title: Sequence-controlled RNA self-processing: computational design, biochemical analysis, and visualization by AFM.
abstract: Reversible chemistry allowing for assembly and disassembly of molecular entities is important for biological self-organization. Thus, ribozymes that support both cleavage and formation of phosphodiester bonds may have contributed to the emergence of functional diversity and increasing complexity of regulatory RNAs in early life. We have previously engineered a variant of the hairpin ribozyme that shows how ribozymes may have circularized or extended their own length by forming concatemers. Using the Vienna RNA package, we now optimized this hairpin ribozyme variant and selected four different RNA sequences that were expected to circularize more efficiently or form longer concatemers upon transcription. (Two-dimensional) PAGE analysis confirms that (i) all four selected ribozymes are catalytically active and (ii) high yields of cyclic species are obtained. AFM imaging in combination with RNA structure prediction enabled us to calculate the distributions of monomers and self-concatenated dimers and trimers. Our results show that computationally optimized molecules do form reasonable amounts of trimers, which has not been observed for the original system so far, and we demonstrate that the combination of theoretical prediction, biochemical and physical analysis is a promising approach toward accurate prediction of ribozyme behavior and design of ribozymes with predefined functions.
authors.affiliation: Institute for Biochemistry, Ernst-Moritz-Arndt University Greifswald, 17487 Greifswald, Germany.
authors.forename: Sonja
authors.initials: S
authors.lastname: Petkovic
authors.affiliation: Institute for Theoretical Chemistry, University of Vienna, A-1090 Vienna, Austria.
authors.forename: Stefan
authors.initials: S
authors.lastname: Badelt
authors.affiliation: ZIK HIKE-Center for Innovation Competence, Humoral Immune Reactions in Cardiovascular Diseases, Ernst-Moritz-Arndt University Greifswald, 17489 Greifswald, Germany smueller@uni-greifswald.de ivo@tbi.univie.ac.at stephan.block@chalmers.se.
authors.forename: Stephan
authors.initials: S
authors.lastname: Block
authors.affiliation: Institute for Theoretical Chemistry, University of Vienna, A-1090 Vienna, Austria.
authors.forename: Christoph
authors.initials: C
authors.lastname: Flamm
authors.affiliation: ZIK HIKE-Center for Innovation Competence, Humoral Immune Reactions in Cardiovascular Diseases, Ernst-Moritz-Arndt University Greifswald, 17489 Greifswald, Germany.
authors.forename: Mihaela
authors.initials: M
authors.lastname: Delcea
authors.affiliation: Institute for Theoretical Chemistry, University of Vienna, A-1090 Vienna, Austria Research Group Bioinformatics and Computational Biology, University of Vienna, A-1090 Vienna, Austria smueller@uni-greifswald.de ivo@tbi.univie.ac.at stephan.block@chalmers.se.
authors.forename: Ivo
authors.initials: I
authors.lastname: Hofacker
authors.affiliation: Institute for Biochemistry, Ernst-Moritz-Arndt University Greifswald, 17487 Greifswald, Germany smueller@uni-greifswald.de ivo@tbi.univie.ac.at stephan.block@chalmers.se.
authors.forename: Sabine
authors.initials: S
authors.lastname: Müller
mesh_terms: D001483:Base Sequence; D018625:Microscopy, Atomic Force / Q000379:methods*; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D012313:RNA / Q000737:chemistry / Q000378:metabolism*; D012323:RNA Processing, Post-Transcriptional
chemical_list: D012313:RNA
keywords: AFM; RNA; circularization; computational design; hairpin ribozyme; self-processing