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@article{Balcerowicz:2021:10.21769/BioProtoc.3950,
author = {Balcerowicz, M and Di, Antonio M and Chung, BYW},
doi = {10.21769/BioProtoc.3950},
journal = {Bio-protocol},
title = {Monitoring real-time temperature dynamics of a short RNA hairpin using Förster resonance energy transfer and Circular Dichroism},
url = {http://dx.doi.org/10.21769/BioProtoc.3950},
volume = {11},
year = {2021}
}
TY - JOUR
AB - RNA secondary structures are highly dynamic and subject to prompt changes in response to the environment. Temperature in particular has a strong impact on RNA structural conformation, and temperature-sensitive RNA hairpin structures have been exploited by multiple organisms to modify the rate of translation in response to temperature changes. Observing RNA structural changes in real-time over a range of temperatures is therefore highly desirable. A variety of approaches exists that probe RNA secondary structures, but many of these either require large amount and/or extensive processing of the RNA or cannot be applied under physiological conditions, rendering the observation of structural dynamics over a range of temperatures difficult. Here, we describe the use of a dually fluorescently labelled RNA oligonucleotide (containing the predicted hairpin structure) that can be used to monitor subtle RNA-structural dynamics by Förster Resonance Energy Transfer (FRET) at different temperatures with RNA concentration as low as 200 nM. FRET efficiency varies as a function of the fluorophores' distance; high efficiency can thus be correlated to a stable hairpin structure, whilst a reduction in FRET efficiency reflects a partial opening of the hairpin or a destabilisation of this structure. The same RNA sequence can also be used for Circular Dichroism spectroscopy to observe global changes of RNA secondary structure at a given temperature. The combination of these approaches allowed us to monitor RNA structural dynamics over a range of temperatures in real-time and correlate structural changes to plant biology phenotypes.
AU - Balcerowicz,M
AU - Di,Antonio M
AU - Chung,BYW
DO - 10.21769/BioProtoc.3950
PY - 2021///
TI - Monitoring real-time temperature dynamics of a short RNA hairpin using Förster resonance energy transfer and Circular Dichroism
T2 - Bio-protocol
UR - http://dx.doi.org/10.21769/BioProtoc.3950
VL - 11
ER -