Triphosphorylation is a ubiquitous 5'modification of RNA in biology and is increasingly used in biotechnology applications. The protocol outlined here enables 5'triphosphorylation of any oligonucleotide prepared by standard automated synthesis. Chemical triphosphorylation works on oligonucleotides containing RNA, DNA, or non-natural nucleic acid.
Here, triphosphorylated left-handed L-RNA, the enantiomer of biological D-RNA, is synthesized purified and used in a ribosome reaction. Triphosphorylation is performed after automated oligonucleotide synthesis and before deprotection. These steps are not shown here, but the researchers should be familiar with standard methods for both.
To begin, ensure the workspace for phosphorylation has a gas manifold with at least two lines and the bubbler connected to a dry argon source with adjustable pressure as the triphosphorylation reaction must be performed under argon. Then attach one milliliter plastic syringes to the lines to facilitate connection to the reaction apparatus. Next, collect the equipment including one milliliter plastic syringes, a three-way polypropylene stopcock, noncoring needles, 1.5 milliliter polypropylene tubes, a small metal spatula, and store them in a sealed container or desiccator with a dessicant at room temperature for at least one day prior to use.
Under argon pressure, withdraw 30 milliliters of anhydrous 1, 4-dioxane from a sealed bottle. Transfer into a dried 30 milliliter glass bottle and add a drying trap. Seal with rubber septa and store in a desiccator with dessicant.
Similarly, prepare 30 milliliters of N, N-dimethylformamide, acetonitrile, and the mixture of three parts dioxane and one part pyridine by volume. Prepare TBAP solution by mixing solid tributylammonium pyrophosphate with dimethylformamide and tributylamine in a 30 milliliter glass bottle as described in the manuscript. Add three drawing traps.
Seal the bottle with a rubber septum under argon and bubble with argon for 30 minutes. After preparing an oligonucleotide with a free 5'hydroxyl on a synthesis column using an automated DNA/RNA synthesizer. Prepare the chamber by removing the plunger from a dry one milliliter syringe.
Cut off the top of the syringe using scissors or a razor blade and attach the syringe to the synthesis column. To the top of the syringe, attach the three-way stopcock and attach the side inlet of the stopcock to the dry argon source with the bubbler so that the top inlet of the stopcock acts as the reagent injection port. Secure this operators to a stand with clamps and seal all upstream joints with wax sealing film.
Then adjust the stopcock so that the injection port is closed and the apparatus is open to the argon source. Close the bubbler and allow argon at low pressure to stream through the action chamber for five minutes. After reopening the bubbler, attach a syringe to the bottom of the synthesis column, which will be the waste syringe.
Next, pull argon through the column repeatedly using the waste syringe and then reattach the syringe with the plunger pushed fully in. To add a reagent or solvent, attach a needle to the dry argon source and insert it into the reagent or solvent bottle septum, taking care not to immerse the needle in the bottle contents. Then assemble a dry syringe with a needle and insert it into the reagent or solvent bottle septum without immersing it in the bottle contents.
Afterward, fill the syringe with argon, withdraw the needle from the septum and expel the argon. After filling the syringe with argon and expelling one more time, fill the syringe with the required volume of solvent or reagent under argon pressure. Adjust the stopcock so that the argon source is closed and the injection port is open.
Once the stop on the apparatus is adjusted quickly, remove the filled syringe and needle from the source bottle, wipe away any solvent stuck to the side or tip of the needle and then insert the needle into the injection port. Following expelled a reagent into the anti-chamber of the operators, remove the needle and close the injection port while reopening the apparatus to the argon source. Gently draw the liquid from the anti-chamber all the way through the synthesis column using the waste syringe so that all liquid is now held in the waste syringe.
Now slowly push the solution back up into the synthesis column, ensuring no gas bubbles are in the column. To mix or agitate, gently pull the solution up and down over the column with the waste syringe. To remove a reagent or solvent from the column, slowly pull the solution into the waste syringe.
After the bulk of the solution has passed into the waste syringe, pull argon through to flush the remaining solvent from the column. Next, remove the wax sealing film around the waste syringe joint. Then remove the syringe and discard the waste solution.
Afterward, replace the waste syringe with a new dry syringe and reseal the joint. After assembling the triphosphorylation apparatus, begin the triphosphorylation reaction by adding 200 microliters of pyridine dioxane to the anti-chamber as demonstrated previously. But do not pull sample onto synthesis column yet.
After dissolving SalPCl in dioxane as described in the manuscript, add it to the anti-chamber and loaded onto the synthesis column using a dry syringe. Then let it react for 15 minutes and then remove and discard the SalPCl solution using a waste syringe as demonstrated previously. Immediately after removing the SalPCl solution, add 250 microliters of TBAP solution to the anti-chamber and loaded onto the synthesis column.
Let it react 20 minutes with agitation and discard as described previously. Afterwards, washed the column with 0.5 milliliters of N, N-dimethylformamide, then 0.5 milliliters of acetonitrile, removing the solvent after each addition. After adding oxidizer solution, removing it and washing the column with acetonitrile again using the same procedure shown, disassemble the apparatus and wash the column with five milliliters of acetonitrile.
Afterward, dry the column. Cleave the triphosphorylated oligonucleotide from the solid support and deprotect using standard protocols as described in the manuscript. Next, dissolved the dried deprotected oligonucleotide in urea loading buffer, heated to 80 degrees Celsius, then load onto a polyacrylamide gel and run for one to two hours at 25 to 35 watts.
At the end of the gel electrophoresis, after removing the gel plate, disassemble it and wrap the gel in polyvinylchloride film. Next, identify the product bands by back shadowing with 254 nanometer UV light and excise the major product band using a razor blade. Crush the excised gel by extruding it through a plastic syringe or mechanically.
For oligonucleotides shorter than 15 nucleotide, elute in three times the gel volume of nuclease free water for at least 12 hours with agitation or shaking. Then remove solid gel pieces by passing the solution through a syringe fitted with a 0.2 micrometer filter and concentrate by lyophilization. Following concentration, remove residual salts and salutes using a disposable size exclusion column and concentrate by lyophilization as demonstrated earlier.
After preparing 10 microliters of an RNA solution containing polymerase ribozyme and L-RNA primer template and triphosphate trinucleotides as described in the manuscript, anneal it by heating it to 90 degrees Celsius for one minute and cooling it to 23 degrees Celsius at 0.2 degrees Celsius per second in a PCR thermocycler. After incubating the RNA solution at 17 degrees Celsius, stop the reaction by adding 10 microliters of twice the concentration of the start buffer as the reaction proceeds, take 10 microliter aliquots and quench with five microliters of 0.5 molar EDTA at pH 8 and process each sample to isolate RNA as described in the manuscript. Dissolve the precipitated RNA in 10 microliters of formalized gel loading buffer and prepare unreacted end labeled primer as described in the text.
Once the samples are prepared, heat to 80 degrees Celsius. Load five microliters of the sample in each well and run the gel at 40 watts for approximately 40 minutes. After removing the gel plate from the stand, scan using a fluorescent or phosphorescent gel scanner to visualize cross chiral L-RNA extension products.
After gel purification, UV back shadowing reveals the presence of 5'triphosphate as a single major product as shown for AAA and CCC DNA trimers and GAA L-RNA trimer. The 5'hydroxyl side product is often visible as a slower migrating band, which was isolated and identified by mass spectrometry for DNA trimers. Additional bands during gel purification correlate with 5'diphosphate, monophosphate, and H-phosphonate side products in mass spectrometry of the unpurified reaction products.
5'triphosphate products show a primary mass peak corresponding to the 5'triphosphate along with 5'di and monophosphate masses. Chemically triphosphate related L-RNA trinucleotides were used with an L-RNA primer and L-RNA template in an RNA polymerization reaction analyzed by PAGE and imaged using a fluorescent gel scanner. The extended product marked by black circles demonstrated the synthesis of an L-RNA version of the hammerhead ribozyme encoded by the template.
Exposure to water during triphosphorylation will reduce yield. Follow steps for preparing dry reagents and assembling the reaction apparatus carefully. Move fluids through the apparatus slowly to avoid break in seals.
Chemical triphosphorylation is essential for preparing non-biologic oligonucleotide triphosphates, including left-handed RNA. This has enabled cross chiral ribozyme synthesis of the mirror image versions of RNA molecules found in biology.
