The overall goal of this procedure is the prediction of RNA secondary structure with single nucleotide resolution. This is accomplished by first chemically modifying the folded RNA with an electrophilic reagent that S isolates single stranded or flexible regions of RNA. The second step is to use reverse transcription to detect sites of the RNA that have been chemically modified.
Next, the CD NA products of reverse transcription are fractionated by capillary electrophoresis. The final step is data processing and normalization. Ultimately, RNA structure software is used to predict RNA secondary structure using the pseudo free energy constraints derived from shape analysis.
This method can help answer key questions in the molecular biology field, such as how RNA structure governs the functions of catalytic and non-current RNAs, and how viral RNA genomes regulates the multi-functionality of their genomes. The implications of this technique extends to our therapy and diagnosis of viral diseases or cancer because gaining the insight into RNA secondary structure might aid the design of structure-based drugs. Although this method provides insights into the structure of RNAs produced by in vitro transcription, it can also be applied in vivo since shape reagents are cell permeable.
Before starting this procedure, prepare RNA by in vitro transcription. Using a commercial transcription kit. RNAs should be stored in TE buffer between minus 20 and minus 80 degrees Celsius in a 0.5 microliter micro centrifuge tube.
Dilute 12 pico moles of RNA to 18 microliters with water and add two microliters of 10 x re naturation buffer mix by tapping the tube followed with brief spinning heat the mixture in the tube to 85 degrees Celsius for one minute, then cool to four degrees Celsius at a rate of 0.1 degrees Celsius per second. Add 100 microliters of water and 30 microliters of five x folding buffer to the micro centrifuge tube. Following this, incubate the contents of the tube at 37 degrees Celsius for 30 to 60 minutes depending on the RNA being folded.
In general, magnesium dependent folding of longer and more structured RNAs require longer incubation times Transfer a 72 microliter aliquot to each of two 0.5 milliliter micro centrifuge tubes, which should be labeled as modified and control respectively. The next step is to add eight microliters of anhydrous dimethyl sulfoxide to the control mix and set aside. Then add eight microliters of 10 x and MIA to the modified mix.
Incubate the modified and control mix at 37 degrees Celsius for 50 minutes. Add eight microliters of three sodium acetate pH 5.28 microliters of 100 millimolar EDTA 240 microliters of cold ethanol, and one microliter of 10 milligrams per milliliter. Glycogen to the modified mixed tube to precipitate the modified RNA refrigerate the contents of the tube for two hours at minus 20 degrees Celsius, and then centrifuge at 14, 000 G for 30 minutes at four degrees Celsius.
Then wash the pellet twice with cold, 70%ethanol centrifuging for five minutes. After each wash, remove the supernatant with the micro pipette and air dry the pellet for five minutes. At room temperature, prepare the modified and control samples for reverse transcription in 0.5 milliliter micro centrifuge tubes.
For the modified reaction mix five microliters of modified RNA six microliters of water and one microliter of a 10 micromolar sci five labeled primer solution. For the control reaction mix five microliters of control RNA six microliters of water and one microliter of a 10 micromolar sci 5.5 labeled primer solution. Place the tubes in a thermal cycler and a kneel the primer to the RNA.
During a kneeling prepare 2.5 x RT mix. For each RT reaction mix well and incubate at 37 degrees Celsius for five minutes before adding to the an kneeling reactions. Once the temperature of the an kneeling reactions reaches 50 degrees Celsius, add eight microliters of 2.5 XRT.
Mix and incubate for 50 minutes at 50 degrees Celsius. Next, hydrolyzed the RNA by adding one microliter of four molar sodium hydroxide and heating to 90 degrees Celsius for three minutes. Cool the reactions on ice and then neutralize them by adding two microliters of two molar hydrochloric acid.
Now combine the modified and control reactions and add 0.1 volume or one 10th of the total reaction volume of three molar sodium acetate and 100 millimolar EDTA 1.5 volume of cold ethanol and one microliter of 10 milligrams per milliliter.Glycogen. This results in the CDNA precipitating out of solution. Refrigerate the solution for two hours, then centrifuge at 14, 000 G for 30 minutes at four degrees Celsius.
Wash the pellet twice with cold, 70%ethanol resuspend, the pelleted CD NA in 40 microliters of deionized form amide by heating to 65 degrees Celsius for 10 minutes, followed by vigorous vortexing for at least 30 minutes for preparation of the sequencing reaction in a 0.5 milliliter micro centrifuge tube mix 40 microliters of the DDA termination mix. Five PICA moles of the DNA template, 4.6 microliters of 10 x sease buffer and 10 microliters of well-read D two labeled primer. To this mixture, add 4.6 microliters of QUIN A and dilute with water to bring the total volume to 82 microliters.
Prepare a second sequencing reaction in the same manner utilizing DDT and lycor IR 800 labeled primer instead. Next, proceed to PCR amplification using the given USB recommended conditions. Add 16 microliters of three molar sodium acetate pH 5.2 16 microliters of 100 millimolar EDTA, one microliter of 10 milligrams per milliliter glycogen, and 480 microliters of 95%ethanol to precipitate the DNA then incubate at four degrees Celsius for 30 minutes and centrifuge at 14, 000 G for 30 minutes at four degrees Celsius.
Resuspend the pelleted CD NA in 100 microliters of deionized form amide by heating to 65 degrees Celsius for 10 minutes, followed by vigorous vortexing for at least 30 minutes. Next, mix 40 microliters of the pooled shape samples with 10 microliters of the pooled sequencing ladders and pipette the total volume of the sample to 96 well sample plates program and prepare the capillary electrophoresis instrument and initiate a run as per the manufacturer's instructions. The four overlapping color coded traces are produced by migration of the four sets of reaction products through the capillary as follows, the Blue trace shows reverse transcription products generated from reverse transcription of NMIA modified RNAs.
The Green Trace shows reverse transcription products from folded but otherwise unmodified RNAs. The Black Trace shows the D-N-A-D-D-G sequencing ladder and the Red Trace shows the DDT sequencing ladder. The raw capillary electrophoresis traces are separated, processed, aligned, and integrated using shape finder software.
The data analysis begins by importing the electropherograms from the capillary electrophoresis system into shape finder. The central data view window provides graphical feedback on each data processing step. The tool inspector window located on the bottom left of the screen shows parameters for the tools selected in the scripting inspector.
The scripting inspector at the top of the screen displays those tools that have been applied to the data, adjust the electropherograms to correct for fluorescent background, spectral overlap between fluorescent channels, mobility shifts, imparted by differently tagged primers signal decay resulting from premature termination of reverse transcription and differences in fluorescence intensity of common products labeled with different fluorophores in shape finders align and integrate tool. Use the setup function to automatically assign identities to individual peaks and correlate this information to the RNA sequence as defined by user input and the two sequencing ladders to incorporate the nucleotide reactivity profiles into the secondary structure algorithm used by RNA structure 5.3 software and or to compare profiles of closely related RNAs. Normalize the shape data in a standardized fashion.
To do this, exclude outliers from subsequent calculations. Determine the effective maximum reactivity and normalize by dividing all reactivity values by the effective maximum. RNA structure software is used to predict experimentally supported RNA secondary structures using the pseudo free energy constraints from shape analysis.
The software provides graphical representations of the lowest energy to DRNA structures, as well as textual representation of these structures. In dot bracket notation, the sequence and the dot bracket annotation should be imported in RNA structure viewer shown here is the 2D structure of the HIV rev response element stem loop two region generated in RNA structure 5.3 using the shape derived reactivity profiles and visualized using a Java lightweight applet. Nucleotides colored blue have low reactivity, whereas nucleotides colored red have higher reactivity Once mastered, the technique can be done in two days if performed properly.
While attempting this procedure, it is important to remember that the resulting RNA model has to be carefully interpreted before reaching final conclusions about the RNA secondary structure Following this procedure. Other methods like the usage of hydroxy radical probing through space cleavage methods can be performed in order to elucidate complex tertiary interactions and eventually allowing researchers to determine the structures of these RNAs in three dimensions.
