The overall goal of this methods article is to introduce the detailed protocols utilized by our lab for studying the molecular recognition of double-stranded RNAs by chemically modified peptide nucleic acids. This method can help answer key questions in the RNA chemical biology field such as detection and targeting of RNA structures. The main advantage of this technique is that the design principles can be applicable to the targeting of any RNA duplex structures.
Demonstrating the procedure will be Desiree Toh, a research associate from my laboratory. To begin this procedure, add four microliters of a 35%glycerol solution to previously prepared RNA PNA samples and mix gently. Using a micropipette and gel loading tips, carefully 20 microliters of each sample to a previously prepared polyacrylamide gel making sure not to introduce any air bubbles.
Run the gel at a constant voltage of 250 volts for five hours. After five hours, turn off the power supply and remove the glass plate from the gel stand. Then remove the gel sealing tape and disassemble the glass plate.
Gently remove the gel from the glass plate and place it in a container filled with 350 milliliters of deionized water. Carefully add 35 microliters of ethidium bromide to the container and place it on the platform shaker on low speed for 30 minutes. After disposing of the ethidium bromide solution, rinse the gel with 1.5 to two liters of distilled water.
Then scan the gel using an imager with a green laser of 532 nanometers and the emission filter set at 610 nanometers. Quantify the gel band intensities using free software. Transfer the appropriate quantity of 2-aminopurine labeled double-stranded RNA into a clean 1.5 milliliter tube.
Then concentrate the RNA solutions using a vacuum concentrator. Next, transfer the appropriate volumes of the targeted PNA for various concentrations from the previously prepared main stock to separate 1.5 milliliter tubes. Concentrate the PNA solutions using the vacuum concentrator.
Add 975 microliters of incubation buffer to the tube containing the dried RNA and mix thoroughly to ensure all the RNA is dissolved. After briefly centrifuging the RNA solution, subject it to annealing by placing the tube into a preheated heat block for 10 minutes. When finished, turn off the heat block and allow the sample to cool to room temperature.
Add 75 microliters of RNA to each of the tubes containing the dried PNA and mix thoroughly. After allowing the samples to sit at room temperature for at least one hour, incubate them at four degrees Celsius overnight. Now, transfer the appropriate quantities of 2-aminopurine labeled single-stranded RNA to separate 1.5 milliliter tubes.
Transfer the appropriate volumes of targeted PNA for various concentrations from the main stock to the respective tubes containing the single-stranded RNA. After drying the samples, add 75 microliters of incubation buffer to each of the tubes and mix thoroughly. Subject each sample to annealing by placing each tube into a preheated heat block for 10 minutes.
After allowing the samples to cool to room temperature, incubate them at four degrees Celsius overnight. Following this, transfer 70 microliters of each sample into a one centimeter square cuvette. Place the cuvette in a fluorescence spectrophotometer and measure the emission over wavelength range of 330 to 550 nanometers.
Once the measurement is complete, remove the buffer from the cuvette. Rinse the cuvette with distilled water and dry with nitrogen gas. After repeating the measurement for all the samples, plot the fluorescence intensity against the wavelength.
Transfer the appropriate quantities of single-stranded RNA to separate 1.5 milliliter tubes. Then transfer the appropriate volumes of targeted PNA from the main stock to the respective tubes containing the single-stranded RNA. After drying the samples, add 130 microliters of incubation buffer to each of the tubes and mix thoroughly.
Subject each sample to annealing by placing each tube into a preheated heat block for 10 minutes. After allowing the samples to cool to room temperature, incubate them at four degrees Celsius overnight. Now, transfer 130 microliters of each sample to an eight microcell cuvette with one cell containing incubation buffer.
Using a UV-Vis spectrophotometer, measure the absorbance of each sample at 260 nanometers. Measure at increasing temperature from 15 to 95 degrees Celsius followed by decreasing temperature from 95 to 15 degrees Celsius at a ramp rate of 0.5 degrees Celsius per minute. PNA oligomers were obtained after two reverse phase HPLC purifications.
The identity of the PNAs can be confirmed by MALDI/TOF analysis. The nondenaturing PAGE data suggest that the Q and L modified PNA can only recognize the double-stranded RNA region with a C-G pair. This specific and enhanced recognition is through the T-A-U-L-G-C and the Q-C-G PNA RNA two based triple formation.
A 2-aminopurine fluorescence titration study showed that a Q and L modified PNA binds to a targeted double-stranded RNA but not single-stranded RNA. PNAs containing Q residues do not show thermal melting transitions suggesting an absence of binding to single-stranded RNA due to the steric clash in the Watson Watson-Crick like Q-G pair. Compared to unmodified PNA P1, PNAs P4 and P5 containing L residues show a lower melting temperature for the corresponding RNA PNA duplexes due to the steric clash in the Watson-Crick like L-G pair.
The UV absorbance detected thermal melting data are consistent with the 2-aminopurine fluorescence titration data which also show that a PNA containing Q and L residues does not strongly bind to single-stranded RNA. Incorporating a Q base is more destabilizing than an L base as a Q base has a more significant steric clash in the formation of a Watson-Crick like Q-G pair. Once mastered, the various experiments can be done in a week if it is performed properly.
Following this procedure, other methods such as probing and targeting RNA structures in cells can be performed in order to answer additional questions like whether we can detect RNA structures and regulate the RNA functions by using double-stranded RNA binding PNAs. After its development, this technique paved the way for researchers in the field of RNA biology and disease to explore RNA structure targeted therapeutic development.
