HYDROLASE STABILITY ENHANCEMENT AND ITS APPLICATION TO SIRNA

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 酶在生物技术中起着重要的作用，被用于催化许多生物相关的反应，但酶的稳定性较差，即酶的催化活性随着与水或有机溶剂接触时间的延长而急剧下降。这种局限性以及对稳定性、结构和功能之间关系的理解不足严重限制了酶的潜力。本研究的主要目的是充分了解和提高酶的稳定性。我们的研究表明，差的酶稳定性本质上是由于在溶剂暴露期间活性位点发生的微小但关键的变化。实验和理论研究将确定这些关键变化的性质，并建立这些变化和酶稳定性之间的因果关系。将开发新的方法来减少活性位点在长时间溶剂暴露期间的变化，以提高酶的稳定性。在酶稳定性研究中获得的技术专长和理解将被应用于超越siRNA面临的类似限制。短干扰RNA是正在开发的短双链核酸，其靶向癌症、病毒感染和其他疾病中的治疗重要基因。这些siRNA将用各种大分子进行化学修饰，以增加其稳定性和效率，并增加其穿过细胞壁膜的能力。将设计新技术来直接测量siRNA在复杂生物流体中的稳定性。 具体目标（SA）： SA 1：从酶可逆结合水的作用和酶动力学的角度研究酶的操作稳定性。将使用核磁共振（NMR）和电子顺磁共振（EPR）研究可逆结合水分子与有机溶剂的交换。将使用EPR测量在有机溶剂中长期酶孵育的影响以及有机溶剂水合对酶极性的影响。EPR自旋标记和可逆结合的沃茨和溶剂分子附近的活性位点的分子动力学模拟将补充实验研究。 SA2：通过将酶固定在材料（如纳米硅酸盐）的极性和亲水性表面上来增强酶在有机溶剂中的稳定性，以确保适当的酶水合作用和稳定性。在不同表面上的酶稳定性测定将显示酶的稳定性和总体水合状态之间的相关性或因果关系。溶剂-酶-材料界面的分子和量子力学模拟将提出关于表面水分子运动和聚集的水合机制。 SA3：通过共冻干和与多种大分子如甲氧基聚乙二醇（PEG）和环糊精的化学修饰，系统地、合理地修饰预先设计的siRNA。HeLa细胞将用这些修饰的siRNA转染以敲低特定基因表达并评估其稳定性、效率和穿过细胞壁膜的能力的可能增加。这项研究将提供物理和化学的见解siRNA的稳定性和细胞膜转移的机制。分子动力学模拟将用于确定修饰的siRNA和天然siRNA之间的结构差异。 SA4：利用我们在Forster共振能量转移（FRET）、EPR和氟NMR方面的专业知识设计新技术，直接测量单链和双链siRNA在复杂生物液体（如血浆、细胞外基质成分和细胞质）中的溶核稳定性。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Enzymes play an essential role in biotechnology where they are used to catalyze a broad range of biorelevant reactions.However enzymes exhibit poor stability, that is, their catalytic activity decreases drastically with prolonged exposure to aqueous or organic solvents. This limitation and the poor understanding of the relationship between stability, structure,and function severely limits the potential of enzymes. The main objectives of this research are to fully understand and enhance enzyme stability. Our studies show that poor enzyme stability is inherently due to minute but critical changes that occur in the active site during solvent exposure. Experimental and theoretical studies will determine the nature of these critical changes and establish causal relationships between these changes and enzyme stability. Novel methodologies will be developed to reduce the changes with in the active site during prolonged solvent exposure in order to enhance enzyme stability. The technical expertise and understanding gained in the study of enzyme stability will be applied to surpass a similar limitations faced by siRNAs. Short interfering RNAs are short double-stranded nucleic acids that are being developed to target therapeuticaly important genes in cancer, viral infections, and other diseases. These siRNA will be chemically modified with a variety of macromolecules to increase their stability and efficiency, and to increase their ability to cross a cell wall membrane. New techniques will be designed to directly measure siRNA stability in complex biological fluids. Proposed Specific Aims (SA): SA1: Study the enzyme's operational stability in terms of the role of the enzyme reversibly bound water and enzyme dynamics. The exchange of reversibly bound water molecules with the bulk of an organic solvent will be studied using nuclear magnetic resonance (NMR) and electron paramagnetic resonance (EPR). The effect of long term enzyme incubation in an organic solvent as well as the effect of organic solvent hydration on enzyme polarity will be measured using EPR. Molecular dynamics simulation of an EPR spin-label and of the reversibly bound waters and solvent molecules near the active site will complement the experimental study. SA2: Enhance the stability of enzymes in organic solvents by immobilizing the enzyme on polar and hydrophilic surfaces of materials such as nanosilicates, to ensure proper enzyme hydration and stability. Enzyme stability determination on different surfaces will show correlation or causation between stability and overall hydration state of the enzyme. Molecular and quantum-mechanical dynamics simulation of the solvent-enzyme-material interface will suggest a hydration mechanism regarding surface water molecule movementand clustering. SA3: Modify systematically and rationally a pre-designed siRNA by co-lyophilization and chemical modification with a variety of macromolecules such as methoxy poly(ethylene glycol) (PEG) and cyclodextrins. HeLa cells will be transfected with these modified siRNA to knockdown a specific gene expression and evaluate possible increase to their stability, efficiency, and ability to cross the cell wall membrane. This study will provide physical and chemical insights into the mechanisms of siRNA stability and cell membrane transfer. Molecular dynamics simulation will be used to determine structural differences between modified and natural siRNA. SA4: Design new techniques involving our expertise with Forster resonance energy transfer (FRET), EPR and fluorine NMR to directly measure single-strand and duplex siRNA nucleolytic stability in complex biological fluids such as blood plasma, extra- cellular matrix components and cellular cytoplasm.