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. 