RESUMO
Dengue virus (DENV) protein NS5 carries two mRNA cap methyltransferase (MTase) activities involved in the synthesis of a cap structure, (7Me)GpppA(2'OMe)-RNA, at the 5'-end of the viral mRNA. The methylation of the cap guanine at its N7-position (N7-MTase, (7Me)GpppA-RNA) is essential for viral replication. The development of high throughput methods to identify specific inhibitors of N7-MTase is hampered by technical limitations in the large scale synthesis of long capped RNAs. In this work, we describe an efficient method to generate such capped RNA, GpppA(2'OMe)-RNA74, by ligation of two RNA fragments. Then, we use GpppA(2'OMe)-RNA74 as a substrate to assess DENV N7-MTase activity and to develop a robust and specific activity assay. We applied the same ligation procedure to generate (7Me)GpppA-RNA74 in order to characterize the DENV 2'-O-MTase activity specifically on long capped RNA. We next compared the N7- and 2'-O-MTase inhibition effect of 18 molecules, previously proposed to affect MTase activities. These experiments allow the validation of a rapid and sensitive method easily adaptable for high-throughput inhibitor screening in anti-flaviviral drug development.
Assuntos
Vírus da Dengue/enzimologia , Dengue/virologia , Avaliação Pré-Clínica de Medicamentos/métodos , Ensaios Enzimáticos/métodos , Metiltransferases/análise , Proteínas não Estruturais Virais/análise , Antivirais/farmacologia , Dengue/tratamento farmacológico , Vírus da Dengue/efeitos dos fármacos , Vírus da Dengue/genética , Vírus da Dengue/metabolismo , Inibidores Enzimáticos/farmacologia , Humanos , Metiltransferases/antagonistas & inibidores , Metiltransferases/genética , Metiltransferases/metabolismo , Capuzes de RNA/genética , Capuzes de RNA/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Viral/genética , RNA Viral/metabolismo , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismoRESUMO
MALDI-TOF mass spectrometry has been used to characterize solid-supported oligonucleotides containing natural and non-natural and non-nucleoside moieties and a variety of internucleosidic linkages including phosphate and phosphite triesters and H-phosphonate diesters. This technique was used to follow the reactions involved in oligonucleotide synthesis; this enabled direct control of the elongation and optimization of the coupling process.
Assuntos
Oligodesoxirribonucleotídeos/química , Oligodesoxirribonucleotídeos/síntese química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Sequência de Bases , Análise de Sequência com Séries de OligonucleotídeosRESUMO
This study presents matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) as a powerful tool to analyze and characterize oligonucleotides covalently linked to a solid support during their synthesis. The analysis of the fragment ions generated either in negative or positive mode allows direct and easy access to the nucleotide sequence and identification of the internucleosidic linkage. The mechanisms of the fragmentation of the solid-supported oligonucleotides induced by MALDI-TOFMS are discussed. Copyright 2000 John Wiley & Sons, Ltd.
