Efficient RNA 5'-adenylation by T4 DNA ligase to facilitate practical applications.

RNA
Yangming Wang, Scott K Silverman

Abstract

We describe a simple procedure for RNA 5'-adenylation using T4 DNA ligase. The 5'-monophosphorylated terminus of an RNA substrate is annealed to a complementary DNA strand that has a 3'-overhang of 10 nucleotides. Then, T4 DNA ligase and ATP are used to synthesize 5'-adenylated RNA (5'-AppRNA), which should find use in a variety of practical applications. In the absence of an acceptor nucleic acid strand, the two-step T4 DNA ligase mechanism is successfully interrupted after the adenylation step, providing 40%-80% yield of 5'-AppRNA after PAGE purification with few side products (the yield varies with RNA sequence). Optimized reaction conditions are described for 5'-adenylating RNA substrates of essentially any length including long and structured RNAs, without need for sequestration of the RNA 3'-terminus to avoid circularization. The new procedure is applicable on the preparative nanomole scale. This 5'-adenylation strategy using T4 DNA ligase is a substantial improvement over our recently reported adenylation method that uses T4 RNA ligase, which often leads to substantial amounts of side products and requires careful optimization for each RNA substrate. Efficient synthetic access to 5'-adenylated RNA will facilitate a range...Continue Reading

References

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Jun 5, 2003·Journal of the American Chemical Society·Yangming Wang, Scott K Silverman
Dec 24, 2003·Biochemistry·Yangming Wang, Scott K Silverman
Feb 6, 2004·Nature Structural & Molecular Biology·Rebecca L Coppins, Scott K Silverman
Mar 3, 2005·Journal of the American Chemical Society·Rebecca L Coppins, Scott K Silverman
Sep 22, 2005·Journal of the American Chemical Society·Whitney E PurthaScott K Silverman

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Citations

Jul 26, 2008·Chemical Communications : Chem Comm·Scott K Silverman
Jan 23, 2009·Nature Methods·Francois VigneaultGeorge M Church
Sep 19, 2007·Chembiochem : a European Journal of Chemical Biology·Elena Zelin, Scott K Silverman
Aug 5, 2011·Chemistry : a European Journal·Xingguo LiangHiroyuki Asanuma
Aug 17, 2020·Molecules : a Journal of Synthetic Chemistry and Natural Product Chemistry·Carolin P M ScheitlClaudia Höbartner

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