Stefan Loic, Lavergne Thomas, Spinelli Nicolas, Defrancq Eric, Monchaud David
Institut de Chimie Moléculaire, Université de Bourgogne (ICMUB), CNRS UMR6302, Dijon, France.
Nanoscale. 2014 Mar 7;6(5):2693-701. doi: 10.1039/c3nr05954e. Epub 2014 Jan 22.
The structure of the double helix of deoxyribonucleic acid (DNA, also called duplex-DNA) was elucidated sixty years ago by Watson, Crick, Wilkins and Franklin. Since then, DNA has continued to hold a fascination for researchers in diverse fields including medicine and nanobiotechnology. Nature has indeed excelled in diversifying the use of DNA: beyond its canonical role of repository of genetic information, DNA could also act as a nanofactory able to perform some complex catalytic tasks in an enzyme-mimicking manner. The catalytic capability of DNA was termed DNAzyme; in this context, a peculiar DNA structure, a quadruple helix also named quadruplex-DNA, has recently garnered considerable interest since its autonomous catalytic proficiency relies on its higher-order folding that makes it suitable to interact efficiently with hemin, a natural cofactor of many enzymes. Quadruplexes have thus been widely studied for their hemoprotein-like properties, chiefly peroxidase-like activity, i.e., their ability to perform hemin-mediated catalytic oxidation reactions. Recent literature is replete with applications of quadruplex-based peroxidase-mimicking DNAzyme systems. Herein, we take a further leap along the road to biochemical applications, assessing the actual efficiency of catalytic quadruplexes for the detection of picomolar levels of surface-bound analytes in an enzyme-linked immunosorbent (ELISA)-type assay. To this end, we exploit an innovative strategy based on the functionalization of DNA by a multitasking platform named RAFT (for regioselectivity addressable functionalized template), whose versatility enables the grafting of DNA whatever its nature (duplex-DNA, quadruplex-DNA, etc.). We demonstrate that the resulting biotinylated RAFT/quadruplex systems indeed acquire catalytic properties that allow for efficient luminescent detection of picomoles of surface-bound streptavidin. We also highlight some of the pitfalls that have to be faced during optimization, notably demonstrating that highly optimized experimental conditions can make DNA pre-catalysts catalytically competent whatever their secondary structures.
六十年前，沃森、克里克、威尔金斯和富兰克林阐明了脱氧核糖核酸（DNA，也称为双链DNA）的双螺旋结构。从那时起，DNA一直吸引着包括医学和纳米生物技术在内的各个领域的研究人员。大自然在DNA的多样化应用方面确实表现出色：除了其作为遗传信息储存库的经典作用外，DNA还可以充当纳米工厂，能够以模仿酶的方式执行一些复杂的催化任务。DNA的催化能力被称为脱氧核酶；在这种情况下，一种特殊的DNA结构，即四重螺旋，也称为四链体DNA，最近引起了相当大的兴趣，因为其自主催化能力依赖于其高级折叠，这使其适合与血红素（许多酶的天然辅助因子）有效相互作用。因此，四链体因其类血红蛋白特性，主要是过氧化物酶样活性，即其进行血红素介导的催化氧化反应的能力，而受到广泛研究。最近的文献中充斥着基于四链体的过氧化物酶模拟脱氧核酶系统的应用。在此，我们在生化应用的道路上更进一步，评估催化四链体在酶联免疫吸附（ELISA）型分析中检测皮摩尔水平表面结合分析物的实际效率。为此，我们利用了一种基于名为RAFT（区域选择性可寻址功能化模板）的多任务平台对DNA进行功能化的创新策略，其多功能性使得无论DNA的性质如何（双链DNA、四链体DNA等）都能进行嫁接。我们证明，所得的生物素化RAFT/四链体系统确实获得了催化特性，能够高效发光检测皮摩尔水平的表面结合链霉亲和素。我们还强调了优化过程中必须面对的一些陷阱，特别是证明了高度优化的实验条件可以使DNA预催化剂无论其二级结构如何都具有催化活性。
