Functional characterization of the SOFA delta ribozyme.
Molecular engineering has led to the development of a novel target-dependent riboswitch that increases deltaribozyme fidelity. This delta ribozyme possesses a specific on/off adapter (SOFA) that switches the cleavage activity from off (a "safety lock") to on solely in the presence of the desired RNA substrate. In this report, we investigate the influence of both the structure and the sequence of each domain of the SOFA module. Analysis of the cleavage activity, using a large collection of substrates and SOFA-ribozyme mutants, together with RNase H probing provided several insights into the nature of the sequence and the optimal design of each domain of the SOFA module. For example, we determined that (1) the optimal size of the blocker sequence, which keeps the ribozyme off in the absence of the substrate, is 4 nucleotides (nt); (2) a single nucleotide difference between the substrate and the biosensor domain, which is responsible for the initial binding of the substrate that subsequently switches the SOFA-ribozyme on, is sufficient to cause non-recognition of the appropriate substrate; (3) the stabilizer, which joins the 5' and 3' ends of the SOFA-ribozyme, plays only a structural role; and (4) the optimal spacer sequence, which serves to separate the binding regions of the biosensor and catalytic domain of the ribozyme on the substrate, is from 1 to 5 nt long. Together, these data should facilitate the design of more efficient SOFA-ribozymes with significant potential for many applications in gene-inactivation systems.
Bergeron
Lucien Junior
LJ
RNA Group/Groupe ARN, Département de Biochimie, Faculté de médecine et des sciences de la santé, Universitéde Sherbrooke, Sherbrooke, Québec, J1H 5N4, Canada.
Reymond
Cédric
C
Perreault
Jean-Pierre
JP
D001483:Base Sequence; D014644:Genetic Variation; D003698:Hepatitis Delta Virus / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006518:Hepatitis Viruses / Q000235:genetics; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D008957:Models, Genetic; D009154:Mutation; D009690:Nucleic Acid Conformation; D012313:RNA / Q000737:chemistry / Q000378:metabolism*; D016337:RNA, Catalytic / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012367:RNA, Viral / Q000737:chemistry / Q000378:metabolism; D016914:Ribonuclease H / Q000378:metabolism; D013379:Substrate Specificity
D016337:RNA, Catalytic; D012367:RNA, Viral; D012313:RNA; D016914:Ribonuclease H