Technical Snippets

Question: Why have we been unable to make a 145mer with multiple 5-carboxy-dC additions?

Discussion:

Our customer was attempting to synthesize a 145mer oligo with multiple additions of 5-carboxy-dC (caC) interspersed throughout. The coupling efficiency based on the instrument trityl monitor’s readings was excellent but no full length product was later revealed. After checking that the recommended deprotection procedure was followed correctly, we set out to mimic the synthesis of a 100mer by conducting the synthesis of a mixed base 12mer oligo with three additions of caC. The support was split into four portions and treated as follows:

These were then deprotected overnight at RT using 0.4 M NaOH MeOH/water 4:1 (v/v) and then desalted and purified on GlenPak™ cartridges. Analysis of the four oligos by ESI MS showed that three were good but the fourth, treated with excess acetic anhydride capping, showed the presence of an addional hydroxyl group at around the 25% level. This suggested that the caC base was activated by capping, allowing a Michael addition of a hydroxyl ion on deprotection.

Resolution:

We suggested remaking the oligo with capping using UniCap (10-4410-xx), a phosphoramidite-based capping reagent. Our customer reported that the oligo synthesis was then successful.

 

Question: Why would my synthesis of oligo-dI be disastrous as determined by trityl yield?

Discussion:

We have never encountered someone who has had problems with the synthesis of oligo-dI. How fast did you see the trityl drop? Was it stepwise from the beginning (x % drop every cycle for instance) or did you see the first few trityls looking just fine and then see a quick drop to nothing? How many bases did you add in the synthesis before you saw the issue? "The trityl was a slow decrease until maybe around base 12-15. We had good color for probably the first 10 or so, with most of the color change happening between bases 10-15. At that point most everything was clear." We have seen some bases that are sensitive to successive cycles do much better with a low water oxidizer. We suggest trying one more time using the dI you have on the machine and 0.5M CSO instead of the standard Iodine based oxidizer (with a 3 minute oxidation time). “We were able to run the poly dI with CSO oxidation and found that the synthesis worked well.”

Resolution:

Inosine is somewhat susceptible to damage by iodine during oxidation. If there are >6 incorporations of inosine within a sequence, use 0.5M CSO in anhydrous acetonitrile (40-4632-xx) with a 3 minute oxidation time for best results.

CNVK

LaB Talk

Coming Soon

We are happy to announce that Glen Research and Maravai LifeSciences have completed an agreement with Nicca Chemical to begin supplying CNVK Phosphoramidite to the research market worldwide, with the exception of Japan. You may remember that CNVK is one of the most effective DNA interstrand crosslinkers so far described.

We are delighted that CNVK Phosphoramidite will be available in late spring. Please watch out for the announcement!

Literature Highlight

RNA Control by Photoreversible Acylation

Willem A. Velema , Anna M. Kietrys, and Eric T. Kool*
Department of Chemistry, Stanford University, Stanford, California 94305, United States
J. Am. Chem. Soc., 2018, 140 (10), pp 3491–3495

Abstract: External photocontrol over RNA function has emerged as a useful tool for studying nucleic acid biology. Most current methods rely on fully synthetic nucleic acids with photocaged nucleobases, limiting application to relatively short synthetic RNAs. Here we report a method to gain photocontrol over RNA by postsynthetic acylation of 2’-hydroxyls with photoprotecting groups. One-step introduction of these groups efficiently blocks hybridization, which is restored after light exposure. Polyacylation (termed cloaking) enables control over a hammerhead ribozyme, illustrating optical control of RNA catalytic function. Use of the new approach on a transcribed 237 nt RNA aptamer demonstrates the utility of this method to switch on RNA folding in a cellular context, and underlines the potential for application in biological studies.