基于单分子生物纳米孔的单碱基分辨miRNA N6腺苷甲基化修饰 (m6A) 测序方法和高通量光学纳米孔测序阵列芯片

miRNA, which is a type of short-length RNA molecule, regulates a series of physiology and pathology activities for a wide variety of life forms. In recent studies, it is evidenced that miRNA, which contains rich base modifications, could be detected in human serum. It is thus beleived that ultra-sensitive detection of miRNA in circulating blood samples would benefit both for fundamental medical studies and clinical diagnosis. At the moment, single molecule nanopore seuqening has been successfully developped and is reaching its preliminary commercialization stage. The ultraportable Minion sequencer from Oxford Nanopore Technologies (UK) could now sequence DNA sizes equivalent to virus genomes and is not limited by the sequencing environment. The portability of nanopore sequencer thus enbales sequencing in Africa or even the first ever successful sequencing trial in the space. Also not limited by the sequencing length, we believe that the nanopore sequencing method is the perfect choice for miRNA epigenetic sequencing applications. With our ownly developped nanopore sequencing method, along with the requirement for miRNA N6-methyladenosine (m6A) epigenetic sequencing, we propose to develope a specific and efficient method to sequence miRNA and its m6A sequences directly. The development of this method fits the topic of the research program and would benefit a variety of related studies within and outside the country.

miRNA是一类广泛参与并调控生命个体生理和病理行为的短链核酸分子，具有极强的基础研究和临床检测价值。随着近年来miRNA在人体血浆中被广泛发现，以及其丰富的碱基修饰现象与生命调控功能被逐步的解读，对miRNA的高灵敏分析和测序手段的需求变得异常紧迫。现阶段，基于纳米孔的单分子测序技术已经逐步成熟并进入产业化初期。以英国牛津纳米孔公司开发的Minion便携式测序仪为代表的一系列纳米孔测序应用已经能够对病毒基因组尺寸的核酸样品进行快速准确的测序，并利用其便携小巧的特性在非洲疫区乃至太空均实现了成功测序。本申报课题拟采用本课题组自行开发的全新的纳米孔测序手段，配合miRNA N6腺苷甲基化修饰（m6A）测序的具体需求，开发一整套单分子测序方法并展示一个原理样机，服务本重大研究计划和一系列相关研究领域。本项目因此具有高度原创性，紧迫性和实用性。对于我国乃至国际范围内的miRNA研究具有重大意义。

纳米孔测序技术是第三代核酸测序技术的代表。纳米孔测序依靠核酸碱基的理化性质直接进行测序读取，具备单分子、单碱基分辨率，无需标记且可以分辨一定的碱基修饰。然而，在本项目立项前，纳米孔测序技术对于短链非编码核酸如miRNA的直接测序和碱基尚未有过报道，也没有任何技术可以实现miRNA的直接单分子测序并分辨其所包含的碱基修饰信息。本申报项目正是以此研究空白为突破口，利用本课题组近年来开发的纳米孔错位测序技术NIPSS为实现手段，展示了采用NIPSS策略的miRNA首次直接单分子测序，并可以直接分辨miRNA上的N6-甲基腺嘌呤。该工作的发表不仅实现了项目主体任务，也是对纳米孔测序领域的一次重要突破，且具备实际的检测意义。在项目执行任务之外，依托纳米孔测序，项目负责团队亦在其它核酸碱基修饰的直接纳米孔测序读取中取得了丰硕的研究成果。包括首次使用纳米孔测序对O6-羧甲基鸟苷实现了高分辨直接单分子测序检测。该方法由于是利用了O6-羧甲基鸟苷自身具有负电荷的特性进行直接读取，读取信号分辨极佳，单分子读取准确率达到了96%以上，具有直接的应用价值。在检测通量的优化上，项目负责人开创了首个无电极纳米孔检测技术DiffusiOptoPhysiology实现了荧光直接汇报的纳米孔检测，大大降低了仪器芯片的复杂性，实现了可抛弃式应用。在纳米孔的工程改造方面，项目负责人开发了若干单分子孔道内部修饰技术，实现了孔道内壁的金属化修饰，启发了纳米孔测序技术在多糖，糖肽，多肽等难测生物分子和其化学修饰的研究新思路。