The overall goal of the following experiment is to ascertain how RNA molecules fold using hydroxyl radical footprint. This is achieved by end labeling, gel purification and pre folding of the RNA, which ensures confirmational integrity of the RNA prior to magnesium mediated folding as a following step, the Fenton reagents are mixed to generate hydroxyl radicals, which can subsequently cleave the RNA backbone according to its solvent accessibility. Next, the RNA cleavage products are separated by denaturing poly acrylamide gel electrophoresis, and visualized by auto radiography.
The band integrals are quantified by semi-automated footprinting analysis software. The ultimate goal is to generate folding isotherms reflecting tertiary structure formation of RNA. This is accomplished by scaling the normalized band integrals to fractional saturation.
The isotherms can subsequently be fitted to the hill equation. The main advantage of hydroxy footprinting is that you obtain tertiary structure information of RNA using inexpensive chemicals due to the higher activity in small size hydroxy radicals are perfect probes to distinguish between solvent accessible and buried nucleotides. This method can help answer key questions in the field of RNA structural biology.
For example, how do ribosomes employ metal ions to reach their active confirmation? Hydroxy radical footprint can be used to understand the fundamentals of RNA specificity and recognition. Besides revealing information about RNA tertiary structure, this method can be used to determine the precise protein binding sites in RNA or in DNA molecules.
The major challenges of this method are to prevent sample degradation by ribonuclease and to optimize iron concentration in order to obtain the right amount of RNA cleavage. Also, the detail analysis of the band intervals can be quite tricky. The main benefit of this video is to help the audience understand the planning stages and successful execution of hydroxy radical footprint experiment Prior to the start of this protocol, prepare all footprint in reagents as described in the written protocol.
Accompanying this video, RNA can be produced DFOs four related and end labeled as outlined there. Purify the radioactively labeled RNA using denaturing gel electropheresis. Recover the purified RNA by two subsequent gel extractions using 0.3 molar sodium acetate.
Then precipitate the RNA by mixing 0.5 milliliters of the aqueous solution with one milliliter of ethanol. Incubate the mixture for one minute on dry ice After spinning the sample, discard the supinate. Wash the RNA pellet with 70%ethanol.
Remove the supinate after additional centrifugation and dry the pellet in a vacuum. Next, dissolve the purified P 32 labeled RNA samples. In 330 microliters of one times reaction buffer pipette 30 microliters of the RNA solution into a new reaction tube.
Precipitate the RNA with ethanol and then dry the pellet in a vacuum once dried, this pellet can be used for generating the reference ladder as described in the written procedure to nature the remaining buffered RNA solution by heating at 95 degrees Celsius for two minutes. Call the samples to room temperature for 15 minutes and follow with a quick spin to bring the condensate back into solution. To begin the footprint experiment, set up 27 reaction tubes for enough samples to fill a 30 well electrophoresis gel.
After including the ladders and cleaved control, determine the final magnesium iron concentrations so that they are evenly spaced on a logarithmic scale over several orders of magnitude around the titration midpoint preparation of the magnesium ions in one times reaction buffer is described in the text separately. Incubate the RNA and the magnesium ion solutions at 50 degrees Celsius for five minutes. Then mix 10 microliters of the RNA solution with 90 microliters of the corresponding amount of magnesium iron solution.
To reach a final volume of 100 microliters, incubate each mixture for 30 minutes of 50 degrees Celsius. Subsequently allow the solutions to equilibrate at 25 degrees Celsius for one hour while RNA folding occurs. As meanwhile, immediately before initiating the hydroxyl radical footprint in reaction, prepare the phantom reaction mix as described in the written protocol.
Prepare the peroxidation reaction by pipetting two microliter droplets each of iron EDTA sodium ascorbate and hydrogen peroxide at the top inside of the reaction tube containing the RNA solution so that the droplets are not touching. Start the footprinting reaction by vigorous mixing. After 15 seconds, stop the reaction by adding 300 microliters of pure cold ethanol.
Turn the tubes three to five times. Finally, precipitate, wash and dry the pellet as performed earlier as an alternative to the per oxidative reaction, the oxidative hydroxyl reaction would be performed by adding five microliters of the freshly prepared Fenton reaction mix to the samples. Incubate the oxidative reaction for 30 minutes at 25 degrees Celsius.
Then add 300 microliters of pure cold ethanol to quench the reaction and mix by turning the tubes. Once again, precipitate. Wash and dry the pellet upon completion of either the per oxidative or oxidative reaction.
Dissolve the dried RNA pellets in eight microliters of gel loading dye two. Confirm that the P 32 labeled RNA is resuspended by using a geer counter prepare a denaturing 8%poly acrylamide sequencing gel according to standard protocols. Using a 30 well comb load the samples, including two references and an cleaved control.
Separate the RNA fragments at 60 to 75 watts for two and a half hours. Expose the dried gel to a storage phospho screen overnight. Scan the phospho screen with an imaging system for filmless auto radiography.
Then transfer the gel image file to the computer for analysis. To begin data analysis, open Safa and open source software for single band fitting and quantification. Load the RNA sequence as a dot TXT file followed by the gel picture as a dot gel file.
Define the lanes and adjust the band intensities. Then choose an anchor lane and perform a gel alignment assignment of bands to nucleotides occurs in reference to the RNAs T one digestion ladder. Quantify the band intensities and use the normalization slash color plot feature to normalize and assign potentially invariant residues.
Save the output as a txt file. SFA outputs a spreadsheet containing columns, representing lanes on the gel and rows representing the individual band integrals corresponding to the RNA fragment. First, the protection sites that display a noticeable change in solvent accessibility have to be identified by comparing the protection profile derived from the no magnesium ion sample to the profile of the endpoint magnesium ion concentration.
The lower the value, the more protected the nucleotide is against attack from the hydroxyl radicals and vice versa. Next, create transition curves of band, intensities of individual or groups of nucleotides versus magnesium iron concentration using a spreadsheet format individually scale these transitions to the fractional saturation function as described in the text portion of this protocol. As a final step, fit the data to the hill equation.
This analysis scales the transition to fractional saturation, determines the transition midpoint and provides a phenomenological test of whether a transition is sigmoidal shown. Here are representative results from P four P six RRNA hydroxyl radical footprint Experiments. Isotherms were derived from sequencing gels analyzed by Safa and then fit as described in the analysis section.
The gel image indicates that background cleavage is minimal and that single nucleotide assignment is possible due to well-defined bands in the T one lane. The hydroxyl radical induced fragmentation of RNA is well above background. The transition from low to high magnesium ions is selectively affiliated with decreasing intensity of individual and groups of bands indicating formation of RNA.
Tertiary structure protection refers to single or groups of contiguous nucleotide whose cleavage changes concomitantly band intensities are quantified and normalized by saffer analysis. The output is a thermal plot visualizing the degree of protection against hydroxyl radicals. The color transition describes the accessibility change upon magnesium iron addition white to red shows more accessible nucleotides, whereas white to blue shows more protected nucleotides.
Each degree of shading is affiliated with a numerical value, which can be plotted as a protection curve and analyzed by a binding model such as the hill equation. In this example, the equilibrium dissociation constant of affiliated with protection 1 5 3 through 1 5 5 is roughly twice that of the corresponding value of protection 1 6 3 through 1 6 4. This hydroxy radical footprint experiment can be done in one and a half days if it's performed properly.
While attempting this procedure, it's important to remember to wear gloves and a lab coat to avoid any RNAs contamination. Also, the final iron concentration depends on the experimental system. Therefore, we recommend performing a dose response experiment before footprinting following this procedure.
Other methods, Such as time resolved hydroxy radical footprint can be performed in order to answer additional questions. For example, at what critical time point do RNA molecules reach a misfolded or active confirmation After its development? This technique paved the way for researchers in the field of structural biology to explore inter as well as interim molecular tertiary interactions of nucleic acids.
After watching this video, you should have a good understanding of how to determine tertiary structure formation of RNA molecules. Don't forget that caate and phosphorous stage two are hazardous precautions, such as wearing gloves and protection goggles, as well as the use of plexiglass shielding are recommended while performing this procedure.
