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

理解和利用信使RNA需要技术来分析原始序列之外的内容，例如mRNA发生的修饰和定义其生命阶段。需要向“大数据”迈出一步，以实现在全转录组水平上对单个转录本和异构体的多种修饰的映射。 mRNA核苷酸修饰被特定蛋白质动态写入、擦除和识别的认识推动了RNA生物学的范式转变。到目前为止，在RNA 1上鉴定出超过160种修饰，它们在RNA生命周期的所有阶段、基因表达和疾病中起着重要作用2，3。新兴的表位转录组学领域试图解释这些神秘的修饰。许多病毒编码它们自己的或捕获宿主修饰编写器以逃避或利用宿主机制，而治疗性RNA必须做同样的事情，以便微调分子在宿主中的活性。对修饰沉积和生物学影响的更深入理解扩展了我们可以对抗病毒感染或微调mRNA治疗的工具包。 里程碑1：学生在纳米孔测序，寡核苷酸合成和建立的测定方面发展专业知识。已建立的方法使用放射性标记或特异性修饰核苷酸的抗体来标记表位转录组。纳米孔直接RNA测序可以揭示全长单分子读数中mRNA加工的复杂性。纳米孔在算法上解释由占据孔的多核苷酸“kmers”产生的信号，以推断链以“一出一入”的方式通过的序列4。因此，虽然RNA可以“直接”读取，但与Illumina测序相比，保真度不足5。因此，大多数RNA测序策略依赖于在充满偏倚的过程中转化为cDNA，这消除了修饰信息。 里程碑二：学生开发了双链RNA-seq（dsRNA-seq），利用病毒RNA依赖性RNA聚合酶复制靶RNA，并将互补RNA链连接到模板的一端。每个测定的核苷酸与未修饰的互补物配对。通过纳米孔测序实现并在Loose实验室7中实现的长读取长度可以为每个测定的RNA分子提供两个读取。复制的RNA将确认正义链的未修饰的身份。在修饰的情况下，与从互补RNA（cRNA）链获得的预期纳米孔“波形”的任何偏差将表示潜在的修饰位点。还将生成新的训练集。通过Hayes实验室8中开发的合成技术，在单个反应中，可以在所有（65，536）序列组合中产生具有侧接四个未修饰核苷酸的修饰核苷酸的寡核苷酸kmer群体。在进行dsRNA-seq之前将这些连接将得到新的kmer“波形”并确认互补读段中的kmer。 里程碑3：学生利用已建立的方法和dsRNA-seq检测宿主和病毒来源mRNA中的修饰核苷酸。这将提供新的基本见解与远端剪接位点或加工变异的修饰的存在之间的相关性。里程碑4和安置：学生应用与阿斯利康合作的里程碑1-3中开发的专业知识和方法。这将补充阿斯利康在开发基于专有RNA技术的治疗方法方面正在进行的工作。这需要产生在其整个长度上具有多种类型修饰的mRNA，以微调分子的宿主代谢。

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.