A unidirectional DNA walker that moves autonomously along a track
A unidirectional DNA walker that moves autonomously along a track
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DOI：
10.1002/anie.200460522
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发表时间：
2004-01-01
影响因子：
16.6
通讯作者：
Reif, JH
中科院分区：
文献类型：
--
作者：
Yin, P;Yan, H;Reif, JH
关于步行者;右侧显示了每个阶段每个锚定末端的碱基序列，以及这些碱基序列如何通过酶的作用转化。步行者的运动取决于交替的酶连接和限制（切割）。在运动开始之前，其位置由 * 指示的步行者驻留在锚定点A处，如图1c的面板0所示。在这种状态下，锚定物A* 和B具有互补的粘性末端，它们可以彼此杂交。然后T4连接酶可以在新杂交的切片的每一端密封切口，从而共价连接两个锚定（A*+ B！A* B）;这是消耗由ATP水解提供的能量的不可逆步骤。A* B的连接产生内切核酸酶PflM I的识别位点。在过程II中，PflM I以这样的方式切割A* B，即步行者移动到锚点B：A* B！A+ B*。然后锚定物B* 的粘性末端可以与锚定物C的互补粘性末端杂交，并且在过程III中将两个锚定物连接以形成B* C。连接产物B* C含有第二内切核酸酶BstAP I的识别位点。在过程IV中，B* C被BstAP I切割以再生锚定B并产生C*。因此，步行者从固定点B移动到C以完成步行者的自主的、编程的运动。步行者的运动是单向的：两个相邻锚定件之间的连接产物只能被切割，使得步行者移动到下游锚定件上（A* B和B* C只能被切割，使得步行者分别保持连接到B和C）。有两个空转步骤：B* 可与A重新连接并通过PflM I的限制再生;类似地，C* 可与B重新连接并通过BstAP I再生。然而，这些空转步伐既不逆转也不阻挡步行者的整体单向运动。一旦B* 与C连接，步行者就再也不能回到A。通过使用变性聚丙烯酰胺凝胶电泳（PAGE）追踪放射性标记的步行者的运动，验证了步行者的自主和单向运动。通过测量标记的DNA片段的大小，可以确定在不同的酶组合存在下步行者到达的位置。图2a是实验设计的示意图。步行者（红色）的5 '端用gP 32标记，在图2a中用红点表示。最初，标记的链（A* 的一部分）是52个核苷酸长。过程I、II、III和IV的完成可以通过分别对应于68、19、57和41个核苷酸的放射性标记的DNA片段的条带的出现来检测。这些条带的出现对应于放射性标记片段在锚定点之间沿轨道沿着的转移。将该系统在378 ℃下在补充有ATP和牛血清白蛋白（BSA）的杂交缓冲液中在不同的酶组合存在下孵育，所述酶组合同时加入到系统中。图2b是变性凝胶的放射自显影图，其显示在每个反应期间形成的产物。泳道1中的系统是没有酶或ATP的对照反应。在泳道2中存在T4连接酶和ATP：预期步行者完成过程I以产生68个核苷酸的放射性标记链，因此对应于形成
of the walker; on the right-hand side the base sequence at the end of each anchorage at each stage is shown, as well as how these base sequences are transformed by the action of enzymes. The motion of the walker depends on alternate enzymatic ligation and restriction (cleavage). Before the motion starts, the walker, whose position is indicated by*, resides at anchorage A, as shown in panel 0 of Figure 1c. In this state anchorages A* and B have complementary sticky ends, which can hybridize with each other. T4 ligase can then seal the nicks at each end of the newly hybridized section, thus joining the two anchorages covalently (A*+ B! A* B); this is an irreversible step that consumes energy provided by the hydrolysis of ATP. The ligation of A* B creates a recognition site for endonuclease PflM I. In process II, PflM I cleaves A* B in such a way that the walker moves to anchorage B: A* B! A+ B*. The sticky end of anchorage B* can then hybridize with the complementary sticky end of anchorage C, and the two anchorages are ligated to form B* C in process III. The ligation product B* C contains a recognition site for the second endonuclease BstAP I. In process IV, B* C is cleaved by BstAP I to regenerate anchorage B and create C*. Thus, the walker moves from anchorage B to C to complete the autonomous, programmed motion of the walker. The motion of the walker is unidirectional: the product of ligation between two neighboring anchorages can only be cleaved such that the walker moves onto the downstream anchorage (A* B and B* C can only be cut such that the walker is left attached to B and C, respectively). Two idling steps are possible: B* can be religated to A and regenerated by restriction by PflM I; similarly, C* can be religated to B and regenerated by BstAP I. However, these idling steps neither reverse nor block the overall unidirectional motion of the walker. Once B* has been ligated to C the walker can never return to A.The autonomous and unidirectional motion of the walker was verified by using denaturing polyacrylamide gel electrophoresis (PAGE) to track the motion of the walker, which was radioactively labeled. The position reached by the walker in the presence of different combinations of enzymes can be determined by measuring the size of the labeled DNA fragment. Figure2a is a schematic drawing of the experimental design. The 5’end of the walker (red) was labeled with gP 32, represented by a red dot in Figure 2 a. Initially, the labeled strand (part of A*) is 52 nucleotides long. The completion of processes I, II, III, and IV can be detected by the appearance of bands corresponding to radioactively labeled DNA fragments of 68, 19, 57, and 41 nucleotides, respectively. The appearance of these bands corresponds to the transfer of the radioactively labeled fragment between the anchorages along the track. The system was incubated at 378C in hybridization buffer supplemented with ATP and bovine serum albumin (BSA) in the presence of different combinations of enzymes, which were added to the system simultaneously. Figure 2b is an autoradiograph of a denaturing gel which shows the products formed during each reaction. The system in lane 1 is the control reaction without an enzyme or ATP. In lane 2 T4 ligase and ATP are present: The walker is expected to complete process I to produce a radiolabeled strand of 68 nucleotides, thus corresponding to the formation