Improved Modeling of RNA/DNA Hybridization

描述(申请人提供)：人类基因组编码大约30,000个基因，选择性剪接导致细胞中大约100,000个不同的RNA。疾病易感性的基因组发现需要RNA-DNA相互作用的热力学知识，以创建使用引物和探针的最佳分析方法。DNA Software Inc.的总体长期使命是利用结合了最先进的核酸折叠和杂交热力学和动力学知识的软件来促进基因组研究。为了增强我们现有的寡核苷酸建模平台(OMP)，我们建议为RNA-DNA杂交测量一个完整的热力学I参数数据库，以促进人类基因组计划的下一步-RNA表达分析。大多数所需的RNA-DNA近邻参数从未被测量过。这项提案将通过进行一系列系统的测量、适当的数据分析和理论建模来解决这一知识差距。这些参数将被纳入OMP的数据库，以提高RNA-DNA杂交预测的准确性。完成RNA-DNA热力学数据库将需要大约320个新的UV热变性测量，除了文献中已有的测量；因此，我们正在寻求一个快速通道建议。在第一阶段，我们将确定84个序列的热力学，以改进现有的匹配RNA-DNA在各种盐条件下的参数，并对RNA-DNA异源双链中的其他基序进行热变性测量，包括内部单个错配、末端错配和末端悬挂。这些初步结果将为第二阶段250多项测量的设计提供指导方针，这将提供一个关于不匹配、终端不匹配、悬垂末端以及凸起、内环和镁离子浓度相关性的完整数据库。同样在第二阶段，我们将把所有这些参数整合到我们的算法中，创建图形用户界面来扩展OMP的能力，并执行实验验证。这些精确的探针/引物设计将促进多种生物技术的发展，如基因特异性逆转录聚合酶链式反应、cRNA表达谱分析、RNA反义技术、mRNA原位杂交、Northern印迹分析等RNA检测方法。这项工作还将改进对细菌和RNA病毒病原体的检测分析，这对国家健康和生物防御具有相当大的兴趣。

DESCRIPTION (provided by applicant): The human genome encodes approximately 30,000 genes, and alternative splicing results in approximately 100,000 different RNAs in the cell. Genomic discoveries for disease susceptibility require thermodynamic knowledge in RNA-DNA interactions to create optimal assays using primers and probes. The overall, long-term mission of DNA Software Inc. is to facilitate genomic investigations with software that incorporates state-of-the-art knowledge of thermodynamics and kinetics of nucleic acid folding and hybridization. To enhance our existing Oligonucleotide Modeling Platform (OMP) platform, we propose to measure a complete thermodynamic i parameter database for RNA-DNA hybridization to facilitate the next step in the Human Genome Project, RNA expression assays. Most of the needed RNA-DNA nearest-neighbor parameters have never been measured. This proposal will address this gap in knowledge by performing a systematic series of measurements, appropriate data analysis and theoretical modeling. The parameters will be incorporated into OMP's database to improve the accuracy of RNA-DNA hybridization predictions. Completion of an RNA-DNA thermodynamic database will require approximately 320 new UV thermal denaturation measurements in addition to those already in the literature; thus, we are pursuing a fast-track proposal. In Phase I, we will determine the thermodynamics of 84 sequences to improve the existing match RNA-DNA parameters under various salt conditions, and perform thermal denaturation measurements on a sampling of other motifs in RNA-DNA heteroduplexes including internal single mismatches, terminal mismatches and dangling ends. These initial results will provide guidelines for the design of over 250 measurements in phase II, which will provide a complete database for mismatches, terminal mismatches, dangling ends, as well as bulges, internal loops and Mg2+ concentration dependence. Also in phase II, we will incorporate all of these parameters into our algorithms, create GUIs to extend OMP's capabilities and perform experimental validations. These accurate probe/primer designs will facilitate a variety of biotechnologies, such as gene-specific reverse-transcription PCR, cRNA expression profiling, RNA antisense technology, in situ hybridization of mRNA, Northern blot analysis, and other RNA detection methods. This work will also allow for improved detection assays for bacterial and RNA virus pathogens, which are of considerable interest for national health and biodefense.