Oligonucleotide-modified nucleotides

寡核苷酸修饰的核苷酸

基本信息

批准号：
237378576
负责人：
Professor Dr. Andreas Marx
金额：
--
依托单位：
Arbeitsgruppe Organische Chemie / Zelluläre Chemie
依托单位国家：
德国
项目类别：
Research Grants
财政年份：
2013
资助国家：
德国
起止时间：
2012-12-31 至 2021-12-31
项目状态：
已结题

来源：
https://gepris.dfg.de/gepris/projekt/237378576?language=en
关键词：
Oligonucleotide modified nucleotides

项目摘要

Nucleic acid-based diagnostics span a wide field reaching from the detection of pathogen-derived nucleic acids (such as viruses) to the analysis of single nucleotide variations in the entire genome such as point mutations and single nucleotide polymorphisms (SNPs). For most analytical approach are based on the polymerase chain reaction (PCR) and require sophisticated equipment. The aim of this project is to further develop methods that allows the detection of a nucleic acid target by the naked eye with single nucleotide precision without requiring PCR-based technology. Such a system would be highly useful for point-of-care testing or the detection of pathogens in the field. DNA polymerase based reactions hold great potential in this regard, since they are catalyzing nucleotide incorporation in a template-dependent fashion with high sequence selectivity. In the first funding period we demonstrated that nucleotides that are modified with large functional entities for signal generation such as DNA constructs with enzymatic activity (i.e. DNAzymes) or proteins (i.e. horse radish peroxidase) are processed by DNA polymerases and sequence selectively incorporated into a growing DNA strand. Thereby the functional entities were connected to solid supports and resulting in sequence-selective signal generation that is detectable by naked eye. These studies were supported by structural investigations on the mechanism of how such large modifications are accepted by DNA polymerases. In future studies we aim at increasing the sensitivity of the systems by exploiting loop-mediated isothermal amplification (LAMP) and antibody-based detection systems. First preliminary results indicate that both approaches are highly promising. Thus, several DNA- and antibody-modified nucleoside triphosphates will be synthesized and thoroughly investigated. Furthermore, we aim at thoroughly investigating the incorporation of the modified nucleotides by DNA polymerases by functional and structural means. This will result in the development of a broader understanding of the mechanisms by which these enzymes process nucleotides that are modified with entities being up to several times larger than the diameter of the DNA polymerases itself. On the other hand, new insights into the complex mechanisms by which DNA polymerases function will be obtained due to planned structural investigations of relevant DNA polymerases that have not been crystallized before with bound modified substrates.

基于核酸的诊断涵盖了广泛的领域，从检测病原体衍生的核酸（例如病毒）到分析整个基因组中的单核苷酸变异，例如点突变和单核苷酸多态性（SNP）。大多数分析方法都基于聚合酶链式反应 (PCR)，并且需要精密的设备。该项目的目的是进一步开发能够通过肉眼以单核苷酸精度检测核酸靶标的方法，而不需要基于 PCR 的技术。这样的系统对于现场检测或病原体检测非常有用。基于 DNA 聚合酶的反应在这方面具有巨大的潜力，因为它们以模板依赖性方式催化核苷酸掺入，具有高序列选择性。在第一个资助期间，我们证明了用大型功能实体修饰的核苷酸，用于产生信号，例如具有酶活性的 DNA 构建体（即 DNAzymes）或蛋白质（即辣根过氧化物酶），由 DNA 聚合酶处理，并且序列选择性地整合到不断生长的细胞中。 DNA 链。从而将功能实体连接到固体支持物上，并产生肉眼可检测到的序列选择性信号。这些研究得到了 DNA 聚合酶如何接受如此大的修饰的机制的结构研究的支持。在未来的研究中，我们的目标是通过利用环介导等温扩增（LAMP）和基于抗体的检测系统来提高系统的灵敏度。初步初步结果表明，这两种方法都非常有前景。因此，将合成并彻底研究几种 DNA 和抗体修饰的核苷三磷酸。此外，我们的目标是通过功能和结构手段彻底研究 DNA 聚合酶对修饰核苷酸的掺入。这将使人们对这些酶处理核苷酸的机制有更广泛的了解，这些核苷酸是用比 DNA 聚合酶本身直径大几倍的实体修饰的。另一方面，由于计划对相关DNA聚合酶进行结构研究，这些酶之前尚未与结合的修饰底物结晶，因此将获得对DNA聚合酶功能的复杂机制的新见解。