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Structural transition of replicable RNAs during in vitro evolution with Qβ replicase
Ryo Mizuuchi1,2, Kimihito Usui3, Norikazu Ichihashi1,4,5
1Komaba Institute for Science, The University of Tokyo, Meguro, Tokyo 153-8902, Japan.
RNA (New York, N.Y.)
|November 7, 2019
Summary
The "fewer unpaired GC rule" helps design replicable single-stranded RNAs (ssRNAs) by minimizing double-stranded RNA (dsRNA) formation. This study validates the rule for longer ssRNAs, showing decreased unpaired G and C bases improve replication efficiency.
Area of Science:
- Molecular Biology
- Virology
- Origins of Life Research
Background:
- Single-stranded RNAs (ssRNAs) serve as genetic material for some viruses and in models of early life.
- A key challenge in ssRNA replication is the formation of non-replicable double-stranded RNA (dsRNA).
- Strong intramolecular secondary structures in ssRNAs can prevent dsRNA formation.
Purpose of the Study:
- To test the validity of the "fewer unpaired GC rule" for designing efficiently replicating ssRNAs.
- To investigate if this rule applies to longer ssRNAs and explains structural evolution.
- To assess the relationship between secondary structure, dsRNA formation, and Qβ replicase efficiency.
Main Methods:
- In vitro evolution of replicable ssRNAs using Qβ replicase.
- Analysis of nine successively evolved long ssRNAs.
- Chemical modification methods to examine ssRNA secondary structures.
- Correlation analysis between mutations, secondary structure, and replication efficiency.
Main Results:
- Evolved ssRNAs demonstrated stepwise improvement in template ability and reduced dsRNA formation.
- Analysis revealed a gradual decrease in unpaired G and C bases during evolution.
- Local structural changes around mutation sites contributed to the reduction of unpaired bases.
- These structural changes correlate with improved replication and reduced dsRNA formation.
Conclusions:
- The "fewer unpaired GC rule" is supported as a valid strategy for designing replicable ssRNAs.
- The rule's applicability is confirmed for longer ssRNAs and in evolutionary contexts.
- Findings offer insights for engineering more complex ssRNA replication systems.
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