基于纳米操纵技术的单分子DNA分子手术:切割、拾取及连接酶分子传递

Single Molecule DNA Nanomanipulation Techniques:Dissection,Pick-up and Molecular Transferring of Ligase

该文利用一种基于原子力显微镜(AFM)抬高模式(Lift mode)的"逐线反馈纳米操纵"技术成功地进行了DNA单分子水平上的切割、拾取及连接酶分子的传递,系统地完成了DNA的分子手术。在切割和拾取过程中,以PBR322/Pst I DNA为研究对象,进行了单分子水平上的切割,实验发现由于DNA分子本身弹性,切割过程极易出现切割端变粗的现象。在分子传递过程中,分别以T4 DNA连接酶和小牛胸腺组蛋白分子为研究对象,实现针尖和基底表面之间的分子传递。同时通过控制针尖运动成功获得连接酶分子点阵排列及小牛胸腺组蛋白方块状纳米结构。结果表明利用此操纵方法可以获得很高的精确度,切割DNA时的空间精确度小于5 nm。系列单分子水平上的分子手术的整合为实现单分子水平上生化反应,甚至构造智能机器提供了可能。

With the appearance and development of scanning probe microscopy techniques, it was possible to manipulate single molecule. As the core material of life and carriers of genetic information, single DNA molecule continues to attract widespread interest among researchers. It is significant to carry out single molecule DNA nanomanipulations including dissection, pick up and molecular transferring. In this paper, dissection, pick-up and molecule transferring were successfully carried out by using the line-by-line feedback nanomanipulation under the AFM Lift mode. Dissection and pick＂ up process were done by using PBR322/Pst I DNA as objective molecule. Experimental results showed the dissection ends of DNA molecule became larger due to the elasticity of DNA molecule. Also, T4 DNA ligase molecules could be transferred from the AFM tips onto the modified mica. Through the tip movement, dot patterns of T4 DNA ligase were successfully obtained by dip pen nan- olithography（DPN）. Then by using calf thymus histone molecules as ink, the square nanostructure was also obtained. Experimental results showed that the nanomanipulation method was accurate and the dissection space resolution less than 5 nm could be achieved. The integration of molecular surgery including dissection, pick up and molecular transferring provided the possibility to achieve single DNA biochemical reaction, even to construct the intelligent machine.