RNA G-quadruplex formation in defined sequence in living cells detected by bimolecular fluorescence complementation

Chemical Science
Hong-He LiuZheng Tan

Abstract

G-quadruplexes are implicated in many essential cellular processes and sequences with potential to form a G-quadruplex are widely present in DNA and RNA. However, it is difficult to know whether a sequence of interest naturally forms a G-quadruplex in living cells. Here we report the detection of a G-quadruplex in defined RNA sequences in living cells in a natural intracellular environment. A G-quadruplex forming sequence in a RNA transcript is tagged at proximity with an aptamer. The two structures are recognized respectively by two probe proteins each of which is fused with a split half of enhanced green fluorescent protein (eGFP). Simultaneous binding of the two proteins to RNA brings the two halves of eGFP into proximity, permitting them to reconstitute into a functional eGFP that emits fluorescence to signal the formation of a G-quadruplex in RNA. We show that a G-quadruplex can form in RNA and can be detected with sequence and structure specificity under both in vitro and in vivo conditions. The results, therefore, provide direct evidence for the formation of RNA G-quadruplexes in live cells and the method provides a useful tool to validate G-quadruplex formation in a specific sequence under a natural cellular condition.

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Citations

Sep 2, 2017·Current Protocols in Cell Biology·Souheila AmorDavid Monchaud
Oct 13, 2020·Nucleic Acids Research·Ke-Wei ZhengZheng Tan
Jan 7, 2022·Journal of the American Chemical Society·Susanta HaldarShozeb Haider

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Methods Mentioned

BETA
electrophoretic mobility shift assay
electrophoresis
flow cytometry
pull-down
PCR
circular dichroism
in vitro transcription

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