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: 8901245
• 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/8901245
• 关键词: Affinity; Antineoplastic Agents; Antiparasitic Agents; Area; Base Pairing; Binding; Biochemical; Biosensor; Biotechnology; Cells; Circular Dichroism; Code; Complex; Crystallography; DNA; DNA Sequence; DNA analysis; DNase-I Footprinting; Development; Disease; Docking; Feedback; Fluorescence; Gene Expression; Genetic Fingerprintings; 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; Untranslated RNA; Work; antimicrobial drug; base; biophysical analysis; biophysical techniques; cancer therapy; design; dimer; drug development; experience; improved; melting; molecular modeling; monomer; novel; research study; skills; stoichiometry; targeted sequencing; 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用序列特异性的、细胞可渗透的合成化合物来调节这些功能。这种缺陷是我们的基因组知识在生物技术和治疗应用中应用的障碍。该提案中描述的项目将消除这一障碍，并将通过新的抗癌和抗寄生虫药物的潜力以及生物技术的新应用对人类健康产生影响。该项目从 AT 特异性小沟结合化合物的 DNA 复合物的广泛库和知识开始。我们假设可以使用 AT 库中的模块并合理地耦合用于 GC 识别的新模块来设计化合物，以对选定的 DNA 靶序列进行广泛的混合序列识别。我们首先使用的 AT 特定化合物具有细胞渗透性，并且是基于包含临床有用化合物的分子平台而设计的。我们的目标化合物保留了这些特征，同时结合了提案目标 1 中正在设计的新 GC 碱基对模块。目标 1 的其余部分描述了全新类型的模块化化合物的制备，这些化合物使用我们已知的 AT 结合单元和新的 GC 识别模块来紧密且特异性地结合 DNA 中的各种混合序列。作为该目标的一部分，我们通过几种新型 DNA 小沟结合物展示了一些非常有希望的初步结果，这些结合物可以特异性识别混合 DNA 序列中的一个或两个 GC 碱基对。这些初步发现证明了我们的模块化设计方法将发挥作用，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。我们将从热熔解筛选开始，将强结合的特定化合物与那些不能很好地与目标 DNA 序列结合的化合物分开。目标的第二部分包括对筛选中最重要的化合物进行更详细的研究。我们将通过生物传感器表面等离子体共振、荧光光谱、DNase I 足迹、质谱和量热方法评估更好的结合剂的相互作用亲和力、化学计量、动力学和协同性。目标 2 的第三部分包括通过高分辨率 NMR 方法确定结构以及可以获得晶体的复杂结构的晶体学确定。我们将对这些化合物抑制重要的 DNA-过渡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，与新药开发具有非常重要的相关性。该项目的主要影响将是成功设计一个基序库，该基序可以以不同的组合进行连接，以识别细胞中广泛的生物学重要的混合 AT 和 GC bp DNA 序列，用于新的基因组应用。