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Scripps Scientists Optimize Ribozyme RNA Polymerase, Perform Protein-less PCR

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NEW YORK (GenomeWeb) – A newly created ribozyme can replicate short RNA templates, acting as a polymerase in a RNA-only version of PCR. Its existence gives credence to the RNA world hypothesis and suggests that nucleic acid "genes" were able to replicate on their own, without protein enzymes.

David Horning and Gerald Joyce of The Scripps Research Institute engineered the ribozyme, building on Joyce's previous work on self-replicating ribozymes.

Starting with a wild type class I polymerase ribozyme evolved in Joyce's lab, they first tweaked its activity with known enhancements before subjecting the ribozyme to "in vitro evolution" where random mutations were introduced and ribozymes were selected for based on their ability to extend a primer.

After 24 rounds of evolution, the ribozymes could complete a 30-nucleotide truncated aptamer and copy purine-rich templates. The most active ribozyme, 24-3, was about 100 times faster at primer extension than the wild type, reaching a rate of 1.2 nucleotides per minute at a fidelity of 92 percent (compared to 96 percent for wild type).

Horning and Joyce published their work yesterday in the Proceedings of the National Academy of Sciences.

They tout their ribozyme as a major step forward in connecting the dots from an RNA world to the DNA-based, protein-driven forms of life seen today.

"The improved polymerase ribozyme is able to synthesize a variety of complex structured RNAs, including aptamers, ribozymes, and, in low yield, even tRNA," the authors wrote. "Furthermore, the polymerase can replicate nucleic acids, amplifying short RNA templates by more than 10,000 fold in an RNA-catalyzed form of the PCR. Thus, the two prerequisites of Darwinian life—the replication of genetic information and its conversion into functional molecules—can now be accomplished with RNA in the complete absence of proteins."

Coined by Nobel laureate Walter Gilbert in a 1986 Nature publication — but assembled piecemeal over decades with contributions from Francis Crick and Oxford University's Leslie Orgel, the "RNA world" hypothesizes that the earliest life forms on Earth were driven by the ability of RNA to not only store and copy information but also catalyze reactions, including its own replication.

Around that same time, several labs began to discover cases of ribozymes acting as RNA polymerases.

Over the years, Joyce's lab has helped contribute to the changing perception of what RNA can do in biology. He's led studies describing self-replicating ligase ribozymes, cross-replicating ribozymes, and "left-handed" chiral ribozyme RNA polymerases.

As reported by GenomeWeb, he's developing those chiral ligase ribozymes into an amplification-based technology for multiplex, quantitative detection of proteins in human serum sample.

While the PNAS paper did not focus on chiral ribozymes, it did focus on directed evolution to improve the activity and efficiency of polymerase ribozymes.  All previous examples have suffered from being slow and a "strong preference" for unstructured, cytidine-rich templates lacking secondary structures, the authors said.

Their newly created ribozyme RNA polymerase 24-3 was a big improvement, they said. It was able to synthesize aptamers faster than its predecessors and other structured, functional RNAs with heterogeneous sequences and secondary structures, such as the F1 ligase ribozyme, that others couldn't even begin to build.

Horning and Joyce were also able to carry out PCR-like exponential amplification of RNA using 24-3. Dubbed "riboPCR," the protocol began with an RNA template, RNA primers, and the four nucleoside triphosphates. The authors noted that they reduced the concentration of magnesium ions to minimize spontaneous RNA cleavage, added polyethylene glycol as a molecular crowding agent to improve activity, and added tetrapropylammonium chloride to lower the melting point of duplex RNA. The ribozyme was able to amplify a 24-nucleotide RNA template 100 fold after 40 cycles, although the four-nucleotide sequence flanked by 10 nucleotide primer sites was affected by wobble pairing.

"This selection should be generalizable to any polymerase selection," Horning told GenomeWeb in an email. "The cross-chiral polymerase reported in 2014 by Sczepanski and Joyce is an excellent candidate for this evolution," but the ligase ribozymes were likely not, he added.

The authors said the results helped make the case for ancient RNA-based life.

"To sustain life, RNA replication must proceed with sufficient yield to support exponential amplification," they wrote. "The improved properties of the 24-3 polymerase enable the residue-by-residue copying and exponential amplification of short RNAs, with a per-cycle amplification efficiency of 1.3 fold."

Horning added that while replicating short RNAs is important, scientists still need to devise sophisticated ribozymes in order to make the case for the RNA world hypothesis.

"The goal is to get to the point where the polymerase can replicate any RNA, including itself — an RNA replicase ribozyme," he said.

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