A New Molecular Lexicon For Sequence-Specific DNA Recognition

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、细胞可渗透的合成化合物调节这些功能。这种缺陷是我们的基因组知识在生物技术和治疗应用中的应用的障碍。本提案所述项目将消除这一障碍，并通过开发新的抗癌和抗寄生虫药物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。我们假设有可能使用AT文库中的模块，并合理地偶联GC识别的新模块，以设计用于所选DNA靶序列的广泛混合序列识别的化合物。我们开始使用的AT特异性化合物是细胞可渗透的，并且是基于包括临床有用化合物的分子平台设计的。我们的目标化合物保持这些功能，同时纳入新的GC碱基对模块，正在设计的目标1的建议。目标1的其余部分描述了制备全新类型的模块化合物，其使用我们已知的AT结合单元与新的GC识别模块紧密且特异性地结合DNA中的广泛混合序列。作为这个目标的一部分，我们提出了一些非常有前途的初步结果与几种新型的DNA小沟粘合剂，可以特异性地识别一个或两个GC碱基对的混合DNA序列。这些初步的发现是一个概念证明，我们的模块化设计方法将工作，该方法可以扩展到更复杂的序列，因为这个项目的发展。该项目的目标2描述了我们分析新药剂的DNA复合物的方法。我们将从一个热熔筛选开始，该筛选将强结合的特异性化合物与那些不能很好地结合靶DNA序列的化合物分开。目标的第二部分包括对筛选出的最重要化合物的更详细研究。我们将用生物传感器-表面等离子体共振、荧光光谱、DNA酶I足迹、质谱和量热法来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方法确定结构和可以获得晶体的复杂结构的晶体学确定。我们将对化合物抑制重要DNA-转化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，该基序库可以以不同的组合连接，以识别细胞中广泛的生物学重要的AT和GC bp混合DNA序列，用于新的基因组应用。

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.