HYDROLASE STABILITY ENHANCEMENT AND ITS APPLICATION TO SIRNA

水解酶稳定性增强及其在 SIRNA 中的应用

• 批准号: 8360149
• 负责人: GABRIEL Luis BARLETTA
• 金额: $ 10.36万
• 依托单位: UNIVERSITY OF PUERTO RICO
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-06-01 至 2012-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8360149
• 关键词: Active Sites; Binding; Biological; Biomedical Research; Biotechnology; Cell Wall; Cell membrane; Chemicals; Complement; Complex; Cyclodextrins; Cytoplasm; Cytoskeleton; Disease; Electron Spin Resonance Spectroscopy; Energy Transfer; Ensure; Enzyme Stability; Enzymes; Ethylene Glycols; Etiology; Exhibits; Exposure to; Fluorine; Freeze Drying; Funding; Gene Expression; Genes; Grant; Hela Cells; Hydration status; Hydrolase; Immobilized Enzymes; Liquid substance; Long-Term Effects; Malignant Neoplasms; Measures; Mechanics; Membrane; Methodology; Modification; Molecular; National Center for Research Resources; Nature; Nuclear Magnetic Resonance; Nucleic Acids; Organic solvent product; Plasma; Play; Principal Investigator; Puerto Rico; Reaction; Research; Research Infrastructure; Resources; Role; Small Interfering RNA; Solvents; Source; Spin Labels; Structure; Surface; Technical Expertise; Techniques; Theoretical Studies; United States National Institutes of Health; Virus Diseases; Water; aqueous; cost; design; ethylene glycol; insight; macromolecule; molecular dynamics; novel; quantum; research study; simulation

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.

该副本是利用资源的众多研究子项目之一 由NIH/NCRR资助的中心赠款提供。对该子弹的主要支持 而且，副投影的主要研究员可能是其他来源提供的 包括其他NIH来源。 列出的总费用可能 代表subproject使用的中心基础架构的估计量， NCRR赠款不直接向子弹或副本人员提供的直接资金。 酶在生物技术中起着至关重要的作用，在生物技术中，它们被用来催化广泛的生物硫化反应。但是，随着延长暴露于水性或有机溶剂的延长，它们的催化活性表现出较差的稳定性。这种局限性以及对稳定，结构和功能之间关系的不良理解严重限制了酶的潜力。这项研究的主要目标是充分理解和增强酶稳定性。我们的研究表明，较差的酶稳定性本质上是由于溶剂暴露期间活跃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，并在这些变化和酶稳定性之间建立因果关系。将开发新的方法，以减少长时间溶剂暴露期间活跃位点的变化，以增强酶稳定性。在酶稳定性研究中获得的技术专长和理解将应用于超过siRNA所面临的类似限制。简短的干扰RNA是短双链核酸，它们正在开发，以靶向癌症，病毒感染和其他疾病的疗法重要基因。这些siRNA将通过多种大分子化学修饰，以提高其稳定性和效率，并提高其穿过细胞壁膜的能力。新技术将旨在直接测量复杂生物流体中的siRNA稳定性。 提出的特定目标（SA）： SA1：根据酶可逆地结合水和酶动力学的作用，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）研究可逆结合的水分子与大部分有机溶剂的交换。长期酶​​在有机溶剂中孵育以及有机溶剂水合对酶极性的影响将使用EPR测量。 EPR自旋标签以及活动位点附近的可逆水域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（例如纳米硅酸盐）的极性和亲水性表面固定在有机溶剂中的稳定性，以确保正确的酶水合和稳定性。不同表面上的酶稳定性测定将显示酶的稳定性与整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动力学模拟将提出有关地表水分子运动和聚类的水合机制。 SA3：通过与多种大分子（例如甲氧基聚（PEG））（PEG）和环糊精的多种大分子（PEG）和环糊精的多种大分子和化学修饰，通过共培养和化学修饰进行系统和合理地修饰。 HeLa细胞将用这些修饰的siRNA转染，以敲除特定的基因表达，并评估其稳定性，效率和越过细胞壁膜的能力的增加。这项研究将提供有关siRNA稳定性和细胞膜转移机制的物理和化学见解。分子动力学模拟将用于确定修饰和天然siRNA之间的结构差异。 SA4：设计涉及Forster共振能量转移（FRET），EPR和氟NMR的专业知识的新技术直接在复杂的生物学流体，例如血浆，外细胞外基质组件和细胞胞质质中直接测量单链和双链siRNA核解度稳定性。