Computer Modeling of Oligonucleotide Reaction Rates

描述(申请人提供)：这项研究计划的目标是测量DNA链与DNA或RNA形成双重结构的反应速度，建立一个确定该反应速度常数的数学模型，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，将测量一系列DNA/DNA反应在单一缓冲条件下的反应速度，这些反应的寡聚物被设计为不具有二级结构。这些低聚物随长度、顺序和G/C含量的不同而不同，并将在从约15℃到熔融温度的温度范围内确定其反应速度。根据初步研究，类似反应的离解速率符合Arrhenius速率方程。我们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，数学模型将被合并到基于电子表格的速率计算器中，该计算器结合了微分方程式解算器，并将计算没有二级结构的寡聚的反应速率和浓度作为时间的函数，并且在第一阶段的缓冲条件下。在第二阶段，将消除第一阶段对双重杂交反应的限制。特别是，没有竞争二级结构的限制将被取消，并将确定有意内置二级结构的寡聚对的反应速率。与第一阶段相似的寡聚对将被修饰，使得其中一条链具有受控的二级结构，该二级结构将以系统的方式进行修饰。分析这些新的发夹结构的反应速率，并与没有发夹的类似双链进行比较，将有助于阐述考虑二级结构的数学模型。除了带有3个小碱基环的发夹，分子信标将被用来模拟具有更大环的寡聚的二级结构的影响。同轴堆积对速率的影响将被考虑在两个较短的寡聚与较长的靶结合的情况下，它们之间有或没有短间隙。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，缓冲液组成对反应速度有显著影响。在钠离子浓度在0.1~1.01M，镁离子浓度在0~6 mM范围内，系统地研究了缓冲液浓度对DNA/DNA反应和DNA/RNA反应的影响。然后，两个阶段的速率数据将被累积到一个数学模型中，该模型将预测反应速率和反应中所有物种的寡聚浓度，并将考虑到研究中使用的所有变量。这个数学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.