RESUMEN
All RNA types are susceptible to ribonuclease (RNase) digestion, which might be a serious problem for several in vitro and in vivo applications. RNase resistance can be reached through chemical modifications or the selection of stable secondary structures via SELEX (systematic evolution of ligands by exponential enrichment). This chapter focuses on the selection of natural RNase-resistant RNAs, enriched by a selection process in the presence of RNase T1. Results of these investigations led to the identification of a particular structural motif, the tetraloop. Further applications could be the advised use of such motifs in order to reach higher stability of RNA molecules.
Asunto(s)
ARN/metabolismo , Ribonucleasas/metabolismo , Animales , Humanos , Conformación de Ácido Nucleico , Ribonucleasa T1/metabolismoAsunto(s)
Azul de Metileno , Ribonucleasa Pancreática/análisis , Cloruro de Tolonio , Secuencia de Bases , Calibración , Indicadores y Reactivos , Cinética , ARN/química , ARN/metabolismo , Proteínas Recombinantes/análisis , Ribonucleasa T1/análisis , Ribonucleasa T1/metabolismo , Ribonucleasa Pancreática/metabolismo , Espectrometría de Fluorescencia/métodos , Espectrofotometría/métodosRESUMEN
An RNA sequence showing high stability with respect to digestion by ribonuclease T1 (RNase T1) was isolated by in vitro selection from an RNA library. Although ribonuclease T1 cleaves single-stranded RNA specifically after guanosine residues, secondary structure calculations predict several guanosines in single-stranded areas. Two of these guanosines are part of a GGCA-tetraloop, a recurring structure element in the secondary structure predictions. Molecular dynamics simulations of the conformation space of the nucleotides involved in this tetraloop show on the one hand that the nucleic acid backbone of the guanosines cannot realise the conformation required for cleavage by RNase T1. On the other hand, it could be shown that an RNA molecule not forced into a tetraloop occupies this conformation several times in the course of the simulation. The simulations confirm the GGCA-tetraloop as an RNase-stable secondary structure element. Our results show that, besides the known prerequisite of a single-stranded RNA, RNase T1 has additional demands on the substrate conformation.
Asunto(s)
Compuestos Orgánicos , ARN/química , Ribonucleasa T1/química , Secuencia de Bases , Simulación por Computador , Colorantes Fluorescentes/química , Modelos Moleculares , Conformación de Ácido Nucleico , Reacción en Cadena de la Polimerasa , ARN/metabolismo , Ribonucleasa T1/metabolismo , TermodinámicaRESUMEN
RNA-oligonucleotides with defined single-stranded stretches were designed to investigate the minimal requirements of a ribonuclease T1 substrate. It could be shown, that RNase T1 cleaves single-stranded RNA after a unique guanosine flanked by two double-stranded areas. However, the turnover of such a G-gap is significantly lower than that of a gap of two, three or four nucleotides.
