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

理解和利用信使RNA需要在原始序列之外进行分析的技术，例如对mRNA的修饰和定义其生命阶段。需要向“大数据”迈进一步，以便能够在整个转录组水平上将多种修饰映射到单个转录本和异构体。人们认识到，特定蛋白质动态地写入、擦除和识别mRNA核苷酸修饰，这推动了RNA生物学的范式转变。到目前为止，在RNA1上发现了160多种修饰，它们在RNA生命周期、基因表达和疾病的所有阶段都扮演着重要角色2，3.新兴的表观转录组学领域试图描述这些神秘的修饰。许多病毒编码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必须做同样的事情以微调宿主中分子的活动。对修饰沉积和生物影响的更多了解扩大了我们抗击病毒感染或微调信使核糖核酸疗法的工具包。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法方面的专业知识。已建立的方法使用放射性标记或针对特定修饰核苷酸的抗体来表征表位转录组。纳米孔直接RNA测序可以揭示全长单分子读数中信使核糖核酸处理的复杂性。纳米孔算法解释由占据孔洞的多核苷酸“kmer”产生的信号，以推断随着链以“一出一入”的方式进行的序列。因此，虽然RNA可以“直接”读出，但与光照测序相比，其保真度较低。因此，大多数RNA测序策略依赖于在充满偏见的过程中转换为cDNA6，这消除了修饰信息。里程碑2：学生开发双链RNA-seq(dsRNA-seq)，复制目标RNA，并利用依赖病毒RNA的RNA聚合酶将互补的RNA链一端连接到模板上。每个被检测的核苷酸都与一个未经修饰的补体配对。通过纳米孔测序实现的长读取长度，并在宽松的实验室7中实现，然后可以为每个被检测的RNA分子提供两个读取。复制的RNA将确认正义链的未修改身份。在修饰的背景下，从互补RNA(CRNA)链获得的预期纳米孔的任何偏离都将意味着潜在的修饰位点。还将生成新的训练集。在Hayes实验室8中开发的合成技术的支持下，在单个反应中，可以用所有(65,536)个序列组合中的四个未修饰核苷酸侧翼修饰的核苷酸来产生寡核苷酸kmer群体。在进行dsRNA-seq之前将这些序列连接起来，将使小说中的kmer出现“曲折”，并在互补阅读中确认kmer。里程碑3：学生使用已建立的方法和dsRNA-seq检测宿主和病毒来源的信使核糖核酸中的修饰核苷酸。这将为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：学生与阿斯利康合作，应用里程碑1-3中开发的专业知识和方法。这将是对阿斯利康正在进行的基于专利RNA技术开发治疗方法的工作的补充。这需要产生在整个长度上具有多种类型修饰的mRNAs，以微调分子的宿主新陈代谢。

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.