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

每个活着的有机体的身份、结构和功能都被编码为DNA中的一系列化学构件(称为碱基)。在高等生物中，DNA碱基被组织成称为染色体的非常长的结构；例如，人类DNA在23条染色体上有60亿个碱基。尽管在阅读整个染色体长度的碱基序列方面取得了进展，但在许多活着的物种中，染色体的某些部分由“难以阅读”的密码组成。在这个项目中，我们将开发一种纳米孔设备，它可以读取超长的DNA分子(100万个碱基)，并通知用户感兴趣的序列在该长度上的位置。纳米孔是膜上的一个小洞，厚度只有几纳米。我们正在设计一种混合纳米孔，它由一种蛋白质分子组成，这种蛋白质分子位于硅基设备的人造薄膜中。这项工作是由英国一个擅长蛋白质结构的结构生物学小组和东北大学的纳米制造小组合作完成的。这项工作的影响将是多方面的：首先，它将允许对生物体(包括人类)中一组特定的DNA区域进行基础研究，这些区域称为微卫星，是基因组DNA上的特定位置，其中2-6个核苷酸序列重复多次。这些微卫星长度的改变直接在DNA、RNA和蛋白质水平上影响细胞功能的许多方面，有可能导致疾病。在谷类作物中，短串联重复序列在基因组中所占的比例要大得多(80%)，并可参与赋予经济上有用的性状，如在不理想的环境中增产。因此，了解这些微卫星区域有助于指导疾病，并对未来的粮食供应产生影响。其他影响包括对各级早期职业人员(本科生、毕业生和博士后)进行分子生物学、纳米技术和DNA相关研究的培训。

The identity, structure, and function of each living organism is encoded as a sequence of chemical building blocks (called bases) in DNA. In higher organisms, DNA bases are organized into very long structures called chromosomes; for example, human DNA has 6 billion bases in 23 chromosomes. Despite the advances in reading the sequence of bases along the lengths of the whole chromosomes, in many living species there are parts of chromosomes composed of "difficult-to-read" codes. In this project, we will develop a nanopore device that can read a DNA molecule of exceptionally long length (1 million bases) and inform the user of the position of sequences of interest along that length. A nanopore is a small hole in a membrane that is only a few nanometers thin. We are designing a hybrid nanopore that is made up of a protein molecule that sits inside a man-made membrane in a silicon-based device. The work is a collaboration between a structural biology group at the the UK whose expertise is in protein structure, and a nanofabrication group at Northeastern University. The impact of this work will be manifold: first, it will allow fundamental research on a particular set of DNA regions in organisms (including humans) called microsatellites, are specific loci on genomic DNA in which a sequence of 2-6 nucleotides is repeated numerous times. Alterations in the lengths of these microsatellites directly impact many aspects of cellular function at the DNA, RNA, and protein levels, potentially causing disease. In cereal crops, STRs constitute a much greater proportion of the genome (80%) and can be involved in conferring economically useful traits such as increased yields in less than ideal environments. Therefore, understanding of these microsatellite regions can help in directing disease, as well as having future impact on food supply. Other impacts include the training of early-career personnel at all levels (undergraduates, graduates, and a post-doctoral associate) on molecular biology, nanotechnology, and DNA-related research.