Methods for enzymatic synthesis of modified nucleic acids (MESNA)

核酸是由核苷酸单体组成的聚合物，DNA中含有ATCG碱基，RNA中含有AUCG碱基（U和T是等效的）。在细胞中，DNA以双螺旋结构存在，被转录成单链信使RNA (mRNA)，然后被翻译成特定的蛋白质（细胞内的功能分子）。辉瑞和Moderna的COVID-19疫苗是编码SARS-CoV-2刺突蛋白的mRNA序列。免疫后，mRNA进入我们的细胞，被翻译成刺突蛋白（抗原），从而产生抗体（一种免疫反应），保护我们免受未来的感染。这两种疫苗都使用合成单体（n1 -甲基伪尿嘧啶）代替U的修饰mRNA，尽管n1 -甲基伪尿嘧啶和U编码相同的信息，但细微的结构差异提高了mRNA在细胞中的寿命并提高了抗原的翻译水平。同样，改良的mrna也被开发出来对抗其他疾病，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。虽然这种方法效果很好，但DNA模板的使用阻止了选择性修饰，因为酶只能使用四种单体（AUCG或等同物）。因此，在特定位置（例如更容易降解的末端区域）包含修饰是无法使用该方法实现的。其他具有重要治疗意义的修饰核酸的例子包括短反义寡核苷酸（ASOs）和小干扰rna （sirna）。ASOs与互补的mRNA结合（碱基配对）以阻断其或诱导切割，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是短修饰的双链rna，与细胞中的蛋白质结合，促进互补靶mrna的分解。ASOs和sirna经过高度修饰，以提高它们对降解核酸的细胞酶的稳定性。由于其高度修饰的结构，ASOs和sirna目前是通过化学固相合成（SPS）生产的。虽然SPS在小规模试验中效果良好，但大规模操作成本极高且非常困难，这意味着为大量患者群体制造所需的aso和sirna是不可行的。所使用的合成单体也非常昂贵，需要大量的化学操作。每一步都需要大量过量的单体，以及其他昂贵的试剂和大量的有机溶剂，其中大多数是有毒的，对环境有害，并且越来越不可持续。在这个项目中，我们将开发新的酶方法，用于修饰核酸（mRNA, ASOs， siRNA和其他治疗药物）的无模板组装。酶法在水中，在温和的条件下，利用良性酶和可再生单体，将提供一个更可持续的，可扩展的和成本效益的替代SPS，同时也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，以接受带有阻断基团的修饰核苷酸单体。阻断基团确保在每一步中只添加一个酶促单体。只有在解块之后才添加下一个单体，这样可以完全控制修改的顺序和位置。我们将利用酶的x射线（3D）结构来指导工程（诱变），对酶的活性位点进行合理的改变，使修饰的核苷酸被接受。我们还将使用更多的随机方法来创建更大的突变酶库。高通量荧光分析将被开发，其中单体的结合导致荧光，使我们能够筛选更多的突变体和选择具有改进特性的变体。工程连接酶也可用于连接较长的RNA链，产生具有选择性修饰的mRNA。

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.