Metal nanocluster-modified nucleotides

在过去的二十年中，金属纳米团簇（NC）在生物成像，生物传感，治疗应用和催化领域取得了重大进展。贵金属NCs如AgNCs和AuNCs，由于其无毒、生物相容性和光稳定性，已经成为比有机染料和量子点更好的发光材料。巯基修饰的纳米金在催化、发光、化学传感等领域有着广泛的应用前景。DNA聚合酶接受染料修饰的脱氧核苷三磷酸（dNTPs）的能力在许多重要的生物技术应用中得到了利用。这些包括下一代测序方法、单分子测序以及DNA和PCR扩增物的标记，例如用于微阵列分析。此外，利用DNA的固有特性与化学引入的功能性的组合，提供了一种新的基于核酸的杂交材料类别的入口。尽管金属NC的令人印象深刻的性能，这些，据我们所知，尚未开发作为dNTP的修饰，随后作为DNA聚合酶的底物。将以合作的方式解决这一问题。我们建议设计新的水溶性纳米团簇修饰的核苷酸，以纳入和标记DNA的DNA聚合酶的作用。我们将合成具有适当布置的硫醇基团的嘌呤和嘧啶，其用作用于获得所需NC缀合物的配体。在开发出有效的合成路线后，将对NC-dNTP和NC-核苷缀合物进行光谱表征。平行地，NC-dNTP缀合物将针对其对DNA聚合酶的作用进行研究，目的是将NC修饰的核苷酸酶促掺入新生DNA中。如果需要，将改造DNA聚合酶以改善NC-dNTP缀合物的底物性质。通过反复的合成以及物理和生物化学研究，将获得基于NC-dNTP的系统，其具有比现在可用的系统更上级的（光）物理和生物化学性质，并且将允许广泛的应用。

In the last two decades, metal nanoclusters (NCs) have made significant advances in the field of bioimaging, biosensing, therapeutic applications, and catalysis. Noble metal NCs like AgNCs and AuNCs, owing to their non-toxicity, biocompatibility, and photostability, have emerged as better luminescent materials over organic dyes and quantum dots. Thiol-capped AuNCs have been extensively studied in the field of catalysis, luminescence, and chemical sensing and exhibit very promising properties, calling for broad applications. The capability of DNA polymerases to accept dye-modified deoxynucleoside triphosphates (dNTPs) is exploited in many important biotechnological applications. These include next-generation sequencing approaches, single molecule sequencing, and labeling of DNA and PCR amplificates e.g. for microarray analysis. Furthermore, utilizing the intrinsic properties of DNA in combination with chemically introduced functionalities, provide an entry to new classes of nucleic acids-based hybrid material. Despite the impressive properties of metal NCs, these have, up to our knowledge, not yet exploited as modification for dNTPs and subsequently as substrate for DNA polymerases. In a collaborative approach will address this issue. We propose to design novel water-soluble nanoclusters modified with nucleotides in order to incorporate and label DNA by the action of DNA polymerases. We will be synthesize purines and pyrimidines with a suitably disposed thiol group used as ligand for obtaining the desired NC conjugates. After development of efficient synthetic routes, the NC-dNTP and NC-nucleoside conjugates will be spectroscopically characterized. In parallel, the NC-dNTP conjugates will be investigated towards their action on DNA polymerases with the aim to enzymatically incorporate the NC-modified nucleotides into the nascent DNA. If required, DNA polymerases will be engineered in order to improve the substrate properties of the NC-dNTP conjugates. By iterative rounds of synthesis and physical and biochemical studies, NC-dNTP-based systems will be obtained that have superior (photo)physical and biochemical properties than the systems available now and will allow broad applications.