Direct real-time single molecule DNA sequencing

描述（由申请人提供）：我们建议开发一种方法，以天然DNA聚合酶使用天然核苷酸的速度和准确性从基因组DNA中直接实时测序单个DNA分子。我们将利用用于DNA复制的真正纳米机器的力量，即天然DNA聚合酶。DNA聚合酶具有精确的原子分辨率3D结构，可以以高保真度和高速度合成非常长的DNA分子，而不像纳米孔测序那样难以设计人工纳米结构来区分近距离和恒定波动的4种碱基类型。具有校对功能的DNA聚合酶的错误率可能低至百万分之一，而像phi29 DNA聚合酶这样的过程聚合酶在一段时间内可以合成多达10万个碱基。从丰富的结构和动力学研究中，我们知道DNA合成的保真度是建立在精细的结构互补性和聚合酶蛋白的活性位点与引物/模板/核苷酸复合物之间的许多特定相互作用的基础上的。伴随着特定相互作用、诱导配合、键裂解/形成和模板易位的动态化学-机械或构象变化确保了高度准确和有序的碱基配对和结合。我们的策略是通过蛋白质工程将传感器设计到聚合酶的表面（而不是活性位点），以监测每种碱基类型结合时微妙而独特的构象变化。利用F"rster共振能量转移（FRET）技术可以精确地测量一个小的距离变化（一到几十埃）。在聚合酶的策略残基上放置多个FRET对或网络将用于实时监测构象变化（比DNA合成速度快10倍）。传感器将提供聚合酶动态结构的多参数信息，这很可能为每一种碱基类型提供独特的特征。模板DNA上的甲基化等化学修饰也可能被检测到。这种方法可以对非常长的DNA分子进行测序，并且可以在几分钟内进行高保真度测序，并且可以在不到一小时内对人类基因组甚至表观基因组进行测序。这将真正实现个性化医疗。

DESCRIPTION (provided by applicant): We propose to develop a method for direct real-time sequencing of single DNA molecules from genomic DNA at the speed and accuracy of the natural DNA polymerases using native nucleotides. We will harness the power of the true nano-machines used in DNA replication, the natural DNA polymerases. Unlike the difficult to engineer man-made nanostructures of nanopore sequencing used to distinguish the 4 base types in close proximity and constant fluctuation, DNA polymerases have precise atomic-resolution 3D structures and can synthesize very long DNA molecules with high fidelity and velocity. The error rate of a DNA polymerase with proof-reading function could be as low as one in a million bases and a processive polymerase such as phi29 DNA polymerase can synthesize up to 100,000 bases in a stretch. From the wealth of structural and kinetics studies, it is well known that the fidelity of DNA synthesis is predicated on the exquisite structural complementarity and the numerous specific interactions between the active site of the polymerase protein and the primer/template/nucleotide complex. The dynamic chemo-mechanical or conformational changes accompanying the specific interactions, induced fit, bond cleavage/formation, and template translocation ensure highly accurate and orderly base pairing and incorporation. Our strategy is to engineer sensors onto the surface (not the active site) of the polymerase by protein engineering to monitor the subtle yet distinct conformational changes accompanying the incorporation of each base type. A small distance change (one to tens of angstroms) can be measured precisely with F"rster resonance energy transfer (FRET) technique. Multiple FRET pairs or networks placed in strategic residues on the polymerase will be used to monitor the conformational changes in real time (10 times faster than the rate of DNA synthesis). The sensors will provide multi-parametric information on the dynamic structures of the polymerase, which very likely will provide a unique signature for each base type incorporated. Chemical modifications such as methylation on the template DNA could also potentially be detected. Such a method could sequence very long DNA molecules and could be sequenced with high fidelity in minutes and a human genome or even epigenome could be sequenced in less than one hour. This will truly enable personalized medicine. PUBLIC HEALTH RELEVANCE: We propose to develop a breakthrough DNA sequencing technology called READS genome technology for direct real-time single molecule sequencing. We aim to develop the new sequencing method and engineer a sequencing platform for ultra-fast and low-cost human genome sequencing so that routine sequencing of individual human genomes can be performed for biomedical applications and personalized medicine.