Skip Navigation

This Article
Right arrow Abstract Freely available
Right arrow Print PDF (74K) Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (22)
Right arrowRequest Permissions
Right arrow Commercial Re-use Guidelines
for Open Access NAR Content
Google Scholar
Right arrow Articles by Katsura, S.
Right arrow Articles by Mizuno, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Katsura, S.
Right arrow Articles by Mizuno, A.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Nucleic Acids Research Pages 4943-4945  

Direct laser trapping of single DNA molecules in the globular state
Introduction
Materials And Methods
   Optical system
   Visualization of DNA molecules
Results
   DNA phase transition from the coiled state to the globular state
   Laser trap of globular DNA
Discussion
Acknowledgements
References

Direct laser trapping of single DNA molecules in the globular state

Direct laser trapping of single DNA molecules in the globular state

Shinji Katsura*, Ken Hirano, Yukiko Matsuzawa, Kenichi Yoshikawa1 and Akira Mizuno

Department of Ecological Engineering, Toyohashi University of Technology, Toyohashi, Aichi 441-8580, Japan and 1Department of Physics, Kyoto University, Kyoto 606-8502, Japan

Received June 29, 1998; Revised and Accepted September 10, 1998

ABSTRACT

A sharply focused laser is able to trap small particles at the laser focal point due to the difference in refractive index of the particles and that of the surrounding medium. This technique, called laser trapping, can be used to manipulate animal or bacterial cells without any contact and has been widely applied in biological research. However, it has been difficult to trap biological macromolecules such as DNA molecules, because these molecules give a low difference in refractive index and cannot overcome Brownian motion. DNA molecules can be transformed to a condensed globular state. This transformation results in a higher refractive index of DNA due to its increased density. We demonstrate in this paper that a single DNA molecule can be optically trapped using a Nd:YAG laser (1064 nm wavelength) upon transformation from the coiled state to the globular state.

INTRODUCTION

Manipulation of single DNA molecules has been recognized as an important technique for biochemistry and molecular biology. Recently, several different methods have been developed to manipulate a single DNA molecule: (i) laser trapping of a DNA molecule anchored to a polystyrene bead (1-3), which affords us one of the most important techniques to transport and stretch individual DNA molecules; (ii) attaching and stretching DNA molecules on a coverslip modified with chemicals (4-5) or using electrical force (6), applied to mapping of restriction enzyme sites on DNA molecules; (iii) attaching and removing with a STM tip (7). In spite of these developments, it has been difficult to trap a single DNA molecule optically without first attaching it to a bead. Optically trapped objects require higher refractive indices than that of the surrounding medium, however, DNA molecules in the coiled state do not have a great enough difference in refractive index.

Condensation of DNA induces a higher refractive index due to increased density. Many studies have been made on DNA condensation using condensing agents (8-11). For example, polyethylene glycol (PEG) in combination with low molecular weight inorganic salt induces DNA transformation from the coiled to the globular state. The transformation is denoted [Psi] (polymer-and-salt-induced) condensation. The globular DNA string is arranged as a hexagonal, closed packed structure (12-13). [Psi] condensation can be induced by several kinds of polymers and salts. It has been reported that the conformation of DNA can be changed from the coiled to the globular state with increasing concentrations of PEG or MgCl2 and the DNA condensation occurs in a right handed manner, represented as [Psi](+) condensation (14).

We demonstrate that a single globular DNA molecule can be optically trapped using a Nd:YAG laser (1064 nm) and that globular transformation of the DNA is essential for laser trapping.

MATERIALS AND METHODS

Optical system

Figure 1 depicts the experimental apparatus for DNA manipulation. Fluorescently stained DNA molecules were observed using a Carl Zeiss inverted microscope (Axiovert 135 TV) equipped with a 100×, 1.3 numerical aperture oil immersion objective lens and with a high sensitivity Hamamatsu SIT (silicon-intensified target) camera. Images were recorded on video tape. The 1064 nm Nd:YAG laser beam, expanded to a diameter of 7.5 mm, was introduced into the microscope through a dichroic mirror. The beam was focused in the object plane of the fluorescence image by the objective lens to trap DNA molecules optically. Approximately 180 mW (measured at the back of the objective lens) of 1064 nm light was used to trap globular DNA.


Figure 1. Schematic diagram of the laser trapping system. The optical trap is placed in the object plane of the objective by adjustment of the collimation of the expanded Nd:YAG laser beam. A globular DNA molecule can be trapped at the focus of the laser and be positioned in the object plane by movement of the x-y stage. DM1 and DM2, dichroic mirrors; ND, neutral density filter; RM, reflective mirror; BF1-BF3, barrier filters. BF1 lets through only the excitation light and BF2 only the emission light. BF3 eliminates scattered light of the Nd:YAG laser.

Visualization of DNA molecules

To determine the dependence of the DNA structure on the concentration of PEG and MgCl2 under our experimental conditions, we observed the fluorescent image under the microscope. The samples were prepared as follows. The PEG (average molecular weight 6000; Nihon Oils and Fats Co. Ltd) was dissolved in a sodium phosphate buffer (pH 7.2) and mixed with T4 phage DNA (166 kb), 4[prime],6-diamidino-2-phenylindole (DAPI), a fluorescent DNA groove-binding dye, and 2-mercaptoethanol (2-ME), an antioxidant to suppress photobleaching. The final concentrations were as follows: 20 mM sodium phosphate buffer, 0.6 µM DNA (nucleotide concentration), 0.6 µM DAPI and 4% (v/v) 2-ME. The concentrations of PEG and MgCl2 were chosen depending on the experimental conditions.

RESULTS

DNA phase transition from the coiled state to the globular state

Figure 2 shows the dependence of the state of DNA molecules on the concentrations of PEG and MgCl2. The state of the DNA was determined by observing its fluorescence image. We observed three distinct regions denoted here as: A, coiled; B, coexistent coiled/globular; C, globular. In region B, the transition between the coiled and the globular DNA could be plainly distinguished by observing the DNA molecules. This behavior of the transition is similar to that in PEG/NaCl systems (15), in which an individual coiled DNA molecule undergoes a first order phase transition to the globular state (16). Magnesium cations in PEG solution more readily induce DNA condensation compared with sodium cations. For instance, in 60 mg/ml PEG solution, a NaCl concentration >450 mM is necessary to change DNA completely into the globular structure (15), whereas a MgCl2 concentration of 40 mM is sufficient for complete transformation. This is an important advantage whenever globular DNA is to be manipulated using electric fields, because higher ionic strengths cause increased heat generation that can perturb the motion of the DNA molecules. Techniques that combine laser trapping and electrostatic forces are effective for precise manipulation, because one can control not only position but also orientation of molecules (17).


Figure 2. Dependence of DNA structure on the concentration of PEG and MgCl2. Open circles indicate the critical concentration required for DNA transformation between coiled and coiled/globular state coexistence. Filled circles indicate the critical concentration for DNA transformation between globular and coiled/globular coexistence. Before observation, the sample was shaken and stood at room temperature for 25 min for complete mixing and then allowed to sit for 10 min. In PEG/MgCl2, the state of the DNA is represented by: (A) coiled; (B) coiled/globular coexistence; (C) globular. Some coiled DNA was observed whenever the PEG concentration was <10 mg/ml.

Laser trap of globular DNA

Globular DNA induced by PEG/MgCl2 was optically trapped using 180 mW of 1064 nm light from a Nd:YAG laser. The infrared laser induces less damage to biological molecules such as DNA compared with visible light from krypton ion or argon ion lasers (17). Under the conditions [PEG] = 60 mg/ml and [MgCl2] = 50 mM, all of the DNA existed in the globular state; no coiled DNA was observed. Figure 3 shows laser trapping of a globular DNA molecule. Free DNA molecules were translated by moving the microscope stage. The globular DNA molecule indicated with white arrows was trapped at the laser focal point as a free DNA molecule moved leftward (Fig. 3a-c) or upward (Fig. 3c-e). At this laser power, it was possible to trap globular DNA, however, coiled DNA could not be trapped. When a coiled DNA molecule arrived at the laser focal point on moving the stage, it was pushed downward by the optical pressure of the laser beam and escaped the trap (data not shown).


Figure 3. Sequential photographs of trapped and free T4 phage DNA molecules in the globular state at t = 0 (a), 0.24 s (b), 0.64 s (c), 0.87 s (d) and 1.14 s (e). Scale bar 5 µm. The position of the optical trap is indicated by white arrows. At t = 0 the microscope stage started to move leftward until t = 0.64 s and the free DNA molecule moved with the stage. During this motion, the optically trapped DNA molecule remained stationary. (f) The trajectories of the free and the trapped DNA molecules are illustrated schematically.

DISCUSSION

We have also succeeded in trapping globular DNA condensed by other agents, such as spermidine and PEG/NaCl (data not shown). These results strongly suggest that condensation of DNA into the globular structure, which may induce a higher refractive index and lower viscosity drag force due to its decreased size, is essential for laser trapping of DNA molecules. Laser trapping of a single supercoiled [lambda] DNA molecule using a krypton ion laser operating at 647 nm was reported by Chiu et al. (18). In their experiment, [lambda] DNA molecules were stained with YOYO dye. We have observed that YOYO dye induces DNA condensation to the globular state within ~30 min in the absence of any condensing agents. This suggests that the mechanism of trapping observed by Chiu et al. might be attributed to YOYO-induced condensation, but the DNA structure is as yet unknown. The globular transformation induced by condensing agents has been extensively studied by means of spectroscopy and microscopy, so that the morphology of and experimental conditions for globular DNA have already been determined (19-21). Laser trapping of a single DNA molecule can possibly be used for mapping. Reversibility of the transformation between the coiled and the globular states is required for this application. The globular DNA in our system could easily be reverted to the coiled state by reducing the PEG or MgCl2 concentration. However, it might be difficult to transform DNA condensed by YOYO reversibly, because YOYO dye binds to DNA molecules with high affinity.

Handling large chromosome size DNA molecules (more than several hundred thousand base pairs) in solution is difficult, because large DNA molecules are easily broken down by mechanical forces, such as shear stress. Recently we reported that globular transformation very effectively suppresses the breakdown of large DNA fragments (22). Large globular DNA fragments can be manipulated using laser trapping without breakage. Furthermore, trapping efficiency increases with increasing fragment size. On reducing the concentration of the condensing agent, the trapped DNA molecule reverts to the coiled form. If a non-uniform electric field is applied during the reversion process, the DNA molecule can be trapped and stretched by the gradient and orientation force due to the non-uniform field. This technique, which we are now developing, enables the preparation of a stretched chromosomal DNA molecule, which might contribute to the development of an in situ hybridization technique for chromosomal DNA and successive preparation of DNA fragments from the terminus. In conclusion, we propose that laser trapping of globular DNA, reversible to the coiled state, will contribute to various fields of biological studies based on single molecules.

ACKNOWLEDGEMENTS

We thank Dr P.M.Goodwin and Dr R.A.Keller of Los Alamos National Laboratory and Dr G.Prieto of Universidad Nacional de Tucuman for valuable discussions and suggestions. This work was supported in part by the Angstrom Technology Partnership of JRCAT, a Grant-in-Aid for Scientific Research of the Ministry of Education Science and Culture Japan (no. 09750875) and JSPS Research Fellowships for Young Scientists.

REFERENCES

1. Perkins,T.T., Quake,S.R., Smith,D.E. and Chu,S. (1994) Science, 264, 822-826. MEDLINE Abstract

2. Smith,S.B., Finzi,L. and Bustamante,C. (1992) Science, 258, 1122-1126. MEDLINE Abstract

3. Nishioka,M., Katsura,S. and Mizuno,A. (1996) Trans. IEE Jpn, 116E, 171-177.

4. Cai,W., Aburatani,H., Stanton,V.P.J., Housman,D.E., Wang,Y.-K. and Schwartz,D.C. (1995) Proc. Natl Acad. Sci. USA, 92, 5164-5168. MEDLINE Abstract

5. Yokota,H., Johnson,F., Lu,H., Robinson,R.M., Belu,A.M., Garrison,M.D., Ratner,B.D., Trask,B.J. and Miller,D.L. (1997) Nucleic Acids Res., 25, 1064-1070. MEDLINE Abstract

6. Kabata,H., Kurosawa,O., Arai,I., Washizu,M., Margarson,S.A., Slass,R.E. and Shimamoto,N. (1993) Science, 262, 1561-1563. MEDLINE Abstract

7. Bustamante,C. (1991) Annu. Rev. Biophys. Biophys. Chem., 20, 415-446.

8. Windom,J. and Baldwin,R.L. (1980) J. Mol. Biol., 144, 431-453.

9. Huey,R. and More,S.C. (1981) Biopolymers, 20, 2533-2552. MEDLINE Abstract

10. Bloomfield,V.A. (1991) Biopolymers, 31, 1471-1481. MEDLINE Abstract

11. Lerman,L.S. (1971) Proc. Natl Acad. Sci. USA, 68, 1886-1890. MEDLINE Abstract

12. Jordan,C.F., Lerman,L.S. and Venable,J.H. (1972) Nature, 236, 67-70.

13. Maniatis,T., Venable,J.H.J. and Lerman,L.S. (1974) J. Mol. Biol., 84, 37-64. MEDLINE Abstract

14. Skuridin,S.G., Damaschun,H., Damaschun,G., Yevdokimov,Yu.M. and Misselwitz,R. (1986) Studia Biophys., 112, 139-150.

15. Vasilevskaya,V.V., Khokhlov,A.R., Matsuzawa,Y. and Yoshikawa,K. (1995) J. Chem. Phys., 102, 6595-6602.

16. Yoshikawa,K. and Matsuzawa,Y. (1996) J. Am. Chem. Soc., 118, 929-930.

17. Nishioka,M., Tanizoe,T., Katsura,S. and Mizuno,A. (1995) J. Electrostatics, 35, 83-91.

18. Chiu,D.T. and Zare,R.N. (1996) J. Am. Chem. Soc., 118, 6512-6513.

19. Bloomfield,V.A. (1996) Curr. Opin. Struct. Biol., 6, 334-341. MEDLINE Abstract

20. Takahashi,M., Yoshikawa,K., Vasilevskaya,V.V. and Khokhlov,A.R. (1997) J. Phys. Chem. B, 101, 9396-9401.

21. Vasilevskaya,V.V., Khokhlov,A.R., Kidoaki,S. and Yoshikawa,K. (1997) Biopolymers, 41, 51-60.

22. Katsura,S., Hirano,K., Yamaguchi,A., Ishii,R., Imayou,H., Matsuzawa,Y. and Mizuno,A. (1997) Conference record of the IEEE Industry Application Society, IEEE 32nd Annual Meeting, 1997. The Institute of Electrical and Electronic Engineers, Inc., New York.

*To whom correspondence should be addressed. Tel: +81 532 44 6919; Fax: +81 532 44 6929; Email: katsura@eco.tut.ac.jp

This page is run by Oxford University Press, Great Clarendon Street, Oxford OX2 6DP, as part of the OUP Journals
Comments and feedback: www-admin{at}oup.co.uk
Last modification: 17 Oct 1998
Copyright©Oxford University Press, 1998.
Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 Biophys. JHome page
C. Douarche, J.-L. Sikorav, and A. Goldar
Aggregation and Adsorption at the Air-Water Interface of Bacteriophage {phi}X174 Single-Stranded DNA
Biophys. J., January 1, 2008; 94(1): 134 - 146.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Print PDF (74K) Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (22)
Right arrowRequest Permissions
Right arrow Commercial Re-use Guidelines
for Open Access NAR Content
Google Scholar
Right arrow Articles by Katsura, S.
Right arrow Articles by Mizuno, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Katsura, S.
Right arrow Articles by Mizuno, A.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?