Basically, purification of RNA is similar as DNA, with DMTr-on and DMTr-off. In RNA treatment, there is one additional silyl deprotection step for removing the protecting groups at 2’-positions.

 

1. Pour the synthesized solid support to an Eppendorf tube (with or without screw cap). Start from half of the support, in case any failure experiment happens.

 

2. Add 1 mL conc. Ammonium hydroxide to the tube. Make sure the support is submerged in the solution. Tightly seal the tube with parafilm, or use the cap with screw to seal it. Heat the sample at 55oC for 16h. Cool down the sample to room temperature. The RNA is now cleaved from the support and all the base-labile protecting groups are removed. (fresh Ammonium hydroxide is important for the complete deprotection.)

 

3. Transfer the supernatant to a new Eppendorf tube. Wash the support with 0.4 mL clean water twice, and combine all the supernatants from every washing.

 

4. Use Speed-vac at 4 oC to evaporate Ammonium hydroxide for ~1h. Don’t heat the sample during evaporation since it may cause de-tritylation. Freeze the RNA sample and lyophilize it to dryness. (the ammonium has to be removed for the successful lyophilization)

 

5. Fully re-dissolve the RNA in 115μL anhydrous DMSO. If necessary, heat the oligo at 65°C for about 5 minutes to get it into solution.Add 60 μL of TEA to the DMSO/RNA solution and mix gently. Add 75μL of TEA.3HF and mix gently. Heat the mixture at 65 oC for 2.5 hours. DO NOT over-heat the sample at more than 65 oC. Cool the sample to room temperature. 

 

If the Glen-pak RNA cartridge is used for the DMTr-on purification, go to step 6a. If HPLC is used for the DMTr-on purification, go to 6b.

 

DMTr-on purification

6a. 1.75mL Glen-Pak RNA Quenching Buffer is added to the sample. Follow the Glen Research Purification of RNA Oligonucleotides (DMT-ON) protocol. https://www.glenresearch.com/media/productattach/import/tbn/GlenPak_UserGuide.pdf

The de-tritylation and washing off failure oligos will happen on the cartridge. The DMTr-off RNA is in solution of MeCN/water mixture. Use speed-vac to evaporate MeCN, for the second purification.

 

6b. Directly add 1.75mL 20mM TEAB buffer pH 7.2 to the RNA solution to dilute DMSO. Inject sample for HPLC purification. Buffer A is water, buffer B is 50% MeCN in water, both containing 20mM TEAB pH 7.2. Collect the full length DMTr-on RNA product (typically washed out at 60% buffer B), lyophilize the sample to dryness. Re-dissolve the RNA in 0.2 mL water, and transfer to a new Eppendorf tube. Wash the tube with 0.2 mL water twice and combine them to maximize the recovery yield. Add 3% trichloroacetic acid in water to the RNA solution for the de-tritylation. Typically 50-100 μl of acid is enough. Monitor the pH of the solution if necessary. pH ~1 is the acidity for de-tritylation to happen, and sometimes white precipitation can be observed. Incubate the sample at room temperature for 5 min and DMTr group should be cleaved off. Add ~100 μl 2M TEAAc or TEAB buffer, pH 7, to the solution to neutralize the TCA acid. Monitor the pH of the solution if necessary. Filter the sample by spin column for second HPLC purification.

 

ps:

a. If possible, 2’-O-TriisopropylsilylOxyMethyl (TOM) protected RNA phosphoramidites can be chosen for higher coupling efficiency, since TOM-group exhibits lower steric hindrance than the traditional 2’-tBDMS. Follow the Glen Research recommended protocol https://www.glenresearch.com/media/productattach/t/b/tb_rna_tom_deprotection_20190625.pdf

b. In some case, DMSO may broaden the HPLC peak and lower the separation efficiency. If so, ethanol precipitation can be used to get rid of DMSO. Into the sample after de-silylation, add 26 μL of 3M Sodium Acetate, mix well by vortexing for 15 seconds. Add 1 mL butanol. Mix well by vortexing for 30 seconds. Cool at -78°C for 30 minutes. Centrifuge for 30 minutes at 12,500rpm. Decant butanol, and rinse with 0.75 mL ethanol, twice. Dry the sample in speed-vac to remove traces of butanol. Re-dissolve the RNA in water for DMTr-on HPLC purification. 

c. The usage of 1.0M TBAF/THF at room temperature can still be used to deprotect 2'-silyl groups, but it's not compatible with Glen-pak cartridge. Removal of THF is necessary for the downstream process.

 

DMTr-off purification

7a. Use Waters preparative HPLC system for DMTr-off purification. DMTr-off RNA is typically washed out at 30% buffer B. Collect the RNA, lyophilize the sample to dryness and re-dissolve in water. Quantify the RNA by UV, and prepare the sample with certain concentrations for the further experiment.

 

7b. Use PAGE gel to do the second purification. Follow our website online protocol for PAGE gel electrophoresis experiment, and recover the RNA from the gel. Remove the unnecessary salt by ethanol precipitation or Glen-pak desalting cartridge/DNA cartridge. Re-dissolve the RNA in water, quantify the sample and prepare for the further experiment.