Name: single-molecule manipulation of g-quadruplexes by magnetic tweezers
Uploaded: 2017-09-19T14:00:00
Description: Watch this Scientific Journal Video about Single-molecule Manipulation of G-quadruplexes by Magnetic Tweezers at JoVE.com

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 comm...

<ol>
	<li>Rhodes, D., Lipps, H. J. <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+and+their+regulatory+roles+in+biology.">G-quadruplexes and their regulatory roles in biology.</a> Nucleic Acids Res. 43 (18), 8627-8637 (2015).</li><li>Brazda, V., Haronikova, L., Liao, J. C., Fojta, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+and+RNA+quadruplex-binding+proteins.">DNA and RNA quadruplex-binding proteins.</a> Int J Mol Sci. 15 (10), 17493-17517 (2014).</li><li>Gonzalez, V., Hurley, L. 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S., Yan, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+distinct+overstretched+DNA+structures+revealed+by+single-molecule+thermodynamics+measurements.">Two distinct overstretched DNA structures revealed by single-molecule thermodynamics measurements.</a> Proc Natl Acad Sci U S A. 109 (21), 8103-8108 (2012).</li><li>Zhang, X., 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=Revealing+the+competition+between+peeled+ssDNA,+melting+bubbles,+and+S-DNA+during+DNA+overstretching+by+single-molecule+calorimetry.">Revealing the competition between peeled ssDNA, melting bubbles, and S-DNA during DNA overstretching by single-molecule calorimetry.</a> Proc Natl Acad Sci U S A. 110 (10), 3865-3870 (2013).</li><li>Chen, H., 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=Improved+High-Force+Magnetic+Tweezers+for+Stretching+and+Refolding+of+Proteins+and+Short+DNA.">Improved High-Force Magnetic Tweezers for Stretching and Refolding of Proteins and Short DNA.</a> Biophys. J. 100 (2), 517-523 (2011).</li><li>Fu, H. X., 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=Transition+dynamics+and+selection+of+the+distinct+S-DNA+and+strand+unpeeling+modes+of+double+helix+overstretching.">Transition dynamics and selection of the distinct S-DNA and strand unpeeling modes of double helix overstretching.</a> Nucleic Acids Res. 39 (8), 3473-3481 (2011).</li><li>Zhang, X., Chen, H., Fu, H., Doyle, P. S., Yan, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+distinct+overstretched+DNA+structures+revealed+by+single-molecule+thermodynamics+measurements.">Two distinct overstretched DNA structures revealed by single-molecule thermodynamics measurements.</a> Proc. Natl. Acad. Sci. U.S.A. 109 (21), 8103-8108 (2012).</li><li>Zhang, X., 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=Revealing+the+competition+between+peeled+ssDNA,+melting+bubbles,+and+S-DNA+during+DNA+overstretching+by+single-molecule+calorimetry.">Revealing the competition between peeled ssDNA, melting bubbles, and S-DNA during DNA overstretching by single-molecule calorimetry.</a> Proc. Natl. Acad. Sci. U.S.A. 110 (10), 3865-3870 (2013).</li><li>Vaughn, J. P., 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=The+DEXH+protein+product+of+the+DHX36+gene+is+the+major+source+of+tetramolecular+quadruplex+G4-DNA+resolving+activity+in+HeLa+cell+lysates.">The DEXH protein product of the DHX36 gene is the major source of tetramolecular quadruplex G4-DNA resolving activity in HeLa cell lysates.</a> J Biol Chem. 280 (46), 38117-38120 (2005).</li><li>Giri, B., 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=G4+resolvase+1+tightly+binds+and+unwinds+unimolecular+G4-DNA.">G4 resolvase 1 tightly binds and unwinds unimolecular G4-DNA.</a> Nucleic Acids Res. 39 (16), 7161-7178 (2011).</li><li>De Vlaminck, I., Dekker, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+magnetic+tweezers.">Recent advances in magnetic tweezers.</a> Annu Rev Biophys. 41, 453-472 (2012).</li><li>Yan, J., Skoko, D., Marko, J. 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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. 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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 ...

The overall goal of this experiment is to directly measure the folding and unfolding of G-quadruplexes, abbreviated G4, and the regulation by binding proteins at the single-molecule level using magnetic tweezers. This message can help answer key questions in G4 field, such as floating dynamics of G4, and the regulation by binding proteins. The main advantage of this technique, is that magnetic tweezers, allow stable measurement, of nanometer sized floating dynamics of a single-molecule over days, by using anti-drift technique.Let's start this with this message, can provide the inside the molecule mechanisms of G4 stability, the helicase activities on G4, it can also use to probe the al-a-do-gees related to G4 binding proteins, or ligans, which may have potential therapeutic applications. In addition, it can also used to probe the outer protein DNA interactions, or protein protein interactions. Begin this procedure with preparation of G4 DNA in the flow channel, as described in the text protocol.G4 DNA was prepared by ligate G4 forming single stranded DNA, two flank single stranded DNA, and a double stranded DNA handle, using T4 DNA ligates. For the magnetic tweezers setup, start the magnetic tweezers program. Here, the magnetic tweezers are controlled by an in-house written lab view program.Next, align the magnet centers before mounting the channel. Use a 10 times objective lens to adjust the X and Y axis of the magnetics, in the optical access of the microscope. Use a computer controlled motorized manipulator to move the magnets along the Z direction, and set the distance between the magnets and coverslip as D equals zero, when the magnetics attach to a coverslip on the microscope.Program the movement of the magnetics through the manipulator to achieve control of the force, including constant force and time varying force. Use a bright light emitting diode light source for back scattered illumination of the bead through the objective. Collect the bead images and a sampling rate of 100 hertz, by a change coupled device camera.For tether formation, gently flow 200 X diluted M280 para magnetic beads in as-say solution into a channel. Incubate the mixture for 10 minutes to allow the beads to bind to biotin labeled DNA molecules immobilized on the SMCC coded surface through the thiol labeled ent. Gently wash away untethered beads using 200 microliters of standard reaction solution.Next, mount the channel onto the microscope stage. Search for the beads on the bottom surface using the 100 times oil immersion objective. 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 and different de-focused plains. Before experiments, use a objective Piezo actuator to obtain images of both reference and tethered beads at different de-focused plains, spaced at 20 to 50 nanometers. Store the images as two separate bead image libraries that are respectively indexed with the de-focused distance.During the experiments, determine the position of the bead in the XY plain, by the bead centroid. Also 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 images.During experiments, use the Pizeo to lock the distance between the objective in 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 the specific image stored in the library. Determine whether the tether is a single double stranded DNA molecule, by applying approximately 65 piconewtons of force. A tether is a single double stranded DNA molecule if it undergoes the characteristic DNA overstretching transition.Repeat the process until a single double stranded DNA tether is found. The relationship of force and magnet position, or so called, the force distance curve, was calibrated based on bead fluctuation. Furthermore, the overstretching transition of double stranded DNA that occurred near 65 piconewtons, could also be used to calibrate the force.For force ramp experiments, perform a force increase scan at a loading rate of 0.2 piconewtons per second, followed by a force decrease scan at minus 0.2 piconewtons per second. After each stretching cycle, hold the DNA molecule at one piconewton for 30 seconds, to allow the single stranded DNA to refold to G4.For force-jump experiments, cycle the force between 54 piconewtons for 30 seconds, under which a folded G4 could be unfolded, and 1 piconewton for 60 seconds allowing a folded G4-15T to refold. Finally, analyze the unfolding force, using an in-house written mat lab program.Shown here, is a typical example of a force increase and force decrease curve of a tethered G4 quadruplex. The extension jump in the force increase scan indicated an unfolding transition of G4, at approximately 52 piconewtons. However, in the presence of RHAU, G4 remained folded during the force increase scan for up to 60 piconewtons, indicating G4 force stabilization by RHAU binding.In the presence of RHAU and ATP, the unfolding force distribution of G4, was shifted to a lower force, indicative of an ATP depended destabilization of G4 by RHAU. Representative traces of the bead height during force-jump cycles, shows that the force applied to the molecule was cycled between 54 piconewtons for 30 seconds, under which a folded G4 could be unfolded, and 1 piconewton for 60 seconds, under which it could refold. The average lifetime a folded G4 at 54 piconewtons, was approximately 6.4 seconds, estimated by fitting the lifetime histogram with an exponential decay function.When RHAU helicase was introduced, the extension of tethered DNA remained at folded level throughout the 30 seconds holding time at 54 piconewtons, indicated that the RHAU strongly stabilizes G4 against mechanical unfolding. Upon addition of RHAU and ATP, the extension was at the level of unfolded single stranded DNA, indicating a destabilization of G4.After watching this video, you should have a good understanding of how to measure the unfolding and refolding dynamics of nuclear ACS junctions using this ultra stabled magnetic tweezers. The allotment of this technique paved the way for researchers in field of nucleic acid structure and binding proteins to explore the regression of structure stability and the folding kinetics, as well as the interaction of nucleic acid and binding proteins.

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

Research

JoVE Journal

Biochemistry

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 ...

Using Three-color Single-molecule FRET to Study the Correlation of Protein Interactions

Single-molecule F&#246;rster resonance energy transfer (smFRET) has become a widely used biophysical technique to study the dynamics of biomolecules. For many molecular machines in a cell proteins have to act together&#160;with interaction partners in a functional cycle to fulfill their task. The extension of two-color to multi-color smFRET makes it possible to simultaneously probe more than one interaction or conformational change. This not only adds a new dimension to smFRET experiments but it also offers the unique possibility to directly study the sequence of events and to detect correlated interactions when using an immobilized sample and a total internal reflection fluorescence microscope (TIRFM). Therefore, multi-color smFRET is a versatile tool for studying biomolecular complexes in a quantitative manner and in a previously unachievable detail.
Here, we demonstrate how to overcome the special challenges of multi-color smFRET experiments on proteins. We present detailed protocols for obtaining the data and for extracting kinetic information. This includes trace selection criteria, state separation, and the recovery of state trajectories from the noisy data using a 3D ensemble Hidden Markov Model (HMM). Compared to other methods, the kinetic information is not recovered from dwell time histograms but directly from the HMM. The maximum likelihood framework allows us to critically evaluate the kinetic model and to provide meaningful uncertainties for the rates.
By applying our method to the heat shock protein 90 (Hsp90), we are able to disentangle the nucleotide binding and the global conformational changes of the protein. This allows us to directly observe the cooperativity between the two nucleotide binding pockets of the Hsp90 dimer.

Here, we present a protocol to obtain three-color smFRET data and its analysis with a 3D ensemble Hidden Markov Model. With this approach, scientists can extract kinetic information from complex protein systems, including cooperativity or correlated interactions.

Single-molecule F&#246;rster resonance energy transfer (smFRET) has become a widely used biophysical technique to study the dynamics of biomolecules. For ...

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 ...

OaAEP1-Mediated Enzymatic Synthesis and Immobilization of Polymerized Protein for Single-Molecule Force Spectroscopy

Chemical and bio-conjugation techniques have been developed rapidly in recent years and allow the building of protein polymers. However, a controlled protein polymerization process is always a challenge. Here, we have developed an enzymatic methodology for constructing polymerized protein step by step in a rationally-controlled sequence. In this method, the C-terminus of a protein monomer is NGL for protein conjugation using OaAEP1 (Oldenlandia affinis asparaginyl endopeptidases) 1) while the N-terminus was a cleavable TEV (tobacco etch virus) cleavage site plus an L (ENLYFQ/GL) for temporary N-terminal protecting. Consequently, OaAEP1 was able to add only one protein monomer at a time, and then the TEV protease cleaved the N-terminus between Q and G to expose the NH2-Gly-Leu. Then the unit is ready for next OaAEP1 ligation. The engineered polyprotein is examined by unfolding individual protein domain using atomic force microscopy-based single-molecule force spectroscopy (AFM-SMFS). Therefore, this study provides a useful strategy for polyprotein engineering and immobilization.

Here, we present a protocol to conjugate protein monomer by enzymes forming protein polymer with a controlled sequence and immobilize it on the surface for single-molecule force spectroscopy studies.

Chemical and bio-conjugation techniques have been developed rapidly in recent years and allow the building of protein polymers. However, a controlled ...

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 ...