jagomart
digital resources
picture1_Scanning Probe Microscopy Pdf 90252 | Huisstede07


 136x       Filetype PDF       File size 0.45 MB       Source: www.nbi.dk


File: Scanning Probe Microscopy Pdf 90252 | Huisstede07
microscopyresearchandtechnique70 2633 2007 combiningopticaltweezersandscanningprobe microscopytostudydna proteininteractions jurgenh g huisstede vinodsubramaniam andmartinl bennink biophysicalengineeringgroupandmesa institutefornanotechnology facultyofscienceandtechnology university of twente ae enschede 7500 the netherlands keywords optical tweezers scanning probe microscopy dnaprotein ...

icon picture PDF Filetype PDF | Posted on 16 Sep 2022 | 3 years ago
Partial capture of text on file.
                                                                                                              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
The words contained in this file might help you see if this file matches what you are looking for:

...Microscopyresearchandtechnique combiningopticaltweezersandscanningprobe microscopytostudydna proteininteractions jurgenh g huisstede vinodsubramaniam andmartinl bennink biophysicalengineeringgroupandmesa institutefornanotechnology facultyofscienceandtechnology university of twente ae enschede the netherlands keywords optical tweezers scanning probe microscopy dnaprotein interactions dig oxygenin single molecule force spectroscopy abstract wepresenttherst results obtained with a new instrument designed and built to study at level this microscope combines allows us locate dna binding proteins on suspended is stretched taut using while scanned along interaction forces between sample are measured thus enables correlate mechanical andfunctional properties bound tension within typical friction micropipette used as naked was found be pn thenetherlands e mail m l utwente nlorv subramaniam nl in many ot afm experi receivedapril acceptedjuly ments change elastic doi jemt result protein studied p...

no reviews yet
Please Login to review.