In this grant proposal "An Automated Antisense Design and Simulation Platform", a comprehensive molecular diagnostics software tool that is specific to the design and simulation of antisense oligonucleotide analog probes will be developed. The application of antisense technologies to the study of human diseases has been proven in the literature for cancer, immune deficiency disorders, diabetes, muscular dystrophy and cardiovascular disease, hepatitis A and C, HIV, SARS-coronovirus, Ebola, Dengue Fever, paramyxoviruses (measles), and the West Nile virus. However, the progress of directed antisense research has been slow due to the absence of antisense oligonucleotide analog thermodynamic and kinetic databases, and because the currently used rule-of-thumb design strategies rarely ever initially produce effective probes. Many researchers are then forced to design a small library of probes against the same genomic target to increase their likelihood of success, which is very costly in both time and financing. The proposed Antisense Design and Simulation Platform will enable the researchers of human diseases by providing a tool for the directed research and evaluation of human and viral genomic targets so that rationally designed antisense oligonucleotide analogs may be used as a molecular diagnostics tool. The developmental strategies for producing the proposed platform are to update existing thermodynamic and kinetic databases and to deploy them on the Antisense Design and Simulation Platform, as outlined in the following specific aims: Aim 1.1: Perform 16 thermodynamic melts each for the fluorophore, biotin and quencher labeled PNA and morpholino antisense probes, and for the phosphorothioate/LNA antisense gap-mer probes to demonstrate the feasibility of applying the nearest-neighbor thermodynamic model to these systems. Aim 1.2: Add the thermodynamic parameters determined in Aim 1.1 to the existing PCR platform Visual OMP to demonstrate the feasibility of applying design and simulation algorithms to modified antisense oligonucleotide analogs. Aim 2.1: Complete the thermodynamic library for the fluorophore, and quencher labeled PNA and morpholino antisense probes, and for the phosphorothioate/LNA antisense gap-mer probes. Aim 2.2: Perform kinetics experiments on modified morpholino/RNA and PNA/RNA duplexes, and develop predictive mathematical models for the rates of Morpholino/RNA and PNA/RNA hybridization and unfolding. Aim 2.3: Engineer a fully automated Antisense Design and Simulation Platform that will allow researchers to design specific and sensitive antisense probes with minimal user inputs. By the end of this project a fully automated Antisense Design and Simulation Platform will have been designed, tested, and debugged, and made available to antisense researchers for the purpose of beta-testing the commercial product.
Public Health Relevance: This project will directly impact public health by providing a comprehensive software tool for the design and simulation of antisense oligonucleotide analog probes, where no other comprehensive antisense software tools exist. The efficacy of applying antisense technologies to human diseases has been proven, using a trial-and-error approach to a number of human diseases, such as: cancer, immune deficiency disorders, diabetes, muscular dystrophy and cardiovascular disease, hepatitis A and C, HIV, SARS-coronovirus, Ebola, Dengue Fever, paramyxoviruses (measles), and the West Nile virus. The proposed Antisense Design and Simulation Platform will benefit the researchers of human diseases by providing an optimized molecular diagnostic tool for the directed research and rational evaluation of human and viral genomic targets.
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