This protocol describes RNA sample preparation and NMR setup for proton R1rho relaxation dispersion measurements to examine confirmational exchange in RNA molecules. Since protons are probed, the method does not need isotopic labeling and can access structural features like shifted base-pairing directly. The pipette is quite modular in the sense that R1rho relaxation dispersion can be measured even if the sample is produced with a different method.
Begin by preparing the plasmid sample and setting up an in vitro transcription and cleavage reaction as described in the text manuscript. Incubate the reaction at 37 degrees Celsius for one hour. Then dilute one microliter of the sample tenfold in loading solution.
And run one microliter on a denaturing PAGE gel. If the cleavage reaction is successful, scale the reaction to the desired volume and run it overnight. Then run another denaturing PAGE gel and assess whether the cleavage reaction has been completed.
In case of incomplete cleavage, heat the reaction solution in a conventional microwave at 450 watts for 15 seconds. Then cool the solution slowly to 37 degrees Celsius for 40 minutes to reanneal the RNA and the cleavage guide. And note the formation of a new precipitate.
Add more inorganic pyrophosphatase and RNase H.Incubate the reaction for one to three hours at 37 degrees Celsius. Then confirm completion of the cleavage reaction with denaturing PAGE. When the RNase H cleavage reaction is completed, quench the reaction by adding EDTA to a 50 millimolar final concentration.
And vortex thoroughly. Filter the solution through a 0.2 micron syringe filter. And concentrate the solution to a volume injectable into an HPLC system for purification.
Before adding the concentrated and purified sample to the NMR tube, clean the tube by flushing with abundant water. Then RNase decontamination reagent, water, and 95%ethanol. After the final rinse with water, leave it to dry.
Rinse the plunger with water and use a lint-free wipe to clean with RNase decontamination reagent and 95%ethanol. After drying the tube and plunger, add 10%D2O to the NMR sample. Use a large pipette tip to transfer the RNA sample into the NMR tube, flowing it along the side of the tube wall.
Insert the plunger into the tube by pushing it down with a fast twisting motion to remove air bubbles, then pull the plunger up slowly without creating new air bubbles. And fix it with paraffin wax film. Create a new dataset based on an aromatic proton carbon HSQC dataset used on fully labeled RNA samples for RNA assignment.
Set the general parameters and RD-specific parameters. Then set proton spin lock power to 1.2 kilohertz for testing. Generate a test vd list with only one entry, zero milliseconds, set TDF1 to one, and update D30 to run a test spectrum.
After setting up a test vd list, update D30 and TDF1. And plot the intensity of the peak for different spin lock lengths. Identify the spin lock length at which the intensity of the original peak decreases to one third.
From the result of the test runs, create the final vd list to be used in the experiment. Select the number of scans so that the weakest peak of the list has a signal to noise ratio of at least 10. The results of several cleavage reactions of tandem transcripts are shown here.
A 20 nucleotide target RNA was generated in a successful reaction. Unsuccessful reactions resulted in incomplete and failed cleavage of the sample. HPLC injection of the RNA sample revealed a peak for pure target RNA at 38 minutes.
While longer and shorter products were well separated from the peak of interest. Analysis of the RNA hairpin showed that the number of observed imino protons matched the number of imino protons expected from a secondary structure simulation. The proton carbon HSQC spectrum of the aromatic resonances of the RNA showed four signals after folding.
And only three signals before folding. In the representative on resonance curves obtained for two different H8 atoms and two different synthetic RNA hairpins, the G6H8 experiences confirmational exchange. While the A4H8 does not.
For G6H8, the colored spin lock powers were selected and off resonance data was recorded. In the CG construct where the exchange is slow, the R1rho row values versus offset plot already displayed a slight asymmetry of the curve, indicating the sign of the chemical shift difference delta omega. This becomes even more apparent in the R2 plus REX plot where the R1 contribution is removed.
On the other hand, the GC construct experiences faster exchange and the R1rho values versus offset plot presented a broader curve where the extraction of delta omega becomes less obvious even in the R2 plus REX plot. Once the RNA dynamics have been elucidated, the states can be characterized and trapped. These trapped states can, for example, be tested in biological assays.
This method allows us to observe functional, relevant base pairing shifts in an active microRNA miRNA complex, loop rearrangements in ribosomal RNA, and base pair stability of on single nucleotide bulges.
