How do I label a periodate oxidized 3' end of an RNA molecule with cystamine?

13 April 2018 3 9K Report

I am trying to label my RNA with cystamine on it's 3' end and I have done periodate oxidation to get an aldehyde from the diols. But the cystamine labeling did not work.

I followed the exact protocol that was described by Chan et. al. in their paper. (Link: http://www.pnas.org/content/pnas/96/2/459.full.pdf).

"The 3' end of TMR–tRNA (100 mM) was next oxidized by incubation in 20 mM NaIO4 at room temperature in the dark for 20 min. Excess NaIO4 was destroyed as described (12). RNA was recovered by NaCl/ethanol precipitation. The oxidized RNA (86 mM) was reductively aminated with cystamine (340 mM) and NaCNBH3 (6 mM) in 100 mM sodium phosphate (pH 6.3) at room temperature overnight (12, 13). "

Only difference is, I had not destroyed excess periodate. I am assuming that shouldn't be too much of a problem.

I used zymo column to clean up the mix. And I used the Ellman's test to quantify the extent of labeling.

What do you think could have been the issue? I have not been able to achieve labeling at all.

Thank you very much.

Nick Gee

I have not looked at the full paper method but a couple of things immediately spring to mind. (i) obviously the destruction of excess periodate has been omitted, with unknown consequences. It has to be a risk, and may need to be checked, as you have an oxidising agent and a reducing agent in the same mix. (ii) pH 6.3. This is unusually low for the amination part of the reaction. I would expect a far higher pH to be used to increase reactivity of amines and to stabilise the schiff's base that is formed. Around pH 9 would be more typical. You should not really use cyanoborohydride below pH 7 for safety reasons too.

Sravya Varali Kovvali

Hi Nick,

Thank you for your valuable suggestions. But I thought a higher pH would probably make it difficult for the aldehyde to react, isn't that true?

Thank you.

Nick Gee

I have generally been using amines with high pKa values, so there is a need to deprotonate the amines by raising the pH. However, as there are several steps in the reaction process the overall pH optimum may have to be determined by trial and error. I cannot immediately find the pKa values for cystamine. I think if you are using aqueous conditions the best pH is likely to be between pH 7-9, but try different pH values and see what works best. I'd be interested to know what you find.

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