title: Rational engineering of the Neurospora VS ribozyme to allow substrate recognition via different kissing-loop interactions.
abstract: The Neurospora VS ribozyme is a catalytic RNA that has the unique ability to specifically recognize and cleave a stem-loop substrate through formation of a highly stable kissing-loop interaction (KLI). In order to explore the engineering potential of the VS ribozyme to cleave alternate substrates, we substituted the wild-type KLI by other known KLIs using an innovative engineering method that combines rational and combinatorial approaches. A bioinformatic search of the protein data bank was initially performed to identify KLIs that are structurally similar to the one found in the VS ribozyme. Next, substrate/ribozyme (S/R) pairs that incorporate these alternative KLIs were kinetically and structurally characterized. Interestingly, several of the resulting S/R pairs allowed substrate cleavage with substantial catalytic efficiency, although with reduced activity compared to the reference S/R pair. Overall, this study describes an innovative approach for RNA engineering and establishes that the KLI of the trans VS ribozyme can be adapted to cleave other folded RNA substrates.
authors.affiliation: Département de Biochimie et Médecine Moléculaire, Université de Montréal, C.P. 6128, Succursale Centre-Ville, Montréal, QC H3C 3J7, Canada.
authors.forename: Julie
authors.initials: J
authors.lastname: Lacroix-Labonté
authors.affiliation: Département de Biochimie et Médecine Moléculaire, Université de Montréal, C.P. 6128, Succursale Centre-Ville, Montréal, QC H3C 3J7, Canada.
authors.forename: Nicolas
authors.initials: N
authors.lastname: Girard
authors.affiliation: Département de Biochimie et Médecine Moléculaire, Université de Montréal, C.P. 6128, Succursale Centre-Ville, Montréal, QC H3C 3J7, Canada.
authors.forename: Pierre
authors.initials: P
authors.lastname: Dagenais
authors.affiliation: Département de Biochimie et Médecine Moléculaire, Université de Montréal, C.P. 6128, Succursale Centre-Ville, Montréal, QC H3C 3J7, Canada pascale.legault@umontreal.ca.
authors.forename: Pascale
authors.initials: P
authors.lastname: Legault
mesh_terms: D001483:Base Sequence; D055162:Biocatalysis; D019295:Computational Biology; D018360:Crystallography, X-Ray; D030562:Databases, Protein; D004722:Endoribonucleases / Q000737:chemistry / Q000378:metabolism*; D004795:Enzyme Stability; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D009491:Neurospora / Q000201:enzymology*; D009690:Nucleic Acid Conformation; D015202:Protein Engineering; D016337:RNA, Catalytic / Q000737:chemistry / Q000378:metabolism*; D013379:Substrate Specificity; D013816:Thermodynamics
chemical_list: D016337:RNA, Catalytic; D004722:Endoribonucleases; C438195:varkud satellite ribozyme