摘要
磁镊是一种高精度的单分子技术,它用磁场对连有生物大分子的超顺磁球产生磁力,通过追踪磁球的位置来测量生物大分子的长度信息.磁镊包括横向磁镊和纵向磁镊.纵向磁镊空间精度高,但昂贵;横向磁镊简单便宜,但由于受其成像原理的限制,一般情况下只能连接较长的DNA等生物大分子,且其空间精度较差,进而限制了其应用范围.为了解决这个问题,本文改进了横向磁镊,用片层光照明的方法使光线主要被磁球散射,从而能够直接观察到吸附在样品槽侧壁上的磁球,这使得测量短连接的底物成为可能.对于实际应用的检测,首先测试了包含270 bp发卡结构的0.5μm双链DNA,用其中发卡结构的"折叠-去折叠"跳变过程证明了改进后的横向磁镊的确可以追踪短DNA等生物大分子.然后,进一步用16μm的λ-DNA检验了实验系统.最后,将新型横向磁镊与普通横向磁镊及纵向磁镊在小力和大力条件下拉伸不同长度DNA的噪声进行了比较,发现改进后的横向磁镊在空间精度上明显优于普通横向磁镊,与纵向磁镊相比也无明显差异.以上结果证明了改进后的横向磁镊的精度优势,并扩展了横向磁镊的应用范围.
Magnetic tweezers are a high precision single-molecule manipulation instrument. A gradient magnetic field is used to generate a force on the order of p N, acting on biomolecule-tethered superparamagnetic beads and to manipulate them.By tracking the bead with an inverted microscope, an imaging system and an image process software, one can obtain the extension length information of the biomolecules, thus can study the mechanism and dynamics of the molecules at a single molecule level. Magnetic tweezers include transverse magnetic tweezers（TMT） which are cheap and simple, and longitudinal magnetic tweezers（LMT） which are expensive and complicated. As the traditional TMT can only track the long biomolecule-tethered beads and their spatial resolution is poorer than that of the LMT according to the error theory of magnetic tweezers and the experimental results, the TMT is not so widely used. To solve this problem, we utilize a light sheet to illuminate the beads only in TMT, and then observe the bead sticking on the lateral surface. The tracking error on the extension axis is 4 nm, which is very small. Then we track and obtain the ＂folding-unfolding＂ state transition trace of a hairpin DNA. The hairpin DNA is inserted into a 0.5 μm ds DNA. This experiment proves its ability to study short DNA, RNA or protein. Instead of the fully folded and unfolded state, we observe a semi-stable state at the 1/3 length of the hairpin. The semi-stable state is precisely at the place of the CG rich area of the hairpin, so the CG rich area should be the reason for the semi-stable state. Then we use the 16 μm λ-DNA to further test the novel TMT system. Having obtained the stretching curve of the ds DNA, we fit the length-force data with the worm-like-chain model. The fitted persistence length of the ds DNA is（47 ± 2） nm, which is consistent with the result in the literature.Finally, we compare the noise of traditional TMT, novel TMT and LMT with that of short and long ds DNA at weak and strong force, and we find that at weak force, the novel TMT distinctly enhances the resolution to the LMT level; while at strong force, the resolution of the novel TMT is about half that of the LMT. The results above prove that（1） the short DNA, RNA or protein can be studied by the novel TMT, which extends the application scope of the instrument;（2） the resolution of TMT is enhanced distinctly under weak and strong force, making the novel TMT competent of more experiments.
作者
马建兵
翟永亮
农大官
李菁华
付航
张兴华
李明
陆颖
徐春华
Ma Jian-Bing;Zhai Yong-Liang;Nong Da-Guan;Li Jing-Hua;Fu Hang;Zhang Xing-Hua;Li Ming;Lu Ying;Xu Chun-Hua(Key Laboratory of Soft Matter Physics,Beijing National Laboratory for Condensed Matter Physics,Institute of Physics Chinese Academy of Sciences,Beijing 100190,China;School of Physical Sciences,University of Chinese Academy of Sciences,Beijing 100049,China;Institute for Advanced Studies,College of Life Sciences,Wuhan University,Wuhan 430072,China)
出处
《物理学报》
SCIE
EI
CAS
CSCD
北大核心
2018年第14期247-254,共8页
Acta Physica Sinica
基金
国家自然科学基金(批准号:11574381
11574382)资助的课题~~
关键词
横向磁镊
片层光照明
单分子
transverse magnetic tweezers
light sheet illumination
single molecule
