Computer Modeling of Oligonucleotide Reaction Rates

寡核苷酸反应速率的计算机建模

• 批准号: 7539142
• 负责人: William J. Kennelly
• 金额: $ 13.57万
• 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 2009-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7539142
• 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; Condition; DNA; DNA Databases; DNA Microarray Chip; DNA Microarray format; Data; Databases; Detection; Development; Diagnostic; Dissociation; Ensure; Equation; Equilibrium; Experimental Designs; Facility Construction Funding Category; Genomics; Goals; Graph; Guanine + Cytosine Composition; Guidelines; Half-Life; Hand functions; Hereditary Disease; Information Storage; 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; Object Attachment; Oligonucleotides; Outcome; Output; Phase; Platelet Factor 4; Polymerase Chain Reaction; Public Health; RNA; RNA Interference; Range; Rate; 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; 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

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. PUBLIC HEALTH RELEVANCE: This project will improve a nucleic acid hybridization simulation and design tool for biochemical by adding kinetic algorithms to the pre-existing thermodynamics. This will enable researchers to develop diagnostic and therapeutic assays, while taking the time-component of a reaction into account and avoiding the formation of undesirable intermediate structures. The specific public health gains from this research will be in the areas of improved methods for analysis of genetic disorders developed with this software, while reducing false positive and false negative outcomes, and improvements in the design of gene therapies.

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 形成双链体结构的反应速率，建立数学模型来确定该反应的速率常数，并将该模型合并到现有软件中以设计和模拟寡核苷酸反应。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，其中寡核苷酸设计为不具有二级结构。这些寡核苷酸将根据长度、序列和G/C含量而变化，并且其反应速率将在约15°C至解链温度的温度范围内确定。根据初步研究，类似反应的解离速率已显示遵循阿累尼乌斯速率方程。将为这些简单模型反应的速率行为构建数学模型。模型将被测试和验证，测试结果将用于改进模型。然后，数学模型将被纳入基于电子表格的速率计算器，该计算器包含微分方程求解器，并将计算没有二级结构的寡核苷酸在第一阶段缓冲条件下的反应速率和浓度作为时间的函数。在第二阶段，将消除第一阶段对双链杂交反应的限制。特别是，将取消无竞争性二级结构的限制，并确定有意内置二级结构的寡核苷酸对的反应速率。与第一阶段中的寡核苷酸对类似的寡核苷酸对将被修改，以便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改。对这些新发夹结构的反应速率的分析以及与没有发夹的类似双链体的比较将有助于制定包括二级结构的数学模型。除了具有小 3 碱基环的发夹之外，分子信标还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结合到较长的靶标（它们之间有或没有短间隙）的情况，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DNA/DNA研究。如初步研究所示，缓冲液成分对反应速率有显着影响。将针对0.1至1.01 M范围内的钠离子浓度和0至6 mM范围内的镁离子浓度，系统地研究缓冲液浓度对DNA/DNA反应和DNA/RNA反应的影响。然后，来自两个阶段的速率数据将累积到数学模型中，该模型将预测反应速率和反应中所有物质的寡核苷酸浓度，并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) 软件包中。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。公共健康相关性：该项目将通过在现有热力学中添加动力学算法来改进核酸杂交模拟和生化设计工具。这将使研究人员能够开发诊断和治疗方法，同时考虑反应的时间成分并避免形成不需要的中间结构。这项研究的具体公共卫生收益将体现在改进使用该软件开发的遗传性疾病分析方法、同时减少假阳性和假阴性结果以及改进基因疗法的设计等领域。