Design synthesis and evaluation of novel nucleotides for use in nanowire-based DNA analysis and diagnostic devices

桑格DNA测序在过去的30年里一直是黄金标准，并在2004年人类基因组计划的完成中发挥了重要作用。虽然该方法提供了高度准确的序列信息，但它相对昂贵，并且需要电泳分离双脱氧核苷酸封端的DNA片段，这限制了其用于大规模平行测序或作为实验室外使用的程序。相比之下，最近的程序如焦磷酸测序和合成测序（SBS）免除了对电泳的需要，并为桑格测序的一些缺点提供了解决方案。SBS通过向引物链添加带有独特且可裂解标签（例如荧光团）的同源核苷酸来顺序地鉴定每个模板碱基。核苷酸掺入伴随着DNA合成的终止。相关的标签识别掺入的核苷酸，然后去除，允许添加和识别下一个核苷酸。理想地，可切割标签应连接至核苷酸的3 '-OH，使得其还充当阻止进一步链延伸的阻断基团。不幸的是，大多数聚合酶通常不能很好地耐受具有修饰的3'-OH基团的dNTP。然而，在现有聚合酶的定向进化以产生具有增强的掺入给定核苷酸类似物的能力的新型酶之后，已经取得了进展。SBS中最常见的方法是将可切割标签连接到核苷酸的碱基上，所述核苷酸的碱基也在3 '位被修饰或阻断。在许多不同的实施例中，选择3’-封闭基团，使得其去除（以产生用于进一步延伸的游离3’-OH）可以通过在单个步骤中使用相同的试剂与标签的切割同时实现。已经报道了几种不同的3'-封闭基团，包括酰基、烯丙基、甲氧基甲基、邻硝基苄基、叠氮基甲基和最近的3'-氨基氧基类似物。QuantuMDx（QMDx）开发了采用纳米线和微流体技术的生物传感器，可用于检测小型手持单元内的生物分析物，包括核酸。这些装置依赖于带电生物分子与附着到纳米线的固定化相互作用伴侣的相互作用，其产生纳米线的电阻的小但可测量的变化。这些设备能够从患者样本中提取和扩增DNA，目前的重点是开发这些设备的方法，这些设备将依赖于使用SBS的DNA序列分析，但关键是允许在护理点进行疾病诊断或基因组分析。用于在QMDx器械内开发的SBS方法的基础依赖于通过其5 '-末端固定到纳米线并退火到DNA模板序列的DNA引物。设想带有适当电荷的可切割标签而不是荧光团（通常用于SBS）的核苷酸将允许鉴定插入的核苷酸。该项目的初始阶段将集中在TTP类似物C5-炔丙基氨基-dUTP上，我们将在其上连接一个含有上述修饰之一的可切割接头，该接头又被附加到带负电荷的标签上。这将使用已建立的酰胺偶联化学，并探索使用不同长度的接头和标签上的有效电荷对类似物被多种DNA聚合酶之一掺入的能力的影响。最初，我们将研究具有未封闭的3'-OH基团的标记核苷酸的掺入，并探索可逆封闭基团的使用。还设想将研究在3'-OH上携带可裂解标签的类似物。在这方面的工作中，QMDx将在该领域正在进行的研究中进行新型聚合酶的进化。TTP类似物的优化之后是A、C和G核苷酸的合成。

Sanger DNA sequencing has been the gold standard for over 30 years and was instrumental in the completion of Human Genome Project in 2004. Whilst the method provides highly accurate sequence information, it is relatively expensive and the need for the electrophoretic separation of dideoxynucleotide-terminated DNA fragments limits its use for large-scale parallel sequencing or as a procedure for use outside the laboratory. In contrast, recent procedures such as pyrosequencing and sequencing by synthesis (SBS) dispense with the need for electrophoresis and offer solutions to some drawbacks of Sanger sequencing. SBS identifies each template base sequentially by the addition of the cognate nucleotide bearing a unique and cleavable tag (e.g fluorophore) to the primer strand. Nucleotide incorporation is accompanied by termination of DNA synthesis. The associated tag identifies the incorporated nucleotide and is then removed allowing the next nucleotide to be added and identified. Ideally the cleavable tag should be attached to the 3'-OH of the nucleotide such that it also functions as a blocking group preventing further chain extension. Unfortunately dNTPs with modified 3'-OH groups are typically not well-tolerated by most polymerases. However, progress has been achieved following directed evolution of existing polymerases to produce novel enzymes with an enhanced ability to incorporate a given nucleotide analogue. The most common approach in SBS has been to attach the cleavable tag to the base of the nucleotide which is also modified or blocked at the 3'-position. In a number of different examples the 3'-blocking group was chosen such that its removal (to produce a free 3'-OH for further extension) could be achieved simultaneously to the cleavage of the tag by using the same reagent in a single step. Several different 3'-blocking groups have been reported and include acyl, allyl, methoxymethyl, o-nitrobenzyl, azidomethyl and recently 3'-aminooxy analogues. QuantuMDx (QMDx) have developed biosensors employing nanowires and microfluidic technologies that can be used for the detection of biological analytes including nucleic acids within small handheld units. These devices rely upon the interaction of a charged biomolecule with an immobilised interacting partner attached to a nanowire which produces a small but measurable change in the electrical resistance of the nanowire. These devices have the capacity to extract and amplify DNA from patient samples and the current focus is to develop methodology for these devices which will rely upon DNA sequence analysis using SBS but crucially allow disease diagnoses or genome analysis at the point of care. The basis of the SBS methodology for development within the QMDx devices relies on a DNA primer immobilized via its 5'-terminus to a nanowire and annealed to the DNA template sequence. It is envisaged that nucleotides bearing a suitably charged cleavable tag rather than a fluorophore (commonly employed in SBS) will allow the identification of the inserted nucleotide. The initial stages of the project will focus on a TTP analogue, C5-propargylamino-dUTP to which will we will attach a cleavable linker containing one of the modifications listed above which in turn is appended to a negatively charged tag. This will use established amide coupling chemistry and explore the use of differing length linkers and effective charge on tag on the abilities of the analogues to be incorporated by one of a variety of DNA polymerases. Initially we will examine the incorporation of tagged nucleotides with unblocked 3'-OH groups and progress to explore the use of reversible blocking groups. It is also envisaged that analogues bearing the cleavable tag on the 3'-OH will be investigated. In this aspect of the work the evolution of novel polymerases will be undertaken by QMDx in ongoing studies in this area. Optimisation of the TTP analogue will be followed by synthesis of the A,C and G nucleotides.