Ligase-Free Synthetic Gene Assembly

合成生物学是一种开创性的研究途径，它横跨所有科学学科，提供新材料、药物、食品和消费品。在研究和商业部门制造和应用生物系统的一个重要组成部分是产生和操作DNA作为遗传信息储存库的能力。为了制造和修饰用于生物技术的蛋白质，可以向生物体提供适当的遗传密码，然而，化学家仍然无法合成足够长的DNA序列来制造完整的基因，从而产生有用的蛋白质产品。在过去，生物学家利用多种酶和一种称为连接的过程来将DNA片段融合成可用的形式。虽然这种方法被证明是有效的，但在应用于多基因的大规模合成时，在时间、效率和成本方面仍然存在局限性。因此，现在有一个明确的机会来开发新的、更快、准确和成本效益高的策略，仅使用合成化学就可以完全合成大的DNA分子。AIMS和OBJECTIVESOur项目的目标是使用容易获得和廉价的化学品将小的、重叠的DNA链串联起来，以组装合成基因。我们将利用我们对这些化学物质在水中的行为和反应的知识，为基因组装过程生成简单、高效的方案。一系列的化学和生化技术将被用来确认我们的链连接的连接方法已经成功。最终，我们将准备一个合成基因，并用它来制造它所编码的蛋白质。我们研究计划的成功是建立在明确的实验证据的基础上，这些证据证明了我们的链连接策略的有效性。我们的目标是通过产生新的和改进的化学构建块来制造短DNA链(称为寡核苷酸)，从而进一步提高连接效率。这些组成的寡核苷酸将被测试，连接条件优化，然后我们将针对我们的最终目标合成基因，以展示完整的概念。潜在的应用和BENEFITSOUR寡核苷酸连接技术可以很容易地应用于DNA组装，以加快和降低基因和基因组制造的成本。我们的方法最初的目标是改善基因合成，然而，它也可以受益于广泛的基于核酸的方法，从分子诊断到医学治疗，甚至在作物保护方面。因为我们的方法提供了快速的化学，所以它将是并行(高通量)监测大量样品的理想选择。它还将为大规模应用提供优势，例如产生更大数量的连接核酸用于基因治疗，甚至用于基于RNA的作物保护剂。这些应用为广泛的用户提供了经济、环境、社会和医疗效益。

CONTEXTSynthetic biology is a pioneering avenue of research that traverses all of the disciplines of science to deliver new materials, pharmaceuticals, food and consumer products. An essential component to fabricating and applying biological systems in both research and commercial sectors is the ability to generate and manipulate DNA as a repository for genetic information. In order to make and modify proteins for biotechnological applications, the appropriate genetic code can be presented to an organism, however, chemists are still unable to synthesise sufficiently long DNA sequences to make entire genes and hence their useful protein products. In the past, biologists harnessed multiple enzymes, and a process called ligation, to fuse DNA segments into usable formats. Whilst this approach proved effective, there are still limitations in terms of time, efficiency and cost when applied to the large-scale synthesis of multiple genes. Hence there is now a clear opportunity to develop new, faster, accurate and cost-effective strategies for the complete synthesis of large DNA molecules using synthetic chemistry alone.AIMS AND OBJECTIVESOur project aims to employ readily available and inexpensive chemicals to connect small, overlapping strands of DNA in tandem to assemble synthetic genes. We will exploit our knowledge of how these chemicals behave and react in water to generate simple, efficient protocols for the gene assembly process. A range of chemical and biochemical techniques will be used to confirm that our ligation approach to strand joining has been successful. Ultimately we will prepare a synthetic gene and use it to make the protein that it encodes. The success of our research plan is based upon clear experimental evidence that demonstrates the validity of our strand ligation strategy. Our goal is to further enhance ligation efficiency by generating new and improved chemical building blocks to make the short DNA strands (known as oligonucleotides). These component oligonucleotides will be tested, the ligation conditions optimised, and then we will target our ultimate goal of a synthetic gene, to demonstrate the complete concept.POTENTIAL APPLICATIONS AND BENEFITSOur oligonucleotide ligation technology can be readily applied to DNA assembly to speed up and reduce the costs of gene and genome manufacture. Our approach will initially be targeted at improving gene synthesis, however, it could also benefit a wide range of nucleic acid-based approaches from molecular diagnostics to medical therapies and even in crop protection. Because our approach offers rapid chemistry, it will be ideal for monitoring large numbers of samples in parallel (high-throughput). It will also offer advantages to large-scale applications, such as the generation of larger quantities of ligated nucleic acids for gene therapies, and even for RNA-based crop protection agents.These applications offer economic, environmental, societal and medical benefits across a wide range of users.