Developing a nucleic acid force field with direct chemical perception for computational modeling of nucleic acid therapeutics

项目摘要/摘要 核酸疗法为治疗那些不能靶向的疾病提供了一条很有希望的途径- 由传统的小分子药物治疗。毒性的降低以及交付和特异性的改进 是通过对自然产生的RNA进行共价修饰而实现的，这些进展最近导致 几种核酸疗法的监管批准。例如，小干扰RNA(SiRNA)沉默 致病信使RNA(MRNAs)的表达。然而，这种策略可能会导致有害的 沉默与siRNA引导链部分互补的非靶标mRNAs。缓解疾病的战略 靶外沉默是双链一端碱基配对的不稳定，其原因是siRNA引导序列的替代 链由乙二醇核酸(GNA)组成，GNA是一种具有非环骨架的核酸类似物。精确的机械装置- 许多共价修饰的结构，以及它们对序列位置和特征的依赖性 结构主题，人们仍然知之甚少。基于物理的原子模拟可以询问 这些对核酸构象系综的修改，并加快了 在实验室里很难合成的分子。这类模拟的成功与否取决于其准确性 力场的一个参数函数，它使用经典物理来估计来自原子的势能 坐标。将基于物理的模拟应用于治疗性核酸的一个主要障碍是 难以开发准确的共价修饰核苷酸的力场参数。这项提议旨在 推进模拟在治疗性RNA的设计和机制理解中的作用 通过开发开放源码工作流来推导力场参数和 为现实的治疗性siRNA运行双重杂交的模拟。在目标1中，原子核的力场 具有任意共价修饰和主链化学的酸将使用系统和 基于Open Force field Initia维护的开放源码软件基础设施的可复制工作流- 主动的。在目标2中，将根据双相熔化的实验测量来验证力场参数。 通过对含有GNA取代的RNA双链进行熔融模拟来确定温度。在《目标3》中， 将应用加权集成增强抽样方法来研究双联结过程 引导含有GNA替换的siRNAs与靶和非靶mRNAs配对。拟议的研究将 支持治疗RNA的可靠原子模拟，扩展了可用于合理设计的工具包 这样的疗法。开发和验证核酸力场参数的系统工作流程将是 容易推广到除了这里研究的siRNA中的那些之外的其他共价修饰，促进类似的 其他治疗平台的研究。这项研究计划将为申请人独立生产做好准备- 通过发展适合学术领导的技术和管理技能来影响研究生涯 研究治疗性核糖核酸的生物物理和药理学特性的计算化学实验室。

PROJECT SUMMARY/ABSTRACT Nucleic acid therapies provide a promising pathway for the treatment of diseases that are not amenable to target- ing by traditional small molecule drugs. Reduction in toxicity and improvements in delivery and specificity have been achieved through covalent modifications to naturally occurring RNAs, and these advances have recently led to the regulatory approval of several nucleic acid therapies. For example, small interfering RNA (siRNA) silences the expression of disease-causing messenger RNAs (mRNAs). However, this strategy can cause deleterious silencing of off-target mRNAs with partial complementarity to the siRNA guide strand. A strategy for mitigation of off-target silencing is destabilization of base pairing in one end of the duplex by substitution of the siRNA guide strand by glycol nucleic acid (GNA), a nucleic acid analogue with an acyclic backbone. The precise mecha- nisms of many covalent modifications, as well as their dependence on features such as sequence position and structural motifs, are still poorly understood. Physics-based atomistic simulations can interrogate the impact of these modifications on the conformational ensembles of nucleic acids and expedite the trial-and-error process for molecules that are difficult to synthesize in the laboratory. The success of such simulations relies on the accuracy of the force field, a parameterized function that uses classical physics to estimate potential energies from atomic coordinates. A major obstacle to the application of physics-based simulations to therapeutic nucleic acids is the difficulty in developing accurate force field parameters for covalently modified nucleotides. This proposal aims to advance the role of simulations in the design and mechanistic understanding of therapeutic RNAs with covalent modifications by developing an open source workflow to derive force field parameters and run simulations of duplex hybridization for realistic therapeutic siRNAs. In Aim 1, a force field for nucleic acids with arbitrary covalent modifications and backbone chemistry will be parameterized using a systematic and reproducible workflow based on open source software infrastructure maintained by the Open Force Field Initia- tive. In Aim 2, the force field parameters will be validated against experimental measurements of duplex melting temperatures by performing melting simulations for RNA duplexes containing substitutions of GNA. In Aim 3, the weighted ensemble enhanced sampling method will be applied to study the duplex association process for guide siRNAs containing GNA substitutions pairing with target and off-target mRNAs. The proposed research will enable reliable atomistic simulations of therapeutic RNAs, expanding the available toolkit for rational design of such therapies. The systematic workflow for developing and validating nucleic acid force field parameters will be easily generalizable to other covalent modifications beyond those in the siRNAs studied here, facilitating similar studies of other therapeutic platforms. This research plan will prepare the applicant for a productive indepen- dent research career by developing technical and management skills appropriate for leadership of an academic computational chemistry lab studying biophysical and pharmacological properties of therapeutic RNAs.