Computer Modeling of Oligonucleotide Reaction Rates

描述（由申请人提供）：本研究提案的目标是测量DNA链与DNA或RNA形成双链体结构的反应速率，建立用于确定该反应速率常数的数学模型，并将该模型纳入现有软件中，用于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为不具有二级结构。这些寡核苷酸的长度，序列和G/C含量将有所不同，并将有他们的反应速率确定在一个范围内的温度从约15？C至熔化温度。基于初步研究，类似反应的解离速率已被证明遵循阿克里尼乌斯速率方程。将为这些简单模型反应的速率行为构建数学模型。将对模型进行测试和验证，并将测试结果用于改进模型。然后将数学模型并入基于电子表格的速率计算器中，该速率计算器包含微分方程求解器，并将计算无二级结构的寡核苷酸在I相缓冲液条件下的反应速率和浓度随时间的变化。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。特别地，将解除没有竞争二级结构的限制，并且将确定具有有意构建的二级结构的寡核苷酸对的反应速率。将对与阶段I中的那些类似的寡核苷酸对进行修饰，使得其中一条链具有受控的二级结构，其将以系统的方式进行修饰。这些新的发夹结构的反应速率的分析和与类似的双链体没有发夹的比较，将有助于阐述的数学模型，包括二级结构。除了具有小的3个碱基环的发夹之外，分子信标将用于模拟具有较大环的寡核苷酸中二级结构的影响。对于两个较短的寡核苷酸与较长的靶标结合的情况，将考虑同轴堆叠对速率的影响，其中两个较短的寡核苷酸之间具有或不具有短间隙。此外，第二阶段还将研究DNA/RNA系统，类似于第一阶段DNA/DNA研究。如初步研究所示，缓冲液组成对反应速率有显著影响。系统研究了缓冲液浓度对DNA/DNA反应及DNA/RNA反应将在钠离子浓度在0.1至1.01 M范围内和镁离子浓度在0至6 mM范围内进行。然后将来自两个阶段的速率数据累积到数学模型中，该模型将预测反应速率和寡核苷酸浓度。反应，并考虑到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，该软件将能够基于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.