We thank Dr. Yih-Fan Chen for help with the axial optical tweezers and for contributing some of his stretching data to this manuscript. This work was sponsored by NSF grant PHY-0957293 and FOCUS grant PHY-0114336.

1. Tweezers Setup
<ol>
	<li>The beam from a 1064 nm laser is split into two orthogonally polarized beams. One is used to manipulate the biomolecule while the other is used for calibration purposes (see Fig. 2).</li>
	<li>The intensity of the manipulation beam is controlled by an acousto-optic deflector (AOD), while the position and focus of each beam is independently controlled by beam steering mirrors and optical telescopes, respectively.</li>
	<li>The beams are then recombined with another polarizing beam splitter and conditioned further by a final set of telescoping optics to slightly overfill the back aperture of the microscope objective. A 50% overfill for the calibration beam leads to a tight optical trap, while a slightly smaller factor for the manipulation beam, approximately 20%, gives a shallower focus that translates into a larger workable region of the linear potential. The beams are tightly focused by a PlanApo 60x/1.4 oil immersion microscope objective onto the sample cell.</li>
	<li>This tweezers setup is combined with a home built brightfield microscope that provides illumination from a Halogen lamp focused onto the sample chamber by a condenser. The brightfield image is separated from the laser light by a dichroic mirror and then imaged on two CCD cameras. One CCD acts as our primary means of acquiring data and is software triggered to yield precise sampling rates, while the other CCD is used to image a single stuck reference microsphere whose position serves as a feedback-control system to compensate for drift in the microscope.</li>
	<li>The sample is coarsely positioned with respect to the objective by an XY stage, and can then be precisely positioned by an embedded three dimensional piezo-stage.</li>
	<li>The forward-scattered and transmitted laser light is collected by the brightfield condenser and focused onto a photodetector. The resulting signal is filtered through an anti-aliasing filter at 100 kHz, amplified and acquired at 200 kHz using a DAQ card.</li>
</ol>
2. Calibrating Apparent Size of the Bead to Axial Position
<ol>
	<li>Load a sample chamber9 with a disperse solution of 800 nm diameter polystyrene microspheres diluted in phosphate buffered saline . Let sit for 10-15 minutes and then lightly flush with buffer to remove excess microspheres.</li>
	<li>Locate a microsphere randomly stuck to the coverglass and adjust the height of the sample chamber until the microsphere is approximately 1 &mu;m below the focus to generate a defocused image.</li>
	<li>To measure the apparent size of the image, first find the centre of the microsphere using the geometric pattern matching function in LabView.</li>
	<li>Next, generate a radial intensity profile of the microsphere by averaging 360&deg; about the center of each cross section. The radial profile, which corresponds to a white ring in each of the brightfield images, can be fitted with a quadratic function to find a brightness peak. The distance between this peak and the center can be used as a measure of the apparent size of the bead.</li>
	<li>Using the calibrated piezostage, gradually increase the axial position of the microsphere and acquire an image at each axial position with the CCD camera.</li>
	<li>Repeat the image analysis for each successive image to correlate the apparent size of the microsphere with its axial location (see Fig. 3). The axial resolution obtained by method is around 1.4 nm7.</li>
</ol>
3. Mapping the Optical Potential of the Manipulation Beam
<ol>
	<li>Begin by collinearly aligning the manipulation beam and the calibration beam. The calibration beam should be around 50 mW at the back aperture of the objective while the manipulation beam should be much weaker, say 10 mW.</li>
	<li>With the manipulation beam switched off, confine a free microsphere within the much stiffer trap of the calibration beam.</li>
	<li>Now, turn on the manipulation beam. Since the manipulation beam is much weaker than the calibration beam, the axial position of the microsphere will be slightly perturbed. The resulting change in axial position can be measured from the defocused brightfield images as already described.</li>
	<li>Turn off the manipulation beam and with the microsphere still trapped in the calibration beam, shift the axial focus of the calibration beam by adjusting the telescopic lens. Turn on the manipulation beam, measure the subsequent displacement of the microsphere and repeat.</li>
	<li>The displacements &Delta;X can be plotted against the axial position of the focus of the calibration beam. The axial position corresponding to the largest displacement of the microsphere determines the center of the linear region of the optical trap (see Fig. 4).</li>
	<li>The final step in obtaining the optical force of the manipulation beam as a function of axial position requires a careful measurement of the stiffness of the calibration beam. To obtain this, move the microsphere, trapped by the calibration beam, to around a micrometer above the surface by adjusting the telescopic lens while the manipulation beam is switched off.</li>
	<li>Record the thermal axial motion of the microsphere by measuring the intensity of transmitted light with a photodetector.</li>
	<li>The autocorrelation of the signal can be fitted to a single exponential decay function to give the time constant of the fluctuations, .&tau;z</li>
	<li>The hydrodynamic friction coefficient &zeta;, of the microsphere can be corrected for the microspheres proximity to a surface5,10 . The stiffness of the calibration trap is then given by &kappa;z = &zeta; / &tau;z (see Supplementary Materials A and B).</li>
	<li>Knowing the stiffness of the calibration trap allows one to convert the previously measured axial displacements into a force &fnof;z =&kappa;z &Delta;X Integration of this curve yields a spatial mapping of the axial optical potential of the manipulation beam (see Fig. 4).</li>
</ol>
4. Stretching a DNA Sample
<ol>
	<li>Mount the chamber9 containing surface tethered DNA molecules (&lt; 1 kbp) and, while observing the brightfield image, place the focus of the manipulation beam slightly above the unstretched microspheres by adjusting the telescope. Then adjust the position of the stage until one of the microspheres is trapped.</li>
	<li>Roughly position the microsphere in the center of the XY plane of the optical trap. Generate a series of square waves in LabView and send them to the AOD to repetitively turn the laser beam on and off. Carefully control the intensity of the laser and the duration of the on state to prevent heating of the sample chamber. Typically, use around 2 mW of laser power (measured at the back aperture of the objective) and send pulses of 200 ms (on) and 500 ms (off). The amplitude of the square wave sent to the AOD should be around 0.5 V.</li>
	<li>Watch the microsphere as the trap is repeatedly turned on and off and note if the laser induces any preferential direction to the microsphere's motion. While iteratively adjusting the microsphere position in both the X and Y direction, by controlling the piezostage, the random motion of the microsphere should become isotropic in the XY plane, though noticeably restricted when the laser is on.</li>
	<li>Next, align the bead in the Z direction. Again pulse the laser beam on and off while, this time, simultaneously measuring the microspheres axial displacement in real time. Center the stage within the linear region, which is the point where the Z displacement is greatest.</li>
	<li>After a careful alignment in the Z direction, it is imperative to check that the trap is still centered in the XY plane. If the XY alignment has changed, both the XY and Z alignment must be repeated until the microsphere is centered properly along all three axes.</li>
	<li>For stretching the DNA, ramp the laser intensity by sending a voltage signal to the AOD, from 0 V to 0.5 V in steps of 0.025 V. In each step, record 400 frames at 100 fps and average them to obtain the axial displacement.</li>
	<li>The force extension curves can now be plotted and fit to a modified WLC model11 (see Supplementary Materials B).</li>
</ol>
5. Representative Results:
We present force extension curves for two DNA sequences: a 1298 bp and a 247 bp sequence, the latter being the shortest sequence we have been able to stretch reproducibly. For short stretches of DNA, the conventional Worm-Like Chain (WLC) model does not fully explain the force extension relationship because at these length scales one must account for finite-size effects and zero force extension arising from boundary constraints. The force extension measurements, therefore, have to be fit using a modified WLC model which has an effective persistence length and a zero force extension as fit parameters, described further in the supplementary materials. For large contour lengths of dsDNA, the effective persistence length is simply the nominal persistence length (&tilde;50 nm) and the zero force extension can be neglected. However, as the contour length becomes shorter the effective persistence length decreases well below 50 nm and the DNA, even under zero force, shows a significant extension.
The data and the corresponding fits of the force extension curves are shown in Fig. 5 for the two sequences. From the modified WLC fits, we have determined the effective persistence lengths to be 35 nm for the 1298 bp DNA and 25 nm for 247 bp DNA. For illustrative purposes, we are presenting single measures of the force extension curves for each sequence. In practice, one would repeat the measurements multiple times and obtain the average results along with the standard errors. It must also be noted that after obtaining each curve it is imperative to ensure that the microsphere remains trapped and properly positioned within the linear region, otherwise the microsphere must be realigned as previously described in the protocol.
<img alt="Figure 1" src="/files/ftp_upload/3405/3405fig1.jpg" /> 
Figure 1 Principle of Axial Optical Tweezers. (Left) Conventional optical tweezers trap near the focus. The bead is then moved lateral to the coverslip to exert tension. (Right) In axial optical tweezers, a microsphere is trapped away from the focus, in the linear region of the optical potential. In this configuration, the polymer is held under constant tension for a range of extensions. Moreover, increasing the laser intensity moves the microsphere in the axial direction.
<img alt="Figure 2" src="/files/ftp_upload/3405/3405fig2.jpg" /> 
Figure 2 Schematic Representation of Axial Optical Tweezers. Laser light (1064nm Nd:YvO4) is split into two beams by passing through a polarizing beam splitter (PBS). The manipulation beam, passes through an acousto-optical deflector (AOD) and the calibration beam can be adjusted independently using telescopic lenses. The two beams are then recombined using another PBS and focused onto the sample by a high N.A. objective. Simultaneously, brightfield illumination used to track the microsphere,passes through the sample, is infrared (IR) filtered and is then imaged by two CCD cameras, one of which takes measurements while the other acts as part of a feedback control system.
<img alt="Figure 3" src="/files/ftp_upload/3405/3405fig3.jpg" /> 
Figure 3 Axial Position Calibration. To calibrate the axial position, we acquire defocused images of a bead stuck to the chamber coverslip at varying axial positions of the stage. The size of the microsphere is given by the distance between its center and the position of peak intensity about the bright ring formed around the image of the microsphere. The inset presents the radial intensity distribution which gives the size of the bead.
<img alt="Figure 4" src="/files/ftp_upload/3405/3405fig4.jpg" /> 
Figure 4 Principle Behind Mapping the Optical Potential. To map out the optical force of the manipulation beam, the axial displacement that it induces on a microsphere trapped within a much stronger calibration beam is measured. The center of the linear region where this displacement is a maximum is located. The Optical Force vs. Axial Position curve can be integrated to find the optical potential.
<img alt="Figure 5" src="/files/ftp_upload/3405/3405fig5.jpg" /> 
Figure 5 Force Extension Curve. Data is shown for a random 1298 bp and 247 bp (inset) segment of dsDNA stretched by axial optical tweezers. The data points were fit to the modified WLC model of Eq. 3 (solid lines) and yielded effective persistence lengths of 34 nm and 25 nm for the 1298bp and 247bp respectively.

<ol>
	<li>Halford, S. E., Welsh, A. J., Szczelkun, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enzyme-mediated+DNA+looping.">Enzyme-mediated DNA looping.</a> Annu. Rev. Biophys. Biomol. Struct. 33, 1-24 (2004).</li><li>Allemand, J. F., Cocco, S., Douarche, N., Lia, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loops+in+DNA:+an+overview+of+experimental+and+theoretical+approaches.">Loops in DNA: an overview of experimental and theoretical approaches.</a> Eur. Phys. J. E. Soft. Matter. 19, 293-302 (2006).</li><li>Kaplan, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+DNA-encoded+nucleosome+organization+of+a+eukaryotic+genome.">The DNA-encoded nucleosome organization of a eukaryotic genome.</a> Nature. 458, 362-366 (2009).</li><li>Garcia, H. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+Consequences+of+Tightly+Bent+DNA:+The+Other+Life+of+a+Macromolecular+Celebrity.">Biological Consequences of Tightly Bent DNA: The Other Life of a Macromolecular Celebrity.</a> Biopolymers. 85, 115-130 (2006).</li><li>Neuman, K. C., Block, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+trapping.">Optical trapping.</a> Rev. Sci. Instrum. 75, 2787-2809 (2004).</li><li>Moffitt, J. R., Chemla, Y. R., Smith, S. B., Bustamante, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+optical+tweezers.">Recent advances in optical tweezers.</a> Annu. Rev. Biochem. 77, 205-228 (2008).</li><li>Chen, Y. F., Blab, G. A., Meiners, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stretching+submicron+biomolecules+with+constant-force+axial+optical+tweezers.">Stretching submicron biomolecules with constant-force axial optical tweezers.</a> Biophys. J. 96, 4701-4708 (2009).</li><li>Chen, Y. F., Wilson, D. P., Raghunathan, K., Meiners, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Entropic+boundary+effects+on+the+elasticity+of+short+DNA+molecules.">Entropic boundary effects on the elasticity of short DNA molecules.</a> Phys. Rev. E. 80, 020903-020903 (2009).</li><li>Chen, Y. F., Chu, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tethered+Particle+Microscopy+(TPM)+Protocol.">Tethered Particle Microscopy (TPM) Protocol.</a> Meiners Lab. , (2011).</li><li>Brenner, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+slow+motion+of+a+sphere+through+a+viscous+fluid+towards+a+plane+surface.">The slow motion of a sphere through a viscous fluid towards a plane surface.</a> Chem. Eng. Sci. 16, 242-251 (1961).</li><li>Marko, J. F., Siggia, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stretching+DNA.">Stretching DNA.</a> Macromolecules. 28, 8759-8770 (1995).</li><li>Greenleaf, W. J., Block, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-molecule,+motion-based+DNA+sequencing+using+RNA+polymerase.">Single-molecule, motion-based DNA sequencing using RNA polymerase.</a> Science. 313, 801-801 (2006).</li><li>Chen, Y. F., Milstein, J. N., Meiners, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein-mediated+DNA+loop+formation+and+breakdown+in+a+fluctuating+environment.">Protein-mediated DNA loop formation and breakdown in a fluctuating environment.</a> Phys. Rev. Lett. 104, 258103-258103 (2010).</li><li>Chen, Y. F., Milstein, J. N., Meiners, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Femtonewton+entropic+forces+can+control+the+formation+of+protein-mediated+DNA+loops.">Femtonewton entropic forces can control the formation of protein-mediated DNA loops.</a> Phys. Rev. Lett. 104, 048301-048301 (2010).</li><li>Raghunathan, K., Milstein, J. N., Juliar, B., Blaty, J., Meiners, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sequence+Dependent+Effects+on+the+Elasticity+of+Short+DNA+Molecules.">Sequence Dependent Effects on the Elasticity of Short DNA Molecules.</a> , (2011).</li></ol>

No conflicts of interest declared.

Conventional optical tweezers rely upon either analog or computer-controlled feedback to apply a constant force on a refractile object. These active feedback systems have difficulty performing under conditions where sudden changes in the extension of the specimen occur, for instance, from the binding of a protein to DNA or the rapid stepping of a molecular motor along a filament. Various passive methods for applying constant forces have recently been developed. One such method, used to resolve the stepping of RNA polymer...

<table border="1">
	<tbody>
		<tr>
			<th>Reagent/Equipment</th>
			<th>Company</th>
			<th>Catalog number</th>
			<th>Comments </th>
		</tr>
		<tr>
			<td>Nd:YVO4 laser</td>
			<td>Spectra Physics</td>
			<td>T40-Z-106C</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Acousto-optic deflector</td>
			<td>IntraAction</td>
			<td>DTD-274HA6</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Microscope Objective</td>
			<td>Olympus</td>
			<td>PlanApo </td>
			<td>60X, NA 1.4</td>
		</tr>
		<tr>
			<td>Piezo stage</td>
			<td>Mad City Labs</td>
			<td>Nano-LP100</td>
			<td>XYZ stage</td>
		</tr>
		<tr>
			<td>CCD camera</td>
			<td>PixeLink</td>
			<td>PL-A741</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Photodetector</td>
			<td>Electro-Optics Tech</td>
			<td>ET-3020</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Polystyrene Beads</td>
			<td>Spherotech</td>
			<td>SVP-08-10</td>
			<td>800nm, streptavidin coated</td>
		</tr>
		<tr>
			<td>Anti-digoxigenin</td>
			<td>Roche</td>
			<td>11333089001</td>
			<td>From sheep</td>
		</tr>
		<tr>
			<td>Primers</td>
			<td>MWG operon</td>
			<td>Custom oligos</td>
			<td>One primer: biotin 
Other : digoxigenin 
</td>
		</tr>
		<tr>
			<td>PCR reagents</td>
			<td>New England Biolabs</td>
			<td>TAQ polymerase, dNTPs</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Coverglass</td>
			<td>Fisher Scientific</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Other chemicals for buffer</td>
			<td>Fisher Scientific</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>
Supplementary Materials
A. Hydrodynamic Friction Coefficient
For determining the hydrodynamic friction coefficient of the microsphere near a surface one can use the following expansion5,10: 
<img src="/files/ftp_upload/3405/3405eq1.jpg" alt="Equation 1" />
where the following shorthand has been introduced: 
<img src="/files/ftp_upload/3405/3405eq2.jpg" alt="Equation 2" />
The friction coefficient is defined in terms of the fluid viscosity &eta; and the radius of the microsphere, with the microsphere's center located a distance &eta; above the surface. The summation converges reasonably well when expanded to about ten terms.
B. Influence of Axial Position on Stiffness Calibration
The calibration of the trap stiffness involves a tradeoff between the accuracy of the calibration, which increases with increasing distance from the surface, and the actual axial position where the trap is used experimentally. In general, the trap is calibrated at around 800-1000 nm from the surface, which is higher than the actual experimental condition.
C. Modified Worm-Like Chain (WLC) Model
The force extension curves can be fit to a modified WLC model that accounts for volume exclusion effects at zero optical force as follows: 
<img src="/files/ftp_upload/3405/3405eq3.jpg" alt="Equation 3" />
where Fopt is the optical force, xo is a fit parameter for the zero force extension,xopt is the extension under force, l is the contour length of the DNA, and l*p is a second fit parameter for an "effective" persistence length. Fwlc is given by the usual WLC model11 
<img src="/files/ftp_upload/3405/3405eq4.jpg" alt="Equation 4" /> 
where &epsilon; is the relative DNA extension.

stretching short sequences of dna with constant force axial optical tweezers

Single-molecule techniques for stretching DNA of contour lengths less than a kilobase are fraught with experimental difficulties. However, many interesting biological events such as histone binding and protein-mediated looping of DNA1,2, occur on this length scale. In recent years, the mechanical properties of DNA have been shown to play a significant role in fundamental cellular processes like the packaging of DNA into compact nucleosomes and chromatin fibers3,4. Clearly, it is then important to understand the mechanical properties of short stretches of DNA. In this paper, we provide a practical guide to a single-molecule optical tweezing technique that we have developed to study the mechanical behavior of DNA with contour lengths as short as a few hundred basepairs.
The major hurdle in stretching short segments of DNA is that conventional optical tweezers are generally designed to apply force in a direction lateral to the stage5,6, (see Fig. 1). In this geometry, the angle between the bead and the coverslip, to which the DNA is tethered, becomes very steep for submicron length DNA. The axial position must now be accounted for, which can be a challenge, and, since the extension drags the microsphere closer to the coverslip, steric effects are enhanced. Furthermore, as a result of the asymmetry of the microspheres, lateral extensions will generate varying levels of torque due to rotation of the microsphere within the optical trap since the direction of the reactive force changes during the extension.
Alternate methods for stretching submicron DNA run up against their own unique hurdles. For instance, a dual-beam optical trap is limited to stretching DNA of around a wavelength, at which point interference effects between the two traps and from light scattering between the microspheres begin to pose a significant problem. Replacing one of the traps with a micropipette would most likely suffer from similar challenges. While one could directly use the axial potential to stretch the DNA, an active feedback scheme would be needed to apply a constant force and the bandwidth of this will be quite limited, especially at low forces.
We circumvent these fundamental problems by directly pulling the DNA away from the coverslip by using a constant force axial optical tweezers7,8. This is achieved by trapping the bead in a linear region of the optical potential, where the optical force is constant-the strength of which can be tuned by adjusting the laser power. Trapping within the linear region also serves as an all optical force-clamp on the DNA that extends for nearly 350 nm in the axial direction. We simultaneously compensate for thermal and mechanical drift by finely adjusting the position of the stage so that a reference microsphere stuck to the coverslip remains at the same position and focus, allowing for a virtually limitless observation period.

Watch this Scientific Journal Video about Stretching Short Sequences of DNA with Constant Force Axial Optical Tweezers at JoVE.com

Stretching Short Sequences of DNA with Constant Force Axial Optical Tweezers

We illustrate the use of a constant force axial optical tweezers to explore the mechanical properties of short DNA molecules. By stretching DNA axially, we minimize steric hindrances and artifacts arising in conventional lateral manipulation, allowing us to study DNA molecules as short as ~100 nm.

Stretching kurze DNA-Sequenzen mit Constant Force Axial Optische Pinzetten

Single-molecule techniques for stretching DNA of contour lengths  less than a kilobase are fraught with experimental difficulties. However, many ...

stretching-short-sequences-dna-with-constant-force-axial-optical

Research

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Bioengineering

13.0K Aufrufe.  University of Michigan. We illustrate the use of a constant force axial optical tweezers to explore the mechanical properties of short DNA molecules. By stretching DNA axially, we minimize steric hindrances and artifacts arising in conventional lateral manipulation, allowing us to study DNA molecules as short as ~100 nm.

Serie: Stretching Short Sequences of DNA with Constant Force Axial Optical Tweezers

Visualization of Recombinant DNA and Protein Complexes Using Atomic Force Microscopy

Atomic force microscopy (AFM) allows for the visualizing of individual proteins, DNA molecules, protein-protein complexes, and DNA-protein complexes. On the end of the microscope's cantilever is a nano-scale probe, which traverses image areas ranging from nanometers to micrometers, measuring the elevation of macromolecules resting on the substrate surface at any given point. Electrostatic forces cause proteins, lipids, and nucleic acids to loosely attach to the substrate in random orientations and permit imaging. The generated data resemble a topographical map, where the macromolecules resolve as three-dimensional particles of discrete sizes (Figure 1) 1,2. Tapping mode AFM involves the repeated oscillation of the cantilever, which permits imaging of relatively soft biomaterials such as DNA and proteins. One of the notable benefits of AFM over other nanoscale microscopy techniques is its relative adaptability to visualize individual proteins and macromolecular complexes in aqueous buffers, including near-physiologic buffered conditions, in real-time, and without staining or coating the sample to be imaged. 
The method presented here describes the imaging of DNA and an immunoadsorbed transcription factor (i.e. the glucocorticoid receptor, GR) in buffered solution (Figure 2). Immunoadsorbed proteins and protein complexes can be separated from the immunoadsorbing antibody-bead pellet by competition with the antibody epitope and then imaged (Figure 2A). This allows for biochemical manipulation of the biomolecules of interest prior to imaging. Once purified, DNA and proteins can be mixed and the resultant interacting complex can be imaged as well. Binding of DNA to mica requires a divalent cation 3,such as Ni2+ or Mg2+, which can be added to sample buffers yet maintain protein activity. Using a similar approach, AFM has been utilized to visualize individual enzymes, including RNA polymerase 4 and a repair enzyme 5, bound to individual DNA strands. These experiments provide significant insight into the protein-protein and DNA-protein biophysical interactions taking place at the molecular level. Imaging individual macromolecular particles with AFM can be useful for determining particle homogeneity and for identifying the physical arrangement of constituent components of the imaged particles. While the present method was developed for visualization of GR-chaperone protein complexes 1,2 and DNA strands to which the GR can bind, it can be applied broadly to imaging DNA and protein samples from a variety of sources.

A tapping mode atomic force microscope (AFM) method for the visualization of plasmid DNA, cytoplasmic proteins, and DNA-protein complexes is described. The method includes alternate approaches for preparing samples for AFM imaging following biochemical manipulation. DNA containing specific protein interacting regions are observed in near-physiologic buffer conditions.

Visualisierung von rekombinanten DNA-und Protein-Komplexe mit Atomic Force Microscopy

Atomic force microscopy (AFM) allows for the visualizing of individual proteins, DNA molecules, protein-protein complexes, and DNA-protein complexes. On ...

Forschung

Biotechnik

Multiplexed Single-molecule Force Proteolysis Measurements Using Magnetic Tweezers

The generation and detection of mechanical forces is a ubiquitous aspect of cell physiology, with direct relevance to cancer metastasis1, atherogenesis2 and wound healing3. In each of these examples, cells both exert force on their surroundings and simultaneously enzymatically remodel the extracellular matrix (ECM). The effect of forces on ECM has thus become an area of considerable interest due to its likely biological and medical importance4-7.
	Single molecule techniques such as optical trapping8, atomic force microscopy9, and magnetic tweezers10,11 allow researchers to probe the function of enzymes at a molecular level by exerting forces on individual proteins. Of these techniques, magnetic tweezers (MT) are notable for their low cost and high throughput. MT exert forces in the range of ~1-100 pN and can provide millisecond temporal resolution, qualities that are well matched to the study of enzyme mechanism at the single-molecule level12. Here we report a highly parallelizable MT assay to study the effect of force on the proteolysis of single protein molecules. We present the specific example of the proteolysis of a trimeric collagen peptide by matrix metalloproteinase 1 (MMP-1); however, this assay can be easily adapted to study other substrates and proteases.

In this article we describe the use of magnetic tweezers to study the effect of force on enzymatic proteolysis at the single molecule level in a highly parallelizable manner.

Multiplexed Einzelmolekül-Kraftspektroskopie Proteolyse Messungen mit magnetischen Pinzette

The  generation and detection of mechanical forces is a ubiquitous aspect of cell physiology,  with direct relevance to cancer metastasis1, atherogenesis2 ...

Quantifying the Mechanical Properties of the Endothelial Glycocalyx with Atomic Force Microscopy

Our understanding of the interaction of leukocytes and the vessel wall during leukocyte capture is limited by an incomplete understanding of the mechanical properties of the endothelial surface layer. It is known that adhesion molecules on leukocytes are distributed non-uniformly relative to surface topography 3, that topography limits adhesive bond formation with other surfaces 9, and that physiological contact forces (&asymp; 5.0 &minus; 10.0 pN per microvillus) can compress the microvilli to as little as a third of their resting length, increasing the accessibility of molecules to the opposing surface 3, 7. We consider the endothelium as a two-layered structure, the relatively rigid cell body, plus the glycocalyx, a soft protective sugar coating on the luminal surface 6. It has been shown that the glycocalyx can act as a barrier to reduce adhesion of leukocytes to the endothelial surface 4. In this report we begin to address the deformability of endothelial surfaces to understand how the endothelial mechanical stiffness might affect bond formation. Endothelial cells grown in static culture do not express a robust glycocalyx, but cells grown under physiological flow conditions begin to approximate the glycocalyx observed in vivo 2. The modulus of the endothelial cell body has been measured using atomic force microscopy (AFM) to be approximately 5 to 20 kPa 5. The thickness and structure of the glycocalyx have been studied using electron microscopy 8, and the modulus of the glycocalyx has been approximated using indirect methods, but to our knowledge, there have been no published reports of a direct measurement of the glycocalyx modulus in living cells. In this study, we present indentation experiments made with a novel AFM probe on cells that have been cultured in conditions to maximize their glycocalyx expression to make direct measurements of the modulus and thickness of the endothelial glycocalyx.

The mechanical characteristics of endothelial glycocalyx were measured by indentation using micron sized spheres on AFM cantilevers. Endothelial cells were cultured in a custom chamber under physiological flow conditions to induce glycocalyx expression. Data were analyzed using a thin film model to determine the glycocalyx thickness and modulus.

Die Quantifizierung der mechanischen Eigenschaften des Endothelial Glykokalyx mit Atomic Force Microscopy

Our  understanding of the interaction of leukocytes and the vessel wall during  leukocyte capture is limited by an incomplete understanding of the ...

Biosensor for Detection of Antibiotic Resistant Staphylococcus Bacteria

A structurally transformed lytic bacteriophage having a broad host range of Staphylococcus aureus strains and a penicillin-binding protein (PBP 2a) antibody conjugated latex beads have been utilized to create a biosensor designed for discrimination of methicillin resistant (MRSA) and sensitive (MSSA) S. aureus species 1,2. The lytic phages have been converted into phage spheroids by contact with water-chloroform interface. Phage spheroid monolayers have been moved onto a biosensor surface by Langmuir-Blodgett (LB) technique 3. The created biosensors have been examined by a quartz crystal microbalance with dissipation tracking (QCM-D) to evaluate bacteria-phage interactions. Bacteria-spheroid interactions led to reduced resonance frequency and a rise in dissipation energy for both MRSA and MSSA strains. After the bacterial binding, these sensors have been further exposed to the penicillin-binding protein antibody latex beads. Sensors analyzed with MRSA responded to PBP 2a antibody beads; although sensors inspected with MSSA gave no response. This experimental distinction determines an unambiguous discrimination between methicillin resistant and sensitive S. aureus strains. Equally bound and unbound bacteriophages suppress bacterial growth on surfaces and in water suspensions. Once lytic phages are changed into spheroids, they retain their strong lytic activity and show high bacterial capture capability. The phage and phage spheroids can be utilized for testing and sterilization of antibiotic resistant microorganisms. Other applications may include use in bacteriophage therapy and antimicrobial surfaces.

Lytic phage biosensors and antibody beads are able to discriminate between methicillin resistant (MRSA) and sensitive staphylococcus bacteria. The phages were immobilized by a Langmuir-Blodgett method onto a surface of a quartz crystal microbalance sensor and worked as broad range staphylococcus probes. Antibody beads recognize MRSA.

Biosensor zum Nachweis von Antibiotika-resistenten Staphylococcus Bakterien

A structurally transformed lytic bacteriophage having a broad host range of Staphylococcus aureus strains and a penicillin-binding protein (PBP 2a) ...

Investigating Receptor-ligand Systems of the Cellulosome with AFM-based Single-molecule Force Spectroscopy

Cellulosomes are discrete multienzyme complexes used by a subset of anaerobic bacteria and fungi to digest lignocellulosic substrates. Assembly of the enzymes onto the noncatalytic scaffold protein is directed by interactions among a family of related receptor-ligand pairs comprising interacting cohesin and dockerin modules. The extremely strong binding between cohesin and dockerin modules results in dissociation constants in the low picomolar to nanomolar range, which may hamper accurate off-rate measurements with conventional bulk methods. Single-molecule force spectroscopy (SMFS) with the atomic force microscope measures the response of individual biomolecules to force, and in contrast to other single-molecule manipulation methods (i.e.&#160;optical tweezers), is optimal for studying high-affinity receptor-ligand interactions because of its ability to probe the high-force regime (&#62;120 pN). Here we present our complete protocol for studying cellulosomal protein assemblies at the single-molecule level. Using a protein topology derived from the native cellulosome, we worked with enzyme-dockerin and carbohydrate binding module-cohesin (CBM-cohesin) fusion proteins, each with an accessible free thiol group at an engineered cysteine residue. We present our site-specific surface immobilization protocol, along with our measurement and data analysis procedure for obtaining detailed binding parameters for the high-affinity complex. We demonstrate how to quantify single subdomain unfolding forces, complex rupture forces, kinetic off-rates, and potential widths of the binding well. The successful application of these methods in characterizing the cohesin-dockerin interaction responsible for assembly of multidomain cellulolytic complexes is further described.

Cellulosomes are multienzyme complexes designed for digesting cellulose. AFM-based SMFS was used to study the mechanical properties and folding configuration of cellulosome-associated protein assemblies. We present a complete workflow for protein immobilization, data acquisition, and data analysis to study the interactions of individual receptor-ligand complexes involved in cellulosome assembly.

Cellulosomes are discrete multienzyme complexes used by a subset of anaerobic bacteria and fungi to digest lignocellulosic substrates. Assembly of the ...

Quantitative Optical Microscopy: Measurement of Cellular Biophysical Features with a Standard Optical Microscope

We describe the use of a standard optical microscope to perform quantitative measurements of mass, volume, and density on cellular specimens through a combination of bright field and differential interference contrast imagery. Two primary approaches are presented: noninterferometric quantitative phase microscopy (NIQPM), to perform measurements of total cell mass and subcellular density distribution, and Hilbert transform differential interference contrast microscopy (HTDIC)&#160;to determine volume. NIQPM is based on a simplified model of wave propagation, termed the paraxial approximation, with three underlying assumptions: low numerical aperture (NA) illumination, weak scattering, and weak absorption of light by the specimen. Fortunately, unstained cellular specimens satisfy these assumptions and low NA illumination is easily achieved on commercial microscopes. HTDIC is used to obtain volumetric information from through-focus DIC imagery under high NA illumination conditions. High NA illumination enables enhanced sectioning of the specimen along the optical axis. Hilbert transform processing on the DIC image stacks greatly enhances edge detection algorithms for localization of the specimen borders in three dimensions by separating the gray values of the specimen intensity from those of the background. The primary advantages of NIQPM and HTDIC lay in their technological accessibility using &#8220;off-the-shelf&#8221; microscopes. There are two basic limitations of these methods: slow z-stack acquisition time on commercial scopes currently abrogates the investigation of phenomena faster than 1 frame/minute, and secondly, diffraction effects restrict the utility of NIQPM and HTDIC to objects from 0.2 up to 10 (NIQPM) and 20 (HTDIC) &#956;m in diameter, respectively. Hence, the specimen and its associated time dynamics of interest must meet certain size and temporal constraints to enable the use of these methods. Excitingly, most fixed cellular specimens are readily investigated with these methods.

We describe the use of a standard optical microscope to perform quantitative measurements of cellular mass, volume, and density through a combination of bright field and differential interference contrast imagery.

Quantitative Optische Mikroskopie: Messung der Cellular biophysikalische Eigenschaften mit einem Standard Optisches Mikroskop

We describe the use of a standard optical microscope to perform quantitative measurements of mass, volume, and density on cellular specimens through a ...

Stretching Micropatterned Cells on a PDMS Membrane

Mechanical forces exerted on cells and/or tissues play a major role in numerous processes. We have developed a device to stretch cells plated on a PolyDiMethylSiloxane (PDMS) membrane, compatible with imaging. This technique is reproducible and versatile. The PDMS membrane can be micropatterned in order to confine cells or tissues to a specific geometry. The first step is to print micropatterns onto the PDMS membrane with a deep UV technique. The PDMS membrane is then mounted on a mechanical stretcher. A chamber is bound on top of the membrane with biocompatible grease to allow gliding during the stretch. The cells are seeded and allowed to spread for several hours on the micropatterns. The sample can be stretched and unstretched multiple times with the use of a micrometric screw. It takes less than a minute to apply the stretch to its full extent (around 30%). The technique presented here does not include a motorized device, which is necessary for applying repeated stretch cycles quickly and/or computer controlled stretching, but this can be implemented. Stretching of cells or tissue can be of interest for questions related to cell forces, cell response to mechanical stress or tissue morphogenesis. This video presentation will show how to avoid typical problems that might arise when doing this type of seemingly simple experiment.

This manuscript presents a technique to apply or release forces on adherent cells or tissues using unidirectional stretching.

Stretching-Cells auf einer mikrostrukturierten PDMS-Membran

Mechanical forces exerted on cells and/or tissues  play a major role in numerous processes. We have developed a device to stretch  cells plated on a ...

Magnetic Tweezers for the Measurement of Twist and Torque

Single-molecule techniques make it possible to investigate the behavior of individual biological molecules in solution in real time. These techniques include so-called force spectroscopy approaches such as atomic force microscopy, optical tweezers, flow stretching, and magnetic tweezers. Amongst these approaches, magnetic tweezers have distinguished themselves by their ability to apply torque while maintaining a constant stretching force. Here, it is illustrated how such a &#8220;conventional&#8221; magnetic tweezers experimental configuration can, through a straightforward modification of its field configuration to minimize the magnitude of the transverse field, be adapted to measure the degree of twist in a biological molecule. The resulting configuration is termed the freely-orbiting magnetic tweezers. Additionally, it is shown how further modification of the field configuration can yield a transverse field with a magnitude intermediate between that of the &#8220;conventional&#8221; magnetic tweezers and the freely-orbiting magnetic tweezers, which makes it possible to directly measure the torque stored in a biological molecule. This configuration is termed the magnetic torque tweezers. The accompanying video explains in detail how the conversion of conventional magnetic tweezers into freely-orbiting magnetic tweezers and magnetic torque tweezers can be accomplished, and demonstrates the use of these techniques. These adaptations maintain all the strengths of conventional magnetic tweezers while greatly expanding the versatility of this powerful instrument.

Magnetic tweezers, a powerful single-molecule manipulation technique, can be adapted for the direct measurements of the twist (using a configuration called freely-orbiting magnetic tweezers) and torque (using a configuration termed magnetic torque tweezers) in biological macromolecules. Guidelines for performing such measurements are given, including applications to the study of DNA and associated nucleo-protein filaments.

Magnetische Pinzette zur Messung von Dreh-und Drehmoment

Single-molecule techniques make it possible to investigate the behavior of individual biological molecules in solution in real time. These techniques ...

Nanomanipulation of Single RNA Molecules by Optical Tweezers

A large portion of the human genome is transcribed but not translated. In this post genomic era, regulatory functions of RNA have been shown to be increasingly important. As RNA function often depends on its ability to adopt alternative structures, it is difficult to predict RNA three-dimensional structures directly from sequence. Single-molecule approaches show potentials to solve the problem of RNA structural polymorphism by monitoring molecular structures one molecule at a time. This work presents a method to precisely manipulate the folding and structure of single RNA molecules using optical tweezers. First, methods to synthesize molecules suitable for single-molecule mechanical work are described. Next, various calibration procedures to ensure the proper operations of the optical tweezers are discussed. Next, various experiments are explained. To demonstrate the utility of the technique, results of mechanically unfolding RNA hairpins and a single RNA kissing complex are used as evidence. In these examples, the nanomanipulation technique was used to study folding of each structural domain, including secondary and tertiary, independently. Lastly, the limitations and future applications of the method are discussed.

Optical tweezers have been used to study RNA folding by stretching individual molecules from their 5&#8217; and 3&#8217; ends. Here common procedures are described to synthesize RNA molecules for tweezing, calibration of the instrument, and methods to manipulate single molecules.

Nanomanipulation von RNA-Einzelmoleküle durch Optische Pinzetten

A large portion of the human genome is transcribed but not translated. In this post genomic era, regulatory functions of RNA have been shown to be ...

Quantifying Fibrillar Collagen Organization with Curvelet Transform-Based Tools

Fibrillar collagens are prominent extracellular matrix (ECM) components, and their topology changes have been shown to be associated with the progression of a wide range of diseases including breast, ovarian, kidney, and pancreatic cancers. Freely available fiber quantification software tools are mainly focused on the calculation of fiber alignment or orientation, and they are subject to limitations such as the requirement of manual steps, inaccuracy in detection of the fiber edge in noisy background, or lack of localized feature characterization. The collagen fiber quantitation tool described in this protocol is characterized by using an optimal multiscale image representation enabled by curvelet transform (CT). This algorithmic approach allows for the removal of noise from fibrillar collagen images and the enhancement of fiber edges to provide location and orientation information directly from a fiber, rather than using the indirect pixel-wise or window-wise information obtained from other tools. This CT-based framework contains two separate, but linked, packages named &#8220;CT-FIRE&#8221; and &#8220;CurveAlign&#8221; that can quantify fiber organization on a global, region of interest (ROI), or individual fiber basis. This quantification framework has been developed for more than ten years and has now evolved into a comprehensive and user-driven collagen quantification platform. Using this platform, one can measure up to about thirty fiber features including individual fiber properties such as length, angle, width, and straightness, as well as bulk measurements such as density and alignment. Additionally, the user can measure fiber angle relative to manually or automatically segmented boundaries. This platform also provides several additional modules including ones for ROI analysis, automatic boundary creation, and post-processing. Using this platform does not require prior experience of programming or image processing, and it can handle large datasets including hundreds or thousands of images, enabling efficient quantification of collagen fiber organization for biological or biomedical applications.

Here, we present a protocol to use a curvelet transform-based, open-source MATLAB software tool for quantifying fibrillar collagen organization in the extracellular matrix of both normal and diseased tissues. This tool can be applied to images with collagen fibers or other types of line-like structures.

Quantifizierung der Fibrillar Collagen Organisation mit Curvelet Transform-Based Tools

Fibrillar collagens are prominent extracellular matrix (ECM) components, and their topology changes have been shown to be associated with the progression ...