HYDROLASE STABILITY ENHANCEMENT AND ITS APPLICATION TO SIRNA

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 酶在生物技术中扮演着重要的角色，它们被用来催化广泛的生物反应，但酶的稳定性很差，也就是说，它们的催化活性随着时间的延长而急剧下降。这种局限性和对稳定性、结构和功能之间关系的缺乏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，较差的酶稳定性本质上是由于在溶剂暴露期间活性部位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，并建立这些变化与酶稳定性之间的因果关系。将开发新的方法来减少长时间接触溶剂时活性部位的变化，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克服siRNAs面临的类似限制。短干扰RNA是一种短双链核酸，目前正在开发用于治疗癌症、病毒感染和其他疾病的重要基因。这些siRNA将被各种大分子化学修饰，以增加它们的稳定性和效率，并增强它们穿越细胞膜的能力。将设计新的技术来直接测量复杂生物体液中的siRNA稳定性。 建议的具体目标(SA)： SA1：从酶的可逆结合水的作用和酶的动力学方面研究酶的操作稳定性。可逆性结合水分子与有机溶剂主体的交换将用核磁共振和电子顺磁共振研究。酶在有机溶剂中长期孵育的影响以及有机溶剂水合作用对酶的极性的影响将用EPR来测量。EPR自旋标记和活性中心附近可逆结合水和溶剂分子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的极性和亲水性表面(如纳米硅酸盐)来增强酶在有机溶剂中的稳定性，以确保适当的酶水合和稳定性。不同表面的酶稳定性测定将显示酶的稳定性与酶的整体水化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揭示地表水分子运动和聚集的水化机理。 SA3：通过与甲氧基聚乙二醇、环糊精等多种大分子的共冷冻干燥和化学修饰，对预先设计的siRNA进行系统合理的修饰。将这些修饰的siRNA导入HeLa细胞，以抑制特定基因的表达，并评估其稳定性、效率和穿越细胞膜的能力可能增加的可能性。这项研究将为siRNA的稳定性和细胞膜转移的机制提供物理和化学上的见解。分子动力学模拟将被用来确定修饰和天然siRNA之间的结构差异。 SA4：利用我们在Forster共振能量转移(FRET)、EPR和氟核磁共振方面的专业知识设计新技术，以直接测量单链和双链siRNA在血浆、细胞外基质成分和细胞胞浆等复杂生物液体中的核溶解稳定性。

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.