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中有ATCG碱基，RNA中有AUCG碱基(U和T等同)。在细胞中，DNA以双螺旋形式存在，并被转录成单链信使RNA(MRNA)，然后被翻译成特定的蛋白质(细胞内的功能分子)。辉瑞和现代新冠肺炎疫苗是编码SARS-CoV-2刺突蛋白的基因序列。在免疫后，信使核糖核酸进入我们的细胞，并被翻译成尖峰蛋白(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。两种疫苗都使用带有合成单体(N1-甲基假性尿苷)的修饰的信使核糖核酸，以代替U。虽然N1-甲基假性尿苷和U编码具有相同的信息，但微小的结构差异提高了信使核糖核酸在细胞中的寿命，并提高了抗原的翻译水平。同样，也在开发改良的mRNAs来对抗其他疾病，如癌症(免疫疗法)。目前，信使核糖核酸疫苗和治疗药物是使用依赖于DNA的聚合酶生产的。虽然这很有效，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或类似物)。因此，使用这种方法不能包括特定位置的修饰(例如，更容易降解的末端区域)。其他具有治疗意义的修饰核酸的例子包括短反义寡核苷酸(ASO)和小干扰RNA(SiRNAs)。ASO与互补的信使核糖核酸(碱基配对)结合，以阻断它或诱导切割，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是一种短的修饰双链RNA，与细胞中的蛋白质结合，促进互补靶标mRNAs的分解。ASO和siRNAs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，ASO和siRNAs目前是通过化学固相合成(SPS)生产的。虽然SPS在小规模上效果很好，但它非常昂贵，而且很难大规模操作，这意味着制造大量患者所需的ASO和siRNA是不可行的。所使用的合成单体的生产成本也非常高，需要进行广泛的化学操作。每一步都需要大量的单体和其他昂贵的试剂，以及大量的有机溶剂，其中大多数是有毒的，对环境造成破坏，并且越来越不可持续。在这个项目中，我们将开发新的无模板组装修饰核酸的酶方法(RNA，ASOS，siRNA和其他疗法)。酶法在温和的条件下在水中工作，利用良性的酶和可再生的单体，将提供一种更可持续、可扩展和成本效益更高的SP的替代品，同时还允许选择性地修饰较长的mRNA。最初，我们将专注于设计无模板的聚合酶和连接酶来接受带有封闭基团的修饰核苷酸单体。封闭基确保在每一步中只酶法添加一种单体。只有在解封之后才会添加下一个单体，从而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)，对酶活性部位进行合理的改变，以便修饰的核苷酸被接受。我们还将使用更随机的方法来创建更大的突变酶文库。高通量荧光分析将被开发，其中单体的掺入导致荧光，使我们能够筛选更多的突变体并选择具有更好性能的变种。这种经过改造的连接酶还可以用来连接较长的RNA链，通过选择性的修饰来产生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.