Oligonucleotide-modified nucleotides

基于核酸的诊断跨越了广泛的领域，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(SNPs)。因为大多数分析方法都是以聚合酶链式反应(PCR)为基础的，需要复杂的设备。该项目的目的是进一步开发允许肉眼以单核苷酸精度检测核酸靶标的方法，而不需要基于聚合酶链式反应的技术。这样的系统将对现场护理测试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很大的潜力，因为它们以模板依赖的方式催化核苷酸掺入，具有很高的序列选择性。在第一个资助期，我们证明了被大的功能实体修饰以产生信号的核苷酸，如具有酶活性的DNA构建体(即DNAzyme)或蛋白质(如马萝卜过氧化物酶)被DNA聚合酶加工并选择性地将序列掺入到正在生长的DNA链中。因此，功能实体被连接到固体载体上，并导致可用肉眼检测的序列选择性信号产生。这些研究得到了对DNA聚合酶如何接受这种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。首先，初步结果表明，这两种方法都非常有希望。因此，将合成几种DNA和抗体修饰的核苷三磷酸，并对其进行深入的研究。此外，我们的目标是通过功能和结构手段深入研究DNA聚合酶对修饰核苷酸的掺入。这将导致对这些酶处理核苷酸的机制有一个更广泛的了解，这些核苷酸被修饰，其实体比DNA聚合酶本身的直径大几倍。另一方面，由于相关DNA聚合酶的计划结构研究将获得对DNA聚合酶功能的复杂机制的新见解，这些DNA聚合酶以前没有与结合的修饰底物结晶。

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.