Generation and enzymatic amplification of high-density functionalized DNA double strands
Generation and enzymatic amplification of high-density functionalized DNA double strands
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DOI：
10.1002/anie.200453926
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发表时间：
2004-01-01
影响因子：
16.6
通讯作者：
Famulok, M
中科院分区：
文献类型：
--
作者：
Jäger, S;Famulok, M
双链DNA（dsDNA）中DNA碱基的特定配对为在纳米尺度上直接产生分子组装提供了有趣的理论基础，并导致人们对利用DNA用于纳米技术、材料科学、计算和生物技术目的的兴趣日益增长。[1]通过利用简单的沃森-克里克配对将常规DNA分子应用于这些目的，已经在产生令人印象深刻的各种拓扑二维和三维几何形状、纳米机械装置和超分子组装体方面取得了相当大的进展。[2]此外，DNA表现出独特的模板性质，这允许其通过DNA聚合酶的酶促复制和扩增，并使得能够通过体外进化开发基于核酸、治疗剂和催化剂的传感器。[3]dsDNA在纳米技术中的应用范围将大大扩展，如果DNA分子可以以碱基特异性的方式进行修饰，并具有额外的化学功能，这些功能可以用于除了沃森-克里克配对之外的其他功能和相互作用。为了获得最大的灵活性，重要的是开发允许在给定DNA链内尽可能多的碱基位置进行修饰而不干扰碱基配对的策略。为了实现这种纳米结构系统的复制和扩增，必须通过体外进化产生耐受广泛的底物和模板的DNA聚合酶。[4]或者，必须找到使酶能够加工通常不被复制的非天然模板和单体的反应条件。这将打开合成更长的DNA构建体（> 100 bp）的可能性，这些构建体通常是合成无法获得的。目前已经合成了一系列化学修饰的2-脱氧核苷三磷酸，并通过引物延伸或聚合酶链式反应（PCR）实现了两种不同修饰核苷酸的模板定向酶促聚合。[5，6]可以使用常规DNA作为模板，通过碱基修饰的脱氧核苷三磷酸（dNTP）的酶促引物延伸来产生其中每个碱基被修饰有额外功能性的单链高密度功能化DNA（fDNA）。[7，8]含有残基1-3和8的单链fDNA（方案1）又可以用作PCR中的模板
The specific pairing of DNA bases in double-stranded DNA (dsDNA) provides an intriguing rationale for the straightforward generation of molecular assemblies on the nanometer scale and has led to growing interest in the exploitation of DNA for nanotechnology, material science, computing, and biotechnology purposes.[1] The application of conventional DNA molecules for these purposes by utilization of simple Watson–Crick pairing has already led to considerable progress in the generation of an impressive variety of topological two-and three-dimensional geometries, nanomechanical devices, and supramolecular assemblies.[2] In addition, DNA exhibits unique template properties which allows for its enzymatic replication and amplification by DNA polymerases and enables the development of sensors based on nucleic acids, therapeutics, and catalysts by in vitro evolution.[3] The scope of applications of dsDNA in nanotechnology would be greatly expanded if DNA molecules could be modified in a base-specific fashion with additional chemical functionalities that could be employed for other functions and interactions apart from Watson–Crick pairing. For maximal flexibility, it would be important to develop strategies that allow for the modification of as many base positions as possible within a given DNA strand without interfering with base pairing. To achieve replication and amplification of such nanostructured systems DNA polymerases that tolerate a broad range of substrates and templates would have to be generated by invitro evolution.[4] Alternatively, reaction conditions would have to be found that enable enzymes to process unnatural templates and monomers that are usually not replicated. This would open up the possibility to synthesize longer DNA constructs (> 100bp) which are usually inaccessible synthetically. As a consequence, a larger sequence space of functionalized DNA molecules, including amplifiable libraries for in vitro selection experiments, could be generated faster and more easily.A series of chemically modified 2о-deoxynucleoside triphosphates has already been synthesized, and the templatedirected enzymatic polymerization of up to two different modified nucleotides was achieved by primer extension or polymerase chain reaction (PCR).[5, 6] Single-stranded highdensity functionalized DNA (fDNA) in which every base is modified with additional functionality can be generated by enzymatic primer extension of base-modified deoxynucleoside triphosphates (dNTPs) using conventional DNA as a template.[7, 8] Single-stranded fDNAs containing residues 1–3 and 8 (Scheme 1) can in turn serve as templates in PCR