Computer Modeling of Oligonucleotide Reaction Rates

描述（由申请人提供）：本研究计划的目标是测量DNA链与DNA或RNA形成双相结构的反应速率，建立数学模型以确定该反应的速率常数，并将该模型纳入现有的设计和模拟寡核苷酸反应的软件中。在第一阶段，将在单一缓冲条件下测量一系列DNA/DNA反应的反应速率，这些反应的低聚物设计为不具有二级结构。这些低聚物会因长度、序列和G/C含量的不同而变化，并且它们的反应速率会在大约15 ？C到熔化温度。根据初步研究，类似反应的解离速率符合阿伦尼乌斯速率方程。将为这些简单模型反应的速率行为建立数学模型。将对模型进行测试和验证，测试结果将用于改进模型。然后将数学模型合并到基于电子表格的速率计算器中，该计算器包含微分方程求解器，并将计算无二级结构的低聚物在I相缓冲条件下的反应速率和浓度作为时间的函数。在II相中，将消除I相对双杂化反应的限制。特别是，没有竞争二级结构的限制将被解除，并且对于有意嵌入二级结构的寡核苷酸对的反应速率将被确定。与第一阶段相似的寡核苷酸对将被修改，使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。对这些新发夹结构的反应速率进行分析，并与没有发夹的类似双相化合物进行比较，将有助于建立包括二级结构的数学模型。除了具有小3碱基环的发夹外，分子信标将用于模拟具有较大环的寡核苷酸的二级结构的影响。当两个较短的寡核苷酸结合到一个较长的目标上，它们之间有或没有短的间隙时，将考虑同轴堆叠对速率的影响。此外，第二阶段还将检查DNA/RNA系统，类似于第一阶段的DNA/DNA研究。初步研究表明，缓冲液的组成对反应速率有显著影响。缓冲浓度的影响的系统研究DNA和DNA / RNA / DNA反应反应将钠离子浓度在0.1到1.01米的范围和镁离子浓度在0到6毫米。率数据从两个阶段将累积成一个数学模型,将预测所有物种的反应速率和低聚糖浓度在反应中,将允许在这项研究中使用的所有变量。该数学模型将作为模块纳入我们的寡核苷酸建模平台（OMP）软件包。然后，该软件将能够根据动力学和热力学原理预测反应中所有可能的结构（例如单体、自二聚体、异二聚体）及其随时间的浓度。

DESCRIPTION (provided by applicant): 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.