严浩继Science后解析人造DNA克隆技术文章登PNAS

　　

生物通报道，来自亚利桑那州大学生物与化学研究所以及纽约大学化学系的科学家已经成功地在活体细胞内克隆人造DNA，该成果发表在最新一期的PNAS上。

 

这是严浩副教授自本年1月发表Sciencewe文章后的又一DNA纳米结构文章。（详细成果参见：华人科学家《科学》发布革新性新技术

http://www.ebiotrade.com/newsf/2008-1/2008114173730.htm）

 

用人工条件模拟天然的生物物质对科学家来说是一项具有很大困难的挑战，也是科学家的终极目标。众所周知，DNA是生命体细胞中携带遗传物质的分子，在天然的细胞中，DNA具有自我复制的能力。

 

今天，尽管人类在DNA纳米技术方面取得了骄人的成绩，但是使用一种通用的方法来复制DNA纳米结构以及模拟DNA的二级结构仍然是我们有待解决的问题，也是科学家们一直都很感兴趣的课题。先前的研究已经利用酶成功地在体外复制DNA，构建出具有天然结构的人造DNA，因此，科学家推测，有可能用病毒作为媒介克隆DNA。

 

在本文中，严浩（生物通译，Hao Yan）带领的研究小组，制备了一种新的系统，在这种系统中，单链DNA纳米结构（Holliday junction or paranemic cross-over DNA）可插入到噬菌体里，通过辅助噬菌体的帮助转化成Blue cell，并具DNA有扩增的功能。使用标准的分子生物学而技术可以很容易地获得大量的克隆纳米生物结构拷贝。

 

对人造克隆的纳米结构是否真的具有扩增的能力可通过多种的途径来验证，比如说，非变性的PAGE电泳，Ferguson分析，内切核酸酶Ⅶ分析。

 

研究者发现，该系统可以在活细胞中复制出具有不动四价键的Holliday junction。

 

人类离人造细胞，人造生命又近了一步。

 

原文摘要：In vivo cloning of artificial DNA nanostructures

 

【Abstract】

Mimicking nature is both a key goal and a difficult challenge for the scientific enterprise. DNA, well known as the genetic-information carrier in nature, can be replicated efficiently in living cells. Today, despite the dramatic evolution of DNA nanotechnology, a versatile method that replicates artificial DNA nanostructures with complex secondary structures remains an appealing target. Previous success in replicating DNA nanostructures enzymatically in vitro suggests that a possible solution could be cloning these nanostructures by using viruses. Here, we report a system where a single-stranded DNA nanostructure (Holliday junction or paranemic cross-over DNA) is inserted into a phagemid, transformed into XL1-Blue cells and amplified in vivo in the presence of helper phages. High copy numbers of cloned nanostructures can be obtained readily by using standard molecular biology techniques. Correct replication is verified by a number of assays including nondenaturing PAGE, Ferguson analysis, endonuclease VII digestion, and hydroxyl radical autofootprinting. The simplicity, efficiency, and fidelity of nature are fully reflected in this system. UV-induced psoralen cross-linking is used to probe the secondary structure of the inserted junction in infected cells. Our data suggest the possible formation of the immobile four-arm junction in vivo.

生物通  张欢

Assistant Professor
Ph.D., New York University, 2001

Office: Biodesign A 124B Lab: Biodesign A 131
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Research Interests

Our research program is highly interdisciplinary which combines Chemistry, Biology, Physics and Material Science. Our goal is to achieve programmed design and assembly of biologically inspired nanomaterials and to explore its applications in nanoelectronics, controlled macromolecular interactions and biosensing. Our research is focused in the following four themes:

1) Bio-Nanotechnology: Design of novel DNA nanostructures, implementation of the designed structure in the construction of patterned DNA arrays and nanomechanical devices. Develop modular methods to achieve biomimetic molecular motors.

2) Nanoelectronics: Utilize rationally designed DNA nanostructure to template nanoelectronic components such as nanoparticles or carbon nanotubes into functional nanodevices.

3) Macromolecule Structure Elucidation: Develop methods to self-assemble 2D and 3D protein arrays for structural determination using Electron Microscopy or X-ray Crystallography.

4) Biomolecular Imaging: Investigation of protein-DNA interactions using high resolution imaging technology such as Atomic Force Microscopy and Electron Microscopy.

Major techniques in our group include: DNA/RNA/Protein manipulation (gel electrophoresis, labeling, hybridization, PCR and footprinting, cloning), electron-beam lithography, Atomic Force Microscopy (AFM), Scanning Electron Microscopy (SEM), Transmission Electron Microscopy (TEM), Electron Microscopy (EM), Fluorescence Spectroscopy, UV-Vis, Circular Dichroism (CD) and chemical synthesis.