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Jack M. Sullivan, Mark N. Schneggenburger; Software Modeling of Hammerhead Ribozyme Kinetics. Invest. Ophthalmol. Vis. Sci. 2011;52(14):1392.
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Convert the kinetic model (KM) of the hammerhead ribozyme (hhRz) into a software program for high throughput screening and optimization of hhRz enzymatic properties.
The hhRz KM required long-hand calculations to obtain kinetic parameters for individual hhRzs (Stage-Zimmermann and Uhlenbeck, 1998 RNA 4:875-889). Programming in Visual Foxpro operationalized this model into a user-friendly utility. Inputs are the target mRNA sequence and the total hhRz antisense flank length and its symmetry around the cleavage site.
The program identifies regions within a target mRNA that contain hhRz cleavage sites (NUH↓: e.g. GUC, GUA, CUU). Target sequence strings chosen for length and symmetry of the hhRz antisense flanks are grouped and numbered for each NUH↓ cleavage site type (e.g. GUC). The program determines the complementary hhRz annealing sequence and calculates all rates (k’s in min-1, n=12) and equilibrium constants (Kd’s in Molar, n=6) of the KM for each hhRz. These parameters depend upon the energetics of Watson-Crick base pairing of the hhRz (enzyme) with an accessible region of the target (substrate) mRNA. The enthalpy and entropy for nearest neighbor base pairs are obtained from published data embedded as lookup tables and the free energies are determined from standard thermodynamic rules and used to calculate the Kd’s and rates. A KM is determined in seconds for all hhRz agents that could cleave a target mRNA. Each Kd and rate are tabulated as vectors for the entire set of hhRz reagents. The outcomes are surveyed for hhRzs with good kinetic properties (e.g. lower Kd for annealing reaction and good cleavage product leaving rates). The hhRz KM program is also useful to investigate rational design changes in the antisense flanks of a given hhRz (e.g. changes in total length, symmetry, composition) to optimize kinetics.
The hhRz KM software tool is useful in silico for high throughput screening of enzymatic properties of whole sets of hhRz agents that could cleave a specific disease target mRNA and for rational optimization of lead candidate therapeutic hhRz agents.
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