Winding single-molecule double-strandedDNA on a nanometer-sized reelHuijuan You1,2, Ryota Iino1,3, Rikiya Watanabe1,3 and Hiroyuki Noji1,3,*

1Department of Applied Chemistry, School of Engineering, The University of Tokyo, Bunkyo-ku,Tokyo 113-8656, 2Department of Biotechnology, Graduate School of Engineering, Osaka University,2-1 Yamadaoka, Suita, Osaka 565-0871 and 3CREST, Japan Science and Technology Agency,The University of Tokyo, Bunkyo-ku, Tokyo 113-8656, Japan

Received March 15, 2012; Revised May 28, 2012; Accepted June 12, 2012

ABSTRACT

A molecular system of a nanometer-sized reel wasdeveloped from F1ATPase, a rotary motor protein.By combination with magnetic tweezers and opticaltweezers, single-molecule double-stranded DNA(dsDNA) was wound around the molecular reel.The bending stiffness of dsDNA was determinedfrom the winding tension (0.96.0 pN) and thediameter of the wound loop (21.48.5 nm). Ourresults were in good agreement with the conven-tional worm-like chain model and a persistencelength of 54 9nm was estimated. This molecularreel system offers a new platform for single-molecule study of micromechanics of sharply bentDNA molecules and is expected to be applicable tothe elucidation of the molecular mechanism ofDNA-associating proteins on sharply bent DNAstrands.

INTRODUCTION

Many signicant proteinDNA interactions involvesharp bending and looping of double-stranded DNA(dsDNA) with curvature radius of 220 nm (1). A remark-able example is the histoneDNA complex in eukaryoticcells; genomic dsDNA is wrapped around histonecomplexes with radii of 4.5 nm. The mechanisticproperties of the histoneDNA complex is thought to beinvolved in the control of transcription activity (2). Mosttranscription factors also deform DNA by bending orlooping DNA strands to regulate gene expression (3).DNA condensation occurs in extremely small viralcapsids (radius of 1550 nm) and is another example ofextensive winding or bending of DNA (4). Thus, revealingthe mechanical properties of dsDNA is crucial for under-standing the molecular mechanisms of DNAproteinsystems, and has been a focal issue in the physicochemicalresearch on DNA (1).

Fundamental aspects of DNA bending mechanics havebeen studied using biochemical bulk measurements (5)and by pioneering single-molecule DNA stretching experi-ments (6,7). DNA bending is well described by theworm-like chain (WLC) model (8,9), in which the mostimportant parameter is the persistence length, Lp, whichcharacterizes the laments resistance to thermal bending.The persistence length of DNA is reported to be 50 nmfor dsDNA, although the Lp of DNA changes dependingon the experimental conditions and the DNA sequenceused.The single-molecule DNA stretching experiment

has high versatility; it offers a unique experimentalplatform that allows one to study not only the mechanicalproperties of DNA but also the conformational dynamicsof DNA associating proteins at the single-molecule level(10). Unidirectional motion of RNA polymerase or diffu-sive translocation of DNA-binding proteins along DNAstrand were analyzed to elucidate their working principles.However, DNA stretching experiments have limitations

for the study of the micromechanics of sharply bent DNAor the molecular mechanism of DNA associating proteinthat induces DNA bending or binds to bent DNA. DNAstretching experiments typically measure the ensemble-averaged stiffness over a long DNA strand, in whichsmall fragments may experience many small bendingevents inuenced by thermal energy. Because thermalenergy supplies only low forces in the order of 0.1 pN(11), it is rarely possible to induce sharp bending of DNAwith a curvature radius in nanometer range in DNAstretch experiments. Therefore, methodology thatcontrols the bending curvature of DNA is required toexplore the micromechanics of sharply bent DNA andits physiological role.Several studies have been carried out to reveal the fun-

damental mechanical features of sharply bent DNA.Biochemical approaches, including DNA ligase-catalyzedcyclization experiments, have been used to quantitativelymeasure the ligation efciency and circularization ofshort DNA fragments against bending force (1214).

*To whom correspondence should be addressed. Tel: +81 3 5841 7252; Fax: +81 3 5841 1872; Email: hnoji@appchem.t.u-tokyo.ac.jp

Published online 5 July 2012 Nucleic Acids Research, 2012, Vol. 40, No. 19 e151doi:10.1093/nar/gks651

The Author(s) 2012. Published by Oxford University Press.This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

These experiments allowed the estimation of the exibilityof DNA based on the ratio of the circularly-ligated tolinearly-ligated fragments in a wide range of fragmentlengths. Atomic force microscopy (AFM) provided moredirect measurements of the distribution of dsDNAbending angles, allowing the estimation of the bendingenergy (15). However, immobilization of DNA on asurface for AFM imaging may have biased the popula-tions of individual DNA forms. The direct measurementof bending force of dsDNA was also attempted by using asingle-stranded DNA as a molecular force sensor, bothends of which were linked to the ends of bent dsDNA(16). However, the precision of the force determinationsuffered from the intrinsic noise and non-linearity ofsingle-molecule Forster resonance energy transfer, onwhich this method relied on for the force estimation. Itseems plausible that the WLC model fails to explainseveral results (12,13,15). Further investigation withregard to this issue is still necessary.In this study, we developed a novel method to wind

individual dsDNA molecules around the rotary motorprotein F1ATPase (F1) to directly measure the forceand elastic energy required to bend dsDNA at nan-ometer-scale curvature radii. F1 is the water-solubleportion of the FoF1ATP synthase (Figure 1a) (17,18).

The minimum ATPase-active complex of the F1 motor isthe a3b3g subcomplex, in which the g subunit rotatesagainst the a3b3 stator in counterclockwise directionupon ATP hydrolysis (19). As a molecular reel for DNAwinding, F1 has an ideal size; the radius of the centralshaft of the g subunit is 1 nm (radius of a3b3 statorring is 5 nm), which is much smaller than the curvatureradius of dsDNA loops induced by histones and transcrip-tion factors and thus suitable for winding dsDNA in freelysuspended conditions. Our measurements showed thatthe curvature diameter of wound DNA decreased from21.4 to 8.8 nm when tension was increased from 0.9 to6.0 pN. The WLC model with the persistence length of549nm well described our data, indicating that tightlybent dsDNA retained its structural integrity.

MATERIALS AND METHODS

DNA construct

The 8688 bp dsDNA was prepared by PCR usingEx-Taq DNA polymerase (TaKaRa, Japan) and thepRA100 plasmid as template (20). A 50-biotin primertogether with a 50-digoxigenin (DIG) primer (SigmaGenosys, USA) were used in PCR reaction. PCRproducts were puried using the Wizard PCR clean-upsystem (Promega, USA). Puried DNA (12.5 mg) wasincubated with 100 ml of streptavidin-coated polystyrenebeads (0.5 mm diameter, 1% wt/vol, Bangs Laboratories,USA) suspension overnight, and unbound DNA wasremoved by washing several times using buffer (100mMKPi, pH 7.0). The number of dsDNA strands bound toeach bead was 1.7 102.

Protein preparation

A mutant of the a3b3g subcomplex of F1ATPase fromthe thermophilic Bacillus PS3, a(His6 in N-terminus/C193S)3b(His10 in N-terminus)3g(S108C/I211C), wasexpressed in Escherichia coli and puried as described pre-viously (21,22) for the single-molecule rotation assay.Amino groups of the anti-digoxigenin (DIG) Fabfragment (Roche, Switzerland) were biotinylated with a5-M excess of biotinPEONHS (Pierce, USA) for 1 h atroom temperature. After removing unreacted biotinPEONHS using a spin column (Bio-Rad, USA), theremaining unreacted amino groups of the Fab fragmentwere reacted using an SPDP cross-linker (Pierce), whichcross-links an amino group and a thiol group. Afterremoving the unreacted cross-linker using a spin column(Bio-Rad), the SPDP-activated Fab fragment was reactedwith cysteine residues that had been genetically introducedinto the g subunit of F1 at a molar ratio of 1:3 (F1 to Fab)overnight at room temperature. The biotinylated FabF1complex was stored at 4C before use (SupplementaryFigure S2).

Microscopy

An inverted microscope (IX71, Olympus, Japan) with a100 objective lens (PlanSAPO NA1.3, Olympus),equipped with home-built magnetic tweezers (23) and

Anti-Dig

F1-ATPasedsDNA 3m

N S

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Trapcenter

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x0x1

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x1+dx

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Figure 1. Experimental setup. (a) The molecular reel was constructedof F1, a magnetic bead, and the Fab fragment on NiNTA glass.A biotinylated anti-DIG Fab fragment (orange) specically linked theg subunit (red) of F1ATPase and the streptavidin-coated magneticbead. A dsDNA molecule (8.7 kb) was bridged between the anti-DIGFab fragment and a streptavidin-coated polystyrene bead trapped usingoptical tweezers. The dsDNA was wound by rotating the magneticbead using the magnetic tweezers. The stretching force was nearlyparallel to the glass surface; the angle of the DNA strand against thecoverglass was

optical tweezers using an infrared laser (1047 nm,Nd:YLF, 1W, IPG Photonics, USA), was used. Bright-eld images were recorded using a CCD camera (MTI)at 30 frames/s and a high-speed camera (FASTCAM-1024PCI, Photron, USA) at 27 000 frames/s for windingexperiments and calibration of trap stiffness, respectively.Bead position was measured as the centroid of the brighteld image of the bead using Image J (National Institutesof Health, USA) and custom-made plug-ins (K. Adachi,Waseda University). Stiffness of the optical trap wasestimated from the distribution of the x- and y-coordin-ates of the trapped bead. Trap stiffness was also measuredfrom displacement of the trapped bead under the bufferow, which exerted drag force on the trapped bead(Supplementary Data).

Construction of reel system

A 6-mm wide ow chamber was constructed from aNi-NTA functionalized coverglass (32 24 mm2) and anunmodied coverglass (18 18 mm2) separated by twospacers 50 mm thick. The bovine serum albumin (BSA)buffer (20mM MOPS/KOH, pH 7.0, 50mM KCl, 2mMMgCl2, 10mg/ml BSA) was infused into the ow chamber.After a 5-min incubation to block non-specic proteinbinding, biotinylated FabF1 (5 pM) in BSA buffer wasinfused into the chamber. After 15min incubation,unbound F1 was washed out of the chamber using BSAbuffer. Next, streptavidin-coated magnetic beads(200700 nm, Thermo Scientic, USA) in BSA bufferwere infused into the chamber and incubated for 1 h.Unbound magnetic beads were removed with buffer.Finally, DNA-coated polystyrene beads (0.5 mm) in BSAbuffer containing 2mM ATP and 5mM BiotinPEGNH2 (MW3400, Creative PEG Works, USA) to blockthe non-specic binding of DNA to magnetic bead, wereinfused. The home-built magnetic tweezers werecontrolled using custom-made software (Celery, Library,Japan) (24). The x- and y-movements of the sample stageof microscope were manipulated using stepping motors(SGSP-13ACTR, Sigma Koki, Japan) controlled bycustom-made software (ActOperator, Sigma Koki).Experiments were carried out at 252C.

RESULTS

Construction of reel system

The designed architecture of the molecular reel is shown inFigure 1a. The molecular reel to wind dsDNA was builtusing the F1 molecule, a magnetic bead, and the Fabfragment (radius of 2.5 nm) of the anti-digoxigenin(DIG) antibody. The anti-DIG antibody was covalentlycross-linked to the g subunit of F1 and connected to themagnetic bead through biotinstreptavidin interaction.The complex of the g subunit, anti-DIG Fab fragment,and the magnetic bead acted as a rotor while the a3b3stator was immobilized on the glass surface. dsDNA mol-ecules of 8.7 kb labeled with DIG and biotin at distal 50-ends were grabbed through the anti-DIG Fab fragment ofthe reel and a streptavidin-coated polystyrene bead thatwas trapped using optical tweezers.

Procedures for constructing the system were carried outas follows (Supplementary Figure S1). First, reelcomplexes were built by infusing the anti-DIG FabF1complex and magnetic beads solutions into a ow cell.The density of F1 on the glass surface was maintained at

In some cases, the system was able to withstand thestretching. When the magnetic tweezers were turned off(gray arrowheads in Figure 2c and d), the magnetic beadexhibited fast backward rotation, turning the samenumber of rotations as was observed in the forciblewind-up process. The speed of this backward rotationwas 0.41.7 rps (n=3). When the tension exerted on themolecular reel complex was completely removed bymoving the stage, F1 showed ATP-driven rotation(green data point in Figure 2d), indicating F1 moleculeswere still active after manipulation. In some cases, F1 didnot resume ATP-driven rotation, likely due to theexposure of F1 to infrared light for optical trapping.However, inactivated F1 molecules still acted as molecularbearings; the magnetic beads showed rotational Brownianmotion after the experiment (data not shown).

Winding tension versus curvature diameter

To draw a curve of winding tension (F) versus curvaturediameter (D) of wound DNA, we determined the tensionexerted on the DNA and the length of the DNA woundaround the reel complex (L). The force was determinedbased on the displacement of the DNA-coated beadfrom the trap center of the optical tweezers. The stiffnessof the optical tweezers used was 0.01 0.05 pN/nm(Supplementary Data). The diameter of wound DNAwas calculated using D=L/n; where n is the numberof forced rotations. Because the displacement of theDNA bead upon winding was partly compensated bythe increase of extension (dz) of the tethered DNAstrand (dz/L=3188%), the total length of the DNAwound around the reel complex (L) is equal to the sumof the displacement of DNA-coated bead (dx) and theincrease of DNA extension (dz). Here, DNA extension

was determined based on the force applied to the DNAin winding experiment, and the forceextension curve ofthe same DNA obtained from the stretching experiment(Figure 3a before correction, Figure 3b after correction).For precise measurement, only the data points from 1.0 to2.0 revolutions were analyzed; data points of 2.0revolutions did not provide reproducible values, likely dueto the low precision of diameter estimation for 2.0 revolutions.

Analysis of tension-diameter data with WLC model

Figure 4a shows the results of eight independent windingexperiments represented by different colors, with windingtension ranging from 0.9 to 6.0 pN and curvature diam-eters ranging from 21.4 to 8.5 nm. Diameters were largerthan the size of the nano-reel (5 nm). Thus, this valuerepresents the mechanical property of dsDNA withoutsteric interaction with the reel. Based on the WLCmodel, the relationship between winding tension andloop diameter of dsDNA can be expressed as follows:

F 2D2

2LpkBTD2

1

where F is the winding tension, D is the curvature diameterof DNA loop, is the bending stiffness of dsDNA (alsoreferred to as exural rigidity), kBT is thermal energy(4.1 pNnm at room temperature) and Lp is the persist-ence length of dsDNA. By tting the averaged data pointsof individual experiments (open squares in Figure 4a), Lpof sharply bent DNA was determined to be 549nm(meanstandard deviation; correlation coefcientR=0.93). This value agrees well with the previouslyreported persistence lengths from stretching experiments

0

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ONOFF

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x0

Figure 2. Time courses of winding experiments. (a) An example of the time course of the displacement of an optically trapped polystyrene beadduring the winding procedure. Trap stiffness was 0.040 pN/nm. dsDNA was stretched at 1.5 pN force for 8 18.5 s. (b) Time course of magneticbead rotation during the winding experiment for (a). The magnetic bead was rotated from 18.5 s at 0.1 rps (orange line). The optically trapped beadwas moved concomitantly with the magnetic bead rotation. After 2.5 revolutions, the magnetic bead detached from the glass surface (showed byasterisk). The optically trapped bead immediately returned to the trap center (x0). (c and d) Time course of another winding experiment. Trapstiffness was 0.022 pN/nm. The magnetic bead was forcibly rotated for ve revolutions. The optically trapped bead was constantly pulled toward therotating magnetic bead. The sudden decrease at 63 s occurred for unknown reason; it might be due to structural relaxation of the DNA wound uparound the molecular reel. The magnetic eld was turned off at 66 s (gray arrowheads), and then, beads showed backward rotation (blue part). At75 s, after reducing the tension exerted on DNA by moving stage, F1 resumed ATP-driven rotation (green line).

e151 Nucleic Acids Research, 2012, Vol. 40, No. 19 PAGE 4 OF 6

(50 nm) (7). This agreement suggests that sharply bentdsDNA retains the same mechanical properties as dsDNAin the relaxed state, implying that deformation of dsDNA,such as local melting or kinking, does not occur under thepresent conditions. Based on our results, the forcerequired to wind dsDNA into a circular loop with a curva-ture diameter of 9 nm, which represents the curvaturediameter of dsDNA around a histone, is estimated to be5.5 pN. The elastic energy (E) of a loop, calculated usingthe equation E=2LpkBT/D, is 38 kBT.

DISCUSSION

We successfully wound single-molecule dsDNA around ananometer-sized reel. As an application of this system, themechanics of sharply bent dsDNA were studied.The results were consistent with the WLC model in therange of forces applied (0.9 6.0 pN). This nding agreeswith the previous studies of the ligase-catalyzed cycliza-tion experiments (14). However, there are also otherstudies reporting that dsDNA strands with curvatureradius of 2 20 nm have higher exibility than the WLCmodel predicts (12,13,15). The higher exibility wasattributed to a local melting bubble (25) or kink (26) for-mation during sharp bending (15). The reason for the dis-crepancy between this study and the previous workssuggesting higher exibility is unknown. One possibleexplanation is the difference in the DNA sequence. Arecent cyclization experiment showed different DNAsequences had different persistence lengths (27) andshort persistence length can explain the high exibility.

Recent theoretical study also showed that the sequenceaffect the tendency of DNA to form kinks. The GC-richsequence is more exible and kink formation can beobserved when a GC-rich fragment is bent to curvatureradius

molecules to modulate catalytic activity (30). However,the lack of quantitative information regarding the mech-anics of DNA molecules under high bending forces limitsthe application of dsDNA as a molecular spring in nano-engineering. We directly measured the restoring force of awound DNA spring. Our method would provide a goodcalibration curve of the curvature radius and bendingforce for DNA spring work. In addition, it should bealso emphasized that, to our knowledge, this is the rstdemonstration of the construction of a molecular reelsystem that can wind up a molecular wire and interconvertthe linear motion and rotary motion. This technique couldbe also applicable for the development of molecular mech-anical systems.

SUPPLEMENTARY DATA

Supplementary Data are available at NAR Online:Supplementary Information 14 and SupplementaryFigures 15.

ACKNOWLEDGEMENTS

The authors thank M. Kino-oka (Osaka University), K.Hayashi (Tohoku University), and all members of theNoji laboratory for valuable comments and discussionsand S. Nishikawa (Nikon, Japan) for technical support.Y.H. thanks the Ministry of Education, Culture, Sports,Science and Technology, Japan, and the InternationalGraduate Program for Frontier Biotechnology,Graduate School of Engineering, Osaka University, forscholarship support.

FUNDING

Funding for open access charge: The Grants-in-aid forScientic Research from Ministry of Education, Culture,Sports, Science and Technology (MEXT) [22247025 toH.N. and 23107720 to R.I.].

Conict of interest statement. None declared.

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