An Antisense Design and Simulation Platform

描述(由申请者提供)：在这份拨款建议书中，将开发一个专门用于反义寡核苷酸模拟探针设计和模拟的综合分子诊断软件工具。反义技术在人类疾病研究中的应用已经在癌症、免疫缺陷疾病、糖尿病、肌肉营养不良和心血管疾病、甲型和丙型肝炎、艾滋病毒、SARS冠状病毒、埃博拉病毒、登革热、副粘病毒(麻疹)和西尼罗河病毒的文献中得到证实。然而，由于缺乏反义寡核苷酸模拟热力学和动力学数据库，以及目前使用的经验法则设计策略很少最初产生有效的探针，定向反义研究进展缓慢。然后，许多研究人员被迫针对相同的基因组目标设计一个小的探针库，以增加他们成功的可能性，这在时间和资金上都非常昂贵。所提出的反义设计和模拟平台将为人类疾病的研究人员提供一个定向研究和评估人类和病毒基因组靶点的工具，从而使合理设计的反义寡核苷酸类似物可以用作分子诊断工具。生产该平台的发展战略是更新现有的热力学和动力学数据库，并将其部署在反义设计和模拟平台上，具体目标如下：目标1.1：对荧光团、生物素和猝灭剂标记的PNA和吗啉立方反义探针以及硫代/LNA反义GAP探针分别进行16次热力学熔融，以证明将最近邻热力学模型应用于这些体系的可行性。目的1.2：将AIM 1.1中确定的热力学参数添加到现有的聚合酶链式反应平台VisualOMP中，以验证设计和模拟算法应用于修饰的反义寡核苷酸类似物的可行性。目的2.1：完成荧光基团、猝灭剂标记的PNA和吗啉基反义探针、硫代/LNA反义GAP探针的热力学文库。目的2.2：对修饰的吗啉/RNA和PNA/RNA双链进行动力学实验，并建立预测Morpholino/RNA和PNA/RNA杂交和去折叠速率的数学模型。目标2.3：设计一个全自动反义设计和模拟平台，允许研究人员以最少的用户输入设计特定和敏感的反义探针。到这个项目结束时，一个完全自动化的反义设计和仿真平台将被设计、测试和调试，并提供给反义研究人员用于商业产品的Beta测试。

DESCRIPTION (provided by applicant): 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.