The overall goal of this report is to illustrate the procedure to obtain oligonucleotides of RNA via solid-phase synthesis. Dodecamers of RNA will be synthesized as models and their purification and characterization will also be demonstrated. Through its visual component, this publication will broaden the scope of the methodologies and facilities used to researchers in various fields.
Providing a visual aid, along with a little protocol will enable researchers to employ these techniques. One advantage of this technique is its amenability to a wide range of modifications. As today's applications become more demanding, it is important to have a standard protocol.
Phosphoramidite chemistry is experiencing a resurgence due to the robustness of the method and its potential for obtaining a oligonucleotides for therapeutic applications. While purchasing oligonucleotides is attractive, price and availability of modifications shift interest towards preparing these oligomers in-house, thus making this procedure of utmost importance. To begin this procedure, weigh each phosphoramidite into oven-dried 10-milliliter amber bottles and cap each bottle with a septum previously perforated with a 20-gauge needle.
Place the bottles immediately under vacuum using a dessicator containing a drying agent. After filling the dessicator with dry argon, remove each bottle and immediately add anhydrous acetonitrile before securing onto a solid-phased peptide synthesizer. Next, remove the septa from each bottle, and place on the synthesizer via a bottle-change function.
To set up the solid-phase synthesis, select the software icon. Select the synthesizer name, and click on OK.Then, create a new synthesis by selecting File, and New Synthesis Order. Following this, use the prompt window to enter the run date, customer, and run ID.Select the instrument, sequence name, and sequence.
Under the Cycle tab, assign the previously created method. Then, choose the End Procedure manual, which leaves the oligonucleotide bound to the CPG resin. Select DMT Off.
Save the file by selecting File and Save As.Then, provide a name for the experiment and click on Save. Next, send the file to begin synthesis by selecting Order and Send Order to Synthesizer. On the prompt window, select a column position and click on OK.On the Synthesizer window, select Trityl Monitor.
Click on Choose Function, and Trityl Monitor every step by typing 1. Begin the synthesis by selecting Synthesizer and Prepare to Start. Place the columns with the desired three-prime end on the positions indicated on the instrument.
Then, click Start and No on the instrument. Once the synthesis is complete, remove the column from the instrument and place it in a round-bottomed flask. Dry the column under reduced pressure for about 0.5 hours.
Transfer the white resin from the column to a 1.6-milliliter centrifuge tube, and add 0.5 milliliters of an aqueous methylamine and ammonia solution. Secure the centrifuge tube cap with parafilm, and heat the contents to 60 degrees Celsius for one and a half hours. Place a heavy object on top of the tubes to ensure the concentration is kept constant.
After cooling to room temperature, briefly centrifuge the sample to spin down contents. Then, transfer the supernatant to a new centrifuge tube. Cool the tube to 70 degrees Celsius by placing in a dry-ice ethanol bath for five minutes, and then concentrate until dry using a speed vac concentrator.
Now, resuspend the solids in 0.4 milliliters of an amine trihydrafluoride solution, and then secure the cap with laboratory film. Heat the solution to 60 degrees Celsius for one and a half hours. Place a heavy object on top of the tubes to ensure constant concentration.
After cooling the solution to room temperature, add 0.04 milliliters of a sodium acetate solution followed by gentle mixing with a pipette tip. Add one milliliter of ethanol and cool the solution to approximately 70 degrees Celsius with a dry-ice ethanol bath for 15 minutes. Following this, centrifuge the solution at 15, 000 RPM at four degrees Celsius for ten minutes.
Use a pipette to extract the supernatant and dry the resulting pellet under reduced pressure. After drying, resuspend the obtained solid and loading buffer, and mix until the solution is homogenous. Then, load the suspension onto a previously prepared polyacrylamide gel.
Apply a current through the gel until the bromethymol blue marker is located between one-half to two-thirds down the gel. After removing the gel from the stand, transfer the gel contents from the glass to the plastic wrap and place over a thin layer chromatography plate covered with silica, to visualize the bands using a UV lamp. Use a marker to delineate the position where the upper band is located, and cut it using a razor blade.
Place the gel containing the RNA into a 50-milliliter conical tube, and crush it to small pieces using a glass rod. Now, suspend the gel residues into an aqueous sodium chloride solution, and shake the suspension at 37 degrees Celsius for 12 hours. Transfer the suspension into a 15-milliliter tube, and then centrifuge at 3400 RPM for ten minutes.
To desalt the sample using a reverse-phase C18 cartridge, first wash the cartridge with acetonitrile, water, aqueous ammonium chloride, and the RNA solution, leaving the gel residues behind. After washing with water, elute the RNA from the column using three milliliters of 60%aqueous methanol solution into three different test tubes. Cool the tubes to 70 degrees Celsius in a dry ice bath for five minutes, and then concentrate the contents to dryness using a speed vac concentrator.
After concentrating the eluant under reduced pressure, redissolve the residue in 0.3 milliliters of RNase-free water. After diluting the solution, deposit one microliter on the UV vis instrument to measure the UV vis spectrum between 200 and 450 nanometers. For MALDI-TOF analysis, first wash a C18 pipette tip with 50%acetonitrile.
Equilibrate the pipette tip with 0.1%trifluoroacetic acid. Then, load the pipette tip with a solution containing 100 to 150 picomoles of sample. After washing the pipette tip with 0.1%TFA and water, elute the sample into a solution containing the desired matrix.
Deposit 0.9 microliters of the solution onto a MALDI plate and allow the sample to dry. For RNA structure analysis, transfer the sample containing RNA into a microcuvette and position in the sample changer of a circular dichroism instrument. Open the nitrogen tank to provide a flow that moves the air floater located on the instrument to approximately 40.
Then, turn on the cooler. Now, turn on the instrument. Open the software by clicking on the Spectrum Manager icon, and then open the Acquisition window by clicking on Spectrum Measurement.
After purging the instrument with nitrogen, acquire the circular dichroism spectra by selecting Measure and Parameters. Under the General tab, select 200-350 nanometers for the scan, CD and HT for the channels, 0.1 nanometers for the data pitch, 100 nanometers per minute for the scanning speed, one nanometer for the bandwidth, and five for the number of accumulations. Under the Cell Unit tab, chose 20 degrees Celsius.
Under the Control tab, select Shutter is opened and closed automatically. Under the Information tab, input the sample name, operator, concentration, and solvent. Under the Data tab, browse to desired folder, and click on OK.Acquire the spectra by clicking on Measure, and Sample Measurement.
Finally, identify the positions of the cuvettes in sample changer one through six, and click on OK.The four strands of RNA were obtained via solid-phase synthesis, and following purification yielded between 300 and 700 nanomoles of each oligonucleotide. The MALDI-TOF chromatograms of the oligonucleotides are shown here. No major difference in the normalized UV vis spectra is observed from comparison of the band at 260 nanometers and canonical and modified oligonucleotides, whether comparing single-stranded samples or duplex structures.
However, minor changes are observed upon measurement of their circular dichroism spectra. In addition, Tm measurements of the three duplex structures displayed a distinct value that was indicative of the destabilization induced by the incorporation of the two prime othyo phenylmethyl modification on one of the strands. It is important to remember that the yield of the solid-phase synthesis is depending on reagent purity.
Moisture will have a negative impact on each step, cause the amount of water present must be reagent at all time. The reported methodology is meant to serve as a general resource for scientists. The technique may vary depending on the desired modification, available instrumentation, or time constraints.
The type of chemistry employed herein represents a very robust methodology that has been in use since the 1980s, and is still heavily used in academic as well as industrial settings. After watching this video, you should be able to perform the synthesis, purification and characterization of your own modified or canonical oligonucleotides of RNA or DNA. Don't forget that working with the necessary reagents can be hazardous, and protective equipment such a safety goggles, gloves, and lab coats should be worn at all times.
All reagents should be handled with care, and inside a fume hood when appropriate.
