The authors thank Meng Pan for proofreading the manuscript. This work is supported by Singapore Ministry of Education Academic Research Fund Tier 3 (MOE2012-T3-1-001) to J.Y.; the National Research Foundation through the Mechanobiology Institute Singapore to J.Y.; the National Research Foundation, Prime Minister&#39;s Office, Singapore, under its NRF Investigatorship Programme (NRF Investigatorship Award No. NRF-NRFI2016-03 to J.Y.; the Fundamental Research Fund for the Central Universities (2017KFYXJJ153) to H. Y.

1. Preparation of G4 DNA for Single-molecule Stretching
<ol>
	<li>Prepare 5&#39;-thiol labeled and 5&#39;-biotin labeled dsDNA handles by PCR using DDNA polymerase on a lambda phage DNA template using 5&#39;-thiol and 5&#39;-biotin primers14 (Figure 1). Both dsDNA handles have high GC content (&#62; 60%) to prevent DNA melting when DNA is held at high forces or during DNA overstretching transition15.</li>
	<li>Purify PCR products using a commercial purification kit and digest with BstXI restriction enzyme according to the manufacturer&#39;s protocol.</li>
	<li>Ligate G4 forming ssDNA, and flank ssDNA and dsDNA handles using T4 DNA ligase according to the manufacturer&#39;s protocol. Purify the ligated product by gel extraction using a commercial purification kit according to the manufacturer&#39;s protocol.</li>
</ol>
2. Preparation of Flow Channel
<ol>
	<li>Coverslip cleaning and surface functionalization
	<ol>
		<li>Place bottom coverslips (#1.5, 22 mm &#215; 32 mm) and top coverslips (#1.5, 20 mm x 20 mm) into cover glass staining jars (each jar can hold 7 pieces of coverslips, the volume is ~ 20 mL). Rinse the coverslips in the jars with distilled water 2-5 times.</li>
		<li>Add ~ 20 mL of 5-40% detergent solution into each jar, and then place in an ultrasonic cleaning bath for 30 min. Rinse with distilled water for &#62; 10 times to remove the detergent.</li>
		<li>Dry the coverslips in the jars in oven (~ 150 &#176;C; CAUTION, hot), or by N2 gas. Store the dried top coverslips in a dry cabinet.</li>
		<li>Use plasma (O2 gas) to clean the coverslips in the jar for 10 min. During the 10 min, prepare 20 mL of 1% (3-Aminopropyl)triethoxysilane (APTES) solution in methanol (CAUTION, toxic/flammable). Use a chemical fume hood to dissolve APTES in methanol. 
		NOTE: Avoid humidity when storing APTES solution. Old APTES often causes problem in surface functionalizing of coverslips.</li>
		<li>Immediately after the plasma cleaning, add all of the 1% APTES solution into the jars and incubate for 1 h. Pour the waste into the waste bottle specific for 1% APTES methanol. Perform the methanol-related steps in the fume hood for flammable chemicals.</li>
		<li>Rinse the jars once with methanol and &#62; 10 times with distilled water, and then dry by oven (~150 &#176;C; CAUTION, hot). Store the APTES-coated bottom coverslips in a dry cabinet if not in use for up to two weeks. 
		NOTE: After each sub-step from 2.1.1 to 2.1.4, the cleaning process can be paused before the next step.</li>
	</ol>
	</li>
	<li>Assemble the flow channel
	<ol>
		<li>Prepare two &#34;spacers&#34;, i.e., parafilm or double-side tape (~ 4 mm &#215; 20 mm) for each channel. Place the two pieces of spacers on a bottom coverslip along the long-edge. Place a top coverslip on the spacer, forming a flow cell in between (~ 10 mm &#215; 20 mm area, Figure 2A, B).</li>
		<li>If parafilm is used as a spacer, place the flow channel on a heater (60-120 &#176;C; CAUTION, hot) for 5-10 s while gently pressing the sides of the top coverslip to stick the two coverslips together by parafilm. The resulting flow channel has a height of ~ 100 &#181;m, and thereby the volume of the channel is ~ 20 &#181;L.</li>
		<li>Seal the long edge of the channel with silicone glue to avoid leakage. Use silicone glue to make a small sink-like structure at each open edge of the flow channel, which serves as an entry and an exit of solution. 
		NOTE: The entry and exit can also be made by other ways, e.g., by adhering small plastic rings using wax.</li>
		<li>Store channels in a dry cabinet for up to 4 weeks.</li>
	</ol>
	</li>
	<li>Tether DNA onto the bottom surface of the flow channel
	<ol>
		<li>Dilute the amino-coated polystyrene beads (diameter: 3 &#181;m) by 200X in distilled 1X phosphate buffered saline (PBS) buffer. Vortex the bead solution and then flow into channels. Incubate the bead solution in channels for ~ 30 min. Remove any unstuck beads by washing with 200 &#181;L of 1X PBS buffer.</li>
		<li>Adjust (increase/decrease) the incubation time to achieve a surface density of 1-5 beads per 50 mm x 50 mm area. Store the channels deposited with the reference beads for up to 3 days.</li>
		<li>Dilute sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (Sulfo-SMCC) powder into 1X PBS solution (~ 0.5 mg/mL). Vortex the solution and then flow into the channel. 
		NOTE: Prepare fresh Sulfo-SMCC solution before use and add it immediately to the channel to avoid hydrolyzation of the SMCC.</li>
		<li>Incubate the SMCC solution in the channel for 30 min. Remove the SMCC solution by washing with a large amount (1 mL, ~ 50X the channel volume) of 1X PBS solution. 
		NOTE: Wash the excess SMCC carefully. This step is very essential for the binding of DNA on the coverslip.</li>
		<li>DNA tethering
		<ol>
			<li>Dilute the thiol-biotin labeled DNA into 1X PBS, with a resulting DNA concentration of ~ 0.3 nM. Gently pipette to mix the solution, flow the DNA solution into the SMCC-coated channel, and incubate for 30 min at room temperature (~ 23 &#176;C).</li>
		</ol>
		</li>
		<li>Gently wash away free DNA with 200 &#181;L of blocking solution that contains 1X PBS with 10 mg/mL bovine serum albumin (BSA) and 0.01% 2-Mercaptoethanol.</li>
		<li>Block the channel surface by incubating the channel in blocking solution (10 mg/mL BSA, 0.01% 2-Mercaptoethanol) for &#62; 2 h; after this step, the channel is ready for experiments. The prepared channel can be kept at 4 &#176;C for ~ 1 day. 
		NOTE: The blocking step is important for reducing the non-specific binding of DNA and magnetic beads to the coverslips.</li>
	</ol>
	</li>
</ol>
3. Magnetic Tweezers Setup and Identification of Single dsDNA Tether
<ol>
	<li>Magnetic tweezers setup
	<ol>
		<li>Start the magnetic tweezers control program. Here, the magnetic tweezers were controlled by an in-house-written LabVIEW program.</li>
		<li>Align the magnet centers before mounting the channel. Use a 4X objective lens to adjust the x- and y-axis of the magnets in the optical axis of the microscope.</li>
		<li>Use a computer-controlled motorized manipulator to move the magnets along the z-direction (Figure 2A) and set the distance as d = 0 (d: distance between the magnets and coverslip) when the magnets attach to a coverslip on the microscope.</li>
		<li>Program the movement of the magnets through the manipulator to achieve control of the force, including constant force (i.e., constant d) and time varying force F(t) (i.e., time varying d(t)).</li>
		<li>Use bright a light-emitting diode (LED) light source for back-scattered illumination of the bead through the objective. Collect the bead images at a sampling rate of 100 Hz by a charge-coupled device (CCD) camera.</li>
	</ol>
	</li>
	<li>Sample setup and identify single dsDNA tether
	<ol>
		<li>Tether formation
		<ol>
			<li>Gently flow in 200X diluted M-280, paramagnetic beads in assay solution (100 mM KCl, 2 mM MgCl2, 10 mM Tris-pH 7.4) into a channel. Incubate for 10 min to allow the beads to bind to biotin-labeled DNA molecules immobilized on the SMCC-coated surface through the thiol-labeled end. Gently wash away un-tethered beads using 200 &#181;L of standard reaction solution. 
			NOTE: The M-280 beads show lower non-specific binding to the surface compared to other commercial magnetic beads such as M-270.</li>
		</ol>
		</li>
		<li>Mount the channel onto the microscope stage. Search for the beads on the bottom surface using the 100X oil immersion objective.</li>
		<li>Select a reference bead on the surface and a moving tethered bead. Build the initial image libraries of both the reference bead and tethered bead at different defocus planes.</li>
		<li>Calibration of beads image
		<ol>
			<li>Before experiments, use an objective piezo actuator to obtain images of both reference and tethered beads at different defocus planes spaced by 20-50 nm, which are stored as two separate bead image libraries and are respectively indexed with the defocus distance (Figure 2D).</li>
		</ol>
		</li>
		<li>x, y, z position determination
		<ol>
			<li>During experiments, determine the position of the bead in the x-y plane by the bead centroid. Determine the height change of the tethered bead by comparing the current bead image with those stored in the library. Use the auto-correlation function analysis of the power spectrum of the Fourier transform of the bead images16.</li>
		</ol>
		</li>
		<li>Anti-drift feedback using reference bead
		<ol>
			<li>During experiments, use the piezo to &#34;lock&#34; the distance between the objective and a specific reference bead image stored in the library through a low frequency feedback control, so that the stuck bead image has the best correlation with a specific image stored in the library (Figure 2D).</li>
		</ol>
		</li>
		<li>Determine whether the tether is a single dsDNA molecule by applying ~ 65 pN force Determine a single dsDNA molecule if the tether undergoes the characteristic DNA overstretching transition17,18,19. Repeat the process until a single dsDNA tether is found. (Please refer to the next section for force calibration and DNA overstretching transition).</li>
	</ol>
	</li>
</ol>
4. Magnetic Tweezers Force Calibration
<ol>
	<li>Directly determine the force (up to 100 pN) for long DNA (48,502 bp &#955;-DNA) molecules using bead fluctuation through: 
	<img alt="Equation 1" src="/files/ftp_upload/56328/56328eq1.jpg" /> 
	, where kB is the Boltzmann constant, T is the temperature in Kelvin scale, &#948;2y is the variance of the bead fluctuation in the direction perpendicular to the magnetic field and the force direction. In this direction, the motion of the bead can be described as a pendulum fluctuation with a length of l + r0, where l is the end-to-end distance along the force direction (extension) of the DNA, and r0 is the radius of the bead10 (Figure 3A).</li>
	<li>Determine the calibration curve for force F as a function of magnets to bead distance d, and F(d) for different beads using the long &#955;-DNA. Usually, the distance between the magnet and the coverslip is used as the DNA tether length can be ignored. Fit the F-d curve by the double-exponential decay function: 
	<img alt="Equation 2" src="/files/ftp_upload/56328/56328eq2.jpg" /> 
	, where the fitting parameters &#945;1, &#945;2, &#947;1, &#947;2 are determined by the magnetic tweezers and the parameter C is determined by the heterogeneous property of the magnetic beads. By shifting the C, the F-d curve obtained from different beads can be overlapped10 (Figure 3B).</li>
	<li>Calibration of parameter C for individual magnetic beads for short DNA experiments.
	<ol>
		<li>Determining the force based on the fluctuation requires recording the bead position at a frequency higher than the corner frequency: 
		<img alt="Equation 3" src="/files/ftp_upload/56328/56328eq3.jpg" /> 
		, where &#947; is the drag coefficient of the bead. For short DNA at high force, it requires recording frequency faster than 100 Hz, therefore, the force cannot be directly measured based on fluctuation with the camera. However, the parameter C can be determined by measuring the force at a low force range (&#60; 15 pN) using fluctuation and fitting the data with a calibrated F-d curve to obtain the parameter C20.</li>
		<li>Alternatively, for experiments with dsDNA, determine the parameter C using DNA overstretching transition at a force of ~ 65 pN (Figure 3C)21,22,23 by recording the d position at which dsDNA showed extension jump (0.6X length increase of contour length). After determining the parameter C, calculate the force for the tethered beads.</li>
	</ol>
	</li>
	<li>Control the loading rate by programming the movement of the magnets through the inverse function of F(d). The magnet should approach the sample double exponentially. 
	NOTE: The relative error of force determined by such an extrapolation method is ~ 10%, and is mainly caused by the bead radius heterogeneity20.</li>
</ol>
5. Single-molecule Manipulation of G4 in the Presence and Absence of Binding Proteins
<ol>
	<li>Force-ramp experiments
	<ol>
		<li>After identifying the dsDNA tether, perform a force-increase scan at a loading rate of 0.2 pN/s followed by a force-decease scan at -0.2 pN/s. After each stretching cycle, hold the DNA molecule at 1 pN for 30 s to allow the ssDNA to refold to G4.</li>
	</ol>
	</li>
	<li>Force-jump experiments
	<ol>
		<li>Cycle the force between 54 pN for 30 s under which a folded G4 could be unfolded, and 1 pN for 60s, under which a folded G4-15T could refold.</li>
	</ol>
	</li>
	<li>Flowing protein solution
	<ol>
		<li>Flow the protein solution when at ~ 20 pN force to avoid attachment of the beads to the coverslip. The DEAH-box RHAU helicase, which showed high specificity to the G4 structure, was used24. The recombinant Drosophila melanogaster RHAU (DmRHAU) helicase was expressed in Escherichia coli and purified as described previously25. The G4 unwinding activity of DmRHAU was assayed on a tetramolecular G4 DNA substrate13.</li>
	</ol>
	</li>
	<li>Analyze the unfolding force using an in-house written Matlab program14.</li>
</ol>

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N., Isaacs, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=G-quadruplexes:+Emerging+roles+in+neurodegenerative+diseases+and+the+non-coding+transcriptome.">G-quadruplexes: Emerging roles in neurodegenerative diseases and the non-coding transcriptome.</a> FEBS Lett. 589 (14), 1653-1668 (2015).</li><li>Balasubramanian, S., Hurley, L. H., Neidle, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+G-quadruplexes+in+gene+promoters:+a+novel+anticancer+strategy?.">Targeting G-quadruplexes in gene promoters: a novel anticancer strategy?.</a> Nat Rev Drug Discov. 10 (4), 261-275 (2011).</li><li>Amato, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toward+the+Development+of+Specific+G-Quadruplex+Binders:+Synthesis,+Biophysical,+and+Biological+Studies+of+New+Hydrazone+Derivatives.">Toward the Development of Specific G-Quadruplex Binders: Synthesis, Biophysical, and Biological Studies of New Hydrazone Derivatives.</a> J Med Chem. 59 (12), 5706-5720 (2016).</li><li>Wells, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-B+DNA+conformations,+mutagenesis+and+disease.">Non-B DNA conformations, mutagenesis and disease.</a> Trends Biochem Sci. 32 (6), 271-278 (2007).</li></ol>

The authors have nothing to disclose.

As described above, a platform for studying the mechanical stability of G4 DNA and the interactions of protein to G4 using single-molecule magnetic tweezers is reported. Accompanying the platform, highly efficient protocols of finding G4 DNA tether, and measurement of the folding-unfolding dynamics and stability of the G4 structure with nanometers special resolution are developed. The focal plane locking enables highly stable anti-drift control, which is important for detecting a small structure transition such as G4 (st...

Four-stranded DNA or RNA G4 structures play critical roles in many important biological processes1. Many proteins are involved in G4 binding and regulation, including telomere binding proteins (telomerase, POT1, RPA, TEBPs, TRF2)1,2, transcription factors (nucleolin, PARP1)3, RNA processing proteins (hnRNP A1, hnRNP A2)4, helicases (BLM, FANCJ, RHAU, WRN, Dna2, Pif1)5, and DNA replication related proteins (Rif1, REV1, PrimPolymerase)6. Protein binding can stabilize or destabilize G4 structures; thus regulating the subsequent biological functions. The stability of G4 was measured by thermal melting using ultraviolet (UV) or circular dichroism (CD) methods7. However, such conditions are not physiological relevant and are difficult to apply to studying the effects of binding proteins7.
The rapid development in single-molecule manipulation technologies has enabled studies of folding and unfolding of a biomolecule, such as a DNA or a protein, at a single-molecule level with nanometer resolution in real time8. Atomic force microscopy (AFM), optical tweezers, and magnetic tweezers are the most commonly used single-molecule manipulation methods. Compared to AFM and optical tweezers9, magnetic tweezers allow stable measurements of folding-unfolding dynamics of a single molecule over days by using an anti-drift technique10,11.
Here, a single-molecule manipulation platform using magnetic tweezers to study the regulation of G4 stability by binding proteins is reported12,13. This work outlines the basic approaches, including sample and flow channel preparation, the setup of magnetic tweezers, and the force calibration. The force control and the anti-drift protocols as described in step 3 allow for long time measurements under various force controls, such as constant force (force clamp) and constant loading rate (force-ramp), and force-jump measurement. The force calibration protocol described in step 4 enables force calibration of &#60; 1 &#181;m short tethers over a wide force range up to 100 pN, with a relative error within 10%. An example of regulation of the stability of the RNA Helicase associated with AU-rich element (RHAU) helicase (alias DHX36, G4R1) that plays essential roles in resolving RNA G4 is used to demonstrate the applications of this platform13.

<table>
	<tbody>
		<tr>
			<td>DNA PCR primers</td>
			<td>IDT</td>
			<td></td>
			<td>DNA preparations</td>
		</tr>
		<tr>
			<td>DNA PCR chemicals</td>
			<td>NEB</td>
			<td></td>
			<td>DNA preparations</td>
		</tr>
		<tr>
			<td>restriction enzyme BstXI</td>
			<td>NEB</td>
			<td>R0113S</td>
			<td>DNA preparations</td>
		</tr>
		<tr>
			<td>coverslips (#1.5, 22*32 mm, and 20*20 mm)</td>
			<td>BMH.BIOMEDIA</td>
			<td>72204</td>
			<td>flow channel preparation</td>
		</tr>
		<tr>
			<td>Decon90</td>
			<td>Decon Laboratories Limited</td>
			<td></td>
			<td>flow channel preparation</td>
		</tr>
		<tr>
			<td>APTES</td>
			<td>Sigma</td>
			<td>440140-500ML</td>
			<td>flow channel preparation</td>
		</tr>
		<tr>
			<td>Sulfo-SMCC</td>
			<td>ThermoFisher Scientific</td>
			<td>22322</td>
			<td>flow channel preparation</td>
		</tr>
		<tr>
			<td>M-280, paramganetic beads,streptavidin</td>
			<td>ThermoFisher Scientific</td>
			<td>11205D</td>
			<td>flow channel preparation</td>
		</tr>
		<tr>
			<td>Polybead Amino Microspheres 3.00 &#956;m</td>
			<td>Polysciences, Inc</td>
			<td>17145-5</td>
			<td>flow channel preparation</td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Sigma</td>
			<td>M6250-250ML</td>
			<td>flow channel preparation</td>
		</tr>
		<tr>
			<td>Olympus Microscopes IX71</td>
			<td>Olympus</td>
			<td>IX71</td>
			<td>Magnetic tweezers setup</td>
		</tr>
		<tr>
			<td>Piezo-Z Stages P-721</td>
			<td>Physik Instrumente</td>
			<td>P-721</td>
			<td>Magnetic tweezers setup</td>
		</tr>
		<tr>
			<td>Olympus Objective lense MPLAPON-Oil 100X</td>
			<td>Olympus</td>
			<td>MPLAPON-Oil 100X</td>
			<td>Magnetic tweezers setup</td>
		</tr>
		<tr>
			<td>CCD/CMOS camera</td>
			<td>AVT</td>
			<td>Pike F-032B</td>
			<td>Magnetic tweezers setup</td>
		</tr>
		<tr>
			<td>Translation linear stage</td>
			<td>Physik Instrumente</td>
			<td>MoCo DC</td>
			<td>Magnetic tweezers setup</td>
		</tr>
		<tr>
			<td>LED</td>
			<td>Thorlabs</td>
			<td>MCWHL</td>
			<td>Magnetic tweezers setup</td>
		</tr>
		<tr>
			<td>Cubic Magnets</td>
			<td>Supermagnete</td>
			<td></td>
			<td>Magnetic tweezers setup</td>
		</tr>
		<tr>
			<td>Labview</td>
			<td>National Instruments</td>
			<td></td>
			<td>Magnetic tweezers setup</td>
		</tr>
		<tr>
			<td>OriginPro/Matlab</td>
			<td>OriginLab/MathWorks</td>
			<td></td>
			<td>Data analysis</td>
		</tr>
	</tbody>
</table>

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.

The experiment setup for stretching a single G4 molecule is shown in Figure 4. A single-stranded G4 forming sequence spanned between two dsDNA handles was tethered between a coverslip and a paramagnetic bead. To find a single dsDNA tethered bead, an overstretching assay was performed by increasing the force at constant loading rates. Three types of measurements were often used for studying the folding and unfolding of biomolecules: (i) constant force measurem...

Watch this Scientific Journal Video about Single-molecule Manipulation of G-quadruplexes by Magnetic Tweezers at JoVE.com

Single-molecule Manipulation of G-quadruplexes by Magnetic Tweezers

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 ...

single-molecule-manipulation-of-g-quadruplexes-by-magnetic-tweezers

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Biochemistry

7.9K Views.  Huazhong University of Science and Technology. 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.

Video: Single-molecule Manipulation of G-quadruplexes by Magnetic Tweezers

High Precision FRET at Single-molecule Level for Biomolecule Structure Determination

A protocol on how to perform high-precision interdye distance measurements using F&#246;rster resonance energy transfer (FRET) at the single-molecule level in multiparameter fluorescence detection (MFD) mode is presented here. MFD maximizes the usage of all &#34;dimensions&#34; of fluorescence to reduce photophysical and experimental artifacts and allows for the measurement of interdye distance with an accuracy up to ~1 &#197; in rigid biomolecules. This method was used to identify three conformational states of the ligand-binding domain of the N-methyl-D-aspartate (NMDA) receptor to explain the activation of the receptor upon ligand binding. When comparing the known crystallographic structures with experimental measurements, they agreed within less than 3 &#197; for more dynamic biomolecules. Gathering a set of distance restraints that covers the entire dimensionality of the biomolecules would make it possible to provide a structural model of dynamic biomolecules.

A protocol for high-precision FRET experiments at the single molecule level is presented here. Additionally, this methodology can be used to identify three conformational states in the ligand-binding domain of the N-methyl-D-aspartate (NMDA) receptor. Determining precise distances is the first step towards building structural models based on FRET experiments.

A protocol on how to perform high-precision interdye distance measurements using F&#246;rster resonance energy transfer (FRET) at the single-molecule ...

Covalent Immobilization of Proteins for the Single Molecule Force Spectroscopy

In recent years, atomic force microscopy (AFM) based single molecule force spectroscopy (SMFS) extended our understanding of molecular properties and functions. It gave us the opportunity to explore a multiplicity of biophysical mechanisms, e.g., how bacterial adhesins bind to host surface receptors in more detail. Among other factors, the success of SMFS experiments depends on the functional and native immobilization of the biomolecules of interest on solid surfaces and AFM tips. Here, we describe a straightforward protocol for the covalent coupling of proteins to silicon surfaces using silane-PEG-carboxyls and the well-established N-hydroxysuccinimid/1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimid (EDC/NHS) chemistry in order to explore the interaction of pilus-1 adhesin RrgA from the Gram-positive bacterium Streptococcus pneumoniae (S. pneumoniae) with the extracellular matrix protein fibronectin (Fn). Our results show that the surface functionalization leads to a homogenous distribution of Fn on the glass surface and to an appropriate concentration of RrgA on the AFM cantilever tip, apparent by the target value of up to 20% of interaction events during SMFS measurements and revealed that RrgA binds to Fn with a mean force of 52 pN. The protocol can be adjusted to couple via site specific free thiol groups. This results in a predefined protein or molecule orientation and is suitable for other biophysical applications besides the SMFS.

This protocol describes the covalent immobilization of proteins with a heterobifunctional silane coupling agent to silicon-oxide surfaces designed for the atomic force microscopy based single molecule force spectroscopy which is exemplified by the interaction of RrgA (pilus-1 tip adhesin of S. pneumoniae) with fibronectin.

In recent years, atomic force microscopy (AFM) based single molecule force spectroscopy (SMFS) extended our understanding of molecular properties and ...

Proteome-wide Quantification of Labeling Homogeneity at the Single Molecule Level

Cell proteomes are often characterized using electrophoresis assays, where all species of proteins in the cells are non-specifically labeled with a fluorescent dye and are spotted by a photodetector following their separation. Single molecule fluorescence imaging can provide ultrasensitive protein detection with its ability for visualizing individual fluorescent molecules. However, the application of this powerful imaging method to electrophoresis assays is hampered by the lack of ways to characterize the homogeneity of fluorescent labeling of each protein species across the proteome. Here, we developed a method to evaluate the labeling homogeneity across the proteome based on a single molecule fluorescence imaging assay. In our measurement using a HeLa cell sample, the proportion of proteins labeled with at least one dye, which we termed &#8216;labeling occupancy&#8217; (LO), was determined to range from 50% to 90%, supporting the high potential of the application of single molecule imaging to sensitive and precise proteome analysis.

Here, we present a protocol to assess the labeling homogeneity for each protein species in a complex protein sample at the single molecule level.

Cell proteomes are often characterized using electrophoresis assays, where all species of proteins in the cells are non-specifically labeled with a ...

Single Cell Analysis Of Transcriptionally Active Alleles By Single Molecule FISH

Gene transcription is an essential process in cell biology, and allows cells to interpret and respond to internal and external cues. Traditional bulk population methods (Northern blot, PCR, and RNAseq) that measure mRNA levels lack the ability to provide information on cell-to-cell variation in responses. Precise single cell and allelic visualization and quantification is possible via single molecule RNA fluorescence in situ hybridization (smFISH). RNA-FISH is performed by hybridizing target RNAs with labeled oligonucleotide probes. These can be imaged in medium/high throughput modalities, and, through image analysis pipelines, provide quantitative data on both mature and nascent RNAs, all at the single cell level. The fixation, permeabilization, hybridization and imaging steps have been optimized in the lab over many years using the model system described herein, which results in successful and robust single cell analysis of smFISH labeling. The main goal with sample preparation and processing is to produce high quality images characterized by a high signal-to-noise ratio to reduce false positives and provide data that are more accurate. Here, we present a protocol describing the pipeline from sample preparation to data analysis in conjunction with suggestions and optimization steps to tailor to specific samples.&#160;

Single molecule RNA fluorescence in situ hybridization (smFISH) is a method to accurately quantify levels and localization of specific RNAs at the single cell level. Here, we report our validated lab protocols for wet-bench processing, imaging and image analysis for single cell quantification of specific RNAs.

Gene transcription is an essential process in cell biology, and allows cells to interpret and respond to internal and external cues. Traditional bulk ...

Single-Molecule Dwell-Time Analysis of Restriction Endonuclease-Mediated DNA Cleavage

This novel total internal reflection fluorescence microscopy-based assay facilitates the simultaneous measurement of the length of the catalytic cycle for hundreds of individual restriction endonuclease (REase) molecules in one experiment. This assay does not require protein labeling and can be carried out with a single imaging channel. In addition, the results of multiple individual experiments can be pooled to generate well-populated dwell-time distributions. Analysis of the resulting dwell-time distributions can help elucidate the DNA cleavage mechanism by revealing the presence of kinetic steps that cannot be directly observed. Example data collected using this assay with the well-studied REase, EcoRV - a dimeric Type IIP restriction endonuclease that cleaves the palindromic sequence GAT&#8595;ATC (where &#8595; is the cut site) - are in agreement with prior studies. These results suggest that there are at least three steps in the pathway to DNA cleavage that is initiated by introducing magnesium after EcoRV binds DNA in its absence, with an average rate of 0.17 s-1 for each step.

Using quantum-dot-labeled DNA and total internal reflection fluorescence microscopy, we can investigate the reaction mechanism of restriction endonucleases while using unlabeled protein. This single-molecule technique allows for massively multiplexed observation of individual protein-DNA interactions, and data can be pooled to generate well-populated dwell-time distributions.

This novel total internal reflection fluorescence microscopy-based assay facilitates the simultaneous measurement of the length of the catalytic cycle for ...

Single Molecule Fluorescence Energy Transfer Study of Ribosome Protein Synthesis

The ribosome is a large ribonucleoprotein complex that assembles proteins processively along mRNA templates. The diameter of the ribosome is approximately 20 nm to accommodate large tRNA substrates at the A-, P- and E-sites. Consequently, the ribosome dynamics are naturally de-phased quickly. Single molecule method can detect each ribosome separately and distinguish inhomogeneous populations, which is essential to reveal the complicated mechanisms of multi-component systems. We report the details of a smFRET method based on the Nikon Ti2 inverted microscope to probe the ribosome dynamics between the ribosomal protein L27 and tRNAs. The L27 is labeled at its unique Cys 53 position and reconstituted into a ribosome that is engineered to lack L27. The tRNA is labeled at its elbow region. As the tRNA moves to different locations inside the ribosome during the elongation cycle, such as pre- and post- translocation, the FRET efficiencies and dynamics exhibit differences, which have suggested multiple subpopulations. These subpopulations are not detectable by ensemble methods. The TIRF-based smFRET microscope is built on a manual or motorized inverted microscope, with home-built laser illumination. The ribosome samples are purified by ultracentrifugation, loaded into a home-built multi-channel sample cell and then illuminated via an evanescent laser field. The reflection laser spot can be used to achieve feedback control of perfect focus. The fluorescence signals are separated by a motorized filter-turret and collected by two digital CMOS cameras. The intensities are retrieved via the NIS-Elements software.

Single molecule fluorescence energy transfer is a method that tracks the tRNA dynamics during ribosomal protein synthesis. By tracking individual ribosomes, inhomogeneous populations are identified, which shed light on mechanisms. This method can be used to track biological conformational changes in general to reveal dynamic-function relationships in many other complexed biosystems. Single molecule methods can observe non-rate limiting steps and low-populated key intermediates, which are not accessible by conventional ensemble methods due to the average effect.

The ribosome is a large ribonucleoprotein complex that assembles proteins processively along mRNA templates. The diameter of the ribosome is approximately ...

Single-Molecule Imaging of EWS-FLI1 Condensates Assembling on DNA

The fusion genes resulting from chromosomal translocation have been found in many solid tumors or leukemia. EWS-FLI1, which belongs to the FUS/EWS/TAF15 (FET) family of fusion oncoproteins, is one of the most frequently involved fusion genes in Ewing sarcoma. These FET family fusion proteins typically harbor a low-complexity domain (LCD) of FET protein at their N-terminus and a DNA-binding domain (DBD) at their C-terminus. EWS-FLI1 has been confirmed to form biomolecular condensates at its target binding loci due to LCD-LCD and LCD-DBD interactions, and these condensates can recruit RNA polymerase II to enhance gene transcription. However, how these condensates are assembled at their binding sites remains unclear. Recently, a single-molecule biophysics method-DNA Curtains-was applied to visualize these assembling processes of EWS-FLI1 condensates. Here, the detailed experimental protocol and data analysis approaches are discussed for the application of DNA Curtains in studying the biomolecular condensates assembling on target DNA.

Here, we describe the use of the single-molecule imaging method, DNA Curtains, to study the biophysical mechanism of EWS-FLI1 condensates assembling on DNA.

The fusion genes resulting from chromosomal translocation have been found in many solid tumors or leukemia. EWS-FLI1, which belongs to the FUS/EWS/TAF15 ...

An Optimized Single-Molecule Pull-Down Assay for Quantification of Protein Phosphorylation

Phosphorylation is a necessary posttranslational modification that regulates protein function and directs cell signaling outcomes. Current methods to measure protein phosphorylation cannot preserve the heterogeneity in phosphorylation across individual proteins. The single-molecule pull-down (SiMPull) assay was developed to investigate the composition of macromolecular complexes via immunoprecipitation of proteins on a glass coverslip followed by single-molecule imaging. The current technique is an adaptation of SiMPull that provides robust quantification of the phosphorylation state of full-length membrane receptors at the single-molecule level. Imaging thousands of individual receptors in this way allows for quantifying protein phosphorylation patterns. The present protocol details the optimized SiMPull procedure, from sample preparation to imaging. Optimization of glass preparation and antibody fixation protocols further enhances data quality. The current protocol provides code for the single-molecule data analysis that calculates the fraction of receptors phosphorylated within a sample. While this work focuses on phosphorylation of the epidermal growth factor receptor (EGFR), the protocol can be generalized to other membrane receptors and cytosolic signaling molecules.

The present protocol describes sample preparation and data analysis to quantify protein phosphorylation using an improved single-molecule pull-down (SiMPull) assay.

Phosphorylation is a necessary posttranslational modification that regulates protein function and directs cell signaling outcomes. Current methods to ...

Single-Molecule Analysis of Sf9 Purified Superprocessive Kinesin-3 Family Motors

A complex cellular environment poses challenges for single-molecule motility analysis. However, advancement in imaging techniques have improved single-molecule studies and has gained immense popularity in detecting and understanding the dynamic behavior of fluorescent-tagged molecules. Here, we describe a detailed method for&#160;in vitro single-molecule studies of kinesin-3 family motors using Total Internal Reflection Fluorescence (TIRF) microscopy. Kinesin-3 is a large family that plays critical roles in cellular and physiological functions ranging from intracellular cargo transport to cell division to development. We have shown previously that constitutively active dimeric kinesin-3 motors exhibit fast and superprocessive motility with high microtubule affinity at the single-molecule level using cell lysates prepared by expressing motor in mammalian cells. Our lab studies kinesin-3 motors and their regulatory mechanisms using cellular, biochemical and biophysical approaches, and such studies demand purified proteins at a large scale. Expression and purification of these motors using mammalian cells would be expensive and time-consuming, whereas expression in a prokaryotic expression system resulted in significantly aggregated and&#160;inactive protein. To overcome the limitations posed by bacterial purification systems and mammalian cell lysate, we have established a robust Sf9-baculovirus expression system to express and purify these motors. The kinesin-3 motors are C-terminally tagged with 3-tandem fluorescent proteins (3xmCitirine or 3xmCit) that provide enhanced signals and decreased photobleaching. In vitro single-molecule and multi-motor gliding analysis of Sf9 purified proteins demonstrate that kinesin-3 motors are fast and superprocessive akin to our previous studies using mammalian cell lysates. Other applications using these assays include detailed knowledge of oligomer conditions of motors, specific binding partners paralleling biochemical studies, and their kinetic state.

This study details purification of KIF1A(1-393LZ), a member of kinesin-3 family, using Sf9-baculovirus expression system. In vitro single-molecule and multi-motor gliding analysis of these purified motors exhibited robust motility properties comparable to motors from mammalian cell lysate. Thus, Sf9-baculovirus system is amenable to express and purify motor protein of interest.

A complex cellular environment poses challenges for single-molecule motility analysis. However, advancement in imaging techniques have improved ...

Single-Molecule Diffusion and Assembly on Polymer-Crowded Lipid Membranes

Cellular membranes are highly crowded environments for biomolecular reactions and signaling. Yet, most in vitro experiments probing protein interaction with lipids employ naked bilayer membranes. Such systems lack the complexities of crowding by membrane-embedded proteins and glycans and exclude the associated volume effects encountered on cellular membrane surfaces. Also, the negatively charged glass surface onto which the lipid bilayers are formed prevents the free diffusion of transmembrane biomolecules. Here, we present a well-characterized polymer-lipid membrane as a mimic for crowded lipid membranes. This protocol utilizes polyethylene glycol (PEG)-conjugated lipids as a generalized approach for incorporating crowders into the supported lipid bilayer&#160;(SLB). First, a cleaning procedure of the microscopic slides and coverslips for performing single-molecule experiments is presented. Next, methods for characterizing the PEG-SLBs and performing single-molecule experiments of the binding, diffusion, and assembly of biomolecules using single-molecule tracking and photobleaching are discussed. Finally, this protocol demonstrates how to monitor the nanopore assembly of bacterial pore-forming toxin Cytolysin A (ClyA) on crowded lipid membranes with single-molecule photobleaching analysis. MATLAB codes with example datasets are also included to perform some of the common analyses such as particle tracking, extracting diffusive behavior, and subunit counting.

Here, a protocol to perform and analyze the binding, mobility, and assembly of single molecules on artificial crowded lipid membranes using single-molecule total internal reflection fluorescence (smTIRF) microscopy is presented.

Cellular membranes are highly crowded environments for biomolecular reactions and signaling. Yet, most in vitro experiments probing protein interaction ...