Direct sequencing of modified nucleotides in viral, host, and therapeutic RNAs

对病毒、宿主和治疗性 RNA 中的修饰核苷酸进行直接测序

• 批准号: 2593525
• 金额: --
• 依托单位: University of Nottingham
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2021
• 资助国家: 英国
• 起止时间: 2021 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2593525
• 关键词: Direct; sequencing; modified; nucleotides; viral

Understanding and exploiting messenger RNA requires techniques to assay beyond the raw sequence, such as the modifications occurring to mRNA and defining its life stages. A step change is needed towards "big data" to enable mapping of multiple modifications to individual transcripts and isoforms at the whole-transcriptome level. The realisation that mRNA nucleotide modifications are dynamically written, erased, and recognised by specific proteins has driven a paradigm shift in RNA biology. There are over 160 modifications thus far identified on RNA1 with fundamental roles in all stages of the RNA life cycle, gene expression, and disease2,3. The emerging field of Epitranscriptomics seeks to characterise these enigmatic modifications. Many viruses encode their own or capture host modification writers to evade or exploit host mechanisms, whilst therapeutic RNA must do the same in order to fine-tune the activities of the molecule in the host. Greater understanding of modification deposition and biological impact expands the toolkit from which we can combat viral infections, or fine-tune mRNA therapeutics. Milestone 1: Student develops expertise in nanopore sequencing, oligonucleotide synthesis, and established assays. Established methods use radio-labelling or antibodies to specific modified nucleotides to characterise the epitranscriptome. Nanopore direct RNA sequencing can reveal complexities of mRNA processing in full-length single molecule reads. Nanopore algorithmically interpret signals resulting from multi-nucleotide "kmers" occupying the pore to infer sequence as strands progress through in a "one-out-one-in" fashion4. As a result, whilst RNA can be read "directly", fidelity is lacking compared to Illumina sequencing5. As a result, most RNA-sequencing strategies rely on conversion to cDNA in a bias-laden process6 which eliminates modification information. Milestone 2: Student develops double stranded RNA-seq (dsRNA-seq) whereby target RNA is copied and the complementary RNA strand joined to the template at one end utilising a viral RNA-dependent RNA polymerase. Each assayed nucleotide is paired to an unmodified complement. Long read-lengths enabled by nanopore sequencing, and achieved in the Loose lab7, can then provide two reads for each RNA molecule assayed. The copied RNA will confirm the unmodified identity of the sense strand. In the context of modifications, any deviations from the expected nanopore "squiggle" acquired from the complementary RNA (cRNA) strand will signify a potential modification site. Novel training sets will also be generated. Enabled by the synthesis techniques developed in the Hayes lab8, in a single reaction, an oligonucleotide kmer population can be generated with a modified nucleotide flanked by four non-modified nucleotides in all (65,536) sequence combinations. Concatenating these prior to subjecting to dsRNA-seq will give both the novel kmer "squiggle" and confirm the kmer in the complementary read. Milestone 3: Student utilises established methods and dsRNA-seq to detect modified nucleotides in mRNA of host and viral origin. This will provide new fundamental insights into correlations between the presence of modifications with distal splice site or processing variants. Milestone 4 and placement: Student applies expertise and methods developed in milestones 1-3 in collaboration with AstraZeneca. This will compliment ongoing work at AstraZeneca in developing treatments based on proprietary RNA technologies. This requires the generation of mRNAs with multiple types of modifications throughout their length, to fine tune host metabolism of the molecule.

了解和利用信使 RNA 需要检测原始序列之外的技术，例如 mRNA 发生的修饰并定义其生命阶段。需要向“大数据”迈出一步，以便能够在全转录组水平上对单个转录本和亚型进行多种修饰映射。 mRNA 核苷酸修饰是动态写入、擦除和被特定蛋白质识别的认识推动了 RNA 生物学的范式转变。迄今为止，RNA1 上已鉴定出 160 多个修饰，这些修饰在 RNA 生命周期、基因表达和疾病的所有阶段都发挥着重要作用2,3。表观转录组学这一新兴领域试图描述这些神秘修饰的特征。许多病毒编码自己的或捕获宿主修饰编写者以逃避或利用宿主机制，而治疗性 RNA 也必须做同样的事情，以便微调宿主中分子的活性。对修饰沉积和生物学影响的更多了解扩展了我们可以对抗病毒感染或微调 mRNA 疗法的工具包。 里程碑 1：学生发展纳米孔测序、寡核苷酸合成和已建立的分析方面的专业知识。已建立的方法使用放射性标记或特定修饰核苷酸的抗体来表征表观转录组。纳米孔直接 RNA 测序可以揭示全长单分子读数中 mRNA 加工的复杂性。纳米孔通过算法解释由占据孔的多核苷酸“kmers”产生的信号，以推断链以“一出一内”方式通过的序列4。因此，虽然 RNA 可以“直接”读取，但与 Illumina 测序相比保真度有所欠缺。因此，大多数 RNA 测序策略依赖于在充满偏差的过程中转换为 cDNA，从而消除了修饰信息。 里程碑 2：学生开发了双链 RNA-seq (dsRNA-seq)，利用病毒 RNA 依赖性 RNA 聚合酶复制目标 RNA，并将互补 RNA 链在一端连接到模板。每个测定的核苷酸都与未修饰的互补体配对。通过纳米孔测序实现的长读长，并在 Loose 实验室中实现，可以为每个分析的 RNA 分子提供两个读长。复制的 RNA 将确认有义链的未修饰身份。在修饰的背景下，与从互补 RNA (cRNA) 链获得的预期纳米孔“曲线”的任何偏差都将意味着潜在的修饰位点。还将生成新的训练集。通过 Hayes 实验室开发的合成技术8，在单个反应中，可以在所有 (65,536) 个序列组合中生成一个修饰的核苷酸，两侧是四个未修饰的核苷酸的寡核苷酸 kmer 群体。在进行 dsRNA-seq 之前将这些连接起来将使新的 kmer 出现“曲线”并确认互补读段中的 kmer。 里程碑 3：学生利用既定方法和 dsRNA-seq 检测宿主和病毒来源 mRNA 中的修饰核苷酸。这将为了解修饰的存在与远端剪接位点或加工变体之间的相关性提供新的基本见解。里程碑 4 和安置：学生应用与阿斯利康合作在里程碑 1-3 中开发的专业知识和方法。这将补充阿斯利康正在开发基于专有 RNA 技术的治疗方法的工作。这需要生成在整个长度上具有多种类型修饰的 mRNA，以微调宿主的分子代谢。