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J Am Chem Soc. 2018 Aug 15;140(32):10067-10070. doi: 10.1021/jacs.8b03784. Epub 2018 Aug 3.

Mimicking Co-Transcriptional RNA Folding Using a Superhelicase.

Author information

1
Department of Biophysics and Biophysical Chemistry , Johns Hopkins School of Medicine , Baltimore , Maryland 21205 , United States.
2
T. C. Jenkins Department of Biophysics , Johns Hopkins University , Baltimore , Maryland 21218 , United States.
3
Department of Biomedical Engineering , Johns Hopkins University , Baltimore , Maryland 21218 , United States.
4
Howard Hughes Medical Institute , Baltimore , Maryland 21205 , United States.

Abstract

Vectorial folding of RNA during transcription can produce intermediates with distinct biochemical activities. Here, we design an artificial minimal system to mimic cotranscriptional RNA folding in vitro. In this system, a presynthesized RNA molecule begins to fold from its 5'-end, as it is released from a heteroduplex by an engineered helicase that translocates on the complementary DNA strand in the 3'-to-5' direction. This chemically stabilized "superhelicase" Rep-X processively unwinds thousands of base pairs of DNA. The presynthesized RNA enables us to flexibly position fluorescent labels on the RNA for single-molecule fluorescence resonance energy transfer analysis and allows us to study real-time conformational dynamics during the vectorial folding process. We observed distinct signatures of the maiden secondary and tertiary folding of the Oryza sativa twister ribozyme. The maiden vectorial tertiary folding transitions occurred faster than Mg2+-induced refolding, but were also more prone to misfolding, likely due to sequential formation of alternative secondary structures. This novel assay can be applied to studying other kinetically controlled processes, such as riboswitch control and RNA-protein assembly.

PMID:
30063835
PMCID:
PMC6169520
[Available on 2019-08-15]
DOI:
10.1021/jacs.8b03784

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