Sequence Specific Targeting of Nucleic Acids Using Intramolecular Triplexes: Energetics and Hydration

使用分子内三链体的核酸序列特异性靶向：能量学和水合

• 批准号: 0616005
• 负责人: Luis Marky
• 金额: $ 67.67万
• 依托单位: University of Nebraska Medical Center
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-07-01 至 2011-12-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0616005&HistoricalAwards=false
• 关键词: Sequence; Specific; Targeting; Nucleic; Acids

The research focuses on the thermodynamics of nucleic acid intramolecular structures - especially, triplet helices because of their implication in the control of cellular processes by endogenous or exogenous mechanisms. The long term objectives are to understand the molecular forces controlling the overall stability of complex intramolecular DNA structures; to quantify the energetics and hydration contributions governing the association of triplexes, and other unusual structures, with their complementary strands, including the role of sequence and cations; and to determine the thermodynamics for their favorable interaction with polycations for cellular delivery purposes. UV and CD spectroscopies are used to verify that each nucleic acid complex contains the appropriate structural features. Further, a combination of T-dependent UV spectroscopy, differential scanning and pressure perturbation calorimetries will be employed to obtain complete thermodynamic profiles for their unfolding reactions as a function of salt, pH and osmolyte concentration. High sensitivity titration calorimetry and density techniques will be used to measure the heat and volume change of association reactions. The complete thermodynamic characterization of these DNA complexes will provide a fundamental understanding of the physical factors that determine their stability as a function of its sequence and solution conditions. These factors are basic to the rational design of gene-targeting reagents, and for their proper cellular delivery, which can be used in therapeutic, diagnostic and biotechnological applications, and for predicting the energetics of sequence-specific local conformational rearrangements in intracellular processes.The broader impacts of this research are in carcinogenesis and gene therapy because of the fundamental importance of developing highly specific and stable agents for targeting oncogenes or their transcribed RNA products via triplex or duplex formation. Another impact is the role of water in the overall properties of biological macromolecules and in their interacting behavior towards one another. Specifically, the correlation of energetics with hydration will improve the picture of how hydration controls the stability, conformation and melting behavior of these novel nucleic acid structures. Furthermore, the resulting hydration data can be used in molecular modeling studies and in theoretical calculations, providing an insight into global water. The educational significance is in the mentoring of undergraduate, graduate students and postdoctoral fellows by training them with a wide variety of biophysical techniques and with the fundamental understanding of biophysics. In addition, the research findings are routinely incorporated into lectures of undergraduate, graduate courses and seminars.

这项研究集中在核酸分子内结构的热力学，特别是三重螺旋，因为它们在通过内源或外源机制控制细胞过程中蕴含着意义。长期目标是了解控制复杂分子内DNA结构总体稳定性的分子作用力；量化控制三链体和其他不寻常结构与其互补链结合的能量和水化贡献，包括序列和阳离子的作用；并确定它们与用于细胞递送目的的聚阳离子有利相互作用的热力学。使用UV和CD光谱来验证每个核酸复合体都包含适当的结构特征。此外，依赖于T的UV光谱、差示扫描和压力微扰量热法将被用来获得它们的展开反应随盐、pH和渗透分子浓度的函数的完整热力学曲线。高灵敏度滴定热法和密度技术将被用来测量缔合反应的热和体积变化。对这些DNA络合物的完整热力学表征将提供对物理因素的基本了解，这些物理因素决定了它们作为其序列和溶液条件的函数的稳定性。这些因素是合理设计基因靶向试剂的基础，也是正确的细胞递送的基础，可用于治疗、诊断和生物技术应用，并用于预测细胞内过程中序列特异性局部构象重排的能量学。本研究的更广泛影响是在癌症发生和基因治疗方面，因为开发高度特异和稳定的试剂，通过三链或双链形成靶向癌基因或其转录的RNA产物具有根本重要性。另一个影响是水在生物大分子的整体性质以及它们相互作用行为中的作用。具体地说，能量学与水合作用的关联将改善水合作用如何控制这些新的核酸结构的稳定性、构象和熔化行为的图景。此外，由此得到的水合数据可用于分子建模研究和理论计算，从而提供对全球水的洞察。其教育意义在于通过对本科生、研究生和博士后研究员进行广泛的生物物理技术培训和对生物物理学的基本理解来指导他们。此外，研究结果通常会被纳入本科生、研究生课程和研讨会的讲座中。