Filetype PDF | Posted on 16 Sep 2022 | 4 years ago
Authentication
digital resources
265x Filetype PDF File size 0.45 MB Source: www.nbi.dk
MICROSCOPYRESEARCHANDTECHNIQUE70:26
33(2007)
CombiningOpticalTweezersandScanningProbe
MicroscopytoStudyDNA–ProteinInteractions
*
JURGENH.G.HUISSTEDE,VINODSUBRAMANIAM,ANDMARTINL.BENNINK
BiophysicalEngineeringGroupandMESAþInstituteforNanotechnology,FacultyofScienceandTechnology,
University of Twente, AE Enschede 7500, The Netherlands
KEYWORDS optical tweezers; scanning probe microscopy; DNA
protein interactions; dig-
oxygenin; single molecule force spectroscopy
ABSTRACT Wepresentthefirst results obtained with a new instrument designed and built to
study DNA
protein interactions at the single molecule level. This microscope combines optical
tweezers with scanning probe microscopy and allows us to locate DNA-binding proteins on a single
suspended DNA molecule. A single DNA molecule is stretched taut using the optical tweezers,
while a probe is scanned along the molecule. Interaction forces between the probe and the sample
are measured with the optical tweezers. The instrument thus enables us to correlate mechanical
andfunctional properties of bound proteins with the tension within the DNA molecule. The typical
friction force between a micropipette used as probe and a naked DNA molecule was found to be
<1 pN. A 16 lm DNA molecule with 10
15 digoxygenin (DIG) molecules located over a 90 nm
range in the middle of the DNAwas used as a model system. By scanning with an antidigoxygenin
(a-DIG) antibody-coated pipette we were able to localize these sites by exploiting the high binding
affinity between this antibody
antigen pair. The estimated experimental resolution assuming an
infinitesimally thin and rigid probe and a single a-DIG/DIG bond was 15 nm. Microsc. Res. Tech.
C
V
70:26
33, 2007. V2006Wiley-Liss,Inc.
INTRODUCTION these studies, however, it is not possible to accurately
Single-molecule approaches yield new information localize these proteins in order to correlate their posi-
about mechanical and dynamic properties of biomole- tion with the force data obtained. To localize proteins
cules. This powerful approach for studying molecular on a single DNA molecule we have developed a new
systemsallowstheusertoassesspropertiesthat,using instrument based on OTcombined with scanning probe
mostothertechniques,arenotaccessible,orthatwould microscopy(SPM).
bemaskedintheensembleaverage. Figure 1a presents the concept of the scanning probe
Several techniques have been developed to measure optical tweezers (SPOT) microscope. A single DNA mol-
or manipulate these single molecules, including optical ecule is stretched taut using the optical tweezers, while
tweezers and atomic force microscopy. Optical tweezers a sharp probe is scanned along the molecule. Interac-
(OT) is a technique that is widely used for single mole- tions between the probe and the DNA and proteins are
cule force spectroscopy capable of measuring forces up sensed by the optical trap in contrast to conventional
to 100 pN with subpiconewton resolution, which makes SPMtechniques,wheretheprobeactsasasensor.One
the technique excellent for measuring forces involved of the controllable parameters influencing the magni-
in stretching individual molecules such as DNA, and tude of the interactions between the probe and the
proteins or complexes thereof, (Bennink et al., 2001; sample is the indentation d of the DNA molecule as
Cui and Bustamante, 2000; Kellermayer et al., 1997) indicated in Figure 1b, which is typically in the range
and measuring forces involved in the action of molecu- of 0
200 nmforlocalization experiments. Animportant
lar motors (Abbondanzieri et al., 2005; Mehta et al., advantage of this microscope with respect to current
1999). SPMtechniques is that the DNA molecule is not influ-
Scanning probe microscopy (SPM), in particular the enced by any interfering surface except for the two
atomic force microscopy (AFM), is capable of obtaining beadstowhichtheDNAendsareattached.Thisconfig-
high resolution images of single biomolecules (Baha- uration allows the study of DNA
protein interactions
tyrova et al., 2004; Czajkowsky and Shao, 1998; Nikova in conditions where the proteins can move freely. Fur-
et al., 2004; Viani et al., 2000). AFM is also used for sin-
gle molecule force spectroscopy studies of various bio-
molecular complexes (Hinterdorfer et al., 1996; Lee *Correspondence to: Dr. Martin L. Bennink or Prof. Vinod Subramaniam, Bio-
physical Engineering Group and MESAþ Institute for Nanotechnology, Faculty
et al., 1994; Muller et al., 1999; Oesterhelt et al., 2000) of Science and Technology, University of Twente, PO Box 217, 7500 AE Enschede,
withatypicalforceresolutionof>5pN. TheNetherlands;E-mail:m.l.bennink@utwente.nlorv.subramaniam@utwente.nl
In many OT and AFM force spectroscopy experi- Received5April2006;accepted14July2006
ments the change in elastic and mechanical properties DOI10.1002/jemt.20382
of DNA as a result of protein binding is studied. In Published online 1 November 2006 in Wiley InterScience (www.interscience.
wiley.com).
C
V
V2006WILEY-LISS,INC.
OPTICALTWEEZERS,SPM,ANDDNA
PROTEINBINDING 27
Fig. 1. (a) Principle of measurement. A single DNA molecule is to a length L is indented with the probe over a distance d an addi-
stretched taut while a lm-sized probe is scanning along the molecule tional force Fdna is generated in the molecule and is a function of the
in order to \feel" the individual proteins. Interactions between the probe position x along the molecule. The magnitude of the interac-
probe and the proteins are detected by the optical tweezers and in tions between the probe and the sample is dependent on the indenta-
combination with the probe position, allows the accurate localization tion d. [Color figure can be viewed in the online issue, which is avail-
of these proteins on the DNA molecule. (b) When a molecule stretched able at www.interscience.wiley.com.]
thermore this microscope enables us to study the effect betweenaprecleanedmicroscope glass and a coverslip.
of tension in the DNA molecule on the functional prop- This creates a flow channel with dimensions of 200 lm
erties of proteins. 35mm350mm.Inthemicroscopeglassthreeholes
Here we demonstrate the feasibility of the SPOT- were powder-blasted (Wensink et al., 2000). Two holes
microscopebyusinganantidigoxygenin(a-DIG)coated (2 mm in diameter) at the end of the flow channel act
micropipette to localize digoxygenin (DIG) molecules as entry and exit points for the flow channel. Inlet and
bound over a range of 90 nm in the middle of a 16 lm outlet tubes necessary to flow the desired solutions in
long DNA molecule. The high binding affinity of this and out were attached on top of these. An additional
antibody
antigen linkage results in strong local inter- entry-hole in the middle of the cell was created to ena-
actions between the probe and the DNA molecule. In ble the injection of the scanning probe. The powder-
addition we use this configuration to determine the blasting procedure used created an entry-hole that has
friction force between the probe and the naked DNA. a conical shape with a smallest diameter of 200 lmat
the bottom side as indicated in Figure 3. The other two
MATERIALSANDMETHODS holes at either end of the flow channel were drilled af-
ExperimentalSetup ter powder-blasting with a diamond drill to provide a
To create a single beam gradient optical trap, a high cylindrical hole.
NA objective was used (Leica N PLAN, NA 1.20, A square glass capillary with an inner diameter of
2
Wetzlar, Germany), resulting in a strongly focused 50 3 50 lm (VitroCom, Mountain Lakes, NJ) was
laser beam. The proximity of the scanning probe to the aligned in between the coverslip and the microscope
DNAmolecule stretched taut by the OT (as schemati- glass such that a suction pipette for the bead immobili-
cally depicted in Fig. 1) interferes with the light trans- zation can be injected into the flow cell with the pipette
mittedthroughthebeadasaforcesignal,asiscommon end located below the entry-hole. This complete sand-
in a conventional OT. Therefore the basis of the SPOT- wich was heated up shortly to 608C using a heating
microscope is a reflection-based OT instrument as plate to provide a waterproof sealing. To prevent leak-
described elsewhere (Huisstede et al., 2005). The com- age through the center hole a small reservoir was
plete configuration of the scanning probe optical tweez- placed on top of the outlet hole. Because the diameter
ers microscope is depicted in Figure 2. of the outlet was 10 times larger than the center entry-
hole, the fluid resistance is highest at the center hole
TheFlowCell andfluidfirst leaves the flow cell through the outlet. A
Aflowcellisrequired to build up a bead
DNA
bead small tube placed in this reservoir actively sucks away
construct (Fig. 3). A single k-DNA molecule (16.4 lm) fluidwheneveritreachesthetube.
end-labeled with biotin is suspended between two
streptavidin-coated 2.67 lm polystyrene beads (Poly- ScanningProbe
sciences, Warrington, PA) according to a procedure pre-
viously described in literature (Bennink et al., 1999). In the SPOT-microscope configuration, and in con-
One bead was immobilized on a suction pipette inte- trast to SPM techniques, the probe is not used as a sen-
grated in the flow cell and one bead was held by the op- sor, but as an actuator, which demands a thin and rigid
tical trap. Moving the micropipette with respect to the probe. The optical tweezers in this instrument are the
optical trap using a piezo-controlled XYZ-stage (P-509, force-measuring element. In the experiments pre-
Physik Instrumente, Karlsruhe, Germany) allowed sented here micropipettes were used as scanning
stretching of the DNA molecule. probes (pipette probe in Fig. 3). Borosilicate micropip-
Tofacilitate the approach of the stretched DNA mole- ettes were pulled from 1.2 mm outer diameter and
cule by a scanning probe some modifications were 0.94 mm inner diameter capillaries (Harvard Appara-
made to the existing flow cell design (Bennink et al., tus GC120TF-15, Holliston, MA) using a Sutter P-87
1999). Twolayers of thermoplastic laboratory film (Par- micropipette puller (Novato, CA) to obtain end diame-
afilm1 ters in the range of 1
2 lm.
) with a channel cut out were sandwiched
MicroscopyResearchandTechniqueDOI10.1002/jemt
28 J.H.G. HUISSTEDE ETAL.
Fig. 2. Schematic layout of the scanning probe optical tweezers chronic mirror (DM) on a second CCD camera (CCD1). A short-pass
(SPOT) microscope. The inset shows the bead
DNA
bead construct filter (SPF) in front of the camera blocks the 1064 nm laser light. The
and the scanning probe. A beam expander (BE) creates a laser beam probe for scanning along the DNA molecule is fixed to a piezo tube
with a diameter of 1 cm that overfills the back-aperture of a 1003 that on its turn is mounted on a translation stage (TS2) to control the
high NA objective. The laser power at this aperture can be tuned by a probe position in the Y and Z direction and a separate translation
half-wave plate (k/2) and a polarizing beam splitter cube (PBS). A stage (TS1) with an integrated piezo stack to accurately control the
beam splitter (BS1) directs the backscattered light onto a position position in the X direction and thus the indentation of the DNA mole-
sensitive detector (PSD) where a second beam splitter in the detection cule by the probe. The integrated piezo stack was used to keep hyster-
path (BS2) enables visualization of the reflection pattern on a CCD esis of the piezo-tube constant during the scanning experiments. The
camera (CCD2). A quarter-wave plate (k/4) placed in front of the high NA objective and a 103 objective were mounted on a sliding
objective converts the incident p-polarized laser light into circularly mechanism to be able to switch objectives where the 103 objective
polarized light, providing an equal trap stiffness in both lateral direc- was used to provide a larger field of view required to be able to inject
tions (Worland et al., 1996). A halogen lamp provides white light illu- the scanning probe into the flow cell.
mination for optical microscopy imaging of the trapped bead via a dia-
HysteresisofthePiezo-Tube SilanizationofPipettes
A piezo-tube was used to scan the probe back and Freshly pulled pipettes were placed upright in a
forth along the DNA molecule while a piezo stack inde- small glass beaker (25 mL), which was placed on a
pendently controlled the indentation. Piezo tubes ex- glass Petri dish. The small glass beaker with the pip-
hibit hysteresis, which makes independent calibration ettes was covered under a 100 mL glass beaker and
of the probe position necessary. baked in an oven at 2008C for at least 4 h. All glass-
The actual position of the tube was determined with ware (except the pipettes) was presilanized by placing
aLEDmountedtotheendofthepiezotubeincombina- them in a 2% APTES (3-aminopropyltriethoxysilane,
tion with a position sensitive detector (PSD, DL100-7- Sigma,St. Louis, MO) solution in 95% aqueous acetone
KER, Pacific Silicon Sensor, Westlake Village, CA) for 1 h. If the pipettes were not freshly pulled they
mountedtotheopticaltable.ToconvertthePSDsignal were cleaned by placing them in a solution of 65%
from volts to microns, optical microscope images of a HNO (nitric acid) for at least a few hours and rinsed
3
micropipette clamped to the piezo tube, as for the scan- several times with acetone.
ning experiments, were obtained with the high NA After the oven was cooled TMSDMA (N,N-dimethyl-
objective as a function of the applied voltage in a direct trimethylsilyamine, Fluka, Seelze, Germany) was in-
current (DC) measurement. A centroid algorithm jected under the 100 mL beaker through a syringe. The
(Wuite et al., 2000) deduced the position of the probe in TMSDMAalmostimmediatelyvaporizedandthevapor
microns. Since the hysteresis is a function of the scan produced a homogeneous hydrophobic silane coating
speed and the scan range, for each experiment the hys- onthepipettes. After 30 min the beaker was opened for
teresis was determined with the settings used. These a short period of time to allow any residual vapor to
curves were used to correct the obtained data in the escape. The silanized pipettes were baked at 2008C
scanningexperiments. overnight.
MicroscopyResearchandTechniqueDOI10.1002/jemt
OPTICALTWEEZERS,SPM,ANDDNA
PROTEINBINDING 29
Fig. 3. Schematic representa-
tion of the flow cell. An additional
entry-hole was drilled at the posi-
tion of the optical trap and the suc-
tion pipette used to immobilize a
2.6 lm bead. This hole allows
injection of a probe used for scan-
ning along a stretched dsDNA
molecule to localize DNA-bound
molecules. [Color figure can be
viewed in the online issue, which
is available at www.interscience.
wiley.com.]
a-DIGCoatedPipette
For the preparation of a-DIG coated pipettes small
volumes are essential to prevent the use of large
amountsofantibody.Glasscapillarieswithaninnerdi-
ameter of 1.5 mm were cleaned by placing them for 1 h
in a 65% HNO solution. Subsequently they were
3
rinsed several times with Milli-Q and finally dried
using nitrogen. Freshly pulled micropipettes (1
2 lm Fig. 4. Schematic representation of a DNA molecule that is biotin-
tip diameter) were silanized with TMSDMA and ylated at each end. In approximately the middle of the molecule there
inserted into the capillaries. A small rubber cap placed is a 268 bp fragment including DIG molecules in a ratio 1:20, result-
at the end of the capillary held the pipette at its back- ing in 10
15 DIGmoleculesavailableoverarangeof90nm.
end.
From a 1 mL syringe (BD Plastipak, Franklin
Lakes, NJ) the rubber cap was taken off from the hang(24.5kbp).Thesefragmentswerepurifiedbyelec-
plunger. Subsequently we placed the rubber cap tro-elution (Bio-trap, Chromtech, Cheshire, UK).
upside down in the syringe tube, creating a small but A 391 bp double-stranded DNA fragment labeled
long container. 200 lL TE-buffer with 100 lg/mL a- with digoxygenin was created by PCR using digoxyge-
DIGwasinjectedinthiscontainer.Uponinsertingthe nin labeled dUTPs (Roche, Basel, Switzerland). Since
capillary with the micropipette in this container the both dUTPanddTTPbindstodATPsomeofthedTTPs
capillary was filled with the a-DIG solution due to can be replaced for DIG-dUTP. The 391 bp fragment
capillary forces. After filling, the capillaries were was incubated with DIG-dUTP and dTTP (DIG-
taken out and closed at their open ends with another dUTP:dTTP ratio: 1:20) and dATP, dGTP, dCTP, and
rubber cap and stored at 48C. Shortly before the Klenow DNA polymerase for PCR. One in 20 dTTPs
experiment an a-DIG coated pipette was taken out of was replaced by a labeled dUTP. The 391 bp fragment
the capillary and rinsed several times with TE-buffer. was digested with SacI and XbaI to create a 268 bp
Next it was mounted into the microscope and directly fragment with a SacI overhang at one end and an XbaI
injected into the flow cell. overhang at the other end. Finally the fragment was
purified by electro-elution. The two long fragments
Digoxygenin-FunctionalizedDNA (22.6 and 24.5 kbp) were mixed together with the 268
Preparation bpfragmentataratioof1:1:20forannealing.Themix-
ture was heated and cooled down slowly and finally
Bacteriophage k-DNAwas end-labeled with biotin as ligated with T4 DNA ligase (NEB) at 168C. A schematic
described (Bennink et al., 1999). This k-DNA was structure of the resulting DNA molecules is shown in
digested with two enzymes, SacI (NEB, Ipswich, UK) Figure 4, where the lengths of the different regions are
andXbaI(NEB)tocreatetwolongfragments,onewith indicated. The contour length of the complete construct
a SacI overhang (22.6 kbp) and one with an XbaI over- is 16.1 lm.
MicroscopyResearchandTechniqueDOI10.1002/jemt
no reviews yet
Please Login to review.
