title: Design, targeting, and initial screening of sTRSV-derived hairpin ribozymes for optimum helix 1 length and catalytic efficiency in vitro.
abstract: Hairpin ribozymes derived from the negative strand of satellite RNAs from the tobacco ringspot virus (sTRSV) can be engineered to target and cleave a variety of heterologous RNAs from both cellular and viral transcripts. Attention to design and targeting rules and optimization of helix 1 length and catalytic efficiency in vitro may increase the efficacy of hairpin ribozymes in reducing the expression of targeted transcripts. Here, principles for the design and targeting of sTRSV-derived hairpin ribozymes are described, as well as methods and materials for optimizing helix 1 length, and for conducting an initial screen of catalytic efficiency to identify promising candidates for further evaluation. Examples are provided for hairpin ribozymes that target human and mouse transforming growth-factor beta (TGF-beta), as well as human polycystic kidney disease gene 1 (PKD1) and JC virus large T-antigen. The tetraloop modification of the sTRSV hairpin ribozyme is considered superior to designs based on the native sTRSV hairpin ribozyme, given its potential to yield considerable improvements in stability and catalytic efficiency.
authors.affiliation: Center for Neurovirology and Cancer Biology, Temple University, Philadelphia, PA, USA.
authors.forename: Max W
authors.initials: MW
authors.lastname: Richardson
authors.forename: Linda
authors.initials: L
authors.lastname: Hostalek
authors.forename: Michelle
authors.initials: M
authors.lastname: Dobson
authors.forename: Jason
authors.initials: J
authors.lastname: Hu
authors.forename: Richard
authors.initials: R
authors.lastname: Shippy
authors.forename: Andrew
authors.initials: A
authors.lastname: Siwkowski
authors.forename: Jonathan D
authors.initials: JD
authors.lastname: Marmur
authors.forename: Kamel
authors.initials: K
authors.lastname: Khalili
authors.forename: Paul E
authors.initials: PE
authors.lastname: Klotman
authors.forename: Arnold
authors.initials: A
authors.lastname: Hampel
authors.forename: Jay
authors.initials: J
authors.lastname: Rappaport
mesh_terms: D001483:Base Sequence; D002384:Catalysis; D005818:Genetic Engineering / Q000379:methods; D005819:Genetic Markers; D006801:Humans; D008969:Molecular Sequence Data; D017860:Nepovirus / Q000235:genetics*; D009690:Nucleic Acid Conformation; D016337:RNA, Catalytic / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012367:RNA, Viral / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity; D013698:Templates, Genetic; D014158:Transcription, Genetic; D016212:Transforming Growth Factor beta / Q000235:genetics
chemical_list: D005819:Genetic Markers; D016337:RNA, Catalytic; D012367:RNA, Viral; D016212:Transforming Growth Factor beta; C418481:hairpin ribozyme