A New Molecular Lexicon For Sequence-Specific DNA Recognition

用于序列特异性 DNA 识别的新分子词典

• 批准号: 8760979
• 负责人: W David Wilson
• 金额: $ 28.12万
• 依托单位: GEORGIA STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-08-01 至 2018-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8760979
• 关键词: Affinity; Antineoplastic Agents; Antiparasitic Agents; Area; Base Pairing; Binding; Biochemical; Biosensor; Biotechnology; Cells; Circular Dichroism; Code; Complex; Crystallography; DNA; DNA Fingerprinting; DNA Sequence; DNA analysis; DNase-I Footprinting; Development; Disease; Docking; Feedback; Fluorescence; Functional RNA; Gene Expression; Genomic DNA; Genomics; Goals; Health; Human; Investigation; Kinetics; Knowledge; Laboratories; Lead; Libraries; Link; Mass Spectrum Analysis; Methods; Minor Groove; Modification; Molecular; Molecular Models; Molecular Structure; Mutate; New Agents; Outcome; Preparation; Probability; Reagent; Relative (related person); Research Design; Resolution; Series; Specificity; Structure; Surface Plasmon Resonance; Techniques; Testing; Therapeutic; Therapeutic Uses; Thermodynamics; Toxic effect; Universities; Work; antimicrobial drug; base; biophysical techniques; cancer therapy; design; dimer; drug development; experience; improved; melting; molecular modeling; monomer; novel; public health relevance; research study; skills; stoichiometry; uptake

DESCRIPTION (provided by applicant): While we currently know much about DNA genomic sequences as well as the functional significance of both coding and non-coding sequences, we have little ability to modulate these functions with sequence-specific, cell-permeable synthetic compounds. This deficiency is a barrier to applications of our genomic knowledge in biotechnology and therapeutic applications. The project described in this proposal will remove that barrier and will have an impact on human health through the potential for new anticancer and antiparasitic drugs as well as new applications in biotechnology. The project starts with an extensive library and knowledge of the DNA complexes of AT specific, minor groove binding compounds. We hypothesize that it is possible to use modules from the AT library and rationally couple novel modules for GC recognition to design compounds for broad, mixed sequence recognition of selected DNA target sequences. The AT specific compounds that we start with are cell permeable and are designed based on a molecular platform that includes clinically useful compounds. Our target compounds maintain these features while incorporating new GC base pair modules that are being designed in Aim 1 of the proposal. The remainder of Aim 1 describes preparation of entirely new types of modular compounds that use our known AT binding units with new GC recognition modules to bind tightly and specifically to a broad array of mixed sequences in DNA. As part of this Aim, we present some very promising preliminary results with several new types of DNA minor groove binders that can specifically recognize one or two GC base pairs in a mixed DNA sequence. These preliminary findings are a proof of concept that our modular design approach will work and that the approach can be expanded to more complex sequences as this project develops. Aim 2 of the project describes our methods for analysis of the DNA complexes of the new agents. We will start with a thermal melting screen that will separate strong-binding, specific-compounds from those that do not bind well to target DNA sequences. The second part of the Aim includes more detailed studies on the most important compounds from the screen. We will evaluate the interaction affinity, stoichiometry, kinetics and cooperativity of the better binding agents with biosensor-surface plasmon resonance, fluorescence spectroscopic, DNase I footprinting, mass spectrometry and calorimetric methods. The third part of Aim 2 includes structure determination by high resolution NMR methods and crystallographic determination of complex structures where crystals can be obtained. We will conduct limited exploratory studies of the ability of the compounds to inhibit important DNA-transition factor complexes and this is an important long term goal with very significant relevance for new drug development. The primary impact of this project will be the successful design of a library of motifs that can be linked in different combinations to recognize a broad array of biologically important mixed AT and GC bp DNA sequences in cells for new genomic applications.

描述（由申请人提供）：虽然我们目前对DNA基因组序列以及编码和非编码序列的功能意义了解很多，但我们几乎没有能力使用序列特异性，可渗透性的合成化合物调节这些功能。这种缺陷是我们在生物技术和治疗应用中应用基因组知识应用的障碍。本提案中描述的项目将消除该障碍，并通过新的抗癌和反寄生虫药物以及生物技术中的新应用对人类健康产生影响。该项目以广泛的库和对特定的，较小的凹槽结合化合物的DNA复合物的了解开始。我们假设可以使用来自AT文库的模块和合理的夫妇新型模块来识别GC识别，以设计化合物，以实现对选定DNA目标序列的广泛，混合序列识别的识别。我们开始使用的特定化合物是细胞渗透的，并且是基于包括临床上有用化合物的分子平台设计的。我们的目标化合物保持了这些功能，同时结合了在提案的AIM 1中设计的新的GC基础对模块。 AIM 1的其余部分描述了准备全新类型的模块化化合物的制备，这些模块化化合物使用我们已知的具有新的GC识别模块的结合单元，以紧密地结合DNA中的一系列混合序列。作为此目标的一部分，我们提供了一些非常有前途的初步结果，并使用几种新型的DNA小凹槽粘合剂可以特异性地识别混合DNA序列中的一个或两个GC碱基对。这些初步发现是一种概念证明，即我们的模块化设计方法将起作用，并且随着该项目的发展，该方法可以扩展到更复杂的序列。该项目的目标2描述了我们分析新药物DNA复合物的方法。我们将从一个热熔融屏幕开始，该屏幕将与不符合目标DNA序列结合的强度结合，特异性化合物分开。目的的第二部分包括对屏幕上最重要的化合物的更详细的研究。我们将评估更好的结合剂与生物传感器表面等离子体共振，荧光光谱，DNase I足迹，质谱和量热法的相互作用亲和力，化学计量学，动力学和协同度。 AIM 2的第三部分包括通过高分辨率NMR方法确定结构，并确定可以获得晶体的复杂结构的晶体学测定。我们将对化合物抑制重要的DNA转变因子复合物的能力进行有限的探索性研究，这是一个重要的长期目标，与新药开发非常重要。该项目的主要影响将是一个可以通过不同组合链接的基库库的成功设计，以识别新基因组应用中细胞中广泛的生物学重要混合和GC BP DNA序列。