Hybrid Nanopores for Single-Molecule Sensing

具有纳米孔的随机感测是一种多功能技术，其可用于通过检测单个分子来识别和定量各种物质（称为分析物）。我们的合作伙伴公司Oxford Nanopore Technologies一直在将随机传感技术融入下一代手持设备。Oxford Nanopore最高度开发的应用是廉价、极其快速的DNA测序，它有望彻底改变生物学的许多领域，包括医学、祖先学和法医学。目前，一种便携式测序仪正在全球数百个地点进行测试。在随机传感中，分析物在进入和离开一个狭窄的小孔时被检测到，小孔的直径被称为纳米孔，与小分子的直径相似，大约是人类头发直径的五万分之一，为通过电流扰动进行检测提供了基础。通常，测量的电流变化为万亿分之一安培。分析物包括药物分子和体内发现的小分子，它们作为疾病的标志物。在DNA测序的情况下，当延伸的DNA链穿过纳米孔时检测单个碱基。蛋白质孔对于随机感测是有利的，因为它们可以以原子精度针对特定应用进行修饰并且以接近均匀的形式制备。到目前为止，已经使用了非常窄的蛋白质孔，因此随机传感仅限于小尺寸的分析物，或者在DNA的情况下，仅限于延伸的聚合物链。在本论文中，我们将致力于制备一类新的功能性纳米孔，即DNA-蛋白质杂化纳米孔。这些孔将由被称为DNA折纸的折叠DNA和蛋白质成分构成。DNA将作为蛋白质的支架，确保新孔的内径比以前使用的孔大15倍。此外，每个孔将具有相同的尺寸，并且将不存在不完整的孔，这是先前未实现的目标。最后，将有可能在精确确定的位置修改新的孔，这是竞争技术（如固态孔）无法做到的。DNA-蛋白质混合纳米孔将通过允许检测可以进入孔的各种大生物分子（包括蛋白质、DNA和聚合糖）来实现随机传感的关键一步。相反，也可以将这些大分子置于混合孔内，在那里它们将充当各种其他分析物的结合位点。在未来的应用中，可以证明有可能用杂交孔对双链DNA进行测序，这将提供优于目前纳米孔测序所需的操作的显著优势。我们的工业合作伙伴Oxford Nanopore将在其手持式传感设备中评估和测试我们最有前途的DNA-蛋白质混合纳米孔，该设备能够并行监测数百个孔的输出，为传感技术在生物战防御，食品认证，动植物育种和医学诊断等领域的逐步变化提供前景。

Stochastic sensing with nanopores is a versatile technology that can be used for the recognition and quantification of a wide range of substances (known as analytes) through the detection of individual molecules. Our partner company, Oxford Nanopore Technologies, has been incorporating stochastic sensing into next-generation hand-held devices. The most highly developed application at Oxford Nanopore is cheap, extremely rapid DNA sequencing, which promises to revolutionise numerous areas of biology including aspects of medicine, ancestry and forensics. Currently, a portable sequencer is being tested at hundreds of sites worldwide. In stochastic sensing, analytes are detected as they enter and leave a single narrow pore perturbing a current that flows through it. The diameters of the pores, known as nanopores, are similar to those of a small molecule, about one-fifty thousandth of the diameter of a human hair, providing the basis for detection by current perturbation. Typically current changes of the order of one trillionth of an ampere are measured. Analytes have included drug molecules and small molecules found in the body that act as markers for disease. In the case of DNA sequencing, individual bases are detected as an extended DNA strand is threaded through a nanopore. Protein pores are advantageous for stochastic sensing, because they can be modified for particular applications with atomic precision and prepared in near homogeneous form. Until now, very narrow protein pores have been used and therefore stochastic sensing has been limited to analytes of small size or, in the case of DNA, to extended polymer chains. In the proposed work, we will endeavour to make a new class of functional nanopores, DNA-Protein hybrid nanopores. These pores will be constructed from folded DNA, known as DNA origami, and protein components. The DNA will act as a scaffold for the protein, ensuring that the new pores are up to fifteen times larger in internal diameter than the pores used before. Further, each pore will be of identical size and no incompletes pores will be present, a goal that has not be achieved previously. Finally, it will be possible to modify the new pores at precisely determined sites, which cannot be done with competing technologies, such as solid-state pores. The DNA-protein hybrid nanopores will enable a critical step forward for stochastic sensing by allowing the detection of a wide range of large biological molecules that can enter the pores, including proteins, DNAs and polymeric sugars. Conversely, it will also be possible to lodge these large molecules within the hybrid pores, where they will act as binding sites for a variety of additional analytes. In a futuristic application, it may prove possible to sequence double-stranded DNAs with hybrid pores, which will provide a significant advantage over the manipulations currently required for nanopore sequencing. Our industrial partner, Oxford Nanopore, will evaluate and test our most promising DNA-protein hybrid nanopores in their hand-held sensing devices, which are capable of monitoring the outputs of hundreds of pores in parallel, offering the prospect of step changes in sensing technology in areas including biological warfare defense, food authentication, plant and animal breeding and medical diagnostics.