A modular system for sequence-specific DNA recognition in the major groove

核酸识别是一个基本的生物学过程。为了定位一个特定的基因，一个自然的或人工的结合伙伴必须指向小槽或大槽，因为在这里，碱基序列变得自由地可访问和可读。在过去的几十年里，复杂的合成小槽和大槽粘合剂已经被创造出来。然而，对于次要凹槽，已经开发出一套完整的低聚酰胺，可以针对任何小碱基序列进行特异性靶向，但对于dna的主要凹槽，这种通用解决方案一直难以实现。三聚体形成寡核苷酸（TFOs）主要局限于同嘌呤；尽管已经设计了许多替代的核酸骨架（PNA， LNA），但大多数都没有保留完整的双链，而是侵入并打开现有的双链。尽管进行了多次尝试，但直到今天，令人沮丧的是，缺乏完全人工的无创序列选择性的ds-DNA主槽结合物，这种结合物可以在没有序列限制的情况下有效地工作。在这个提议中，我们想要描绘这样一个通用的模块化的DNA双链识别单元集的发展，它通过在主槽内形成三联体来操作。每个模块包含一个强大的碱基对粘合剂连接到一个PNA元件。因此，主槽中所有可用的供体和受体都由碱基对结合物形成氢键；进一步的稳定来自于与磷酸二酯主链的吸引相互作用以及杂环系统之间广泛的pi堆叠。与适当碱基对结合物连续排列的共价模块连接导致新的PNA衍生物与选定的短ds DNA片段完美互补。因此，仅4个不同的模块（AT-, TA-, CG-， gc -粘合剂）就足以在标准肽合成器上进行迭代耦合。构建基于主槽内一般碱基对识别的序列选择性DNA配体对超分子化学家来说是一个巨大的挑战。在本提案中，重点在于新模块的构建，偶联和DNA结合特性，涉及与分离寡核苷酸的体外结合研究。第二个项目阶段（另一个3年）预计将在体内应用优化的模块（细胞培养，如果可能的话，动物）。对特定DNA片段的故意干扰可用于基因表达的位点特异性调节、蛋白质结合的调节、DNA损伤的靶向、诱变和同源重组的增强，从而为基因特异性操作DNA提供了工具。另一方面，这种新的tfo可用于在体外和体内将切割或交联剂、转录因子或核酸酶递送到DNA上的选定位点。

Nucleic acid recognition is a fundamental biological process. For the purpose of adressing a specific gene, a natural or artificial binding partner must be directed towards the minor or major groove, because here the base sequence becomes freely accessible - and readable. In the past decades, sophisticated synthetic minor and major groove binders have been created. However, while for the minor groove a complete set of oligoamides has been developed which allows specific targeting of any small base sequence, such a universal solution has been elusive for DNAs major groove.Triplex forming oligonucleotides (TFOs) are largely limited to homopurines; and although a number of alternative nucleic acid skeletons have been designed (PNA, LNA), most of these do not preserve the intact duplex, but invade and open the existing double strand. Despite several attempts, there is until today, a frustrating lack of entirely artificial non-invasive sequence-selective major groove binders for ds-DNA which operate efficiently without sequence constraints.In this proposal we want to delineate the development of such a general modular set of recognition units for DNA double strands, which operate by triplex formation inside the major groove. Each module contains a powerful base pair binder attached to a PNA element. All available donors and acceptors in the major groove are thus hydrogen-bonded by the base pair binders; further stabilization comes from attractive interactions with the phosphodiester backbone as well as from extensive pi-stacking among the heterocyclic ring systems. Covalent module connection with a consecutive arrangement of appropriate base pair binders leads to a new PNA derivative which is perfectly complementary to a selected short ds DNA fragment. Thus, only 4 different modules (AT-, TA-, CG-, GC-binder) suffice for the iterative coupling on a standard peptide synthesizer. The construction of synthetic sequence-selective DNA ligands which operate by general base-pair recognition inside the major groove represents a formidable challenge for supramolecular chemists; in this proposal, the emphasis lies on the construction, coupling and DNA binding properties of the new modules involving in-vitro binding studies with isolated oligonucleotides. A second project stage (another 3 years) is envisioned, which will apply the optimized modules in vivo (cell culture and, if possible, animals). Deliberate interference with specific DNA fragments can be used for site-specific modulation of gene expression, modulation of protein binding, targeting of DNA damage, mutagenesis and enhancement of homologous recombination, thus providing a tool for gene-specific manipulation of DNA. Such new TFOs may be used, on the other hand to deliver cleaving or cross-linking agents, transcription factors or nucleases to a chosen site on the DNA, both in vitro and in vivo.