title: Selection, design, and characterization of a new potentially therapeutic ribozyme.
abstract: An in vitro selection was designed to identify RNA-cleaving ribozymes predisposed for function as a drug. The selection scheme required the catalyst to be trans-acting with phosphodiesterase activity targeting a fragment of the Kras mRNA under simulated physiological conditions. To increase stabilization against nucleases and to offer the potential for improved functionality, modified sequence space was sampled by transcribing with the following NTPs: 2'-F-ATP, 2'-F-UTP, or 2'-F-5-[(N-imidazole-4-acetyl) propylamine]-UTP, 2'-NH2-CTP, and GTP. Active motifs were identified and assessed for their modified NMP and divalent metal dependence. The minimization of the ribozyme's size and the ability to substitute 2'-OMe for 2'-F and 2'-NH2 moieties yielded the motif from these selections most suited for both nuclease stability and therapeutic development. This motif requires only two 2'-NH2-Cs and functions as a 36-mer. Its substrate sequence requirements were determined to be 5'-Y-G-H-3'. Its half-life in human serum is >100 h. In physiologically relevant magnesium concentrations [approximately 1 mM] its kcat = 0.07 min(-1), Km = 70 nM. This report presents a novel nuclease stable ribozyme, designated Zinzyme, possessing optimal activity in simulated physiological conditions and ready for testing in a therapeutic setting.
authors.affiliation: Ribozyme Pharmaceuticals Incorporated, Boulder, Colorado 80301, USA. zinnens@rpi.com
authors.forename: Shawn P
authors.initials: SP
authors.lastname: Zinnen
authors.forename: Kristal
authors.initials: K
authors.lastname: Domenico
authors.forename: Mike
authors.initials: M
authors.lastname: Wilson
authors.forename: Brent A
authors.initials: BA
authors.lastname: Dickinson
authors.forename: Amber
authors.initials: A
authors.lastname: Beaudry
authors.forename: Victor
authors.initials: V
authors.lastname: Mokler
authors.forename: Andrew T
authors.initials: AT
authors.lastname: Daniher
authors.forename: Alex
authors.initials: A
authors.lastname: Burgin
authors.forename: Leonid
authors.initials: L
authors.lastname: Beigelman
mesh_terms: D001483:Base Sequence; D015195:Drug Design; D004355:Drug Stability; D015723:Gene Library; D007700:Kinetics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides; D016337:RNA, Catalytic / Q000138:chemical synthesis / Q000737:chemistry / Q000235:genetics* / Q000627:therapeutic use; D014158:Transcription, Genetic
chemical_list: D009838:Oligodeoxyribonucleotides; D016337:RNA, Catalytic