RESUMO
The systematic evolution of ligands by exponential enrichment (SELEX) enables the identification of ssDNA or RNA sequences binding to different target molecules, highly specific and with high affinity. In this chapter, we describe a selection strategy with ssDNA for a histidine-tagged protein that could be either performed hands-on manually or fully automated by an appropriate robotic selection platform.
Assuntos
Aptâmeros de Nucleotídeos , Técnica de Seleção de Aptâmeros , Aptâmeros de Nucleotídeos/química , DNA de Cadeia Simples/genética , Histidina/genética , Ligantes , Proteínas/genéticaRESUMO
The receptor binding domain (RBD) of the spike glycoprotein of the coronavirus SARS-CoV-2 (CoV2-S) binds to the human angiotensin-converting enzyme 2 (ACE2) representing the initial contact point for leveraging the infection cascade. We used an automated selection process and identified an aptamer that specifically interacts with CoV2-S. The aptamer does not bind to the RBD of CoV2-S and does not block the interaction of CoV2-S with ACE2. Nevertheless, infection studies revealed potent and specific inhibition of pseudoviral infection by the aptamer. The present study opens up new vistas in developing SARS-CoV2 infection inhibitors, independent of blocking the ACE2 interaction of the virus, and harnesses aptamers as potential drug candidates and tools to disentangle hitherto inaccessible infection modalities, which is of particular interest in light of the increasing number of escape mutants that are currently being reported.
RESUMO
The receptor binding domain (RBD) of the spike glycoprotein of the coronavirus SARS-CoV-2 (CoV2-S) binds to the human angiotensin-converting enzyme 2 (ACE2) representing the initial contact point for leveraging the infection cascade. We used an automated selection process and identified an aptamer that specifically interacts with CoV2-S. The aptamer does not bind to the RBD of CoV2-S and does not block the interaction of CoV2-S with ACE2. Nevertheless, infection studies revealed potent and specific inhibition of pseudoviral infection by the aptamer. The present study opens up new vistas in developing SARS-CoV2 infection inhibitors, independent of blocking the ACE2 interaction of the virus, and harnesses aptamers as potential drug candidates and tools to disentangle hitherto inaccessible infection modalities, which is of particular interest in light of the increasing number of escape mutants that are currently being reported.
Assuntos
Antivirais/farmacologia , Aptâmeros de Nucleotídeos/farmacologia , Tratamento Farmacológico da COVID-19 , SARS-CoV-2/efeitos dos fármacos , Glicoproteína da Espícula de Coronavírus/metabolismo , Enzima de Conversão de Angiotensina 2/metabolismo , Antivirais/química , Aptâmeros de Nucleotídeos/química , Sítios de Ligação/efeitos dos fármacos , COVID-19/metabolismo , Descoberta de Drogas , Células HEK293 , Humanos , Ligação Proteica/efeitos dos fármacos , Domínios e Motivos de Interação entre Proteínas/efeitos dos fármacos , SARS-CoV-2/química , SARS-CoV-2/fisiologia , Técnica de Seleção de Aptâmeros , Glicoproteína da Espícula de Coronavírus/químicaRESUMO
Aptamer selection is a laborious procedure, requiring expertise and significant resources. These characteristics limit the accessibility of researchers to these molecular tools. We describe a selection procedure, making use of a robotic system that allows the fully automated selection of RNA and 2'deoxy-2'-fluoro pyrimidine RNA aptamers. The platform offers a rapid access to aptamers for basic research and development, therefore opening the path to aptamer-based systemic analysis of proteomes in biological settings.
