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.

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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><tbody><tr><td>Reagent/Equipment</td><td>Company</td><td>Catalog number</td><td>Comments </td></tr><tr><td>Nd:YVO4 laser</td><td>Spectra Physics</td><td>T40-Z-106C</td><td></td></tr><tr><td>Acousto-optic deflector</td><td>IntraAction</td><td>DTD-274HA6</td><td></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></td></tr><tr><td>Photodetector</td><td>Electro-Optics Tech</td><td>ET-3020</td><td></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></td></tr><tr><td>Coverglass</td><td>Fisher Scientific</td><td></td><td></td></tr><tr><td>Other chemicals for buffer</td><td>Fisher Scientific</td><td></td><td></td></tr><tr><td colspan="4">&lt;p class=&quot;jove_title&quot;&gt;Supplementary Materials&lt;/p&gt;</td></tr><tr><td colspan="4">&lt;p class=&quot;jove_step&quot;&gt;A. Hydrodynamic Friction C&amp;#8211;fficient&lt;/p&gt;</td></tr><tr><td colspan="4">&lt;p class=&quot;jove_content&quot;&gt;For determining the hydrodynamic friction c&amp;#8211;fficient of the microsphere near a surface one can use the following expansion&lt;sup&gt;5,10&lt;/sup&gt;: &lt;img src=&quot;/files/ftp_upload/3405/3405eq1.jpg&quot; alt=&quot;Equation 1&quot;/&gt;&lt;/p&gt;</td></tr><tr><td colspan="4">&lt;p class=&quot;jove_content&quot;&gt;where the following shorthand has been introduced: &lt;img src=&quot;/files/ftp_upload/3405/3405eq2.jpg&quot; alt=&quot;Equation 2&quot;/&gt;&lt;/p&gt;</td></tr><tr><td colspan="4">&lt;p class=&quot;jove_content&quot;&gt;The friction c&amp;#8211;fficient is defined in terms of the fluid viscosity &amp;#951; and the radius of the microsphere, with the microsphere&#039;s center located a distance &amp;#951; above the surface. The summation converges reasonably well when expanded to about ten terms.&lt;/p&gt;</td></tr><tr><td colspan="4">&lt;p class=&quot;jove_step&quot;&gt;B. Influence of Axial Position on Stiffness Calibration&lt;/p&gt;</td></tr><tr><td colspan="4">&lt;p class=&quot;jove_content&quot;&gt;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.&lt;/p&gt;</td></tr><tr><td colspan="4">&lt;p class=&quot;jove_step&quot;&gt;C. Modified Worm-Like Chain (WLC) Model&lt;/p&gt;</td></tr><tr><td colspan="4">&lt;p class=&quot;jove_content&quot;&gt;The force extension curves can be fit to a modified WLC model that accounts for volume exclusion effects at zero optical force as follows: &lt;img src=&quot;/files/ftp_upload/3405/3405eq3.jpg&quot; alt=&quot;Equation 3&quot;/&gt;&lt;/p&gt;</td></tr><tr><td colspan="4">&lt;p class=&quot;jove_content&quot;&gt;where &lt;em&gt;F&lt;sub&gt;opt&lt;/sub&gt;&lt;/em&gt; is the optical force, &lt;em&gt;x&lt;sub&gt;o&lt;/sub&gt;&lt;/em&gt; is a fit parameter for the zero force extension,&lt;em&gt;x&lt;sub&gt;opt&lt;/sub&gt;&lt;/em&gt; is the extension under force, &lt;em&gt;l&lt;/em&gt; is the contour length of the DNA, and l*&lt;sub&gt;p&lt;/sub&gt; is a second fit parameter for an &quot;effective&quot; persistence length. &lt;em&gt;F&lt;sub&gt;wlc&lt;/sub&gt;&lt;/em&gt; is given by the usual WLC model&lt;sup&gt;11&lt;/sup&gt; &lt;img src=&quot;/files/ftp_upload/3405/3405eq4.jpg&quot; alt=&quot;Equation 4&quot;/&gt; where &lt;em&gt;&amp;#949;&lt;/em&gt; is the relative DNA extension.&lt;/p&gt;</td></tr></tbody></table>

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.

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

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

13.0K Views.  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.

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

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.

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

A Simple, Robust, and High Throughput Single Molecule Flow Stretching Assay Implementation for Studying Transport of Molecules Along DNA

We describe a simple, robust and high throughput single molecule flow-stretching assay for studying 1D diffusion of molecules along DNA. In this assay, glass coverslips are functionalized in a one-step reaction with silane-PEG-biotin. Flow cells are constructed by sandwiching an adhesive tape with pre-cut channels between a functionalized coverslip and a PDMS slab containing inlet and outlet holes. Multiple channels are integrated into one flow cell and the flow of reagents into each channel can be fully automated, which significantly increases the assay throughput and reduces hands-on time per assay. Inside each channel, biotin-&#955;-DNAs are immobilized on the surface and a laminar flow is applied to flow-stretch the DNAs. The DNA molecules are stretched to &#62;80% of their contour length and serve as spatially extended templates for studying the binding and transport activity of fluorescently labeled molecules. The trajectories of single molecules are tracked by time-lapse Total Internal Reflection Fluorescence (TIRF) imaging. Raw images are analyzed using streamlined custom single particle tracking software to automatically identify trajectories of single molecules diffusing along DNA and estimate their 1D diffusion constants.

This protocol demonstrates a simple, robust and high throughput single molecule flow-stretching assay for studying one-dimensional (1D) diffusion of molecules along DNA.

We describe a simple, robust and high throughput single molecule flow-stretching assay for studying 1D diffusion of molecules along DNA. In this assay, ...

Biochemistry

Single-molecule Manipulation of G-quadruplexes by Magnetic Tweezers

Non-canonical nucleic acid secondary structure G-quadruplexes (G4) are involved in diverse cellular processes, such as DNA replication, transcription, RNA processing, and telomere elongation. During these processes, various proteins bind and resolve G4 structures to perform their function. As the function of G4 often depends on the stability of its folded structure, it is important to investigate how G4 binding proteins regulate the stability of G4. This work presents a method to manipulate single G4 molecules using magnetic tweezers, which enables studies of the regulation of G4 binding proteins on a single G4 molecule in real time. In general, this method is suitable for a wide scope of applications in studies for proteins/ligands interactions and regulations on various DNA or RNA secondary structures.

A single-molecule magnetic tweezers platform to manipulate G-quadruplexes is reported, which allows for the study of G4 stability and regulation by various proteins.

Non-canonical nucleic acid secondary structure G-quadruplexes (G4) are involved in diverse cellular processes, such as DNA replication, transcription, RNA ...

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.

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.

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 ...

Direct Observation of Enzymes Replicating DNA Using a Single-molecule DNA Stretching Assay

We describe a method for observing real time replication of individual DNA molecules mediated by proteins of the bacteriophage replication system. Linearized &lambda; DNA is modified to have a biotin on the end of one strand, and a digoxigenin moiety on the other end of the same strand. The biotinylated end is attached to a functionalized glass coverslip and the digoxigeninated end to a small bead. The assembly of these DNA-bead tethers on the surface of a flow cell allows a laminar flow to be applied to exert a drag force on the bead. As a result, the DNA is stretched close to and parallel to the surface of the coverslip at a force that is determined by the flow rate (Figure 1). The length of the DNA is measured by monitoring the position of the bead. Length differences between single- and double-stranded DNA are utilized to obtain real-time information on the activity of the replication proteins at the fork. Measuring the position of the bead allows precise determination of the rates and processivities of DNA unwinding and polymerization (Figure 2).

We describe a method for observing real time replication of individual DNA molecules mediated by proteins of the bacteriophage replication system.

We describe a method for observing real time replication of individual DNA molecules mediated by proteins of the bacteriophage replication system. ...

Biology

Use of Dual Optical Tweezers and Microfluidics for Single-Molecule Studies

Visual biochemistry is a powerful technique for observing the stochastic properties of single enzymes or enzyme complexes that are obscured in the averaging that takes place in bulk-phase studies. To achieve visualization, dual optical tweezers, where one trap is fixed and the other is mobile, are focused into one channel of a multi-stream microfluidic chamber positioned on the stage of an inverted fluorescence microscope. The optical tweezers trap single molecules of fluorescently labeled DNA and fluid flow through the chamber and past the trapped beads, stretches the DNA to B-form (under minimal force, i.e., 0 pN) with the nucleic acid being observed as a white string against a black background. DNA molecules are moved from one stream to the next, by translating the stage perpendicular to the flow to enable the initiation of reactions in a controlled manner. To achieve success, microfluidic devices with optically clear channels are mated to glass syringes held in place in a syringe pump. Optimal results use connectors permanently bonded to the flow cell, tubing that is mechanically rigid and chemically resistant, and which is connected to switching valves that eliminate bubbles that prohibit laminar flow.

Visual, single-molecule biochemistry studied through microfluidic chambers is greatly facilitated using glass barrel, gas-tight syringes, stable connections of tubing to flow cells, and elimination of bubbles by placing switching valves between the syringes and tubing. The protocol describes dual optical traps that enable visualization of DNA transactions and intermolecular interactions.

Visual biochemistry is a powerful technique for observing the stochastic properties of single enzymes or enzyme complexes that are obscured in the ...

High-Speed Magnetic Tweezers for Nanomechanical Measurements on Force-Sensitive Elements

Single-molecule magnetic tweezers (MTs) have served as powerful tools to forcefully interrogate biomolecules, such as nucleic acids and proteins, and are therefore poised to be useful in the field of mechanobiology. Since the method commonly relies on image-based tracking of magnetic beads, the speed limit in recording and analyzing images, as well as the thermal fluctuations of the beads, has long hampered its application in observing small and fast structural changes in target molecules. This article describes detailed methods for the construction and operation of a high-resolution MT setup that can resolve nanoscale, millisecond dynamics of biomolecules and their complexes. As application examples, experiments with DNA hairpins and SNARE complexes (membrane-fusion machinery) are demonstrated, focusing on how their transient states and transitions can be detected in the presence of piconewton-scale forces. We expect that high-speed MTs will continue to enable high-precision nanomechanical measurements on molecules that sense, transmit, and generate forces in cells, and thereby deepen our molecular-level understanding of mechanobiology.

Here, we describe a high-speed magnetic tweezer setup that performs nanomechanical measurements on force-sensitive biomolecules at the maximum rate of 1.2 kHz. We introduce its application to DNA hairpins and SNARE complexes as model systems, but it will be also applicable to other molecules involved in mechanobiological events.

Single-molecule magnetic tweezers (MTs) have served as powerful tools to forcefully interrogate biomolecules, such as nucleic acids and proteins, and are ...

Studying TRF1/2 Interactions with Telomeric DNA Using Single-Molecule Assay

Analyzing Telomeric Protein-DNA Interactions Using Single-Molecule Magnetic Tweezers

Telomeres, the protective structures at the ends of chromosomes, are crucial for maintaining cellular longevity and genome stability. Their proper function depends on tightly regulated processes of replication, elongation, and damage response. The shelterin complex, especially Telomere Repeat-binding Factor 1 (TRF1) and TRF2, plays a pivotal role in telomere protection and has emerged as a potential anti-cancer target for drug discovery. These proteins bind to the repetitive telomeric DNA motif TTAGGG, facilitating the formation of protective structures and recruitment of other telomeric proteins. Structural methods and advanced imaging techniques have provided insights into telomeric protein-DNA interactions, but probing the dynamic processes requires single-molecule approaches. Tools like magnetic tweezers, optical tweezers, and atomic force microscopy (AFM) have been employed to study telomeric protein-DNA interactions, revealing important details such as TRF2-dependent DNA distortion and telomerase catalysis. However, the preparation of single-molecule constructs with telomeric repetitive motifs continues to be a challenging task, potentially limiting the breadth of studies utilizing single-molecule mechanical methods. To address this, we developed a method to study interactions using full-length human telomeric DNA with magnetic tweezers. This protocol describes how to express and purify TRF2, prepare telomeric DNA, set up single-molecule mechanical assays, and analyze data. This detailed guide will benefit researchers in telomere biology and telomere-targeted drug discovery.

Author Spotlight: Advanced Single-Molecule Techniques for Investigating Telomeric Protein-DNA Interactions

This protocol demonstrates using single-molecule magnetic tweezers to study interactions between telomeric DNA-binding proteins (Telomere Repeat-binding Factor 1 [TRF1] and TRF2) and long telomeres extracted from human cells. It describes the preparatory steps for telomeres and telomeric repeat-binding factors, the execution of single-molecule experiments, and the data collection and analysis methods.

Telomeres, the protective structures at the ends of chromosomes, are crucial for maintaining cellular longevity and genome stability. Their proper ...

CMG-Mediated DNA Unwinding Observed by ssDNA-RPA Tract Growth

Single-Molecule Real-Time Visualization of DNA Unwinding by CMG Helicase

Faithful genome duplication is essential for preserving the genetic stability of dividing cells. DNA replication is carried out during the S phase by a dynamic complex of proteins termed the replisome. At the heart of the replisome is the&#160;CDC45-MCM2-7-GINS (CMG) helicase, which separates the two strands of the DNA double helix such that DNA polymerases can copy each strand. During genome duplication, replisomes must overcome a plethora of obstacles and challenges. Each of these threatens genome stability, as failure to replicate DNA completely and accurately can lead to mutations, diseases, or cell death. Therefore, it is of great interest to understand how CMG functions in the replisome during both normal replication and replication stress. Here, we describe a total internal reflection fluorescence (TIRF) microscopy assay using recombinant purified proteins, which allows for real-time visualization of surface-tethered stretched DNA molecules by individual CMG complexes. This assay provides a powerful platform to investigate CMG behavior at the single-molecule level, allowing helicase dynamics to be directly observed with real-time control over reaction conditions.

Author Spotlight: Unraveling the Dynamics of Eukaryotic DNA Replication Through Single-Molecule Visualization

This protocol demonstrates performing a single-molecule assay for live visualization of DNA unwinding by CMG helicase. It describes (1) preparing a DNA substrate, (2) purifying fluorescently labeled Drosophila melanogaster CMG helicase, (3) preparing a microfluidic flow cell for total internal reflection fluorescence (TIRF) microscopy, and (4) the single-molecule DNA unwinding assay.

Faithful genome duplication is essential for preserving the genetic stability of dividing cells. DNA replication is carried out during the S phase by a ...