华裔教授《自然》子刊：最新技术解开奥秘

　　生物通报道：来自伊利诺斯大学厄巴纳香槟分校（University of Illinois at Urbana-Champaign），霍德华休斯医学院，以及埃默里大学（Emory University）的研究人员利用一种高度敏感的技术发现了一种铅特异性脱氧核酶（a lead-specific DNAzyme）利用“锁-钥匙”模式反应的机制，这不同于其它的脱氧核酶，由于存在锌离子或镁离子，同样的脱氧核酶利用的是“诱导契合（induced fit）”模式机制，这与核酶（ribozyme)是相似的。

这一研究成果公布在《Nature Chemical Biology》杂志上，文章的通讯作者是伊利诺斯大学的鲁毅（Yi Lu，音译）教授，以及HHMI知名的研究员Taekjip Ha教授，前者早年毕业于北京大学，主要研究兴趣在于研究蛋白和核酸分子作用过程中金属离子的功能（简介见下）。

Yi Lu表示，“锁-钥匙机制解释了为什么这种铅特异性的脱氧核酶能进行如此灵敏和选择性强的应答”，“加深结构改变和反应之间关系的了解对于我们进一步研究脱氧核酶如何工作的，以及设计更有效的感应器来说都是十分重要的。”

早在20世纪80年代，科学家们发现RNA分子能催化酶反应，并将之命名为核酶，之后又发现DNA也可以作为一种酶作用，命名为脱氧核酶(deoxyribozyme或DNAzyme)，其发现是人类对于酶的认识的又一次重大飞跃。

核酸类酶（nucleic acid enzymes）仅仅只有四种核苷分子——不用于蛋白有20种，因此也许需要补充一些辅助分子（cofactors），其中金属离子就是一种天然的选择，而且实际上大部分核酸类酶都需要金属离子辅助生理条件下的酶反应（因此也称为金属酶）。

金属酶利用不同的模式行使功能，包括金属依赖性结构改变（诱导契合模式），以及另外一些不需要结构改变的模式（锁-钥匙模式）。相反，大部分核酶都需要在酶反应之前发生结构改变。

研究人员在一种称为单分子荧光共振能量转移（single-molecule fluorescence resonance energy，介绍见下）技术的基础上在靶标分子上加上了两种染料分子——绿色和红色，然后用激光激活，这样一些能量从绿色染剂转移到了红色染剂，转移的多少依赖于两种染剂的距离。

Ha表示，“两者强度的改变比例就说明了两种染剂分子的相对运动”，“通过模拟这两种染剂的明亮程度，我们就可以在纳米精度上检测结构改变了。”

这样研究人员发现，当存在锌离子或镁离子的时候，脱氧核酶就会发生结构改变，之后即进行剪切反应（类似于许多蛋白和核酶），但是当存在铅的时候，这种剪切反应就不需要结构变化。

Lu表示，“这证明了铅特异性酶利用的是锁-钥匙反应机制”，“这种脱氧核酶好像预先接受了铅。”

“我们认为这一研究结果说明更快更灵敏的感应器是属于锁-钥匙模式机制的”，“下一步我们将需要其它采用锁-钥匙模式的金属离子特异性脱氧核酶，而且，我们希望研究金属绑定位点的结构细节，以及观测在催化过程中，它们是如何变化的。”
（生物通：张迪）

原文摘要：
Nature Chemical Biology
Published online: 28 October 2007 | doi:10.1038/nchembio.2007.45
Dissecting metal ion–dependent folding and catalysis of a single DNAzyme
『Abstract』

荧光共振能量转移( FRET)是用于对生物大分子之间相互作用定性、定量检测的一种有效方法。根据所基于的荧光显微镜配置不同而有不同的应用侧重，可在溶液，细胞悬液，多细胞，单细胞，细胞膜，细胞器等不同层次对生物大分子间的相互作用距离，动力学特性等进行研究。

一般系综测量结果表示的是大量由一种或多种对象组成的一个整体所表现出来的平均效应和平均值。这一平均效应掩盖了许多特殊的信息。而这些特殊的信息有时是非常重要的，尤其在研究具有非均匀特性的凝聚相物质和生物大分子结构时。

而相比之下，单分子检测就可做到对体系中单个分子的行为进行研究，可以得到在特定时刻，特定分子的特殊位置和行为，因为在某一时刻，集团中的任何成员只能处于一种状态。将此再与时间相关，还可得到单个分子的行为的分布状况。这样我们就可以同时得到所研究的对象的整体行为和个体行为了，然后将数据综合处理，得到更为全面的信息。

单分子检测技术有别于与一般的常规检测技术，观测到的是单个分子的个体行为，而不单分子检测技术是大量分子的综合平均效应。近年来随着相关学科的技术进步，单分子研究已经在从分子生物学到细胞生物学等生命科学领域有了迅速的发展和应用。

附：
Yi Lu
Associate Professor of Chemistry
Department of Chemistry
University of Illinois at Urbana-Champaign


A322 Chemical & Life Sciences Lab
600 South Mathews Avenue
Urbana, IL 61801
Phone: [phone]
Fax: [phone]
Email: [e-mail]

Education

B.S. 1986 Beijing University, P.R. China
Ph.D. 1992 University of California, Los Angeles
Postdoc. 1992-1994 California Institute of Technology

Yi Lu received his B.S. from Beijing University, P. R. China in 1986 and his Ph.D. from University of California, Los Angeles in 1992. After two years of postdoctoral research at California Institute of Technology, he joined the faculty at Illinois in 1994. Professor Lu has received the National Science Foundation CAREER award, the National Science Foundation Special Creativity Extension, the National Institute of Health FIRST award, the Arnold and Mabel Beckman Young Investigator Award, the Research Corporation Cottrell Scholar Award, the Camille Dreyfus Teacher-Scholar Award, and an Alfred P. Sloan Research Fellowship. Professor Yi Lu's research interests are in bioinorganic chemistry.

Research
We are interested in elucidating the role of metal ions in proteins and nucleic acid enzymes (DNA and RNA with enzymatic activities), designing metalloenzymes with novel structures and functions, and exploring the use of the enzymes in biotechnological and pharmaceutical applications. To achieve these goals, we are developing new approaches that combine the advantages of both chemical and biochemical systems. We employ knowledge and techniques from various disciplines including biochemistry, inorganic chemistry, organic chemistry, biophysical chemistry as well as molecular biology and biomedical engineering. The multidisciplinary nature of our research projects will allow students to successfully tackle difficult problems in their thesis research and to better prepare for their future independent career.

Metalloprotein Design and Engineering
Metalloenzymes are one of the most important groups of enzymes in nature. Metal ions help catalyze some of the most difficult reactions and fine-tune the reactivity at a level unmatched by non-metalloenzymes. While much progress has been made on the study of native metalloenzymes, little is known about how to design a metalloenzyme with desired structure and activity. We have been using stable, easy-to-produce, and well-characterized proteins as a scaffold for designing and engineering artificial metalloenzymes that either have similar structural and functional properties of much more complex native enzymes, or possess new structure or reactivity that is unprecedented in nature. We have designed and engineered a copper center into azurin and cytochrome c peroxidase that mimics the CuA and CuB site, respectively, in cytochrome oxidase (a terminal oxidase of the aerobic respiratory chain). We have also designed and engineered a manganese-binding site in cytochrome c peroxidase that closely resemble the manganese-binding site in manganese peroxidase (a heme enzyme that has great promise in providing renewable energy and in destroying environmental pollutants). Through rational design, cytochrome c peroxidase was also redesigned to closely mimic a cytochrome P450, a heme enzyme that is responsible for many regio-, stereo- and enantio-selective chemical transformations in biological system and in chemical synthesis.

DNA and RNA Enzymes : A New Class of Metalloenzymes
Another major area of our research is the characterization of metal-binding sites in DNA and RNA enzymes and the design of effective nucleic acid enzyme drugs against HIV virus and other retroviral diseases. The majority of these nucleic acid enzymes require divalent metal ions for its structure and function. It has been demonstrated that metal ions are essential for the catalytic function of nucleic acid enzymes. Therefore, these enzymes constitute a new class of metalloenzymes and studying metallo-nucleic acid enzymes is a new frontier in biochemistry and chemistry. The exact role of metal ions in nucleic acid enzyme catalysis and the structure of the catalytic metal-binding site are unknown. Spectroscopic study of metal-binding sites in ribozymes can provide detailed structural and mechanistic information on ribozyme catalysis. We have developed a new technique for large-scale purification of RNA so that routine spectroscopic study can be carried out. This method combines the high capacity and automation of column chromatography with the high resolution of gel electrophoresis. By using phosphorothioate approach, we became the first group to report the spectroscopic characterization of a catalytically active metal-binding site in a ribozyme. Finally, to increase both the metal-binding affinity for spectroscopy study and the nucleic enzyme reactivity for pharmaceutical application, we have utilized in vitro selection approach and have selected a group of Zn2+-dependent DNA enzymes that has one of the highest activity and metal ion affinity from a library of 1014-1015 random sequences.

Representative Publications

Sigman, J.A., Kwok, B.C., Gengenbach, A., and Lu, Y. (1999) "Design and Creation of a Cu(II)-binding site in Cytochrome c Peroxidase that Mimics the CuB-heme center in Terminal Oxidases," J. Am. Chem. Soc. 121, 8949-8950.
Gengenbach, A., Syn, S., Wang, X., and Lu, Y. (1999) "The Redesign of Cytochrome c Peroxidase into a Manganese Peroxidase: The Role of Tryptophans in Peroxidase Activity," Biochemistry 38, 11425-11432.
Sigman, J.A., Pond, A.E., Dawson, J.H., and Lu, Y. (1999) "Engineering Cytochrome c Peroxidase into Cytochrome P450: A Proximal Effect on Heme-Thiolate Ligation," Biochemistry 38, 11122-11129.
Wang, X., Berry, S.M., Xia, Y., and Lu, Y. (1999) "The Role of Histidine Ligands in the Structure of Purple CuA Azurin," J. Am. Chem. Soc. 121, 7449-7450.
Wang, X. and Lu, Y. (1999) "Proton NMR Investigation of the Heme Active Site Structure of an Engineered Cytochrome c Peroxidase that Mimics Manganese Peroxidase," Biochemistry 38, 9146-9157.
Cunningham, L., Li, J., and Lu, Y. (1998) "Spectroscopic Evidence for Inner Sphere Coordination of Metal Ions to the Active Site of a Hammerhead Ribozyme," J. Am. Chem. Soc. 120, 4518-4519.