Alternative States (Multi-State) Workflow
RNA sequences often form multiple secondary structures, some of which are functionally important, and others that need to be avoided in the biological milieu. Evidence for alternative structures often comes from single-molecule biophysics measurements or NMR, but it’s hard to model the structures, even at nucleotide resolution. This new workflow allows for rapid detection and structure modeling of secondary structure ensembles.
Carry out the mutate-and-map experiment described in this workflow.
Model sets of possible base pairs in REEFFIT and determine which single mutants might stabilize putative alternative structures. [Coming soon: fast cluster analysis in Biers]
Test alternative structures through compensatory mutation/rescue, read out through chemical mapping. See notes at Primerize.
Make predictions or the behavior of the structure-stabilizing mutants in your alternative functional assay (e.g., single molecule FRET measurements), and test them.
REEFFIT is computationall expensive. It is not yet well optimized for RNAs beyond about 50-100 nts in length; computations for molecules of that size or with numerous mutants remain challenging, even on high-performance clusters.
REEFFIT typically detects helices that are present at >10% population in the starting sequence. Make sure to get bootstrapping error estimates to evaluate the significance of low population helices.
Tian, S., and Das, R. (2016)
RNA structure through multidimensional chemical mapping
Quarterly Review of Biophysics 49 (e7): 1 - 30. | Paper | Link
Cordero, P., and Das, R. (2015)
Rich structure landscapes in both natural and artificial RNAs revealed by mutate-and-map analysis
PLOS Computational Biology 11 (11): e1004473. | Paper | Link
Tian, S., Cordero, P., Kladwang, W., and Das, R. (2014)
High-throughput mutate-map-rescue evaluates SHAPE-directed RNA structure and uncovers excited states
RNA 20 (11): 1815 - 1826. | Paper | Link
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