Effects of various promoter derived sequences on the cleavage kinetic of an hammerhead ribozyme directed against cyclin E1 mRNA.
Hammerhead ribozymes (HRz), catalytic RNA molecules capable of inducing the site-specific cleavage of a phosphodiester bond within an RNA molecule, are typically introduced into target cells by specific constructs (such as viral vectors) able to drive their expression from defined expression cassettes (promoter). This strategy implies the presence of promoter-derived sequences bound to the hammerhead ribozyme structure, a fact which can unpredictably affect HRz cleavage efficiency and eventually the biological effect. We explored the effects of promoter-derived sequences on the cleavage kinetics of an HRz targeted against a relevant cell cycle regulator, i.e. cyclin E1, implicated in the pathogenesis of several human diseases including in-stent restenosis and hepatocellular carcinoma. Sequences derived form the most commonly used promoters (CMV, T7, Pol I and Pol III promoters) were added to the minimal HRz structure and their effects on the cleavage kinetic constants k(cat) and K(m) evaluated in vitro under single turn-over conditions, using a mathematical model we recently developed. The different promoter derived sequences variably affected HRz cleavage efficiency (k(cat)/K(m)) with those derived from the pol III and from a truncated form of T7 promoter (T7-S), impairing maximally and minimally k(cat)/K(m), respectively. Additionally, the extra sequences tend to increase K(m) and to reduce k(cat). The extent of this effect depends both on the secondary RNA structure and on the length of the added sequences. In conclusion, these data, together with further work in cultured cells, can lead to the selection of optimal expression cassettes thus contributing to improve HRz efficacy, bringing these molecules closer to practical applications.
Department of Molecular Pathology, University Hospital of Tübingen, Liebermeisterstr. 8, D-72076, Tübingen, Germany.
Johannes
J
Platz
Mario
M
Grassi
Anne
A
Kuhn
Reinhard
R
Kandolf
Giorgia
G
Racchi
Rossella
R
Farra
Barbara
B
Dapas
Andrea
A
Pascotto
Carlo
C
Giansante
Nicola
N
Fiotti
Gianfranco
G
Guarnieri
Gabriele
G
Grassi
D001483:Base Sequence; D019927:Cyclin E / Q000235:genetics / Q000378:metabolism*; D018390:Gene Targeting / Q000379:methods; D006801:Humans; D008962:Models, Theoretical; D009690:Nucleic Acid Conformation; D015513:Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D016337:RNA, Catalytic / Q000494:pharmacology*; D012333:RNA, Messenger / Q000378:metabolism*
C475820:CCNE1 protein, human; D019927:Cyclin E; D015513:Oncogene Proteins; D016337:RNA, Catalytic; D012333:RNA, Messenger; C427204:hammerhead ribozyme