Technical Brief – 5'-Phosphorylation of RNA

PC Spacer Phosphoramidite
PC Spacer-77mer RNA DMT-ON Crude
PC Spacer-77mer RNA DMT-ON Purified
5-Phosphate 77mer RNA - UV Treatment for 20 minutes

The presence of a 5' phosphate on RNA oligonucleotides is critical for a variety of biological functions. It is a required structural component for RIG-1 to recognize single-stranded viral RNA and turn on the innate immune response.1

Similarly, 5'-phosphorylation of siRNA and piRNA is also required for the RNAi machinery to properly load and suppress gene translation (in the case of double-stranded siRNA) and protect germline cells from genetic damage due to transposons (in the case of single-stranded piRNA).2

For more practical applications, 5'-phosphorylated RNA allows long RNA oligonucleotides to be synthesized through enzymatic ligation of shorter, more easily purified RNA oligonucleotides.3

The efficient production of pure, 5'-phosphorylated RNA oligonucleotides by chemical synthesis has been hampered by the inability to purify these oligonucleotides by reverse phase cartridges or HPLC. While there are a number of options for the DMT-On purification of DNA,4 they all require treatment with base following the removal of the DMT which leads to both strand scission and 2'-3' migration of phosphodiester linkages within the RNA.

However, a researcher at Horizon Discovery/Dharmacon, Dr. Shoeb Khan, approached us with an elegant solution to this problem using an existing product of ours - the photocleavable PC Spacer. He suggested that by coupling the PC Spacer to the 5' terminus of the RNA during synthesis, the RNA oligo could be cleaved and deprotected while maintaining the 5' trityl on the PC Spacer which then could be used as a reverse-phase handle to purify the full-length oligo from the failures. After purification, the entire PC Spacer is removed by simple irradiation with long UV light to reveal a pure, 5' phosphorylated RNA oligo. We were happy to provide Dr. Khan with reagents for testing his procedure, which worked remarkably well. Dr. Khan has generously shared his results and protocol below.


• 5'-Phosphate 77 bases RNA was synthesized on an Akta Oligopilot synthesizer.

• RNA synthesis was carried out using 2'-TBDMS chemistry.

• DMT-ON method selected.

• Cleavage & deprotection by AMA treatment, 20 min at 65°C.

• 2'-Deprotection by TEA:3HF for 2hr at 65 °C.

• Purified by preparative Waters RP-HPLC.

• Removal of PC-spacer by UV at 365nm for 20min, then additional 20min.

• The oligo was then ethanol precipitated to remove the remnants of the tritylated PC Spacer and analyzed.

We are happy to supply detailed information about Dr. Khan's method on request. On a smaller synthesis scale, we recommend the use of GlenPak™ RNA cartridge purification as an alternative to HPLC.

5-Phosphate 77mer RNA - UV Treatment for additional 20 minutes
5-Phosphate 77mer RNA - ESI MS


1. A. Pichlmair, et al., Science, 2006, 314, 997-1001.

2. M. Ha, and V.N. Kim, Nat Rev Mol Cell Biol, 2014, 15, 509-24.

3. G.C. Walker, O.C. Uhlenbeck, E. Bedows, and R.I. Gumport, Proc Natl Acad Sci U S A, 1975, 72, 122-6.

4. The Glen Report, 2011, 23, 10-11.