Why Stop At One? Researchers Make 10,000 Ribozyme Mutants At Once

Researchers in Japan have created over 10,000 mutants of a ribozyme simultaneously, helping them understand which chemical bases are important for ribozyme activity.

AsianScientist (Aug. 16, 2016) – With the help of a powerful DNA sequencer, researchers in Japan have created over 10,000 mutants of a ribozyme simultaneously, helping them understand which chemical bases are important for ribozyme activity. Their work was published in Angewandte Chemie.

Ribozymes, or ribonucleic acid enzymes, are RNA molecules that catalyze chemical reactions. Much like DNA, RNA is a linear molecule made up of the four chemical bases A, C, G and U.

Scientists typically try to gain a better understanding of the structure and properties of a ribozyme by introducing mutations in their RNA sequence. However, the currently available techniques allow only several dozen mutants to be studied at a time, which is a relatively small number.

Drs. Shungo Kobori and Yohei Yokobayashi, who are researchers in the Nucleic Acid Chemistry and Engineering Unit at the Okinawa Institute of Science and Technology Graduate University (OIST), have now developed an efficient and unbiased approach to studying ribozyme mutants that overcomes these limitations.

“Instead of selecting specific mutations,” Yokobayashi explained, “we decided to make and test as many mutants as possible of a specific ribozyme.”

3D structure of a ribozyme. Credit: OIST
3D structure of a ribozyme. Credit: OIST


A ‘single’ mutant is a ribozyme that differs in only one base from the original ribozyme, while a ‘double’ mutant differs in two bases from the original ribozyme. Even for the small ribozyme that the researchers studied, which is made up of made of 48 bases, there were a staggering 10,296 single and double mutants in total.

“With the help of a powerful DNA sequencer at OIST, we generated and then measured the chemical activity of all the single and double mutants of a variant of the ‘twister’ ribozyme, which is found in the genome of rice,” Yokobayashi said.

“Thanks to this comprehensive approach, we now have a better understanding of which bases are more important for this ribozyme activity.”

A key finding is that the ribozyme is highly robust against mutations.

“This result is surprising,” Yokobayashi commented, “because the ribozyme we studied has quite a compact and complex structure.”

Deeper understanding of ribozymes could also have significant practical implications, he said. Ribozymes can be engineered to control gene expression in living cells and viruses, with potential applications in gene therapy and regenerative medicine, he added.


The article can be found at: Kobori & Yokobayashi (2016) High-Throughput Mutational Analysis of a Twister Ribozyme.

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Source: OIST.
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