RESUMEN
Many reports have suggested that target-activated ribozymes hold potential value as detection reagents. We show that a "half"-ribozyme ligase is activated similarly by three unstructured oligoribonucleotides representing the major sequence variants of a hepatitis C virus 5'-untranslated region (5'-UTR) target and by a structured RNA corresponding to the entire 5'-UTR. Half-ribozyme ligation product was detected both in an ELISA-like assay and in an optical immunoassay through the use of hapten-carrying substrate RNAs. Both assay formats afford a limit of detection of approximately 1 x 10(6) HCV molecules (1.6 attomol, 330 fM), a sensitivity which compares favorably to that provided by standard immunoassays. These data suggest that target-activated ribozyme systems are a viable approach for the sensitive detection of viral nucleic acids using high-throughput platforms.
Asunto(s)
Hepatitis C/diagnóstico , ARN Catalítico/metabolismo , ARN Viral/análisis , Secuencia de Bases , Ensayo de Inmunoadsorción Enzimática/métodos , Hepatitis C/virología , Inmunoensayo/métodos , Cinética , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Sensibilidad y EspecificidadRESUMEN
An allosteric ribozyme is an RNA-based enzyme (ribozyme) whose catalytic activity is modulated by molecular recognition of a protein. The direct coupling of a detectable catalytic event to molecular recognition by an allosteric ribozyme enables simple assays for quantitative protein detection. Most significantly, the mode of development and molecular recognition characteristics of allosteric ribozymes are fundamentally different from antibodies, providing them with functional characteristics that complement those of antibodies. Allosteric ribozymes can be developed using native proteins and, therefore, are often sensitive to protein conformation. In contrast, antibodies tend to recognize a series of adjacent amino acids as a consequence of antigen presentation and typically are not sensitive to protein conformation. Unlike antibody development, the development of allosteric ribozymes is a completely in vitro process that allows the specificity of an allosteric ribozyme to be tightly controlled. These significant differences from antibodies allow the pre-programmed development of conformation-state-specific protein detection reagents that can be used to investigate the activation-state of signal transduction components.
Asunto(s)
Proteínas/metabolismo , ARN Catalítico/metabolismo , Regulación Alostérica , Sitio Alostérico , Secuencia de Bases , Catálisis , Clonación Molecular , ADN Complementario/genética , ARN Polimerasas Dirigidas por ADN/metabolismo , Evolución Molecular Dirigida/métodos , Transferencia Resonante de Energía de Fluorescencia , Biblioteca de Genes , Cinética , Modelos Químicos , Modelos Moleculares , Conformación de Ácido Nucleico , Reacción en Cadena de la Polimerasa , Unión Proteica , Conformación Proteica , Proteínas/química , ARN/química , ARN/genética , ARN/metabolismo , ARN Catalítico/química , ARN Catalítico/genética , Proteínas ViralesRESUMEN
We describe a strategy for the ultra-sensitive detection of nucleic acids using "half" ribozymes that are devoid of catalytic activity unless completed by a trans-acting target nucleic acid. The half-ribozyme concept was initially demonstrated using a construct derived from a multiple turnover Class I ligase. Iterative RNA selection was carried out to evolve this half-ribozyme into one activated by a conserved sequence present in the hepatitis C virus (HCV) genome. Following sequence optimization of substrate RNAs, this HCV-activated half-ribozyme displayed a maximal turnover rate of 69 min(-1) (pH 8.3) and was induced in rate by approximately 2.6 x 10(9)-fold by the HCV target. It detected the HCV target oligonucleotide in the zeptomole range (6700 molecules), a sensitivity of detection roughly 2.6 x 10(6)-fold greater than that previously demonstrated by oligonucleotide-activated ribozymes, and one that is sufficient for molecular diagnostic applications.
Asunto(s)
Hepacivirus/genética , ARN Catalítico/metabolismo , ARN Viral/análisis , Concentración de Iones de Hidrógeno , ARN Catalítico/genética , Factores de TiempoRESUMEN
Ribozymes are catalytically active RNA molecules that cleave other RNA molecules in a sequence-specific fashion, with significant turnover. The successful design and synthesis of ribozymes with modifications to increase their stability in biological fluids, while maintaining catalytic activity, has been instrumental in moving this technology from the laboratory into clinical trials. With the entry of ribozymes into the clinical setting, the need has arisen for reagents and/or assays to detect these drugs in tissues. We have developed a monoclonal antibody to the 2(')-deoxy-2(')-C-allyl uridine modification present in our synthetic hammerhead ribozymes. The monoclonal antibody, termed CA1USR, is a murine IgG1(k), whose epitope appears to involve both the 2(')-C-allyl modification, and the uridine base. Use of CA1USR for immunohistochemical detection of ribozymes in the tissues of mice which were administered two structurally different ribozymes has demonstrated its utility as a reagent for in vivo localization of ribozymes containing the 2(')-C-allyl uridine modification.