Bilateral NSF/BIO-BBSRC: Engineering Tunable Portal Hybrid Nanopores for High-Resolution Sequence Mapping

基因组技术是现代生活的一个重要组成部分，通常用于医学，法医学和作物科学。然而，尽管DNA测序技术迅速发展，但包括人类基因组在内的大部分基因组区域的特征很差。在这项提案中，我们的目标是开发一种新的混合纳米孔技术用于DNA分析，并探索使用这种方法来改善这些定义不明确或“黑暗”的基因组区域的分析的可能性。这些“黑暗”区域定义不明确，主要是由于存在多个重复。这些“重复元素”很难用现有技术进行分析。这是因为目前的方法依赖于DNA聚合酶活性，其易于在富含AT的重复序列上“卡顿”和“打滑”，并且在富含GC的重复序列上“停滞”。纳米孔技术是一种用于在非常长的DNA片段（>100 Kb）的近碱基对分辨率下作图的不依赖于聚合酶的方法。事实上，人们希望这项技术将朝着全基因组从头测序的方向发展。然而，尽管有很大的希望，这些技术仍在开发中，目前准确率在60-90%之间。虽然蛋白质纳米孔，如α-溶血素蛋白，是容易再现和可调的，但它们通常是膜孔的衍生物。因此，它们被支撑在相对脆弱的脂质样膜中，需要去污剂进行处理和高（微摩尔）DNA浓度进行信号检测。相反，超薄层固态（SS）纳米孔更坚固，只需要纳摩尔量的DNA。杂化纳米孔可以联合收割机结合两种系统的优点：即（i）可重复地产生直径为1-3 nm的可调纳米孔，（ii）稳健的性质，导致信号检测所需的低（纳摩尔）DNA浓度。然而，目前的蛋白质纳米孔需要去污剂和大量的化学修饰以整合到SS纳米孔中。我们建议研究天然DNA纳米孔，从嗜热病毒的门户蛋白，用于混合纳米孔生产的适用性。这种蛋白质是病毒基因组包装过程中DNA通过的纳米孔，因此自然地处理设计用于捕获和定向转移dsDNA的特征。此外，这种生物纳米孔是热稳定的、高度可溶的、易于生物工程目的处理的，并且易于以高纯度大量生产。此外，可用的高分辨率X-射线结构的门户蛋白允许设计的门户变异与修改的DNA transition properties.In这个提案中，我们特别旨在探索和优化整合的门户蛋白到SS nanopores和定义的DNA transition dynamics。生物工程方法将用于优化门户蛋白3微秒/碱基对转换速度。杂交纳米孔将被校准用于精确分析含有多个重复的DNA序列。

Genome technology is an important part of modern life and is used routinely in medicine, forensic and crop science. However, despite rapid advances in DNA sequencing technology, large regions of genomes, including the human genome, are poorly characterised. In this proposal we aim to develop a new hybrid nanopore technology for DNA analysis and explore the possibility of using this method for improved analysis of these poorly defined or 'dark' genomic regions.These 'dark' regions are poorly defined largely due to the presence of multiple repeats. These 'repetitive elements' are difficult to analyse with currently available technology. This is because current methods rely on DNA polymerase enzyme activity which is prone to 'stuttering' and 'slipping' on AT rich repeats and 'stalling' on GC rich repeats.Nanopore technology is a polymerase-independent method for mapping at near-basepair resolution of very long DNA fragments (>100 Kb). Indeed it is hoped that this technology will be advanced towards the de novo sequencing of whole genomes. However, despite great promise, these technologies are still in development and currently are between 60-90% accurate. While protein nanopores, such as the alpha-haemolysin protein, are easily reproducible and tunable, they are generally derivatives of membrane pores. Thus they are supported in relatively fragile lipid-like membranes, requiring detergent for handling and high (micromolar) DNA concentration for signal detection. Conversely, ultra-thin layer solid-state (SS) nanopores are more robust and require only nanomolar quantities of DNA. However, they are difficult to fabricate routinely with the tiny diameter (<4nm) required for high-resolution DNA mapping.Hybrid nanopores can combine the advantages of both systems: namely (i) the reproducible production of tunable nanopores with diameters of 1-3 nm with (ii) robust properties that results in low (nanomolar) DNA concentrations required for signal detection. However, current protein nanopores require detergent and substantial chemical modification for integration into SS nanopores. We propose to investigate the suitability of a natural DNA nanopore, the portal protein from a thermophilic virus, for hybrid nanopore production. This protein is the nanopore through which DNA passes during packaging of the viral genome and so naturally processes the characteristics designed for capture and directional transition of dsDNA. Additionally this bionanopore is thermostable, highly soluble, tractable for bioengineering purposes and easy to produce in large highly pure quantities. Furthermore, the available high-resolution X-ray structure of the portal protein allows the design of portal variants with modified DNA transition properties.In this proposal, we specifically aim to explore and optimise the integration of the portal protein into SS nanopores and define the DNA transition dynamics. Bioengineering methods will be used to optimise the portal protein for 3 microseconds/basepair transition speeds. The hybrid nanopore will be calibrated for accurate analysis of DNA sequences containing multiple repeats.