title: In vitro evolution of flexizymes that function under the conditions in translation system.
abstract: Flexizyme is an artificial ribozyme capable of acylating any kinds of tRNAs with virtually no limitation of acid donor substrates. Although the flexizyme system is a highly flexible tRNA acylation tool, it requires relatively high Mg(2+) concentrations (>50 mM) for the full function. For instance, lowering the Mg(2+) concentration to 10 mM, where the in vitro translation system generally functions, resulted in significant decrease in activity. To overcome this limitation, we implanted a new random domain into a part of flexizyme to aim at selecting a new functional domain that would exhibit acylation activity at lower Mg(2+) concentrations. Indeed, in vitro selection of active species from such an RNA pool afforded a new flexizyme, called mdFx, showing a 3-fold higher yield at 10 mM Mg(2+) concentration. Thus, this new flexizyme opens a new avenue to construct an mdFx-transcription-translation coupled system.
authors.affiliation: Department of Advanced Interdisciplinary Studies, Graduate School of Engineering, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8904, Japan.
authors.forename: Masaki
authors.initials: M
authors.lastname: Ohuchi
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Murakami
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Suga
mesh_terms: D001483:Base Sequence; D019020:Directed Molecular Evolution; D008969:Molecular Sequence Data; D016337:RNA, Catalytic / Q000737:chemistry*; D046249:Transfer RNA Aminoacylation
chemical_list: D016337:RNA, Catalytic