The overall goal of this video is to demonstrate the procedure from synthesis of the modified nucleoside phosphates to their complete biochemical characterization. This is accomplished by first converting the suitably protected nucleoside precursor to the corresponding tri phosphate by application of a four step one pot procedure. The second step is to purify the resulting tri phosphate first by a precipitation and then by reverse phase high performance liquid chromatography.
Next, the tri phosphate analog is characterized by nuclear magnetic resonance and matrix assisted laser DESORPTION ionization time of flight or NMR and maloff to ensure high purity. The final step is primer extension reactions and terminal deoxy nucleotide transferase or TDT polymerization reactions. Ultimately, the reaction products, and thus the substrate acceptance of the tri phosphate are analyzed by gel electrophoresis.
The advantage of this method compared to other synthetic strategies like the Borge approach, is that fewer side products are being formed, and thus the purification is easier. Generally, individuals new to this technique will struggle as it is quite a lengthy and intricate procedure that requires knowledge in multiple fields. We first had the idea for this method to be filmed when we synthesized modified phosphates and found it rather challenging to find a detailed procedure.
Another advantage of this method is that it can be used to tackle other questions in the field of modified nucleic acids. Indeed, it can be used to select for DNA enzymes that catalyze a broad range of reactions. In addition, this method can be used for the functional tagging of nucleic acids.
Visual demonstration of this method is critical as synthesis and purification steps are difficult to learn because tri phosphates are polar and activated derivatives that must be handled with care. To begin co evaporate the suitably three-prime hydroxyl protected nucleoside twice with two milliliters of anhydrous purine before drying under vacuum overnight. At the same time, dry tribunal ammonium pyrophosphate under vacuum overnight the following day dissolved the nucleoside in a minimum of 200 microliters of dry pyridine.
Then add 400 microliters of dry dioxane as a COS solvent. Finally, add the cyclic phosphorus three derivative and allow it to react at room temperature for 45 minutes. Next, prepare a solution of dry butyl ammonium pyrophosphate in 170 microliters of dry dimethylformamide and 58 microliters of freshly distilled tri butyl amine.
Add the resulting solution to the reaction mixture and allow it to react at room temperature for 45 minutes. Then prepare a solution of 160 micromolar iodine in 980 microliters of purine and 20 microliters of water. Add the solution to the reaction mixture.
In order to oxidize the alpha phosphorus center. Allow the resulting dark solution to stir at room temperature for 30 minutes. Use a 10%aqueous solution of sodium thio sulfate to quench the excess iodine.
Remove the solvent under vacuum. Then add five milliliters of water and allow the mixture to stand at room temperature for 30 minutes to hydrolyze the cyclic tri phosphate moty. At this stage, the protecting groups are usually removed.
Consequently, add 30%Imodium hydroxide to the crude sample. Stir for 1.5 hours at room temperature before removing the solvent under vacuum. Next, add two milliliters of water and split the mixture into tube tubes.
Add 12 milliliters of a 2%solution of sodium per chlorate in acetone and centrifuge for 30 minutes before repeating the procedure. One more time. The reagents used in the synthesis from the tri phosphate will precipitate forming an oily residue and allowing for the separation of the solvents, and thus simplifying the ensuing reverse phase HPLC purification substantially after air drying of the oily residue.
Record a P 31 NMR spectrum of the crude sample according to standard procedures. Following NMR dissolved the oily residue in four milliliters of distilled water. Begin HPLC purification with preparation of 50 millimolar triethyl ammonium bicarbonate in ultrapure water as EENT A and in 50%acetyl nitrile as eent B as detailed in the text protocol, Degas both EENT under vacuum and stirring for 20 minutes.
Prepare an analytical sample by dissolving 10 microliters of the crude tri phosphate in 300 microliters of distilled water. Then using a gradient ranging from 0%B to 100%B in 40 minutes. Inject the sample into an HPLC system equipped with a semi preparative reverse Phase C 18 column.
Adjust the HPLC program according to the retention time of the tri phosphate and the diphosphate. Purify the crude mixture using these conditions and by removing early fractions that might contain some diphosphate, combine all the fractions that contain the product and freeze dry. Then co evaporate several times with ultrapure water.
Assess the purity of the tri phosphate by NMR and maloff by application of standard protocols. Typical yields obtained by application of this protocol lie in the range of 30 to 70%depending on the substrate for five prime and labeling of the primer. Mix the primer and reagents as detailed in the text protocol and incubate the labeling reaction at 37 degrees Celsius for 30 minutes.
During the labeling reaction, prepare a G 10 cidex column by plugging a one milliliter pipette tip with siloized glass wool and filling the tip with G 10 solution. Spin down and wash three times with 500 microliters of double distilled water and one final wash with 50 microliters of double distilled water. Following incubation of the labeling reaction, heat deactivate the kinase for 10 minutes at 70 degrees Celsius.
Next, run the reaction mixture through the G 10. To remove the free radioactive label gel, purify the radio labeled primer and recover by application of the crush and soak method, ethanol precipitate and G 10 desalt the eluded oligonucleotide. For the primer extension reaction, combine one PICA mole of radiolabeled primer, P one 10 PICA moles of primer, P one and 10 PICA moles of template, T one in 10 x reaction buffer and kneel in hot water.
Then gradually cool down to room temperature over 45 minutes. Put the tube on ice and add the nucleoside phosphate or DNTP cocktail and one unit of the DNA polymerase ice. Complete the reaction with water for a total volume of 20 microliters incubate at the optimal working temperature of the enzyme.
Then add 20 microliters of stop solution and heat the samples at 95 degrees Celsius for five minutes. Cool, the samples down at zero degrees Celsius. Before resolving by denaturing poly acrylamide gel electrophoresis.
Visualize the bands by phospho imager for TDT polymerization, dilute single stranded unlabeled primer P two and radio labeled primer in one microliter of TDT buffer. Put the tube on ice and add the DNTP cocktail and the TDT polymerase then incubate at 37 degrees Celsius for one hour. Following incubation, add 10 microliters of the stop solution before heating the samples at 95 degrees Celsius for five minutes, and then cooling down at zero degrees Celsius.
After resolving the sample by denaturing poly acrylamide gel electrophoresis, visualize the bands using a phospho imager. Shown here is a typical crude P 31 NMR spectrum of a modified DNTP where the characteristic signals of the phosphorus centers can be observed. In addition, signals stemming from the diphosphate and monophosphate side.
Products along with higher phosphates are always observed at this stage. Reverse phase HPLC analysis of the crude mixture is shown here. The main peak corresponds to the five prime phosphate, while the main byproduct, the five prime diphosphate displays a slightly lower retention time.
Finally, after a thorough reverse phase, HPLC purification, the modified DNTP is characterized by NMR and maldi to shown here is the maloff spectrum of the sulfa ide, modified DUTP analog. The sodium adduct can be seen at mass overcharge of 712.54, and no trace of diphosphate is present. Both the P 31 NMR and proton NMR spectra are crucial for assessing the purity of the modified dps.
Since the presence of undesired dye and mono phosphates gives distinctive signals illustrated, here is the outcome of primary extension reactions with trans proline modified DUTP histamine modified DATP and valeric acid modified DCTP used either as lone modifications as combinations of two modified DN dps or together, along with a lone natural DGTP. Finally, representative TDT mediated polymerization reactions with different modified DUTP analogs were analyzed with denaturing poly acrylamide gel electrophoresis. In this context, cis proline modified DUTP and peptide modified DUTP are the best substrates for TDT.
Since the tailing efficiencies are comparable or exceed those of the natural DTTP instead, urea modified DUTP is a rather poor substrate in this context. Since little poly disperse, longer sized oligonucleotides can be observed Once mastered, this technique can be done in about one week if it is performed properly. While attempting this procedure, it is important to keep in mind that nuclear cytric phosphates are nature's activated building blocks, and therefore care should be taken while handling them After it was created.
Researchers in the field of nucleic acids use this method to explore combinatorial selections to generate modified abers and catalytic nucleic acids. After watching this video, you should have a good understanding of how to synthesize and characterize modified phosphates using the four step one pop procedure, followed by reverse phase HPLC purification and primary extension reactions. Some experimental details that are not normally found in experimental sections will become apparent.
This method can be used in combination with other techniques such as PCR to answer additional questions. Indeed, it can be used to assess the compatibility of modified tri phosphates with methods of in VIO selection.
