Computer Modeling of Oligonucleotide Reaction Rates

这项研究计划的目标是测量DNA链与DNA或RNA反应形成的速度 双链结构，建立用于确定该反应的速率常数的数学模型，以及 将该模型整合到现有的设计和模拟寡核苷酸反应的软件中。 在第一阶段，将测量一系列DNA/DNA反应在单一缓冲液条件下的反应速度 低聚物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会有所不同 含量，并将在大约15摄氏度到 熔化温度。根据初步研究，类似反应的离解速率为 结果表明符合阿累尼乌斯速率方程。我们将为以下情况构建数学模型： 这些简单的模型反应。将对模型进行测试和验证，测试结果将用于 改进模型。然后将数学模型合并到基于电子表格的费率中 包含一个微分方程式解算器的计算器，它将计算反应速率和浓度如下 对于无二级结构的低聚物，在I相缓冲条件下，时间的函数。 在第二阶段，将取消第一阶段对双重杂交反应的限制。尤其是， 没有竞争二级结构的限制将被取消，寡聚的反应速率将被确定 故意内置二级结构的配对。与第一阶段相似的寡核苷酸对将被修改为 其中一条链具有受控的二级结构，这将以系统的方式进行修改。一个 对这些新型发夹结构的反应速率进行了分析，并与没有这种结构的类似双链进行了比较 发夹将有助于数学模型的阐述，以包括二级结构。除了……之外 带有3个小碱基环的发夹，分子信标将用于模拟二级结构的影响 有更大环路的寡头。同轴堆叠对速率的影响将在以下情况下考虑 寡核苷酸与较长的靶子结合，它们之间有或没有较短的间隙。此外，第二阶段还将审查 DNA/RNA系统，类似于第一阶段DNA/DNA研究。缓冲区组成对以下各项有重大影响 初步研究显示的反应速度。缓冲液浓度对黄曲霉毒素影响的系统研究 在钠离子浓度范围内，将进行DNA/DNA反应和DNA/RNA反应 0.1至1.01M和0至6 mm范围内的镁离子浓度。来自两个阶段的速率数据 然后累积到一个数学模型中，该模型将预测反应和寡聚的速度 反应中所有物种的浓度，并将考虑到研究中使用的所有变量。这是数学上的 该模型将作为一个模块并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、杂二聚体) 基于动力学和热力学原理的反应及其浓度随时间的变化。

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.