title: Design of hairpin ribozyme variants with improved activity for poorly processed substrates.
abstract: Application of ribozymes for knockdown of RNA targets requires the identification of suitable target sites according to the consensus sequence. For the hairpin ribozyme, this was originally defined as Y⁻² N⁻¹ *G+¹ U+² Y+³ B+⁴, with Y = U or C, and B = U, C or G, and C being the preferred nucleobase at positions -2 and +4. In the context of development of ribozymes for destruction of an oncogenic mRNA, we have designed ribozyme variants that efficiently process RNA substrates at U⁻² G⁻¹ *G+¹ U+² A+³ A+⁴ sites. Substrates with G⁻¹ *G+¹ U+² A+³ sites were previously shown to be processed by the wild-type hairpin ribozyme. However, our study demonstrates that, in the specific sequence context of the substrate studied herein, compensatory base changes in the ribozyme improve activity for cleavage (eight-fold) and ligation (100-fold). In particular, we show that A+³ and A+⁴ are well tolerated if compensatory mutations are made at positions 6 and 7 of the ribozyme strand. Adenine at position +4 is neutralized by G⁶ →U, owing to restoration of a Watson-Crick base pair in helix 1. In this ribozyme-substrate complex, adenine at position +3 is also tolerated, with a slightly decreased cleavage rate. Additional substitution of A⁷ with uracil doubled the cleavage rate and restored ligation, which was lost in variants with A⁷, C⁷ and G⁷. The ability to cleave, in conjunction with the inability to ligate RNA, makes these ribozyme variants particularly suitable candidates for RNA destruction.
authors.affiliation: Max Planck Institute for Molecular Physiology, Department I, Dortmund, Germany  Ernst-Moritz-Arndt Universität Greifswald, Institut für Biochemie, Germany.
authors.forename: Irene
authors.initials: I
authors.lastname: Drude
authors.forename: Anne
authors.initials: A
authors.lastname: Strahl
authors.forename: Daniel
authors.initials: D
authors.lastname: Galla
authors.forename: Oliver
authors.initials: O
authors.lastname: Müller
authors.forename: Sabine
authors.initials: S
authors.lastname: Müller
mesh_terms: D001483:Base Sequence; D006860:Hydrogen Bonding; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D016337:RNA, Catalytic / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics; D013379:Substrate Specificity; D051176:beta Catenin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D016337:RNA, Catalytic; D012333:RNA, Messenger; D051176:beta Catenin; C418481:hairpin ribozyme