title: Engineering a ribozyme cleavage-induced split fluorescent aptamer complementation assay.
abstract: Hammerhead ribozymes are self-cleaving RNA molecules capable of regulating gene expression in living cells. Their cleavage performance is strongly influenced by intra-molecular loop-loop interactions, a feature not readily accessible through modern prediction algorithms. Ribozyme engineering and efficient implementation of ribozyme-based genetic switches requires detailed knowledge of individual self-cleavage performances. By rational design, we devised fluorescent aptamer-ribozyme RNA architectures that allow for the real-time measurement of ribozyme self-cleavage activity in vitro The engineered nucleic acid molecules implement a split Spinach aptamer sequence that is made accessible for strand displacement upon ribozyme self-cleavage, thereby complementing the fluorescent Spinach aptamer. This fully RNA-based ribozyme performance assay correlates ribozyme cleavage activity with Spinach fluorescence to provide a rapid and straightforward technology for the validation of loop-loop interactions in hammerhead ribozymes.
authors.affiliation: Department of Biosystems Science and Engineering, ETH Zurich, Mattenstrasse 26, CH-4058 Basel, Switzerland.
authors.forename: Simon
authors.initials: S
authors.lastname: Ausländer
authors.affiliation: Department of Biosystems Science and Engineering, ETH Zurich, Mattenstrasse 26, CH-4058 Basel, Switzerland.
authors.forename: David
authors.initials: D
authors.lastname: Fuchs
authors.affiliation: Department of Biosystems Science and Engineering, ETH Zurich, Mattenstrasse 26, CH-4058 Basel, Switzerland.
authors.forename: Samuel
authors.initials: S
authors.lastname: Hürlemann
authors.affiliation: Department of Biosystems Science and Engineering, ETH Zurich, Mattenstrasse 26, CH-4058 Basel, Switzerland.
authors.forename: David
authors.initials: D
authors.lastname: Ausländer
authors.affiliation: Department of Biosystems Science and Engineering, ETH Zurich, Mattenstrasse 26, CH-4058 Basel, Switzerland Faculty of Science, University of Basel, Mattenstrasse 26, CH-4058 Basel, Switzerland fussenegger@bsse.ethz.ch.
authors.forename: Martin
authors.initials: M
authors.lastname: Fussenegger
mesh_terms: D052157:Aptamers, Nucleotide / Q000737:chemistry*; D005456:Fluorescent Dyes / Q000737:chemistry; D005816:Genetic Complementation Test / Q000379:methods*; D005818:Genetic Engineering / Q000379:methods; D016337:RNA, Catalytic / Q000235:genetics / Q000378:metabolism*
chemical_list: D052157:Aptamers, Nucleotide; D005456:Fluorescent Dyes; D016337:RNA, Catalytic