Computer Modeling of Oligonucleotide Reaction Rates

寡核苷酸反应速率的计算机建模

• 批准号: 7899692
• 负责人: William J. Kennelly
• 金额: $ 7.7万
• 依托单位: DNA SOFTWARE, INC.
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-09-01 至 2010-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7899692
• 关键词: Accounting; Address; Affect; Algorithms; Area; Arts; Base Pairing; Behavior; Binding; Biochemical; Biological Assay; Biological Models; Buffers; Classification; Complex; Computer Simulation; Computer software; DNA; DNA Databases; DNA Microarray Chip; Data; Data Storage and Retrieval; Databases; Detection; Development; Diagnostic; Dissociation; Ensure; Equation; Equilibrium; Experimental Designs; Genomics; Goals; Graph; Guanine + Cytosine Composition; Guidelines; Half-Life; Hand functions; Hereditary Disease; Investigation; Kinetics; Knock-out; Knowledge; Laboratory Research; Laws; Length; Lifting; Literature; Magnesium; Measurement; Measures; Methods; Mission; Modeling; Molecular; NASBA Analysis; Nucleic Acid Folding; Nucleic Acid Hybridization; Nucleic Acids; Oligonucleotides; Outcome; Output; Phase; Platelet Factor 4; Public Health; RNA; RNA Interference; Reaction; Research; Research Personnel; Research Proposals; Running; Series; Simulate; Sodium; Sodium Chloride; Software Design; Solutions; Structure; System; Techniques; Temperature; Test Result; Testing; Therapeutic; Thermodynamics; Time; Training; Work; base; data modeling; design; dimer; flexibility; fluorophore; gene function; gene therapy; high throughput screening; improved; magnesium ion; mathematical model; melting; monomer; predictive modeling; prototype; reaction rate; research study; simulation; sodium ion; success; tool

This research proposal has the goal of measuring the rate of reaction of DNA strands with DNA or RNA to form a duplex structure, building a mathematical model for determining the rate constant of that reaction, and incorporating that model into existing software for design and simulation of oligonucleotide reactions. In phase I the rate of reaction will be measured at a single buffer condition for a series of DNA/DNA reactions with oligos designed not to have secondary structure. These oligos will vary by length, sequence, and G/C content and will have their reaction rates determined over a range of temperatures from about 15 ¿C to the melting temperature. Based on preliminary studies, the dissociation rates for similar reactions have been shown to follow the Arrhenius rate equation. Mathematical models will be constructed for the rate behavior of these simple model reactions. The models will be tested and validated and the testing results will be used to improve the models. The mathematical models will then be incorporated into a spreadsheet based rate calculator that incorporates a differential equation solver and will compute reaction rates and concentrations as a function of time for oligos with no secondary structure and at the buffer conditions of phase I. In phase II the constraints from phase I on duplex hybridization reactions will be removed. In particular, the restriction of no competing secondary structures will be lifted and reaction rates will be determined for oligos pairs with secondary structure intentionally built in. Oligo pairs similar to those in phase I will be modified so that one of the strands has a controlled secondary structure, which will be modified in a systematic way. An analysis of the reaction rates of these new hairpin structures and comparison with the similar duplexes without hairpins will facilitate the elaboration of the mathematical model to include secondary structure. In addition to hairpins with small 3 base loops, molecular beacons will be used to model the effect of secondary structure in oligos with larger loops. The effect of coaxial stacking on rates will be considered for cases where two shorter oligos bind to a longer target with or without a short gap between them. In addition, phase II will also examine DNA/RNA systems, similar to the phase I DNA/DNA studies. Buffer composition has a significant impact on the reaction rate as shown in preliminary studies. A systematic study of the effect of buffer concentration on DNA/DNA reactions and DNA/RNA reaction will be carried out for sodium ion concentrations in the range of 0.1 to 1.01 M and magnesium ion concentrations in the range of 0 to 6 mM. The rate data from both phases will then be accumulated into a mathematical model which will predict the rate of reaction and oligo concentrations of all species in the reaction and will allow for all variable used in the study. This mathematical model will be incorporated as a module in our oligonucleotide modeling platform (OMP) software package. The software will then be capable predict all possible structures (e.g. monomers, self-dimers, heterodimers) in a reaction and their concentration over time, based on both kinetics and thermodynamics principles.

这项研究计划的目标是测量DNA链与DNA或RNA形成 - 双链体结构，建立用于确定该反应的速率常数的数学模型，以及 将该模型结合到现有软件中以设计和模拟寡核苷酸反应。 在阶段I中，将在单一缓冲液条件下测量一系列DNA/DNA反应的反应速率 其中寡核苷酸被设计成不具有二级结构。这些寡核苷酸的长度、序列和G/C 含量，并将在约15 º C至约20 º C的温度范围内确定其反应速率。 熔化温度根据初步研究，类似反应的解离速率已被 显示遵循阿克里乌斯速率方程。数学模型将被构建为速率行为， 这些简单的反应模型。将对模型进行测试和验证，测试结果将用于 改进模型。然后，将数学模型纳入基于电子表格的费率中 计算器，其包含微分方程求解器，并将计算反应速率和浓度， 对于没有二级结构的寡聚物和在I相的缓冲条件下，是时间的函数。 在阶段II中，将去除来自阶段I的对双链体杂交反应的限制。特别是 将取消没有竞争二级结构的限制，并确定寡核苷酸的反应速率 与有意内置的二级结构成对。将修改与第一阶段相似的寡核苷酸对， 其中一条链具有受控的二级结构，它将以系统的方式进行修饰。一个 分析这些新的发夹结构的反应速率，并与不具有发夹结构的类似双链体进行比较。 发夹结构将促进数学模型的详细阐述以包括二级结构。除了 发夹与小3碱基环，分子信标将用于模拟二级结构的影响， 具有较大环的寡核苷酸。同轴堆叠对速率的影响将被考虑的情况下，两个较短的 寡核苷酸与较长的靶结合，它们之间有或没有短的间隙。此外，第二阶段还将审查 DNA/RNA系统，类似于I期DNA/DNA研究。缓冲液组成对 初步研究表明，反应速率。系统研究了缓冲液浓度对 DNA/DNA反应和DNA/RNA反应将在钠离子浓度为 0.1至1.01 M，镁离子浓度范围为0至6 mM。 然后将被累积到一个数学模型中，该模型将预测反应速率和寡聚物。 反应中所有物质的浓度，并将考虑研究中使用的所有变量。该数学 模型将作为一个模块纳入我们的寡核苷酸建模平台（OMP）软件包。 然后，该软件将能够预测所有可能的结构（例如单体、自二聚体、异二聚体）， 根据动力学和热力学原理，计算反应及其浓度随时间的变化。