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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一个广泛的领域，从检测病原体衍生的核酸（如病毒）到分析整个基因组中的单核苷酸变异，如点突变和单核苷酸多态性（snp）。对于大多数分析方法是基于聚合酶链反应（PCR）和需要复杂的设备。该项目的目的是进一步开发方法，使裸眼检测核酸靶标具有单核苷酸精度，而不需要基于pcr的技术。这种系统对于即时检测或现场检测病原体非常有用。基于DNA聚合酶的反应在这方面具有很大的潜力，因为它们以模板依赖的方式催化核苷酸结合，具有高序列选择性。在第一个资助期，我们证明了被用于产生信号的大功能实体修饰的核苷酸，如具有酶活性的DNA结构（即DNAzymes）或蛋白质（即马萝卜过氧化物酶），由DNA聚合酶处理，并选择性地将序列整合到生长的DNA链中。因此，将功能实体连接到固体支架上，并产生可通过肉眼检测的序列选择性信号。这些研究得到了对DNA聚合酶如何接受如此大的修饰机制的结构调查的支持。在未来的研究中，我们的目标是通过利用环介导的等温扩增（LAMP）和基于抗体的检测系统来提高系统的灵敏度。初步结果表明，这两种方法都非常有前途。因此，几种DNA和抗体修饰的核苷三磷酸将被合成和彻底研究。此外，我们旨在通过功能和结构手段彻底研究DNA聚合酶与修饰核苷酸的结合。这将导致对这些酶处理核苷酸的机制有更广泛的理解，这些核苷酸被修饰的实体比DNA聚合酶本身的直径大几倍。另一方面，由于计划对相关DNA聚合酶进行结构研究，将获得对DNA聚合酶功能复杂机制的新见解，这些DNA聚合酶以前没有用结合修饰底物结晶。