into RNA using T7 RNA polymerase and have been incorporated into random sequence RNA pools for use in in vitro selection experiments.

These results demonstrate that ribozyme activity can be regulated in much the same way that protein enzymes are regulated in contemporary biochemistry. This ability of RNA to be controlled by various potential metabolic intermediates contributes an additional layer of sophistication to ribozyme catalysis and increases the plausibility that a complex metabolism based solely on RNA could have once existed.



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