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.
Many eukaryotic and viral mRNAs, in which the first transcribed nucleotide is an adenosine, are decorated with a cap-1 structure, (7Me)G5'-ppp5'-A(2'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 (7Me)GpppA-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 GpppAC(n) and (7Me)GpppAC(n) (1 < or = n < or = 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 < or = n < or = 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.
Peyrane
F
F
Centre 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.
Selisko
B
B
Decroly
E
E
Vasseur
J J
JJ
Benarroch
D
D
Canard
B
B
Alvarez
K
K
D000241:Adenosine / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D003570:Cytidine Triphosphate / Q000378:metabolism; D019915:DNA Primase; D006147:Guanine / Q000378:metabolism; D006801:Humans; D008780:Methyltransferases / Q000378:metabolism*; D009843:Oligoribonucleotides / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D012314:RNA Cap Analogs / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D013698:Templates, Genetic
D009843:Oligoribonucleotides; D012314:RNA Cap Analogs; D006147:Guanine; D003570:Cytidine Triphosphate; D008780:Methyltransferases; C055097:mRNA (guanine(N7))-methyltransferase; C055099:mRNA (nucleoside-O(2'))-methyltransferase; D019915:DNA Primase; D000241:Adenosine