This work was supported by an NSF grant (MCB-1054449) and a collaborative interdisciplinary Pilot Research Program award from The RNA Institute at University at Albany to PTXL.

1. Preparation of RNA Molecules for Single-molecule Tweezing
<ol>
	<li>Cloning the sequence of interest. Clone the DNA sequence corresponding to the RNA structure into a vector.</li>
	<li>Synthesis and purification of the transcription template. Synthesize a transcription template by PCR. [place table 1 here] Next, purify the PCR products using a PCR purification kit, and concentrate the sample to &#62;200 ng/&#181;l. Because the purified DNA is used for transcription, RNase free water should be used in the elution and concentration steps.</li>
	<li>In vitro transcription. Synthesize RNA by in vitro transcription at 37 &#176;C overnight to maximize RNA yield. [place table 2 here] After transcription, add 1 &#181;l DNase I to digest the DNA template for 15 min at 37 &#176;C. Purify the RNA using a silica spin column.</li>
	<li>Synthesis of the biotinylated handle A. First, produce the unmodified handle A by PCR using primers Af and Ar and concentrate the amplified DNA to &#62;200 ng/&#181;l. Next, incorporate biotin modifications into the PCR product using a primer extension reaction. [place table 3 here] NOTE: The biotinylated handle A (biotin-HA) can be directly used in the annealing without purification. As the biotin-HA is to be annealed with RNA, it is critical to use RNase free water in both steps.</li>
	<li>Synthesis of the digoxigenin-modified handle B. Synthesize digoxigenin modified handle B (dig-HB) by PCR using primer Bf and digoxigenin-modified oligo Dig-Br (Figure 3). Next, purify the PCR product and concentrate the sample to &#62;200 ng/&#181;l.</li>
	<li>Annealing RNA to the handles. Anneal the RNA with handles. [place tables 4 and 5 here]</li>
	<li>Purify annealed sample. Add 3 volumes of ethanol of the annealed sample and mix well. Store the mixture at -70 &#176;C for at least 1 hr. Spin down at 15,800 x g and discard supernatant. Dry the pellet using a lyophilizer. Dissolve the pellet in 100 &#181;l RNase free water. NOTE: The sample is ready to be used and can be stored at -20 &#176;C.</li>
</ol>
2. Calibration and Operation of the Optical Tweezers
<ol>
	<li>Calibrate Pixel Size of Video Monitor
	<ol>
		<li>Use the optical trap to trap a bead with known diameter (size standard).</li>
		<li>Capture images of a trapped bead using imaging software (Figure 4).</li>
		<li>Determine the diameter of the bead using the imaging software based on the centroid method.</li>
		<li>Repeat this procedure to determine diameters of beads of different size.</li>
		<li>Fit the measured and known diameters of the beads by a linear regression to obtain the physical size of a pixel.</li>
	</ol>
	</li>
	<li>Verify Distance Calibration
	<ol>
		<li>Use the optical trap to trap a bead.</li>
		<li>Determine the pixel position of its centroid by the image capture software and record its position using the Minitweezers.</li>
		<li>Steer the bead to different positions and repeat the position determination.</li>
		<li>Convert the X and Y positions of the bead from pixels to m unit using the calibrated pixel size.</li>
		<li>Compare the X and Y between positions measured by video and by the Minitweezers. The two sets of values should agree. As the image resolution is &#62; 0.1 &#181;m, move the bead over 1 &#181;m between measurements to ensure accuracy. If the distance calibration is not comparable to the one stored in the Minitweezers, recalibrate the instrument.</li>
	</ol>
	</li>
	<li>Trap Stiffness Calibration
	<ol>
		<li>Capture a bead using the optical trap and hold it still.</li>
		<li>Record the force of the trap using a bypass fast data acquisition at a rate &#62; 5 kHz for 3 sec.</li>
		<li>Generate a power spectrum from the recording of the bead motion using software. Fit the power spectrum to a Lorentzian (Figure 5)38: 
		<img alt="Equation 1" fo:content-width="2in" src="/files/ftp_upload/51542/51542eq1.jpg" width="200" /> (Eq. 1) 
		in which S(f) is the power at the frequency of f, fc is the corner frequency, and S0 is the asymptotic power. The corner frequency, fc, is the frequency where the power of thermal motion has decreased to half of the asymptotic value. As fc varies with laser power and bead size, calibrate each trapped bead individually.</li>
		<li>Compute the spring constant of the trap, &#954;, from fc: 
		<img alt="Equation 2" fo:content-width="1in" src="/files/ftp_upload/51542/51542eq2.jpg" width="100" /> (Eq. 2) 
		in which &#947; is the drag coefficient of the bead (Eq. 3). Use the spring constant to determine extension changes of an RNA from force change.</li>
	</ol>
	</li>
	<li>Stokes&#8217; Law Test
	<ol>
		<li>Capture a bead using the optical trap. Move the bead back-and-force at different speed.</li>
		<li>Record the motion of the bead and force using the Minitweezers.</li>
		<li>Compute the radius of the bead. 
		NOTE: The frictional force, Fd, generated by the motion of a spherical particle in a fluid can be described by the Stokes&#8217; law: 
		<img alt="Equation 3" fo:content-width="1.5in" src="/files/ftp_upload/51542/51542eq3.jpg" width="150" /> (Eq. 3) 
		in which r is the radius of the sphere, v is the velocity, and h is the dynamic viscosity of the fluid, which can be found in reference tables. Using the measured force as Fd, the radius of the bead can be computed using Eq. 3 (Figure 6) and should agree with that determined by the image acquisition software. The Stokes&#8217; law test effectively tests the overall calibrations and performance of the instrument.</li>
	</ol>
	</li>
</ol>
3. Nanomanipulation of Single RNA Molecules
<ol>
	<li>Making fluidics.
	<ol>
		<li>Drill six holes (2 mm diameter each) on a No. 2 cover glass (Figure 7a) and cut three slits (2 mm width) into double-sided polyimide tape (Figure 7b) using a laser engraver.</li>
		<li>Make a flow chamber by sandwiching two cover glass with two layers of double-sided Kapton tape. Insert a micropipette and two bypass tubes between the tape (Figure 7c).</li>
		<li>Mount the flow chamber onto a metal frame by matching fluidic holes (Figure 7d).</li>
		<li>Connect the fluidic channels of the chamber to 10 ml syringes filled with buffer of choice via polyethylene tubings (Figure 7e).</li>
		<li>Mount the entire chamber onto the optical tweezers between the two objectives. Ensure that the front side of the chamber with the fluidic channels faces to the right. Retract the right objective and plug the brass pins of the frame (Figure 7e) to mounting holes on the tweezers. Tighten screws to fix the chamber position.</li>
	</ol>
	</li>
	<li>Mixing the annealed sample and beads. Mix the annealed sample with anti-digoxigenin-coated beads (dig-beads), and let the mixture to stay at room temperature for 5-10 min. Typically, 1 &#181;l of annealed sample is mixed with 5 &#181;l dig-beads in 1 ml of buffer. NOTE: The amount of the beads to be loaded into the flow chamber should be chosen to ensure a reasonable quantity of the beads to be delivered from the bypassing tube. The ratio of the annealed sample to the beads cannot be easily quantified. In an ideal case, most beads have no RNA such that only a small fraction of beads may have only one RNA molecule per bead. As the annealed samples and the bead suspension are difficult to quantify, the best and only way currently is to vary the mixing ratio and test the mixture on the tweezers.</li>
	<li>Deliver beads to the reaction site in the flow chamber (i.e., a few micrometers on top of the micropipette tip, Figure 7c).
	<ol>
		<li>Load a suspension of streptavidin-coated beads (strep-beads) into a 1 ml syringe.</li>
		<li>Connect the syringe to the fluidic tubing leading to the bottom channel of the flow chamber (Figure 7a).</li>
		<li>Push the syringe to flow the strep-beads into the chamber.</li>
		<li>Move the entire chamber using motor control software to place the optical trap near the opening of the bottom bypass tube (Figure 7b). Then operate the optical trap by a trackball to capture a strep-bead.</li>
		<li>Move the bead close to the tip of the micropipette using the motor control software.</li>
		<li>Pull the syringe connected to the micropipette to suck the bead onto the micropipette.</li>
		<li>Apply flow gently by pressing the syringe to the middle channel to flush out extra strep-beads.</li>
		<li>Similarly, deliver the mixture of sample and the dig-beads (see step 3.2) to the top channel of the chamber.</li>
		<li>Capture a dig-bead and bring it close to the strep-bead using the optical trap.</li>
		<li>Clean the middle channel by applying flow. NOTE: The strep- and dig-beads are chosen to be different sizes so that they can be distinguished visually under the microscopic view (Figure 4).</li>
	</ol>
	</li>
	<li>Fishing&#8221; a single-molecule tethering between a pair of beads.
	<ol>
		<li>Place the dig-bead vertically on top of the strep-bead (Figure 4).</li>
		<li>Move the dig-bead down towards the strep-bead by steering the trap. Open a force-distance plot window on computer to visualize force changes.</li>
		<li>Upon contact of the two beads, move the dig-bead vertically away from the strep-bead.</li>
		<li>If no specific contact is made, the force remains almost zero. If the two beads are connected by molecules, the force increases significantly when the two beads separate. This procedure, commonly referred to as &#8220;fishing&#8221;, is often performed multiple times on each pair of beads to find an effective single-molecule tether.</li>
	</ol>
	</li>
</ol>

The authors have nothing to disclose.

Modification and Troubleshooting in Preparation of Tweezing Samples
Choice of Cloning Vector. Although the general scheme described here (Figure 3) does not require a special cloning vector, the vector is preferred not to have intrinsic T7 or T3 promoter for the ease of transcription such that a promoter can be introduced via PCR at desired positions.
Sequence and Lengths of the Handles. There is no specific sequen...

The development of the optical tweezers technique has long been accompanied with its application in biological research. When the optical trapping effect was first discovered, Arthur Ashkin observed that bacteria in contaminated water could be trapped at the laser focus1. Since then, bacteria trapping has become an unintentional, fun experiment for generations of biophysics students as well as a serious research tool to study microbial physiology2,3. The optical trapping technique uses focused laser beam to immobilize a microscopic object1. Practically, a laser trap functions as an optical spring, which also measures force (F) on the trapped object by its displacement from the center of the trap (&#916;X). Within a short range, F =&#160; &#954; x&#160; &#916;X, in&#160;&#954; is the spring constant of the trap. The optical trap can be used to exert force in picoNewton (pN) precision to a microscopic object and measure its position with nanometer (nm) accuracy. In the past two decades, optical tweezers have become one of the most used single-molecule techniques in biophysics. The technique has been employed to study folding and mechanics of DNA4-6, RNA7-9, and proteins10,11. Optical tweezers have also been used to observe DNA replication12, RNA transcription13, and protein synthesis14,15, as well as many other biomolecular events16-18.
Single-molecule approaches are employed in RNA structural research mainly to explore the rugged folding energy landscape of RNA. An RNA sequence can usually fold into multiple stable, mutually exclusive structures, due to the simple chemical composition and base paring rules of RNA. It has long been known that RNA structural polymorphism, such as that occurs in riboswitches19,20, plays an important role in gene regulation. A recent genome-wide survey has revealed that temperature variations as small as a few degrees greatly influence structure and protein synthesis of a large portion of the cellular transcriptome21. This example hints that the biological role of alternative RNA folding is perhaps more important and pervasive than previously assumed. Structural polymorphism, however, poses a challenge for the traditional biochemical and biophysical approaches, which examine average properties of many molecules. For example, the &#8220;on&#8221; and &#8220;off&#8221; conformations of a riboswitch are mutually exclusive. An averaged structure derived from heterogeneous structures is not likely to resemble either of the biologically relevant conformers. Moreover, untranslated RNA and mRNA usually form structures. Their interaction with proteins and regulatory RNAs commonly requires unfolding of existing structure as part of the interaction. Therefore, studying RNA unfolding/refolding becomes a pertinent issue of RNA biology. To meet such a challenge, the single-molecule approach has been employed to unravel RNA structural polymorphism by studying one molecule at a time22-27.
As compared to the popular single-molecule fluorescence method, tweezers based mechanical unfolding offers an advantage that conformations of individual molecules can be manipulated by applied force and be measured with nanometer precision. This nanomanipulation capability can be utilized to monitor the unfolding/folding of individual structural domains such that the hierarchical folding of a large RNA can be dissected28. Alternatively, a single strand can be directed to fold into one of several conformers; or an existing structure can be mechanically induced to refold into a different conformation29. Under biological conditions, an RNA structure can be altered upon temperature change or ligand binding. The capability of directly manipulating molecular structure opens a new venue of RNA structural study. In principle, other mechanical techniques, such as atomic force microscope and magnetic tweezers, can also be used to study folding of single RNA molecules. However, such applications are limited largely due to the relatively low spatial resolution30.
The employment of optical tweezers allows RNA structures to be unfolded by mechanical force.
The advantage of mechanical unfolding is several fold. Force can be used to unfold both secondary and tertiary structures, whereas metal ions and ligand induced folding are mainly limited to tertiary structures. Temperature and denaturant can significantly affect the activities of water and solutes. In contrast, force is applied locally to perturb molecular structures; the effect of force on the surrounding environment is negligible. In addition, thermal melting is best used to study folding thermodynamics of small RNA structures, whereas optical tweezers have been used to study RNA structures with various sizes, ranging from a seven-base-pair tetraloop hairpin28 to a 400-nucleotide ribozyme31. Furthermore, RNA structures can be mechanically unfolded at mesophilic temperatures. In contrast, to unfold RNAs in a thermal melting experiment, temperature is typically raised well above physiological temperatures, which significantly increases RNA hydrolysis, especially in the presence of Mg2+ ions.
It is important to note that the mechanical effect of force depends on how it is applied to a structure. The applied force tilts the folding energy landscape. For example, when force is applied to a hairpin, the base pairs are sequentially broken, one at a time (Figure 1a). The ripping fork progresses along the helical axis, which is perpendicular to the applied force. In contrast, when a minimal kissing complex is under tension, the two kissing base pairs, which are parallel to the applied force, share the force load (Figure 1b). The different geometries of the hairpin and kissing complex relative to the applied force result in their different mechanical response, which can be utilized to distinguish secondary and tertiary folding28,32. The theoretic aspect of mechanical unfolding has been previously reviewed8,9,30. This work outlines the basic approaches to set up and perform a single-molecule mechanical unfolding assay.
Experimental Setup. In our mechanical pulling experiment, the RNA sample for tweezing consists of the RNA of interest flanked by two double-stranded DNA/RNA handles (Figure 2)7. The entire molecule can be tethered to two surface-coated micron-size beads (Spherotech) via streptavidin-biotin and digoxigenin-anti-digoxigenin antibody interactions, respectively. One bead is held by a force-measuring optical trap, whereas the other is held on the tip of a micropipette. The relative distance between the beads can be changed by either steering the trap or moving the micropipette. Using this approach, a single RNA molecule tethering the beads can be stretched and relaxed.
Preparation of Samples. The synthesis of RNA samples for tweezing includes a few steps (Figure 3). First, the DNA sequence corresponding to the RNA of interest is first cloned into a plasmid vector. Next, three PCR reactions are performed to generate the two handles and a template for transcription. The transcription template encompasses the handle regions and the inserted sequences. The full length RNA is synthesized by in vitro transcription. Last, the RNA and chemically modified handles are annealed together to generate the molecules for tweezing.
Calibration and Operation of Tweezers. The basic design of the Minitweezers used in this work follows that of the dual-beam optical tweezers33. With many improvements, the Minitweezers display extraordinary stability as compared to the first generation optical tweezers. A number of research groups in several countries use the Minitweezers in their single-molecule research14,15,34-37. The details of construction, calibration, and operation of the device, including instruction videos, are available at the &#8220;Tweezers Lab&#8221; website (http://tweezerslab.unipr.it). Here, improvements and calibration procedures that need to be performed on daily bases are described in detail.

<table border="0" cellpadding="0" cellspacing="0" width="745">
	<tbody>
		<tr height="20">
			<td height="20" style="height:21px;width:249px;">Minitweezers</td>
			<td style="width:211px;">Steven B. Smith Engineering</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"><a href="http://tweezerslab.unipr.it/">http://tweezerslab.unipr.it</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">Data acquisition card</td>
			<td style="width:211px;">National Instruments</td>
			<td style="width:119px;">USB-6351</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">Video card</td>
			<td style="width:211px;">National Instruments</td>
			<td style="width:119px;">PCI-1407</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">Labview programming software</td>
			<td style="width:211px;">National Instruments</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">NI Vision Builder</td>
			<td style="width:211px;">National Instruments</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">Laser engraver</td>
			<td style="width:211px;">Epilogue laser systems</td>
			<td style="width:119px;">Zing-16</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;"></td>
			<td style="width:211px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">PCR purification kit</td>
			<td style="width:211px;">Qiagen</td>
			<td style="width:119px;">28104</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:249px;">MEGAscript T7 kit</td>
			<td style="width:211px;">Life Technologies</td>
			<td style="width:119px;">AM1334</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">MEGAclear kit</td>
			<td style="width:211px;">Life Technologies</td>
			<td style="width:119px;">AM1908</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">Biotin-11-UTP</td>
			<td style="width:211px;">Thermo Fisher</td>
			<td>FERR0081</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">T4 DNA polymerase</td>
			<td style="width:211px;">New England Labs</td>
			<td style="width:119px;">M0203</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:249px;">Streptavidin coated microsphere</td>
			<td style="width:211px;">Spherotech</td>
			<td style="width:119px;">SVP-20-5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">Antidigoxigenin coated microsphere</td>
			<td style="width:211px;">Spherotech</td>
			<td style="width:119px;">DIGP-20-2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">Size standard microspheres</td>
			<td style="width:211px;">Spherotech</td>
			<td style="width:119px;">PPS-6K</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">Kapton tape</td>
			<td style="width:211px;">Kaptontape.com</td>
			<td>KPPTDE-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">No. 2 cover glass</td>
			<td style="width:211px;">Thermo Fisher</td>
			<td style="width:119px;">12-543D</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Limited by space, nanomanipulation of single RNA molecules is demonstrated by showing only mechanical unfolding examples of RNA hairpins and an RNA with tertiary structure.
Manipulate Single Hairpin Molecules
Once a tether is established between the beads, extension of and force on the tether can be manipulated using a user-defined protocol. Four common manipulation protocols are commonly used.
Force-ramp Experiment</...

Watch this Scientific Journal Video about Nanomanipulation of Single RNA Molecules by Optical Tweezers at JoVE.com

Nanomanipulation of Single RNA Molecules by Optical Tweezers

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 ...

nanomanipulation-of-single-rna-molecules-by-optical-tweezers

Research

JoVE Journal

Bioengineering

14.7K Views.  University at Albany, State University of New York. Optical tweezers have been used to study RNA folding by stretching individual molecules from their 5’ and 3’ ends. Here common procedures are described to synthesize RNA molecules for tweezing, calibration of the instrument, and methods to manipulate single molecules. 

Video: Nanomanipulation of Single RNA Molecules by Optical Tweezers

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.

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 ...

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 ...

In vitro Synthesis of Native, Fibrous Long Spacing and Segmental Long Spacing Collagen

Collagen fibrils are present in the extracellular matrix of animal tissue to provide structural scaffolding and mechanical strength. These native collagen fibrils have a characteristic banding periodicity of ~67 nm and are formed in vivo through the hierarchical assembly of Type I collagen monomers, which are 300 nm in length and 1.4 nm in diameter. In vitro, by varying the conditions to which the monomer building blocks are exposed, unique structures ranging in length scales up to 50 microns can be constructed, including not only native type fibrils, but also fibrous long spacing and segmental long spacing collagen. Herein, we present procedures for forming the three different collagen structures from a common commercially available collagen monomer. Using the protocols that we and others have published in the past to make these three types typically lead to mixtures of structures. In particular, unbanded fibrils were commonly found when making native collagen, and native fibrils were often present when making fibrous long spacing collagen. These new procedures have the advantage of producing the desired collagen fibril type almost exclusively. The formation of the desired structures is verified by imaging using an atomic force microscope.

In vitro Synthesis of Native, Fibrous Long Spacing and Segmental Long Spacing Collagen

Simple and reproducible procedures are described for making three structurally distinct collagen assemblies from a common commercially available Type I collagen monomer. Native type, fibrous long spacing or segmental long spacing collagen can be constructed by varying the conditions to which the 300 nm long and 1.4 nm diameter monomer building block is exposed.

Collagen fibrils are present in the  extracellular matrix of animal tissue to provide structural scaffolding and  mechanical strength. These native ...

Optical Detection of E. coli Bacteria by Mesoporous Silicon Biosensors

A label-free optical biosensor based on a nanostructured porous Si is designed for rapid capture and detection of Escherichia coli K12 bacteria, as a model microorganism. The biosensor relies on direct binding of the target bacteria cells onto its surface, while no pretreatment (e.g.&#160;by cell lysis) of the studied sample is required. A mesoporous Si thin film is used as the optical transducer element of the biosensor. Under white light illumination, the porous layer displays well-resolved Fabry-P&#233;rot fringe patterns in its reflectivity spectrum. Applying a fast Fourier transform (FFT) to reflectivity data results in a single peak. Changes in the intensity of the FFT peak are monitored. Thus, target bacteria capture onto the biosensor surface, through antibody-antigen interactions, induces measurable changes in the intensity of the FFT peaks, allowing for a &#39;real time&#39; observation of bacteria attachment.
The mesoporous Si film, fabricated by an electrochemical anodization process, is conjugated with monoclonal antibodies, specific to the target bacteria. The immobilization, immunoactivity and specificity of the antibodies are confirmed by fluorescent labeling experiments. Once the biosensor is exposed to the target bacteria, the cells are directly captured onto the antibody-modified porous Si surface. These specific capturing events result in intensity changes in the thin-film optical interference spectrum of the biosensor. We demonstrate that these biosensors can detect relatively low bacteria concentrations (detection limit of 104 cells/ml) in less than an hour.

Optical Detection of E. coli Bacteria by Mesoporous Silicon Biosensors

A label-free optical biosensor for rapid bacteria detection is introduced. The biosensor is based on a nanostructured porous Si, which is designed to directly capture the target bacteria cells onto its surface. We use monoclonal antibodies, immobilized onto the porous transducer, as the capture probes. Our studies demonstrate the applicability of these biosensors for the detection of low bacterial concentrations within minutes with no prior sample processing (such as cell lysis).

A label-free optical biosensor based on a nanostructured porous Si is designed for rapid capture and detection of Escherichia coli K12 bacteria, as 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.

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

Fluorescence Biomembrane Force Probe: Concurrent Quantitation of Receptor-ligand Kinetics and Binding-induced Intracellular Signaling on a Single Cell

Membrane receptor-ligand interactions mediate many cellular functions. Binding kinetics and downstream signaling triggered by these molecular interactions are likely affected by the mechanical environment in which binding and signaling take place. A recent study demonstrated that mechanical force can regulate antigen recognition by and triggering of the T-cell receptor (TCR). This was made possible by a new technology we developed and termed fluorescence biomembrane force probe (fBFP), which combines single-molecule force spectroscopy with fluorescence microscopy. Using an ultra-soft human red blood cell as the sensitive force sensor, a high-speed camera and real-time imaging tracking techniques, the fBFP is of ~1 pN (10-12 N), ~3 nm and ~0.5 msec in force, spatial and temporal resolution. With the fBFP, one can precisely measure single receptor-ligand binding kinetics under force regulation and simultaneously image binding-triggered intracellular calcium signaling on a single live cell. This new technology can be used to study other membrane receptor-ligand interaction and signaling in other cells under mechanical regulation.

We describe a technique for concurrently measuring force-regulated single receptor-ligand binding kinetics and real-time imaging of calcium signaling in a single T lymphocyte.

Membrane receptor-ligand interactions mediate many cellular functions. Binding kinetics and downstream signaling triggered by these molecular interactions ...

Delivery of Proteins, Peptides or Cell-impermeable Small Molecules into Live Cells by Incubation with the Endosomolytic Reagent dfTAT

Macromolecular delivery strategies typically utilize the endocytic pathway as a route of cellular entry. However, endosomal entrapment severely limits the efficiency with which macromolecules penetrate the cytosolic space of cells. Recently, we have circumvented this problem by identifying the reagent dfTAT, a disulfide bond dimer of the peptide TAT labeled with the fluorophore tetramethylrhodamine. We have generated a fluorescently labeled dimer of the prototypical cell-penetrating peptide (CPP) TAT, dfTAT, which penetrates live cells and reaches the cytosolic space of cells with a particularly high efficiency. Cytosolic delivery of dfTAT is achieved in multiple cell lines, including primary cells. Moreover, delivery does not noticeably impact cell viability, proliferation or gene expression. dfTAT can deliver small molecules, peptides, antibodies, biologically active enzymes and a transcription factor. In this report, we describe the protocols involved in dfTAT synthesis and cellular delivery. The manuscript describes how to control the amount of protein delivered to the cytosolic space of cells by varying the amount of protein administered extracellularly. Finally, the current limitations of this new technology and steps involved in validating delivery are discussed. The described protocols should be extremely useful for cell-based assays as well as for the ex vivo manipulation and reprogramming of cells.

We describe how to deliver proteins and cell-impermeable small molecules into cultured mammalian cells by a simple co-incubation protocol with a reagent that causes endocytic organelles to become leaky.

Macromolecular delivery strategies typically utilize the endocytic pathway as a route of cellular entry. However, endosomal entrapment severely limits the ...

Label-free Single Molecule Detection Using Microtoroid Optical Resonators

Detecting small concentrations of molecules down to the single molecule limit has impact on areas such as early detection of disease, and fundamental studies on the behavior of molecules. Single molecule detection techniques commonly utilize labels such as fluorescent tags or quantum dots, however, labels are not always available, increase cost and complexity, and can perturb the events being studied. Optical resonators have emerged as a promising means to detect single molecules without the use of labels. Currently the smallest particle detected by a non-plasmonically-enhanced bare optical resonator system in solution is a 25 nm polystyrene sphere1. We have developed a technique known as Frequency Locking Optical Whispering Evanescent Resonator (FLOWER) that can surpass this limit and achieve label-free single molecule detection in aqueous solution2. As signal strength scales with particle volume, our work represents a &#62; 100x improvement in the signal to noise ratio (SNR) over the current state of the art. Here the procedures behind FLOWER are presented in an effort to increase its usage in the field.

We have developed a label-free biosensing system based on optical resonator technology known as Frequency Locking Optical Whispering Evanescent Resonator (FLOWER) that is capable of detecting single molecules in solution. Here the procedures behind this work are described and presented.

Detecting small concentrations of molecules down to the single molecule limit has impact on areas such as early detection of disease, and fundamental ...

Optical Control of Living Cells Electrical Activity by Conjugated Polymers

Hybrid interfaces between organic semiconductors and living tissues represent a new tool for in-vitro and in-vivo applications. In particular, conjugated polymers display several optimal properties as substrates for biological systems, such as good biocompatibility, excellent mechanical properties, cheap and easy processing technology, and possibility of deposition on light, thin and flexible substrates. These materials have been employed for cellular interfaces like neural probes, transistors for excitation and recording of neural activity, biosensors and actuators for drug release. Recent experiments have also demonstrated the possibility to use conjugated polymers for all-optical modulation of the electrical activity of cells. Several in-vitro study cases have been reported, including primary neuronal networks, astrocytes and secondary line cells. Moreover, signal photo-transduction mediated by organic polymers has been shown to restore light sensitivity in degenerated retinas, suggesting that these devices may be used for artificial retinal prosthesis in the future. All in all, light sensitive conjugated polymers represent a new approach for optical modulation of cellular activity.
In this work, all the steps required to fabricate a bio-polymer interface for optical excitation of living cells are described. The function of the active interface is to transduce the light stimulus into a modulation of the cell membrane potential. As a study case, useful for in-vitro studies, a polythiophene thin film is used as the functional, light absorbing layer, and Human Embryonic Kidney (HEK-293) cells are employed as the biological component of the interface. Practical examples of successful control of the cell membrane potential upon stimulation with light pulses of different duration are provided. In particular, it is shown that both depolarizing and hyperpolarizing effects on the cell membrane can be achieved depending on the duration of the light stimulus. The reported protocol is of general validity and can be straightforwardly extended to other biological preparations.

A method for stimulation of in-vitro cell cultures electrical activity with visible light, based on the use of organic semiconducting polymers is described.

Hybrid interfaces between organic semiconductors and living tissues represent a new tool for in-vitro and in-vivo applications. In particular, conjugated ...

Development of Microfluidic Devices to Study the Elongation Capability of Tip-growing Plant Cells in Extremely Small Spaces

In vivo, tip-growing plant cells need to overcome a series of physical barriers; however, researchers lack the methodology to visualize cellular behavior in such restrictive conditions. To address this issue, we have developed growth chambers for tip-growing plant cells that contain a series of narrow, micro-fabricated gaps (~1 &#181;m) in a poly-dimethylsiloxane (PDMS) substrate. This transparent material allows the user to monitor tip elongation processes in individual cells during microgap penetration by time-lapse imaging. Using this experimental platform, we observed morphological changes in pollen tubes as they penetrated the microgap. We captured the dynamic changes in the shape of a fluorescently labeled vegetative nucleus and sperm cells in a pollen tube during this process. Furthermore, we demonstrated the capability of root hairs and moss protonemata to penetrate the 1 &#181;m gap. This in vitro platform can be used to study how individual cells respond to physically constrained spaces and may provide insights into tip-growth mechanisms.

We describe a method to investigate the capability of tip-growing plant cells, including pollen tubes, root hairs, and moss protonemata, to elongate through extremely narrow gaps (~1 &#181;m) in a microfluidic device.

In vivo, tip-growing plant cells need to overcome a series of physical barriers; however, researchers lack the methodology to visualize cellular behavior ...