Annealing/Folding RNA - Method
RNA folding of long RNA
acc. to (Iserman 2020 BioRxiv PMID: 32587965)
Methods: Briefly, In vitro transcribed 5'-End or Frameshifting-region RNA (1200 ng in 40μL nuclease-free water) was denatured at 95°C for 2 minutes followed by snap cooling on ice for 2 minutes. RNA was folded by adding 20 μL of 3.3x SHAPE folding buffer [333mM HEPES (pH 8.0), 333mM NaCl, 33mM MgCl2] and incubating at 37°Cfor 20minutes.
Folding/Annealing of Oligos
How we should do it
Intramolecular folding of Oligos
- 2 uM in H2O
- 95°C, 2 min
- snap cool on ice
- ad same volume 2x Re-folding buffer (20 mM Tris (pH 7.6), 200 mM NaCl, 20 mM MgCl2)
- incubate at 37°C for 20 min
We should do it the same way for in vitro transcribed and loger RNA
folding of 2 complementary Oligos
- fluorescently labeled Oligo - 2 uM in H2O / non-labelled oligo 6 uM (to avoid non-paired labeled)
- ad same volume 2x Re-folding buffer (20 mM Tris (pH 7.6), 200 mM NaCl, 20 mM MgCl2)
- 95°C, 2 min
- slowly cool down to room temperature
Testing for annealing of the complementary oligos: run 8% PAA gel in 0.4x TBE.
Using these refolded RNAs for MST measurements
Dilute the RNAs in the reaction buffer to reach the desired sensitivity for MST.
Cy5 - picoRed MST: LED power 20% - 12000 fluorescence counts equals about 20nM
Cy5 - redMST: LED power xxxx
Cy3 - greenMST: LED power xxxx