Nucleic Acids Research Advance Access originally published online on January 26, 2007
Nucleic Acids Research 2007 35(4):e26; doi:10.1093/nar/gkl1119
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Nucleic Acids Research, 2007, Vol. 35, No. 4 e26
© 2007 The Author(s).
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
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High-yield production of short GpppA- and 7MeGpppA-capped RNAs and HPLC-monitoring of methyltransfer reactions at the guanine-N7 and adenosine-2'O positions
1Centre National de la Recherche Scientifique and Universités d’Aix-Marseille I et II, UMR 6098, Architecture et Fonction des Macromolécules Biologiques, AFMB-CNRS-ESIL, Case 925, 163 avenue de Luminy, 13288 Marseille Cedex 9, France and 2LCOBS, UMR 5625 CNRS-UMII, CC 008, Université Montpellier II, Place Eugène Bataillon, 34095 Montpellier Cedex 5, France
*To whom the correspondence should be addressed. Tel: +33 491 828630; Fax: +33 491 828646; Email: Karine.Alvarez{at}afmb.univ-mrs.fr
Received August 31, 2006. Revised October 27, 2006. Accepted December 7, 2006.
Many eukaryotic and viral mRNAs, in which the first transcribed nucleotide is an adenosine, are decorated with a cap-1 structure, 7MeG5'-ppp5'-A2'OMe. The positive-sense RNA genomes of flaviviruses (Dengue, West Nile virus) for example show strict conservation of the adenosine. We set out to produce GpppA- and 7MeGpppA-capped RNA oligonucleotides for non-radioactive mRNA cap methyltransferase assays and, in perspective, for studies of enzyme specificity in relation to substrate length as well as for co-crystallization studies. This study reports the use of a bacteriophage T7 DNA primase fragment to synthesize GpppACn and 7MeGpppACn (1 
n 9) in a one-step enzymatic reaction, followed by direct on-line cleaning HPLC purification. Optimization studies show that yields could be modulated by DNA template, enzyme and substrate concentration adjustments and longer reaction times. Large-scale synthesis rendered pure (in average 99%) products (1 n 7) in quantities of up to 100 nmol starting from 200 nmol cap analog. The capped RNA oligonucleotides were efficient substrates of Dengue virus (nucleoside-2'-O-)-methyltransferase, and human (guanine-N7)-methyltransferase. Methyltransfer reactions were monitored by a non-radioactive, quantitative HPLC assay. Additionally, the produced capped RNAs may serve in biochemical, inhibition and structural studies involving a variety of eukaryotic and viral methyltransferases and guanylyltransferases.
Present address: D. Benarroch, Molecular Biology Program, Sloan-Kettering Institute, New York, NY 10021, USA
The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors.
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