NANO LETTERS
Confinement Spectroscopy: Probing Single DNA Molecules with Tapered Nanochannels
2009 Vol. 9, No. 4 1382-1385
Fredrik Persson, Pawel Utko, Walter Reisner, Niels B. Larsen, and Anders Kristensen* DTU Nanotech, Department of Micro- and Nanotechnology, Technical UniVersity of Denmark, DTU Building 345 East, DK-2800 Kongens Lyngby, Denmark Received October 6, 2008; Revised Manuscript Received January 19, 2009
ABSTRACT We demonstrate a confinement spectroscopy technique capable of probing small conformational changes of unanchored single DNA molecules in a manner analogous to force spectroscopy, in the regime corresponding to femtonewton forces. In contrast to force spectroscopy, various structural forms of DNA can easily be probed, as indicated by experiments on linear and circular DNA. The extension of circular DNA is found to scale according to the de Gennes exponent, unlike for linear DNA.
Biological molecules are often subject to conformational restrictions, deformation, or spatial confinement. Small tension, in the femtonewton (fN) regime, resulting from those restrictions can influence biological functions of the molecules.1 However, despite a rapid progress in single-molecule force spectroscopy techniques,2 weak conformational changes cannot be easily detected by those methods, as their operational range is typically limited to the piconewton (pN) regime. Such techniques, based on optical3 and magnetic4 traps, as well as on atomic force microscopy,5 rely on anchoring the molecule to mechanical probes which hinders their applicability to a large set of sequential measurements or for integration in laboratory-on-a-chip devices. In addition, proper anchoring can be difficult to achieve, for example, with circular DNA molecules. Here, we introduce a confinement spectroscopy technique capable of probing small conformational changes of unanchored single DNA molecules, of various structural forms. This is achieved by confining DNA in nanofluidic channels of spatially varying dimensions, yielding detailed molecule extension versus confinement curves in the regime corresponding to femtonewton forces. In order to probe small conformational changes of the DNA, we study its response to a continuously varying spatial confinement. This is in contrast to the existing force spectroscopy techniques3-5 where the effect of direct force, applied to the molecule via beads or linkers, is detected. The extension of a confined DNA molecule results from a balance between entropy and excluded volume interactions within the DNA.6 Hence, no chemical anchoring to the DNA or * Corresponding author,
[email protected]. 10.1021/nl803030e CCC: $40.75 Published on Web 03/16/2009
2009 American Chemical Society
elaborate external setups are required for applying force, while the probed extensions correspond to sub-100-fN forces. Figure 1 shows a schematic layout of our device, with an array of 60 nm deep nanochannels in the center. Their width w increases from 100 to 1000 nm over a distance of 450 µm, while the depth remains constant. Such a tapered design allows us to study the DNA response to varying spatial confinement, determined by the molecule position along the channel. The tapering is sufficiently gentle so that, in the absence of an electric field or hydrodynamic flow, the molecule remains stationary in the nanochannel (i.e., there is no significant entropic recoil).7 The effect of channel tapering on DNA extension is discussed in the Supporting Information. Previous experiments using straight (nontapered) fluidic nanochannels have shown that DNA confinement is a useful tool for measuring DNA size as well as for mapping the interaction sites of restriction enzymes and transcription factors.8-10 In addition, studies performed on straight channels of different cross sections have provided basic DNA extension versus confinement characteristics.11,12 However, due to the small number of channel cross sections available, they were limited to a handful data points. In contrast, tapered nanochannels used in our confinement spectroscopy experiments enable continuous probing of the same single DNA molecule at varying confinement while maintaining identical environmental conditions (ionic strength, pH, etc.), in analogy to force spectroscopy techniques. We note that acquiring a similar set of data by means of straight channels of fixed dimensions would be highly challenging and time-consuming: (i) To obtain a comparable resolution
Figure 1. (center) Schematic layout of our devices. An array of 60 nm deep channels (F) is connected at both ends to two 50 µm wide nanoslits (I and II) of the same depth. The channel width increases from 100 to 1000 nm over a distance of 450 µm. The nanoslits (I and II) serve as an intermediate step reducing shear forces on the DNA while transferring it from the 1 µm deep inlet channels that are connected to eight buffer reservoirs (1-8). (right) Illustrations of a DNA molecule confined at different positions along a tapered nanochannel. (left) A scanning electron microscopy micrograph of a test nanochannel array at its narrow end. In the actual devices, the spacing between the adjacent channels is 7 µm.
in transverse confinement, a large set of straight nanochannels would be required. (ii) Probing the same single molecule under identical environmental conditions would then require its controllable manipulation between a large number of channels. (iii) Different fluidic resistance in such interconnected channels would further hinder operation of the device. Hence, sensitive spectroscopic measurements, which can be performed using our method, cannot be easily reproduced using nanochannels of fixed cross sections. Conventional single-molecule force spectroscopy applies tension to opposing end points of the investigated molecule, whereas confinement spectroscopy, by definition, applies compression force leading to molecular elongation in the nonconfined directions. The two approaches may be correlated by employing the wormlike chain model13 for calculating the force corresponding to a measured molecular extension. Confinement spectroscopy may provide a more global and homogeneous stress on all parts of the molecule, as compared to local variations in stress caused by terminal beads or other force transducers. Since this technique is fully compatible with lab-on-a-chip systems, it also enables simultaneous investigation of both the effects of confinement and the effects of local variations and/or gradients of the surrounding media. In this report, we demonstrate two of the numerous applications of our method. In particular, we probe circular DNA, a structural form found both in bacteria and in the mitochondria of eukariotes, that is otherwise difficult to investigate with standard force spectroscopy techniques due to anchoring problems. We also show that confinement spectroscopy could be a convenient way to study ligand binding to DNA, a highly relevant issue, e.g., in DNA folding,14 DNA digestion,15 and in cancer treatment.16 Figure 2a shows fluorescence images of a λ-bacteriophage DNA molecule, stained with the dye molecule YOYO-1 at a dye ratio of 10:1 ([base pair]:[dye molecule]), taken at different transverse confinements along the nanochannel. (See Supporting Information for details on sample preparation, experimental setup, and data analysis.) The molecule extenNano Lett., Vol. 9, No. 4, 2009
Figure 2. (a) Fluorescence images of a λ-DNA molecule confined at different spatial positions along the same tapered nanochannel. The geometric average increases from left to right: DAv ≈ 97, 118, 155, 194, 215, 240 nm, respectively. The scale bar is 5 µm. (b) Molecule extension r as a function of geometric average DAv measured for linear λ-DNA (upper trace, 48.5 kbp, L10:1 ) 19.8 µm) and circular charomid DNA (lower trace, 42.2 kbp, L10:1 ) 17.2 µm), stained with YOYO-1 at a dye ratio of 10:1 ([base pair]: [dye molecule]). Each data set comprises data taken for 10 independent single molecules of respective type. A single data point represents the extension of a given molecule, determined and averaged from 200 consecutive fluorescence images recorded at a given location along the channel. Error bars indicate standard deviation. The thin solid lines are fits r ∝ DAv-R to the data points (not including the error bars), yielding R ) 0.85 ( 0.01 and 0.65 ( 0.01 for the upper and lower traces, respectively. (c) Relative extension r/L10:1 vs geometric average DAv on a log-log scale.
sion r clearly increases as the channel becomes narrower. More detailed extension versus confinement characteristics are shown in Figure 2b, for both linear λ-DNA (48.5 kbp) and circular charomid DNA (42.2 kbp). The degree of confinement is represented by the cross-sectional geometric 1383
average DAv ) (hw)1/2, where h and w are the height and the width of the channel, respectively.17 The r(DAv) characteristics in Figure 2b indicate a power law dependence r ∝ DAv-R, with exponents R ≈ 0.85 ( 0.01 and 0.65 ( 0.01 for the linear and circular DNA, respectively. This becomes more apparent in Figure 2c where the same data are plotted in a log-log scale. Such a power law dependence is in qualitative agreement with the scaling relation6
( )
r=L
weffP DAv
2
1/3
(1)
developed for confined self-avoiding polymers of contour length L and effective width weff, in the regime where DAv is larger than the DNA persistence length P. This is also consistent with recent experiments on linear λ-DNA confined in straight channels of different cross sections,11 where a power law scaling r ∝ DAv-0.85(0.05 was found. The apparent discrepancy between exponents R obtained for linear and circular DNA could be associated with the higher contour density of extended circular DNA, leading to greater self-interaction. Due to an increase of the contour density along the channel by roughly a factor of 2, the selfavoidance effects become increasingly important with respect to the interactions between the DNA and channel walls.18 In a recent work, Levy et al. presented results concerning partly folded DNA confined in straight nanochannels.19 They reported an approximately 30% greater extension for a folded DNA segment as opposed to an unfolded fragment, which was attributed to a greater self-exclusion in such a segment. For circular DNA we find the extension to be within 5% of the linear DNA for channel dimensions similar to those presented in ref 19. A more detailed theoretical study is necessary to clarify the exact origin of the observed discrepancy. However, its very presence demonstrates that confinement spectroscopy can rapidly yield data on subtle effects of confinement for polymers of complex topology, as well as additional insights into the controversy regarding the importance of self-exclusion in such systems.12,18-20 In the worm-like chain model,13 a semiflexible polymer in the low force regime can be approximated by a simple spring force versus extension relation f ) κr, with a spring constant13 κ ) 3kBT/(2LP). Here kB is Boltzmann’s constant and T the temperature. Assuming the above model and P ) 57 nm, the minimum relative extension r/L ≈ 0.25 observed for the λ-DNA trace in Figure 2c would correspond to an applied force of f ≈ 25 fN. Accordingly, r/L ≈ 0.65 would yield f ≈ 65 fN. In comparison, forces applied in the optical tweezer experiments are typically in the piconewton regime. Resolutions down to 25 fN can be reached, e.g., by applying force to a trapped bead via a second laser source.21 Sufficiently low extensions of a single molecule, required to probe its small conformational changes, are however difficult to achieve in those methods. In a recent study, Meiners et al.22 investigated λ-DNA molecules by means of an optical tweezer, reaching the sub-piconewton force regime. However 1384
Figure 3. (a) Molecule extension r as a function of geometric average DAv measured for a linear T4 bacteriophage-DNA (166.7 kbp, L ) 56.7 µm). The DNA is stained with YOYO-1 molecules at two different dye ratios [base pair]:[dye molecule]: 5:1 (upper trace) and 20:1 (lower trace), respectively. The thin solid lines are fits r ∝ DAv-R to the data points (not including the error bars), yielding R ) 0.83 ( 0.01 and 0.78 ( 0.01 for the upper and lower trace, respectively. Inset shows a YOYO-1 analogous molecule bound to DNA,23 as visualized using the open-source viewer JMol. (b) The same data as in (a) plotted on a log-log scale.
despite a high spatial and force resolution, the DNA could only be probed at large relative extensions of r/L > 0.5. Figure 3 shows extension versus confinement characteristics obtained for linear T4-bacteriophage DNA (166.7 kbp) stained with YOYO-1 at two different dye ratios: 5:1 and 20:1. At a fixed confinement, longer extension is observed for molecules with higher dye concentration. In addition, both traces indicate r ∝ DAv-R dependence with R ) 0.83 ( 0.01 and R ) 0.78 ( 0.01 for 5:1 and 20:1 data, respectively. The difference in exponents might once again indicate a subtle effect due to self-avoidance. Since each YOYO molecule screens the intrinsic negative charge of DNA, the effect of self-avoidance could be increased for a less stained molecule,18 possibly influencing the exponent as previously argued. Classic de Gennes theory for confined polymers6 predicts linear dependence of the molecule extension on its contour length, see eq 1. Even though YOYO-1 affects, due to electrostatic screening effects, both the persistence length and the effective width of the DNA molecule,24,25 the large increase in the contour length results predominantly from intercalation.26 Since a single intercalation event in the DNA increases L by a length of one base pair,26 raising the concentration of bis-intercalating YOYO-1 (where each molecule contributes with two intercalation events) from 20:1 to 5:1 should thus yield a 27% increase in molecule extension (L5:1/L20:1 ) L(1 + (2/5))/L(1 + (2/20)) ≈ 1.27). This is in good agreement with results shown in Figure 3, where an increase in r of roughly 30% can be observed over the whole DAv range. Nano Lett., Vol. 9, No. 4, 2009
In summary, we present confinement spectroscopy experiments performed on single DNA molecules trapped in fluidic nanochannels of continuously varying transverse dimensions. The two probed structural forms of DNA (linear and circular) indicate different response to spatial confinement, thus suggesting a greater self-interaction for the circular DNA. Detailed understanding of the specific dependencies may be important for understanding differences in DNA interactions (with, e.g., drugs and proteins) in prokaryotes and eukaryotes. In addition, the effect of YOYO intercalation onto DNA is investigated and shown to scale with the increase in contour length. On the basis of those examples, we demonstrate that, unlike existing force spectroscopy techniques, the confinement spectroscopy method is well suited for sensitive singlemolecule studies in a regime corresponding to sub-100-fN forces. It also enables straightforward integration into laboratory-on-a-chip systems and/or measurement parallelization in well-controlled environmental conditions. Apart from probing different structural forms of DNA and DNA-ligand complexes, confinement spectroscopy could also be used to study DNA-protein interactions. The local nanometer scale “bulging” at the DNA-protein interaction site would result in high local sensitivity to geometrical confinement,1 thereby enabling protein-binding analysis with the confinement spectroscopy technique. This method could also provide a new insight to the fields of spore DNA and viral DNA translocation.27 In either case, coating the channels with polymer brushes could provide a method to simulate soft surroundings present in vivo. Acknowledgment. The authors acknowledge financial support by the Danish Research Council for Technology and Production (FTP), Grant Number 274-050375. For assistance in fabrication, the staff and facilities of the DTU Danchip cleanroom are acknowledged. Supporting Information Available: Details on sample preparation, experimental setup, and data analysis. This material is available free of charge via the Internet at http:// pubs.acs.org.
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