Footprinting and Structure Probing with T1 and RNase 1- (Devika Gates)
THIS IS STILL IN THE PROCESS OF BEING WRITTEN-Don't use yet!!
It should be noted that concentrations of RNA, Rnases and NMIA will have to be determined for each individual RNA for best results.
Buffers and reagents:
10x T1 structure buffer
100mM Tris ph7
10xRNase1 Structure Buffer
100mM Tris ph7
0.7M Sodium Acetate pH5.2
100% Ethanol (200proof)
Structure probing/Footprinting with protein
1) mix together:
12µl 10X Structure Buffer (either T1 or RNase 1)
0.6µl Heparin stock
0.6µl (total 2µg) kinased RNA
2) Heat to 95°C for 2 min, snap cool on ice for 5 min
3) Set up 7 tubes, into each tube aliquot:
9µl of RNA mix
1.1µl of protein storage buffer or protein in protein storage buffer
1.6µl of water
repeat each tube with a different concentration of protein
4) incubate at RT for 15 minutes
5) To each tube add: 1.3µl of enzyme diluted into 1x of it's matching buffer (2.5U/1.3µl when added becomes 0.25U for T1 Rnase or 0.3U for Rnase1)
6) incubate at RT for 15 minutes
Samples were incubated on ice for 5 minutes, and spun at max speed for 15 minutes at 4°C.
Pellets were resuspended in a final concentration of 18 nM of radiolabeled reverse primer (5’–CAGGTC AAAGGTTGCCTCG–3’) and 0.83 mM dNTPs (the reverse primer was kinased using Polynucleotide Kinase and [γ-P32] ATP).
Samples were incubated at 65°C for 5 minutes, 35°C for 5 minutes and on ice for one minute.
Reverse transcription was carried out by adding appropriate amounts of 5X First Strand buffer, 0.1 M DTT, and 200U of SuperScript III.
Samples were incubated at 52°C for 1 hour and at 70C for 15 minutes.
Samples were then phenol chloroformed, EtOH precipitated and the pellet was resuspended in 20μL of low TE.
An equal volume of 2X Denaturing dye was added to the samples, which were then incubated at 95°C for 2 minutes and run on an 8% denaturing gel (8% 19:1 acrylamide:bisacrylamide, 1X TBE and 7.5 M urea)
The SHAPE (Selective 2’ Hydroxyl Acylation by Primer Extension) assay
SHAPE was done as described above, except RNA and heparin were snap annealed in
HE buffer (10 mM HEPES pH 8.0 and 1 mM EDTA pH 8.0)
1X Folding mix buffer (Recipe for 3X buffer is 333 mM HEPES pH 8.0, 20 mM MgCl2, 333 mM NaCl) was added and the RNA sample was incubated at 37°C for 20 minutes.
RNA samples were aliquoted (volume????) and 1μL 120nM NMIA in neat DMSO were added to samples (Concentration of NMIA???). Neat DMSO was added to a sample as a control (free RNA).
Reactions were incubated at 37°C for 45 minutes. Inactivation buffer was added and reverse transcription was done as described above.
Quantification and normalization of T1, RNase 1 and SHAPE gels-
Footprinting and structure probing data was quantified using the SAFA program (Das, R., Laederach, A., Pearlman, S. M., Herschlag, D., and Altman, R. B. (2005) RNA 11, 344-354).
Data was normalized for each lane as previously described (Low, J. T., and Weeks, K. M. (2010) Methods 52, 150-158).
The sequence and text files were imported to the RNAStructure programs (available from http://rna.urmc.rochester.edu/rnastructure.html ). The slope (m) and intercept (b) chosen were 2.6kcal/mol and –0.8kcal/mol, respectively. Minor adjustments in the structure were made.
Difference plots were made by subtracting normalized reactivities of the protein lane from the no protein lane. The average of the difference was then added and the values were plotted. The sequence and SHAPE text files were prepared as previously described.