TriLink | January 2012 eNewsletter

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Research Update

Advancements in RNA Click Chemistry

Click chemistry is increasingly popular method for oligonucleotide modification. Click chemistry uses a copper catalyzed 1, 3 dicycloaddition (CuAAC) reaction to efficiently conjugate two ligands together, one containing an azide moiety and the other a terminal alkyne to form a triazole. This irreversible and highly efficient reaction has been successfully applied to DNA chemistry for a broad range of applications from labeling to ligation.

Until recently, click chemistry has been limited to DNA. Unfortunately, the conditions described for use with DNA were too harsh to be used with the much more labile RNA molecule. As one would predict, the lengthy reaction time of 5 hours in the presence of Copper (Cu¹) led to the complete degradation of RNA. El-Sagheer and Brown were the first to describe the application of click chemistry to RNA chemistry in 2010.(1) They labeled select positions of RNA oligos with 5-position azido and alkynyl modified uridines and cytidines, respectively, to assemble RNA hammerhead ribozymes. In this approach hybridization of the stems of the ribozyme brought the azide and alkynyl groups in close proximity. The reaction was conducted for one hour to crosslink the stems, which allowed for functional ribozymes to be produced. While this was the first example of click chemistry on RNA, this protocol is limited in scope to reactions that bring the azide and alkyne groups in close proximity.

A more recent publication by Paredes and Das has greatly expanded the range of applications and reagents that can be used with RNA.(2) They describe a more general protocol that resolved the RNA degradation issues by a combination of a decrease in reaction time to only 30 minutes, an increase in concentration of Cu¹ to 15 equivalents and the addition of a Cu¹ stabilizing reagent to the reaction. The authors used a solution of 20% acetonitrile in buffer to stabilize the copper. A number of azido and alkynyl modified RNA oligonucleotides were prepared using commercial reagents. These modifications were placed at the termini as well as within the oligonucleotide through 5-position modified uridines and cytidines. All were shown to be reactive and the RNA remained intact. Examples of oligo modifications that enable click chemistry include: 5′ Hexynyl, 5′ Bromohexyl, Azidobutyrate and 5′ Iodo-dT.

TriLink’s team of experts is available to help you understand how you can make this powerful chemistry a part of your routine RNA conjugations or to synthesize your modified long RNA transcript or custom modified oligonucleotide.

Ask An Expert I Custom Oligos I RNA Oligos

Featured Question

Q. Can you add a 3′ azido to my long RNA transcripts?

A. Yes, Paredes and Das also demonstrated a very simple approach to add 3′ terminal azides to RNA enzymatically.(2) The approach is elegant in that it can be applied to any RNA molecule: short synthetic oligonucleotides, as well as long RNA transcripts. They labeled the 3′ termini of RNA with 3′ azido-2′,3′-ddATP (Cat #N-4007) using poly(A)polymerase. This technique efficiently adds a single azido modified adenosine to the end of the RNA molecule which is now ready for click chemistry with an alkynyl bearing ligand. The conjugation method described by the authors is straightforward and used the following specifications:
1. The removal of oxygen is important to ensure that oxidative degradation is minimized. It was critical that all stock solutions were degassed just prior to use by bubbling argon through the solution for 15 minutes.
2. A reaction mixture was prepared with the following reagents and concentrations and dissolved in 20% acetonitrile/100 mM Tris-HCl, pH 7.5:
a. 1 mM azido modified RNA
b. 3 mM alkynyl modified ligand
c. 15 mM sodium ascorbate
d. 15 mM CuSO₄
3. The reaction was carried out at room temperature for 30 minutes with shaking.
4. The reaction mixture can be loaded directly onto a denaturing PAGE gel for purification.

This technique will work with any RNA, whether it is a short synthetic oligonucleotide or a long RNA transcript. Any RNA currently in the freezer that has a free 3′ hydroxyl terminus can be readily modified for use in click chemistry.

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