Seelig Georg, Yurke Bernard, Winfree Erik
Department of Applied Physics, California Institute of Technology, Pasadena, California 91125, USA.
J Am Chem Soc. 2006 Sep 20;128(37):12211-20. doi: 10.1021/ja0635635.
Practically all of life's molecular processes, from chemical synthesis to replication, involve enzymes that carry out their functions through the catalytic transformation of metastable fuels into waste products. Catalytic control of reaction rates will prove to be as useful and ubiquitous in nucleic-acid-based engineering as it is in biology. Here we report a metastable DNA "fuel" and a corresponding DNA "catalyst" that improve upon the original hybridization-based catalyst system (Turberfield et al. Phys. Rev. Lett. 90, 118102-1-118102-4) by more than 2 orders of magnitude. This is achieved by identifying and purifying a fuel with a kinetically trapped metastable configuration consisting of a "kissing loop" stabilized by flanking helical domains; the catalyst strand acts by opening a helical domain and allowing the complex to relax to its ground state by a multistep pathway. The improved fuel/catalyst system shows a roughly 5000-fold acceleration of the uncatalyzed reaction, with each catalyst molecule capable of turning over in excess of 40 substrates. With k(cat)/K(M) approximately 10(7)/M/min, comparable to many protein enzymes and ribozymes, this fuel system becomes a viable component enabling future DNA-based synthetic molecular machines and logic circuits. As an example, we designed and characterized a signal amplifier based on the fuel-catalyst system. The amplifier uses a single strand of DNA as input and releases a second strand with unrelated sequence as output. A single input strand can catalytically trigger the release of more than 10 output strands.
实际上，生命的所有分子过程，从化学合成到复制，都涉及酶，这些酶通过将亚稳态燃料催化转化为废物来执行其功能。事实将证明，反应速率的催化控制在基于核酸的工程中与在生物学中一样有用且普遍存在。在此，我们报告了一种亚稳态DNA“燃料”和一种相应的DNA“催化剂”，它们在原始的基于杂交的催化剂系统（Turberfield等人，《物理评论快报》90，118102 - 1 - 118102 - 4）的基础上提高了两个多数量级。这是通过鉴定和纯化一种具有动力学捕获的亚稳态构型的燃料来实现的，该构型由侧翼螺旋结构域稳定的“亲吻环”组成；催化剂链通过打开一个螺旋结构域并允许复合物通过多步途径弛豫到其基态来发挥作用。改进后的燃料/催化剂系统显示出未催化反应的加速约5000倍，每个催化剂分子能够转化超过40个底物。由于k(cat)/K(M)约为10(7)/M/分钟，与许多蛋白质酶和核酶相当，这种燃料系统成为一种可行的组件，能够实现未来基于DNA的合成分子机器和逻辑电路。例如，我们设计并表征了一种基于燃料 - 催化剂系统的信号放大器。该放大器使用单链DNA作为输入，并释放一条具有不相关序列的第二条链作为输出。一条单输入链可以催化触发释放超过10条输出链。
