NDLI: Ligation of RNA Oligomers by the Schistosoma mansoni Hammerhead Ribozyme in Frozen Solution

Content Provider	Springer Nature Link
Author	Lie, Lively Biliya, Shweta Vannberg, Fredrik Wartell, Roger M.
Copyright Year	2016
Abstract	The interstitial liquid phase within frozen aqueous solutions is an environment that minimizes RNA degradation and facilitates reactions that may have relevance to the RNA World hypothesis. Previous work has shown that frozen solutions support condensation of activated nucleotides into RNA oligomers, RNA ligation by the hairpin ribozyme, and RNA synthesis by a RNA polymerase ribozyme. In the current study, we examined the activity of a hammerhead ribozyme (HHR) in frozen solution. The Schistosoma mansoni hammerhead ribozyme, which predominantly cleaves RNA, can ligate its cleaved products (P1 and P2) with yields up to ~23 % in single turnover experiments at 25 °C in the presence of Mg2+. Our studies show that this HHR ligates RNA oligomers in frozen solution in the absence of divalent cations. Citrate and other anions that exhibit strong ion-water affinity enhanced ligation. Yields up to 43 % were observed in one freeze–thaw cycle and a maximum of 60 % was obtained after several freeze–thaw cycles using wild-type P1 and P2. Truncated and mutated P1 substrates were ligated to P2 with yields of 14–24 % in one freeze–thaw cycle. A pool of P2 substrates with mixtures of all four bases at five positions were ligated with P1 in frozen solution. High-throughput sequencing indicated that 70 of the 1024 possible P2 sequences were represented in ligated products at 1000 or more read counts per million reads. The results indicate that the HHR can ligate a range of short RNA oligomers into an ensemble of diverse sequences in ice.
Starting Page	81
Ending Page	92
Page Count	12
File Format	PDF
ISSN	00222844
Journal	Journal of Molecular Evolution
Volume Number	82
Issue Number	2-3
e-ISSN	14321432
Language	English
Publisher	Springer US
Publisher Date	2016-02-20
Publisher Place	New York
Access Restriction	One Nation One Subscription (ONOS)
Subject Keyword	Ribozyme RNA ligation RNA world Dehydration Evolutionary Biology Microbiology Plant Sciences Plant Genetics & Genomics Animal Genetics and Genomics Cell Biology
Content Type	Text
Resource Type	Article
Subject	Genetics Molecular Biology Ecology, Evolution, Behavior and Systematics

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4	Project PI / Joint PI	Principal Investigator and Joint Principal Investigators of the project	Dr. B. Sutradhar bsutra@ndl.gov.in Prof. Saswat Chakrabarti will be added soon
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