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.

[Dengue virus: viral targets and antiviral drugs]. / Le virus de la dengue : cibles virales et antiviraux.

This work reviews the opportunities and scientific bases in the development of anti-dengue drugs. The timeliness of anti-dengue drug development is addressed in the context of the growing impact of dengueworldwide and existing strategies to fight the virus. The antiviral approach in therapy or prophylaxis during an epidemic as well as the impact of recent technological advances in drug-discovery and antiviral chemotherapy on the development of anti-dengue drugs are discussed. An analysis of current sources of synthetic or natural drugs is provided. Finally, we summarize the current knowledge on dengue virus proteins, which are currently considered the most viable as drug targets, as the envelop protein E and non-structural proteins NS3 and NS5 carrying protease, helicase, RNA triphosphatase, methyltransferase and RNA-dependent RNA polymerase activities. Other viral proteins proposed to be part of the replication complex and the complex itself are considered as potential targets of anti-dengue drugs. State-of-the-art methods are listed, that are expected to allow the discovery, design, and characterisation of anti-dengue drugs effective against the four serotypes.