Screening of molecular interactions using reporter hammerhead ribozymes.
The characterization of molecular interactions is a central task in modern life sciences. Applications such as drug screening in pharmaceutics or the elucidation of biomolecular interactions in molecular biology rely on efficient methods to search for interacting partners. Here, we describe a novel technique that utilizes hammerhead ribozymes to signal molecular interactions. The ribozyme is modified by a domain that specifically binds to a target molecule such as a protein. Upon binding of the target, the catalytic activity of the ribozyme is changed, allowing for detection of the presence as well as the occurrence of interactions of the targeted ligand. The assay can be performed in high-throughput format by employing double-labeled ribozyme substrates, hence being well suited for drug-screening applications. The detection proceeds rapidly and in real-time. Moreover, the technique neither requires labeling of the target molecule nor the potential interaction partners or analytes since an indirect readout is facilitated by switching the catalytic activity of a reporter ribozyme. The assay can be utilized to sense a broad variety of biomolecular interactions, and is very sensitive due to signal amplification by the ribozyme reaction.
Hartig
Jörg S
JS
Department of Chemistry, University of Konstanz, Konstanz, Germany.
Famulok
Michael
M
D052157:Aptamers, Nucleotide / Q000737:chemistry / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D054303:HIV Reverse Transcriptase / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D025941:Protein Interaction Mapping; D016337:RNA, Catalytic / Q000378:metabolism*; D013917:Thrombin / Q000737:chemistry / Q000378:metabolism*
D052157:Aptamers, Nucleotide; D016337:RNA, Catalytic; C427204:hammerhead ribozyme; C081676:thrombin aptamer; D054303:HIV Reverse Transcriptase; D013917:Thrombin