Laser Trapping of an Individual DNA Molecule Folded Using Various Condensing Agents
Laser Trapping of an Individual DNA Molecule Folded Using Various Condensing Agents
复制标题
使用各种凝聚剂折叠的单个 DNA 分子的激光捕获
DOI：
10.1021/ja993038o
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发表时间：
1999
影响因子：
15
通讯作者：
A. Mizuno
中科院分区：
文献类型：
--
作者：
Y. Matsuzawa;K. Hirano;K. Mori;S. Katsura;and Kenichi Yoshikawa;A. Mizuno
Conformational change of DNA from a coiled state into a globular state is induced by various condensing agents, such as PEG (poly (ethylene glycol)) in the presence of low-molecular weight cation, inorganic multivalent cation, polyamine, cationic surfactant, etc. 1 The DNA strand is formed into a hexagonal arrangement 2-4 and shaped into a toroidal or a rod structure by condensing agents, 5, 6 so that the effective volume of the DNA decreases on the order of∼ 10-4. Thus far, most studies concerning laser trapping of a biopolymer have been carried out by anchoring the polymer chain to a micrometer-sized latex bead. 7 Recently, however, effective laser trapping without the use of a latex bead has been shown to be possible for compacted DNA. 8, 9 Even though various mechanisms by which different chemical agents induce DNA condensation have been examined, the optimum conditions for noninvasive trapping of macromolecules remain unclear. In the present contribution, we report the difference in effectiveness of trapping or manipulating globular DNA for three different condensing agents: PEG6000 (poly (ethylene glycol) 6000, from Nihon Oils and Fats Co Ltd.)-MgCl2, spermidine (Nacalai Tesque Inc.), and CTAB (cetyltrimethylammonium bromide, Tokyo Kasei Kogyo Co., Ltd.). T4 phage DNA (166 kbps, NipponGene) was used in the present study. The DNA was mixed with the condensing agents, DAPI (4′, 6-diamidino-2-phenylindol), as a fluorescent dye, and 2-ME (2-mercaptoethanol), as an anti-oxidant for suppression of photobleaching, in a 20 mM MOPS (3-(N-morpholino) propanesulfonic acid) buffer (pH 7.2). The final concentrations were as follows: 20 mM MOPS buffer, 0.3 µM DNA in nucleotide, 0.6 µM DAPI, 2%(v/v) 2-ME. The samples were maintained for 2 h at room temperature. The critical concentrations necessary to induce DNA condensation completely under each condition were measured by changing the concentration of condensing agents 10 and were found to be [PEG]) 60 mg/mL,[MgCl2]) 30 mM;[spermidine]) 159 µM;[CTAB]) 50 µM. Figure 1 exemplifies trapping on globular DNA using a Nd: YAG laser (1064 nm). In response to the mechanical motion of the microscope stage, the trapped DNA (depicted by the closed arrow) remained in the same position, showing no noticeable change in conformation, whereas the free DNA (depicted by the opened arrow) moved from right to left (Figure 1a-c). When the rate of stage motion exceeded a critical value, the trapped DNA was released from the laser focal point (Figure 1d). 11 Optically trapped DNA can be manipulated in any horizontal direction (as shown in Figure 1). Horizontal force (F) can be estimated by Stokes’ law as follows: where η, R, and V are the viscosity of the solvent, the hydrodynamic radius of the globular DNA, and the critical velocity, respectively. The viscosity of the solvent was measured using a TOKIMEC Visconic ELD viscometer at 22 (0.2 C, and the values obtained in PEG-MgCl2, spermidine, and CTAB are 2.77, 1.06, and 0.95 mPas, respectively. The size of the globular DNA as judged from the fluorescence image is overestimated due to a blurring effect in the fluorescence image. From the measurement of the Brownian motion on the individual DNA molecules using fluorescence microscopy, the time-dependent translational dis-
