Biotechnological mass production of DNA origami
Biotechnological mass production of DNA origami
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DOI：
10.1038/nature24650
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发表时间：
2017-12-07
期刊：
影响因子：
64.8
通讯作者：
Dietz, Hendrik
中科院分区：
文献类型：
--
作者：
Praetorius, Florian;Kick, Benjamin;Dietz, Hendrik
DNA纳米技术，特别是DNA折纸，使自下而上的自组装微米级，三维结构与纳米精确的功能（1-12）。这些结构是可定制的，因为它们可以是位点特异性功能化的（13）或被构造成表现出类似机器的（14，15）或逻辑门控行为（16）。由于目前生产方法的限制，它们的使用仅限于只需要少量材料（微克量级）的应用。但是，如果可以使用更多的材料，则可以实现许多提议的应用，例如作为治疗剂或在复杂材料中（3，16 -22）。在DNA折纸中，纳米结构由许多短的单链钉合寡核苷酸固定在适当位置的非常长的单链支架分子组装而成。只有噬菌体衍生的支架分子适合于规模化和有效的大规模生产（23）;较短的钉合链通过昂贵的固相合成（24）或酶促过程（25）获得。在这里，我们表明，几乎任意长度和几乎任意序列的单链DNA可以通过使用噬菌体产生单链前体DNA，其含有与自切除“盒”交错的靶链序列，每个盒包含两个Zn 2+依赖性DNA切割DNA酶，以可扩展和具有成本效益的方式产生。我们使用摇瓶培养物生产了几种DNA折纸所需的所有DNA单链，并在升规模的搅拌罐生物反应器中展示了宏观量的DNA折纸纳米棒的端到端生产。我们的方法与现有的DNA折纸设计框架兼容，并保留了DNA折纸对象的模块化和可寻址性，这对于使用官能团进行自定义修改是必要的。由于所有的生产和纯化步骤都适合规模化，我们预计我们的方法将扩大DNA纳米技术在许多科学和技术领域的范围。
DNA nanotechnology, in particular DNA origami, enables the bottom-up self-assembly of micrometre-scale, three-dimensional structures with nanometre-precise features(1-12). These structures are customizable in that they can be site-specifically functionalized(13) or constructed to exhibit machine-like(14,15) or logic-gating behaviour(16). Their use has been limited to applications that require only small amounts of material (of the order of micrograms), owing to the limitations of current production methods. But many proposed applications, for example as therapeutic agents or in complex materials(3,16-22), could be realized if more material could be used. In DNA origami, a nanostructure is assembled from a very long single-stranded scaffold molecule held in place by many short single-stranded staple oligonucleotides. Only the bacteriophage-derived scaffold molecules are amenable to scalable and efficient mass production(23); the shorter staple strands are obtained through costly solid-phase synthesis(24) or enzymatic processes(25). Here we show that single strands of DNA of virtually arbitrary length and with virtually arbitrary sequences can be produced in a scalable and cost-efficient manner by using bacteriophages to generate single-stranded precursor DNA that contains target strand sequences interleaved with self-excising 'cassettes', with each cassette comprising two Zn2+-dependent DNA-cleaving DNA enzymes. We produce all of the necessary single strands of DNA for several DNA origami using shaker-flask cultures, and demonstrate end-to-end production of macroscopic amounts of a DNA origami nanorod in a litre-scale stirred-tank bioreactor. Our method is compatible with existing DNA origami design frameworks and retains the modularity and addressability of DNA origami objects that are necessary for implementing custom modifications using functional groups. With all of the production and purification steps amenable to scaling, we expect that our method will expand the scope of DNA nanotechnology in many areas of science and technology.