Methods for enzymatic synthesis of modified nucleic acids (MESNA)

修饰核酸的酶促合成方法 (MESNA)

• 批准号: BB/X008991/1
• 负责人: Jason Micklefield
• 金额: $ 69.79万
• 依托单位: University of Manchester
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2023
• 资助国家: 英国
• 起止时间: 2023 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FX008991%2F1
• 关键词: Methods; enzymatic; synthesis; modified; nucleic

Nucleic acids are polymers comprising of nucleotide monomers, with ATCG bases in DNA or AUCG bases in RNA (U & T are equivalent). In cells, DNA exists as a double helix and is transcribed to single-stranded messenger RNA (mRNA), which is then translated to create specific proteins (functional molecules within cells). The Pfizer and Moderna COVID-19 vaccines are mRNA sequences coding for the SARS-CoV-2 spike protein. Upon immunisation the mRNA enters our cells and is translated to produce the spike protein (antigen) leading to the production of antibodies (an immune response) that protect us from future infection. Both vaccines use modified mRNA with a synthetic monomer (N1-methylpseudouridine) in place of U. Although N1-methylpseudouridine and U code for the same information, the slight structural differences improve mRNA longevity in the cell and boost translation levels of the antigen. Similarly, modified mRNAs are also being developed to combat other diseases such as cancer (immunotherapies). Currently mRNA vaccines and therapeutics are produced using a DNA-dependant polymerase enzyme. Whilst this works well, the use of DNA templates prevent selective modification as the enzyme can only use four monomers (AUCG or equivalent). Therefore, inclusion of modifications at specific positions (e.g. terminal regions more prone to degradation) is unachievable using this method.Other examples of therapeutically important modified nucleic acids include short antisense oligonucleotides (ASOs) and small interfering RNAs (siRNAs). ASOs bind to a complementary mRNA (base-pairing) to block it or induce cleavage, preventing translation of detrimental proteins associated with a disease (e.g. genetic disorders or cancer). siRNA are short modified double stranded RNAs that associate with proteins in the cell and promote breakdown of complementary target mRNAs. ASOs and siRNAs are highly modified to improve their stability to cellular enzymes that degrade nucleic acids. Given their highly modified structures, ASOs and siRNAs are currently produced by chemical solid-phase synthesis (SPS). Although SPS works well on a small-scale, it is extremely costly and very difficult to operate at large-scale, which means that manufacture of ASOs and siRNAs required for large patient populations is not feasible. The synthetic monomers used are also very expensive to produce and require extensive chemical manipulation. Large excesses of monomers are required at each step, along with other costly reagents, and large volumes of organic solvents, most of which are toxic, damaging to the environment and increasingly unsustainable.In this project we will develop novel enzymatic methods for template-free assembly of modified nucleic acids (mRNA, ASOs, siRNA & other therapeutics). Enzymatic methods operate in water, under mild conditions, utilising benign enzymes and renewable monomers and will provide a more sustainable, scalable, and cost-effective alternative to SPS, while also allowing selective modification of longer mRNA. Initially, we will focus on engineering template-free polymerase and ligase enzymes to accept modified nucleotide monomers with blocking groups. A blocking group ensures only one monomer is enzymatically added in each step. Only after deblocking is the next monomer added, which gives complete control over the sequence and position of modifications. We will use X-ray (3D) structures of the enzymes to guide engineering (mutagenesis), making rational changes to the enzyme active site so that modified nucleotides are accepted. We will also use more random approaches to create larger libraries of mutant enzymes. High-throughput fluorescent assays will be developed, where incorporation of a monomer leads to fluorescence, allowing us to screen larger numbers of mutants and select variants with improved properties. The engineered ligase enzymes can also be used to join longer RNA strands to produce mRNA with selective modifications.

核酸是由核苷酸单体组成的聚合物，其中DNA中具有ATCG碱基或RNA中具有AUCG碱基（U & T是等效的）。在细胞中，DNA以双螺旋形式存在，并被转录为单链信使RNA（mRNA），然后被翻译为特定的蛋白质（细胞内的功能分子）。辉瑞和Moderna COVID-19疫苗是编码SARS-CoV-2刺突蛋白的mRNA序列。在免疫后，mRNA进入我们的细胞并被翻译产生刺突蛋白（抗原），从而产生抗体（免疫反应），保护我们免受未来的感染。这两种疫苗都使用合成单体（N1-甲基假尿苷）代替U修饰的mRNA。虽然N1-甲基假尿苷和U编码相同的信息，但轻微的结构差异改善了细胞中mRNA的寿命，并提高了抗原的翻译水平。类似地，修饰的mRNA也正在开发用于对抗其他疾病，如癌症（免疫疗法）。目前，mRNA疫苗和治疗剂是使用DNA依赖性聚合酶生产的。虽然这很有效，但DNA模板的使用防止了选择性修饰，因为酶只能使用四个单体（AUCG或等同物）。治疗上重要的修饰核酸的其它实例包括短反义寡核苷酸（ASO）和小干扰RNA（siRNA）。ASO与互补mRNA结合（碱基配对）以阻断其或诱导切割，从而防止与疾病（例如遗传疾病或癌症）相关的有害蛋白质的翻译。siRNA是短的修饰的双链RNA，其与细胞中的蛋白质缔合并促进互补靶mRNA的分解。ASO和siRNA被高度修饰以提高它们对降解核酸的细胞酶的稳定性。鉴于其高度修饰的结构，ASO和siRNA目前通过化学固相合成（SPS）产生。尽管SPS在小规模上效果良好，但其成本极高且很难大规模操作，这意味着制造大患者群体所需的ASO和siRNA是不可行的。所使用的合成单体的生产也非常昂贵，并且需要大量的化学操作。每一步都需要大量的单体，沿着其他昂贵的试剂，以及大量的有机溶剂，其中大部分是有毒的，对环境有害，越来越不可持续。在这个项目中，我们将开发新的酶促方法，用于修饰核酸（mRNA，ASO，siRNA和其他治疗剂）的无模板组装。酶促方法在温和条件下在水中操作，利用良性酶和可再生单体，并将提供SPS的更可持续、可扩展和具有成本效益的替代方案，同时还允许选择性修饰较长的mRNA。最初，我们将专注于工程无模板聚合酶和连接酶接受修饰的核苷酸单体与封闭基团。封闭基团确保在每个步骤中仅酶促添加一种单体。只有在去封闭后才加入下一个单体，这可以完全控制修饰的顺序和位置。我们将使用酶的X射线（3D）结构来指导工程（诱变），对酶活性位点进行合理的改变，以便接受修饰的核苷酸。我们还将使用更随机的方法来创建更大的突变酶库。将开发高通量荧光测定，其中单体的掺入导致荧光，使我们能够筛选大量的突变体并选择具有改进特性的变体。工程化的连接酶也可用于连接较长的RNA链以产生具有选择性修饰的mRNA。