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。为了寻址特定的基因，天然或人工结合的配对必须指向小槽或大槽，因为在这里碱基序列变得可自由访问和阅读。在过去的几十年里，已经创造了复杂的合成次要和主要沟槽粘结剂。然而，虽然对于次要的沟槽已经开发了一套完整的寡酰胺，它允许针对任何小的碱基序列进行特定的靶向，但是对于DNA的主要沟槽，这样的通用解决方案一直是难以捉摸的。形成三链的寡核苷酸(TFOS)主要限于高嘌呤；尽管已经设计了一些替代的核酸骨架(PNA，LNA)，但大多数这些骨架不能保持完整的双链，而是入侵和打开现有的双链。尽管进行了几次尝试，但令人沮丧的是，直到今天，仍然缺乏完全人工的、非侵入性的、无序列限制的DS-DNA主槽结合子。在这个提案中，我们想要描述这样一组通用的DNA双链识别单元的发展，它在主槽内通过三链形成工作。每个模块都包含一个连接到PNA元件上的强大的碱基对联结剂。因此，主槽中所有可用的给体和受体都是通过碱基对粘结剂氢键连接的；进一步的稳定来自于与磷酸二酯主链的有吸引力的相互作用以及杂环环系统中广泛的pi堆积。与连续排列的适当碱基对结合的共价模块连接导致了一种新的PNA衍生物，它与选定的短DS DNA片段完全互补。因此，只有4个不同的模块(AT-、TA-、CG-、GC-结合剂)就足以在标准的多肽合成器上进行迭代偶联。合成序列选择性DNA配体的构建对超分子化学家来说是一个巨大的挑战；在这个建议中，重点放在新模块的构建、偶联和DNA结合特性上，这些新模块涉及到与分离的寡核苷酸的体外结合研究。第二个项目阶段(另一个3年)是设想的，这将在体内应用优化的模块(细胞培养，如果可能，还包括动物)。对特定DNA片段的有意干扰可以用于基因表达的定点调控、蛋白质结合的调控、DNA损伤的靶向、突变和同源重组的增强，从而为DNA的基因特异性操作提供了工具。另一方面，这种新的TFOS可用于在体外和体内将裂解或交联剂、转录因子或核酸酶输送到DNA上选定的位置。

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.