NANO LETTERS
Initiating Conformation Transitions of Individual YOYO-Intercalated DNA Molecules with Optical Trapping
2003 Vol. 3, No. 10 1387-1389
Christopher L. Kuyper, Greg P. Brewood, and Daniel T. Chiu* Department of Chemistry, UniVersity of Washington, Seattle, Washington 98195-1700 Received July 24, 2003; Revised Manuscript Received August 12, 2003
ABSTRACT This letter reports that the commonly used DNA intercalator dye YOYO-1 can act as a condensing agent under moderately acidic pH conditions (pH 5.7) for DNA molecules ranging in size from ∼2.7 kbp (pUC19) to ∼105 kbp (T5). With atomic force microscopy, individual YOYOintercalated λ-DNA condensates (∼48.5 kbp) were shown to exhibit a toroidal structure that ranged in diameter from 100 to 150 nm with a maximum height of ∼6 nm. Once these molecules are compacted, optical trapping can be used to initiate the conformational transitions of such single, condensed DNA molecules with excellent time resolution.
DNA molecules with micrometer-scale contour lengths can be tightly packed into nanometer-sized structures using several different polycations and proteins. In nature, for example, lambda phage virus tightly packs 48.5 kilobase pairs (kbp) DNA (contour length ∼17 µm) into the phage head, which measures only ∼50 nm in diameter. Whereas observations in vivo give important insight into DNA packaging processes, in vitro studies provide more detailed information about the structural properties of DNA condensates, which typically form highly ordered toroidal, or donutlike, structures.1 DNA condensation is believed to be induced primarily by an electrostatic neutralization of the negatively charged DNA backbone with biologically relevant DNA-binding multivalent cations such as spermidine3+ or spermine4+, by which repulsive energies are decreased sufficiently to allow for tight packing.1 To investigate the processes underlying DNA condensation, most previous studies have relied on static visualization techniques (i.e., electron microscopy (EM) and atomic force microscopy (AFM)) to obtain high-resolution images of DNA condensates.1-3 Although these approaches provide highinformation-content static images of condensed DNA, little information is offered on the dynamics of the condensation process. The kinetics of DNA condensation and decondensation has been investigated with bulk measurements using dynamic light scattering.4,5 With stop-flow measurements, time resolution in the microsecond regime can be achieved, but studies are limited to DNA fragments of fewer than ∼30 kbp because larger fragments tend to be sheared by the turbulent flow. More recently, single-molecule fluorescence * Corresponding author. E-mail:
[email protected]. Phone: (206) 543-1655. Fax: (206) 685-8665. 10.1021/nl0345668 CCC: $25.00 Published on Web 09/11/2003
© 2003 American Chemical Society
imaging of DNA condensation and decondensation by protamine6 and poly(ethylene glycol)7 has been reported. Optical trapping has been used to translate a DNA/histone H1 complex between high and low ionic strength solution, allowing for direct observation and reversible control of the DNA packing and unpacking process.8 Because of slow mass transfer, which limited these approaches, condensation processes were observed over seconds to minutes. To conduct experiments on the single-molecule level, fluorescent dyes intercalated into the DNA are used for visualization, and nonfluorescent small-molecule condensing agents are used to induce condensation.6 In certain cases, however, the use of fluorescent dyes to visualize condensation by smallmolecule condensing agents can inhibit the packing of DNA.9 Here, we report that the commonly used DNA fluorescent intercalator dye YOYO-1 can act as a condensing agent under moderately acidic pH conditions. Individual YOYO-intercalated λ-DNA molecules (∼48.5 kbp), for example, were collapsed into toroidal structures ranging from 100 to 150 nm in diameter at pH 5.7. Furthermore, using optical trapping, we can initiate discrete conformational transitions of single, condensed YOYO-intercalated DNA molecules to an extended random-coil state that occurs over a time period of ∼150 ms. Owing to the excellent fluorogenic properties of YOYO, the pH-induced condensation of YOYO-intercalated DNAs offers unique advantages and possibilities in studying this process at the single-molecule level; YOYO acts both as a condensing agent and as a fluorescent intercalator. In addition, awareness and understanding of the ability of YOYO to alter significantly the conformation of DNA molecules is important because the dye is a widely used
Figure 1. (A) Extended, random-coil λ-DNA molecule in pH 8 solution. (B) Optically trapped, condensed single pUC19 DNA (∼2.7 kbp), (C) λ-DNA (∼48.5 kbp), and (D) T5 DNA (∼105 kbp) in pH 5.7 solution (scale bar for (A-D): 1 µm). (E) Chemical structure of YOYO-1. The DNA solution contained 10 mM Tris, 1 mM EDTA, 2 mM NaCl, and 0.1% glucose. To reduce the photobleaching of YOYO, 125 µg/mL glucose oxidase and 25 µg/ mL catalase were present in the solution. Dilute HCl and NaOH were used to adjust the pH of the solutions.
intercalator exploited for visualizing and detecting DNA. Separation and fragment sizing of YOYO-labeled DNA, for example, can be critically affected by large changes in DNA conformations.10 With fluorescence microscopy, we have studied the behavior of a wide range of YOYO-intercalated DNA molecules, with sizes ranging from ∼2.7 to ∼105 kbp. Figure 1A shows a fluorescence image of a single λ-DNA molecule in the random-coil state at pH 8. This image appeared blurry because the λ-DNA molecule, driven by Brownian motion, contracted and extended rapidly in solution in comparison with our image integration time (∼50 ms). In this randomcoil form, the λ-DNA measures 3-5 µm in length, but molecules can extend to up to 15-20 µm under shear flow conditions. In contrast, condensed DNAs exhibited no observable internal motion and had noticeably faster rates of translational diffusion in free solution because of their decreased size. Figure 1B-D demonstrates the 3D optical trapping of single pUC19 DNA (∼2.7 kbp), λ-DNA (∼48.5 kbp), and T5 DNA (∼105 kbp), in which the single trapped DNAs were immobilized in free solution. We have observed the pH-induced condensation of YOYO-intercalated DNA (nM-µM bp of DNA) over a wide range of YOYO/bp ratios (e.g., 1:50, 1:20, 1:1, and 3:1) and in different buffers (10 mM Tris and 10 mM borate). Condensation, however, was not achieved until the pH of the solution was lowered to ∼5.7. Typically, YOYO (Figure 1E) is intercalated at ratios of 1:20 to 1:5 dye/bp for studies that require fluorescent staining of DNA.10,11 Because of the poor fluorescence properties of DNA, dynamic light scattering (DLS) was used to determine whether pH-induced condensation occurred without the presence of YOYO. DLS studies revealed essentially no difference in the internal motions of the DNA over a range of scattering angles for λ-DNA (∼45 µM bp) without YOYO at pH 8 and 5.7, which indicated that no condensation occurred in the absence of YOYO. Last, we observed no titration of YOYO or λ-DNA within the range of pH 9 to 3 in a solution of 10 mM Tris, 1 mM EDTA, and 2 mM NaCl. In previous reports, DNA 1388
Figure 2. (A) Contact-AFM image of a toroidal condensate of λ-DNA. AFM was performed in aqueous solution (10 mM Tris, 1 mM EDTA, 2 mM NaCl, pH 5.7). Red arrows correspond to those shown in B (scale bar: 50 nm). (B) Cross-sectional view of the toroid shown in A. This toroid had a maximum height of ∼6 nm above the relatively flat surface of the mica substrate.
structural changes have been controlled by pH when DNA was condensed with spermidine12 and spermine,13 but changes occurred when the pH was basic instead of acidic. Studies into the mechanism behind this phenomenon are still ongoing. To gain detailed structural information about the condensates, contact-mode AFM images were obtained in aqueous solution (10 mM Tris, 1 mM EDTA, 2 mM NaCl, pH 5.7) on an unmodified, atomically flat mica surface. A close-up of a single DNA toroid (Figure 2A) showed that it had a diameter of ∼135 nm and a maximum height of ∼6 nm (Figure 2B). We have observed toroids that ranged from 100 to 150 nm in diameter. Assuming a contour length of the intercalated λ-DNA of ∼20 µm14 and an approximate radius of 1 nm, we found the calculated volume for a single molecule to be ∼60 × 103 nm3. If we treat the DNA toroid as a donutlike disk with an average height of ∼5 nm, an outside radius of ∼67 nm, and an inside radius of ∼20 nm, we find the volume to be ∼65 × 103 nm3. This toroid, therefore, appeared to be composed of a tightly packed single DNA molecule. The AFM images we obtained for the pHinduced YOYO-intercalated DNA condensate showed remarkable similarities to those of spermidine-induced single DNA condensates described in previous reports using AFM.15 In addition to structural characteristics of YOYO/DNA condensates, kinetic studies on the decondensation process can be carried out by using optical trapping to initiate the transition of an individual YOYO-intercalated λ-DNA molecule (∼48.5 kbp) from a tightly packed, condensed state to an extended, random-coil state. Optical trapping has emerged as a useful method for manipulating single DNA molecules in solution, with the help of microbeads as handles.16,17 The direct trapping and manipulation of single globular DNAs in solution was first reported by Zare and co-workers.18 Here, we used optical trapping to initiate the conformational transition of an individual YOYO-intercalated λ-DNA molecule from a tightly packed, condensed globular state to an extended, random-coil state. Figure 3A-D shows our experimental approach and illustrates the microfluidic system that we used to facilitate the exchange of solution around a single optically trapped DNA molecule. Figure 3A shows Nano Lett., Vol. 3, No. 10, 2003
Figure 3. (A) Fluorescence micrograph showing the flow pattern around the side notch of a circular microchamber; fluorescent beads (1 µm) were used as flow tracers (scale bar: 50 µm). (B-D) Schematics illustrating the procedure we used to initiate the decondensation of single DNA molecules, in which condensed DNAs in a pH 5.7 solution were first introduced into the circular microchamber (B), then optically trapped and translated into the side notch (C), and finally released from the trap after a pH 8 solution flushed the microchamber and replaced the pH 5.7 solution within the side notch by diffusion (D). (E-H) Successive fluorescence images showing the decondensation of a λ-DNA molecule (scale bar: 3 µm). Slight blurring occurred in panels F-H because the image acquisition rate of our camera was slower than the internal motion exhibited by the DNA molecule. After t ) 0, each frame constituted a time lapse of 50 ms.
the flow pattern, which was visualized using 1-µm fluorescent beads as tracers. Under moderate flow rates, the side notch acts as a dead volume where there is little fluid movement and the beads remain relatively stationary. The circular microchamber was designed both to slow the flow within the chamber and to minimize flow detachment, which usually occurs near sharp bends and barriers. To initiate the decondensation of single YOYO-intercalated λ-DNA molecules, we used the following procedure: (1) A pH 5.7 solution containing globular DNA molecules was introduced into a microfluidic channel (Figure 3B). (2) A single DNA molecule was optically trapped and transported from the channel to a region of dead volume with no flow (Figure 3C). (3) A pH 8 buffer solution was driven rapidly through the channel to replace the pH 5.7 solution while the single DNA was stably trapped in the dead volume (Figure 3D). (4) Decondensation of the trapped DNA was initiated by closing the shutter in the beam path of the trapping laser. (5) Immediately after shuttering the laser beam, the DNA molecule underwent a globule-to-randomcoil transition (Figure 3E-H). For this single YOYOintercalated λ-DNA molecule (1:10 dye/bp DNA), the observed transition occurred over a period ∼150 ms. This particular DNA appeared to recoil such that one end of the molecule flopped back into the plane of view (Figure 3H). Integrated fluorescence intensity values of an individual floppy DNA at pH 8 and a globular condensed DNA at pH 5.7 were found to be identical within our error of measurement, which further indicated that each condensate consisted of a single DNA molecule. In addition to trapping individual DNA molecules, we also have trapped DNA aggregates; however, these aggregates did not exhibit the floppy internal
Nano Lett., Vol. 3, No. 10, 2003
motions characteristic of single molecules upon release from the optical trap in a pH 8 solution. With a high-speed, high-sensitivity camera and a faster laser shutter, time resolution in the microsecond regime should be achievable. This method made use of a key advantage of the single-molecule approach, which is the possibility to study kinetic events without the need for synchronization because only one molecule was probed at a time. The constraint on optical-based single-molecule studies, however, is the need for a good fluorophore to provide the optical readout. The observation that YOYO, which is an excellent fluorophore, also acted as a good condensing agent was the key factor that permitted the direct study of DNA condensation at the single-molecule level using our technique. Although this technique is limited to the study of DNA decondensation, the use of optical trapping for controlling single-molecule DNA conformational transformations should offer new possibilities for monitoring the dynamics of such transitions, phenomena that are central to many biological processes from DNA packing to gene expression. Acknowledgment. We thank Professor J. M. Schurr for insightful discussions and assistance with the light-scattering studies. C.L.K. thanks the NSF for a graduate research fellowship. This work was supported by the National Institutes of Health (GM65293). Supporting Information Available: A slow-motion video showing the decondensation of a λ-DNA molecule. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Bloomfield, V. A. Curr. Opin. Struct. Biol. 1996, 6, 334-341. (2) Liu, D.; Wang, C.; Li, J.; Lin, Z.; Zukun, T.; Bai, C. J. Biomol. Struct. Dyn. 2000, 18, 1-9. (3) Hud, N. V.; Downing, K. H.; Balhorn, R. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 3581-3585. (4) Porschke, D. Biochemistry 1984, 23, 4821-4828. (5) He, S.; Arscott, P. G.; Bloomfield, V. A. Biopolymers 2000, 53, 329341. (6) Brewer, L. R.; Corzett, M.; Balhorn, R. Science 1999, 286, 120123. (7) Yoshikawa, K.; Matsuzawa, Y. J. Am. Chem. Soc. 1996, 118, 929930. (8) Yoshikawa, Y.; Nomura, S.-i. M.; Kanbe, T.; Yoshikawa, K. Chem. Phys. Lett. 2000, 330, 77-82. (9) Yoshinaga, N.; Akitaya, T.; Yoshikawa, K. Biochem. Biophys. Res. Commun. 2001, 286, 264-267. (10) Shortreed, M. R.; Li, H.; Huang, W.-H.; Yeung, E. S. Anal. Chem. 2000, 72, 2879-2885. (11) Larsson, A.; Carlsson, C.; Jonsson, M.; Albinsson, B. J. Am. Chem. Soc. 1994, 116, 8459-8465. (12) Wilcoxon, J.; Schurr, J. M.; Warren, R. A. J. Biopolymers 1984, 23, 767-774. (13) Makita, N.; Yoshikawa, K. Biophys. Chem. 2002, 99, 43-53. (14) Quake, S. R.; Babcock, H.; Chu, S. Nature 1997, 388, 151-154. (15) Lin, Z.; Wang, C.; Feng, X.; Liu, M.; Li, J.; Bai, C. Nucleic Acids Res. 1998, 26, 3228-3234. (16) Ashkin, A. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 4853-4860. (17) Mehta, A. D.; Rief, M.; Spudich, J. A.; Smith, D. A.; Simmons, R. M. Science 1999, 283, 1689-1695. (18) Chiu, D. T.; Zare, R. N. J. Am. Chem. Soc. 1996, 118, 6512-6513.
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