Determination of hammerhead ribozyme kinetic constants at high molar ratio ribozyme-substrate |
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Authors: | Grassi Gabriele Grassi Mario Kuhn Anne Kandolf Reinhard |
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Institution: | (1) Department of Molecular Pathology, Institute for Pathology, University of Tübingen, Liebermeisterstrasse 8, D-72076, Tübingen, Germany. e-mail: gegrassi@med.uni-tuebingen.de, DE;(2) Department of Chemical, Environmental and Raw Materials Engineering – DICAMP, University of Trieste, Piazzale Europa 1, I-34127 Trieste, Italy, IT |
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Abstract: | Hammerhead ribozymes provide valuable tools in the field of gene therapy due to their cleavage specificity and the broad
range of RNA targets. A major prerequisite for the selection of suitable ribozymes for in vivo application is represented by in vitro determination of ribozyme cleavage kinetic constants. From the experimental cleavage data, kinetic constants are usually
calculated under the assumption of rapid conversion of the substrate into the ribozyme-substrate complex. However, this condition
is often not satisfied for ribozymes carrying additional RNA stretches, due to cloning strategies or necessary for ribozyme
expression in the cell. To overcome this problem, we propose a mathematical model which is able to calculate ribozyme kinetic
constants in the case of non-rapid conversion of substrate into ribozyme-substrate complex. In addition, our system gives
the opportunity to evaluate the nature of the S conversion into ES through the determination of a model parameter. The validity of the proposed model is restricted to the hypothesis of a ribozyme
excess over the substrate at the beginning of the cleavage reaction and to the absence of any mass exchange with the external
environment.
Received: 1 February 2001 / Revised version: 1 September 2001 / Published online: 23 August 2002 |
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