G. N. L. C. acknowledges support from the National Institute of Mental Health of the National Institutes of Health (NIH) under award number F31MH132306. S. L. is supported by the Robertson Foundation, the International Rett Syndrome Foundation, and the NIH (award number R01GM149862).

The details of the reagents and the equipment used in the study are listed in the Table of Materials.
1. Preparation of biotinylated DNA
<ol>
	<li>Prepare a 120 &#181;L volume reaction containing 20 &#181;g of &#955; DNA, 33 &#181;M of biotin-dCTP, 33 &#181;M of biotin-dUTP, 33 &#181;M of biotin-dATP, 33 &#181;M of dGTP, 10 units of Klenow enzyme, and 1x NEB Buffer 2. 
	NOTE: This procedure specifically utilizes linear double-stranded (ds) methylation-free bacteriophage &#955; genomic DNA (48,502 bps), which contains 12-nucleotide&#160;(nt) 5&#39; single-stranded (ss) DNA overhang23. However, the protocol can be adapted to make use of any length or sequence of linear dsDNA provided it contains at least several nts of ss 5&#39; overhangs, the length and nt sequence dictating the number of biotinylated nts installed on each end for downstream tethering between optical traps. Additionally, the reaction can be scaled down. For example, 5 &#181;g of DNA can be used in a 30 &#181;L volume reaction. Additionally, the DNA substrates generated in this way are not torsionally constrained. For protocols to create torsionally constrained DNA, please refer to the previously published reports24,25.</li>
	<li>Incubate the reaction at room temperature for 15 min.</li>
	<li>Add 10 mM of EDTA and incubate at 75&#160;&#176;C for 20 min.</li>
	<li>Perform ethanol precipitation to obtain the biotinylated DNA product.
	<ol>
		<li>Add 0.1x volume of 3 M sodium acetate and 3x volume of ice cold 100% ethanol. Keep at -20&#160;&#176;C for at least 1 h up to overnight. Centrifuge at 13,500 x g for 30 min at 4&#160;&#176;C.</li>
		<li>Carefully remove the supernatant without disturbing the pellet and add 1 mL of 75% ethanol. Centrifuge at 13,500 x g for 1 min at 4 &#176;C.</li>
		<li>Repeat the supernatant removal, ethanol addition, and centrifugation steps.</li>
		<li>Carefully remove all the supernatant and let the pellet air dry for 10 min. Dissolve the pellet in 100-200 &#181;L of TE buffer or ddH2O (can incubate at 37&#160;&#176;C to facilitate dissolving). Measure the concentration using a Nanodrop spectrophotometer.</li>
	</ol>
	</li>
</ol>
2. Preparation of channels in a flow cell
NOTE: This procedure is based on a commercially available microfluidic chip (see Table of Materials) but can be adapted to any smCFFM instrument with a multi-channel flow cell.
<ol>
	<li>Passivate channels (ch.) 4 and 5 (or any ch. that will contain proteins) (see schematic of channels in Figure 1A). Add 500 &#181;L of 0.1% BSA to each channel and flow at 0.8 bar (~0.45 &#181;L/s for tubing with a 0.010 inch inner diameter) for 8 min. Remove solution.</li>
	<li>Add 500 &#181;L of 0.05% Pluronic-F127 to each channel and flow at 0.8 bar for 8 min. Remove the solution and rinse out the syringes with 1x PBS. Add 2 mL of 1x PBS to each channel and flow at 0.8 barfor 27 min.</li>
	<li>Vortex the stock solution of streptavidin-coated beads (this procedure uses 3.13 &#181;m size beads) and add 2.15 &#181;L of the bead solution to 1 mL of 1x PBS. Add to ch. 1. 
	NOTE: The size and concentration of the streptavidin-coated beads can be adjusted according to user preference.</li>
	<li>Add 30-80 ng of biotinylated &#955; DNA to 1 mL of 1x PBS. Add to ch. 2. 
	NOTE: The concentration of biotinylated DNA can be adjusted to user preference. Greater amounts of DNA will increase the frequency of forming DNA tethers but also raise the frequency of tethering multiple molecules of DNA at once, which can add to the difficulty of the experiment.</li>
</ol>
3. Nap1-mediated in situ nucleosome formation
<ol>
	<li>Add 500 &#181;L of protein-free&#160;image buffer to ch. 3. 
	NOTE: The buffer composition in ch. 3 is flexible and should be appropriate for both nucleosomes and the specific protein(s) of interest. Optionally, users may choose to add oxygen scavenging systems for reduced photobleaching of fluorophores and/or competitor DNA to remove histone octamers that are not properly wrapped into nucleosomes.</li>
	<li>Add 2 nM S. cerevisiae Nap1 and 2-5 nM LD655-labeled H4 histone octamer (HO) to 500 &#181;L of 1x HR buffer18. Add to ch. 4. 
	NOTE: Histone octamers and Nap1 can be commercially purchased or prepared in-house (see Table of Materials). The number of nucleosomes to be formed along each tethered DNA depends on the octamer concentration as well as the time spent in this channel. The user&#39;s desired and approximate nucleosome density along each DNA molecule can be modulated by adjusting either or both of these parameters. Additionally, the user may choose to label histone octamers with their preferred fluorophore.</li>
	<li>Optional: Add chromatin-binding protein or other protein of interest to ch. 5 in an appropriate buffer.</li>
	<li>Flow all channels at 1.0 bar (~0.55 &#181;L/s in the current experimental setup) for 30 s to flush the flow cell channels with samples.</li>
	<li>Set the flow to 0.2 bar (~0.16 &#181;L/s in the current experimental setup). Move the optical traps (OTs) to ch. 1 and catch a suitable pair of streptavidin-coated beads.</li>
	<li>Move the OTs to ch. 3, conduct a force calibration for the bead pair, turn on the red confocal laser (or another laser line of choice), and calibrate the imaging area.</li>
	<li>Set the flow to 0.2 bar and move OTs to ch. 2. Catch biotinylated DNA.</li>
	<li>Move the OTs to ch. 3 and stop the flow. Increase the distance between the OTs to stretch the DNA and monitor the associated force-distance (FD) curve to confirm the presence of a single DNA tether (as opposed to multiple tethers). The curve should follow the worm-like-chain model for dsDNA at the given contour length26.</li>
	<li>Adjust the distance between the OTs so that the DNA tension reads approximately 1 pN. 
	NOTE: 1 pN of tension is low enough to allow nucleosomes to remain wrapped by DNA27 but is high enough to place the entire DNA molecule within the line scanning imaging axis.</li>
	<li>Turn on line scanning to start collecting a kymograph with the red laser on. 
	NOTE: Kymographs or 2D scans of tethered DNA can be obtained, as per user preference.</li>
	<li>Move the OTs to ch. 4 with the flow off. Facilitated by Nap1, HOs are loaded onto DNA and wrapped to form nucleosomes in real-time. Nucleosome loading is visualized as fluorescent trajectories appearing on the DNA within kymographs. Concurrently, nucleosome formation on the DNA can be monitored by the force increase as more HOs are loaded when the positions of OTs are held constant. 
	NOTE: Fluorescence excitation laser can be turned off during the nucleosome formation steps to avoid photobleaching of fluorophores. Additionally, non-specific binding of histone octamers to DNA wherein the octamers are not wrapped into nucleosomes could occur using this protocol. These binding events will not generate a &#34;rip&#34; when the DNA is stretched, as described in the Unwrapping Nucleosomes (step 4) protocol below, and can sometimes be washed off by a gentle buffer flow, particularly if the buffer contains free competitor DNA.</li>
	<li>When the approximate desired number of nucleosomes is formed, start collecting data.</li>
</ol>
4. Unwrapping nucleosomes
<ol>
	<li>Complete step 2 and step 3.</li>
	<li>After forming a DNA tether containing nucleosomes, move the OTs to ch. 3.</li>
	<li>Prepare for nucleosome unwrapping experiments by reducing the distance between OTs until the force is approximately zero. Zero the force.</li>
	<li>Begin moving the OT 1 away from the OT 2 at a constant speed of 0.1 &#181;m/s. On a kymograph, the bead in OT 1 will visually move farther from the bead in OT 2. On the FD curve, the force will rise as the distance increases. As nucleosomes unwrap, there will appear &#34;rips&#34; or transitions in the force (~8-37 pN) that signify the unraveling of the inner wrap of individual nucleosomes27,28,29. 
	NOTE: The force at which unwrapping occurs depends on the pulling rate, which is expected for non-equilibrium processes. Sometimes, multiple nucleosomes unwrap at once, producing a larger distance change in the transition on the FD curve. Additionally, the unraveling of the outer wrap of the nucleosome is not readily visible in this experimental setting (see the Discussion section).</li>
</ol>
5. Visualizing protein binding to chromatin
<ol>
	<li>Complete step 2 and step 3.</li>
	<li>Add 10 nM of Cy3-labeled linker histone H1.4 to 500 &#181;L of image buffer in ch. 5. 
	NOTE: Any protein of interest can be added to ch. 5 at a concentration of choice (typically below 100 nM for single-molecule visualization).</li>
	<li>After forming a DNA tether containing nucleosomes, turn on the green laser in addition to the red laser and move OTs to ch. 5 for real-time imaging of H1 binding to chromatin.</li>
	<li>Continue collecting kymographs or scans until sufficient statistics have been achieved (at least ~10-20 kymographs depending on the frequency of events and experimental difficulty).</li>
</ol>

In Situ Nucleosome Reconstitution for Single-Molecule Studies

<ol>
	<li>Kornberg, 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=Chromatin+structure:+A+repeating+unit+of+histones+and+DNA.">Chromatin structure: A repeating unit of histones and DNA.</a> Science. 184, 868-871 (1974).</li><li>Luger, K., Mader, A. W., Richmond, R. K., Sargent, D. F., Crystal Richmond, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=structure+of+the+nucleosome+core+particle+at+2.8+A+resolution.">structure of the nucleosome core particle at 2.8 A resolution.</a> Nature. 389, 251-260 (1997).</li><li>Zhou, K., Gaullier, G., Luger, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nucleosome+structure+and+dynamics+are+coming+of+age.">Nucleosome structure and dynamics are coming of age.</a> Nat Struct Mol Biol. 26, 3-13 (2019).</li><li>McGinty, R. K., Tan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Principles+of+nucleosome+recognition+by+chromatin+factors+and+enzymes.">Principles of nucleosome recognition by chromatin factors and enzymes.</a> Curr Opin Struct Biol. 71, 16-26 (2021).</li><li>Fierz, B., Poirier, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biophysics+of+chromatin+dynamics.">Biophysics of chromatin dynamics.</a> Annu Rev Biophys. 48, 321-345 (2019).</li><li>Selvin, P. R., Ha, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Single-molecule techniques: A laboratory manual. , (2008).</li><li>Chua, G. N. L., Liu, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=When+force+met+fluorescence:+Single-molecule+manipulation+and+visualization+of+protein-DNA+interactions.">When force met fluorescence: Single-molecule manipulation and visualization of protein-DNA interactions.</a> Annu Rev Biophys. , (2024).</li><li>Hashemi Shabestari, M., Meijering, A. E. C., Roos, W. H., Wuite, G. J. L., Peterman, E. J. G. <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+biological+single-molecule+applications+of+optical+tweezers+and+fluorescence+microscopy.">Recent Advances in biological single-molecule applications of optical tweezers and fluorescence microscopy.</a> Methods Enzymol. 582, 85-119 (2017).</li><li>Ashkin, A., Dziedzic, J. M., Bjorkholm, J. E., Chu, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Observation+of+a+single-beam+gradient+force+optical+trap+for+dielectric+particles.">Observation of a single-beam gradient force optical trap for dielectric particles.</a> Opt Lett. 11, 288 (1986).</li><li>Neuman, K. C., Block, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+trapping.">Optical trapping.</a> Rev Sci Instrum. 75, 2787-2809 (2004).</li><li>Hansen, J. C., van Holde, K. E., Lohr, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mechanism+of+nucleosome+assembly+onto+oligomers+of+the+sea+urchin+5+S+DNA+positioning+sequence.">The mechanism of nucleosome assembly onto oligomers of the sea urchin 5 S DNA positioning sequence.</a> J Biol Chem. 266, 4276-4282 (1991).</li><li>Peterson, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Salt+gradient+dialysis+reconstitution+of+nucleosomes.">Salt gradient dialysis reconstitution of nucleosomes.</a> CSH Protoc. , (2008).</li><li>Leicher, R., Liu, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probing+the+interaction+between+chromatin+and+chromatin-associated+complexes+with+optical+tweezers.">Probing the interaction between chromatin and chromatin-associated complexes with optical tweezers.</a> Methods Mol Biol. 2478, 313-327 (2022).</li><li>Segal, E., 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=A+genomic+code+for+nucleosome+positioning.">A genomic code for nucleosome positioning.</a> Nature. 442, 772-778 (2006).</li><li>Ioshikhes, I. P., Albert, I., Zanton, S. J., Pugh, B. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nucleosome+positions+predicted+through+comparative+genomics.">Nucleosome positions predicted through comparative genomics.</a> Nat Genet. 38, 1210-1215 (2006).</li><li>Lowary, P. T., Widom, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+DNA+sequence+rules+for+high+affinity+binding+to+histone+octamer+and+sequence-directed+nucleosome+positioning.">New DNA sequence rules for high affinity binding to histone octamer and sequence-directed nucleosome positioning.</a> J Mol Biol. 276, 19-42 (1998).</li><li>Chipev, C. C., Wolffe, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromosomal+organization+of+Xenopus+laevis+oocyte+and+somatic+5S+rRNA+genes+in+vivo.">Chromosomal organization of Xenopus laevis oocyte and somatic 5S rRNA genes in vivo.</a> Mol Cell Biol. 12, 45-55 (1992).</li><li>Crickard, J. B., Moevus, C. J., Kwon, Y., Sung, P., Greene, E. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rad54+drives+ATP+hydrolysis-dependent+DNA+sequence+alignment+during+homologous+recombination.">Rad54 drives ATP hydrolysis-dependent DNA sequence alignment during homologous recombination.</a> Cell. 181, 1380-1394.e18 (2020).</li><li>Vlijm, R., 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=Comparing+the+assembly+and+handedness+dynamics+of+(H3.3-H4)2+tetrasomes+to+canonical+tetrasomes.">Comparing the assembly and handedness dynamics of (H3.3-H4)2 tetrasomes to canonical tetrasomes.</a> PLoS One. 10, e0141267 (2015).</li><li>Mazurkiewicz, J., Kepert, J. F., Rippe, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+mechanism+of+nucleosome+assembly+by+histone+chaperone+NAP1.">On the mechanism of nucleosome assembly by histone chaperone NAP1.</a> J Biol Chem. 281, 16462-16472 (2006).</li><li>Nakagawa, T., Bulger, M., Muramatsu, M., Ito, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multistep+chromatin+assembly+on+supercoiled+plasmid+DNA+by+nucleosome+assembly+protein-1+and+ATP-utilizing+chromatin+assembly+and+remodeling+factor.">Multistep chromatin assembly on supercoiled plasmid DNA by nucleosome assembly protein-1 and ATP-utilizing chromatin assembly and remodeling factor.</a> J Biol Chem. 276, 27384-27391 (2001).</li><li>Andrews, A. J., Chen, X., Zevin, A., Stargell, L. A., Luger, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+histone+chaperone+Nap1+promotes+nucleosome+assembly+by+eliminating+nonnucleosomal+histone+DNA+interactions.">The histone chaperone Nap1 promotes nucleosome assembly by eliminating nonnucleosomal histone DNA interactions.</a> Mol Cell. 37, 834-842 (2010).</li><li>Wu, R., Taylor, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nucleotide+sequence+analysis+of+DNA.+II.+Complete+nucleotide+sequence+of+the+cohesive+ends+of+bacteriophage+lambda+DNA.">Nucleotide sequence analysis of DNA. II. Complete nucleotide sequence of the cohesive ends of bacteriophage lambda DNA.</a> J Mol Biol. 57, 491-511 (1971).</li><li>King, G. A., Peterman, E. J., Wuite, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unravelling+the+structural+plasticity+of+stretched+DNA+under+torsional+constraint.">Unravelling the structural plasticity of stretched DNA under torsional constraint.</a> Nat Commun. 7, 11810 (2016).</li><li>Paik, D. H., Roskens, V. A., Perkins, T. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Torsionally+constrained+DNA+for+single-molecule+assays:+An+efficient,+ligation-free+method.">Torsionally constrained DNA for single-molecule assays: An efficient, ligation-free method.</a> Nucleic Acids Res. 41, e179 (2013).</li><li>Bustamante, C. J., Chemla, Y. R., Liu, S., Wang, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+tweezers+in+single-molecule+biophysics.">Optical tweezers in single-molecule biophysics.</a> Nat Rev Methods Primers. 1, 25 (2021).</li><li>Diaz-Celis, C., 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=Assignment+of+structural+transitions+during+mechanical+unwrapping+of+nucleosomes+and+their+disassembly+products.">Assignment of structural transitions during mechanical unwrapping of nucleosomes and their disassembly products.</a> Proc Natl Acad Sci USA. 119, e2206513119 (2022).</li><li>Leicher, R., 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=Single-molecule+and+in+silico+dissection+of+the+interaction+between+Polycomb+repressive+complex+2+and+chromatin.">Single-molecule and in silico dissection of the interaction between Polycomb repressive complex 2 and chromatin.</a> Proc Natl Acad Sci USA. 117, 30465-30475 (2020).</li><li>Chua, G. N. L., 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=Differential+dynamics+specify+MeCP2+function+at+methylated+DNA+and+nucleosomes.">Differential dynamics specify MeCP2 function at methylated DNA and nucleosomes.</a> bioRxiv. , (2023).</li><li>Brower-Toland, B. D., 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=Mechanical+disruption+of+individual+nucleosomes+reveals+a+reversible+multistage+release+of+DNA.">Mechanical disruption of individual nucleosomes reveals a reversible multistage release of DNA.</a> Proc Natl Acad Sci USA. 99, 1960-1965 (2002).</li><li>McCauley, M. 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=Human+FACT+subunits+coordinate+to+catalyze+both+disassembly+and+reassembly+of+nucleosomes.">Human FACT subunits coordinate to catalyze both disassembly and reassembly of nucleosomes.</a> Cell Rep. 41, 111858 (2022).</li><li>Mihardja, S., Spakowitz, A. J., Zhang, Y., Bustamante, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+force+on+mononucleosomal+dynamics.">Effect of force on mononucleosomal dynamics.</a> Proc Natl Acad Sci USA. 103, 15871-15876 (2006).</li><li>Li, S., 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=Nucleosome-directed+replication+origin+licensing+independent+of+a+consensus+DNA+sequence.">Nucleosome-directed replication origin licensing independent of a consensus DNA sequence.</a> Nat Commun. 13, 4947 (2022).</li><li>Li, S., 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=Origin+recognition+complex+harbors+an+intrinsic+nucleosome+remodeling+activity.">Origin recognition complex harbors an intrinsic nucleosome remodeling activity.</a> Proc Natl Acad Sci USA. 119, e2211568119 (2022).</li><li>Harada, B. T., 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=Stepwise+nucleosome+translocation+by+RSC+remodeling+complexes.">Stepwise nucleosome translocation by RSC remodeling complexes.</a> Elife. 5, e10051 (2016).</li><li>Luger, K., Rechsteiner, T. J., Richmond, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+and+purification+of+recombinant+histones+and+nucleosome+reconstitution.">Expression and purification of recombinant histones and nucleosome reconstitution.</a> Methods Mol Biol. 119, 1-16 (1999).</li><li>Rogge, R. A., 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=Assembly+of+nucleosomal+arrays+from+recombinant+core+histones+and+nucleosome+positioning+DNA.">Assembly of nucleosomal arrays from recombinant core histones and nucleosome positioning DNA.</a> J Vis Exp. (79), e50354 (2013).</li><li>Leicher, R., 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=Single-stranded+nucleic+acid+binding+and+coacervation+by+linker+histone+H1.">Single-stranded nucleic acid binding and coacervation by linker histone H1.</a> Nat Struct Mol Biol. 29, 463-471 (2022).</li></ol>

The authors declare no competing interests.

The described protocol provides several advantages for nucleosome reconstitution including minimizing reagents and time as well as enabling chaperone-dependent nucleosome formation along any (potentially native) DNA sequence. Moreover, the in situ method allows simpler experimental workflow and a convenient transition into single-molecule assays of nucleosome mechanics and protein-chromatin interaction. On the other hand, limitations of this approach include the inability to direct nucleosome positioning along t...

The primary packaging unit of eukaryotic chromatin is the nucleosome, in which ~147 base pairs (bps) of DNA are wrapped around an octamer of core histones proteins1,2. In addition to genome packaging, the nucleosome architecture serves as another rich layer of biophysical regulation that can be harnessed by chromatin-binding proteins when performing their various functions3,4. Experimentally accessing and measuring the physical characteristics of nucleosomes has been technically challenging since these units perform at minuscule scales (e.g., nanometer lengths, piconewton forces), and thus, their probing requires sufficient sensitivity and precision to meaningfully inform function. Moreover, chromatin-binding proteins often engage with their substrates transiently, and most ensemble approaches lack appropriate temporal resolution to inform the kinetics of these interactions5. Fortunately, the advent of single-molecule techniques has made it possible to visualize and manipulate individual proteins and their interactions in real-time, revealing mechanistic information about these molecular events occurring at the nanoscale6. In particular, single-molecule correlative force and fluorescence microscopy (smCFFM) harnesses both high-resolution fluorescence detection and force manipulation tools to simultaneously resolve the mechanics, composition, and coordination of chromatin and chromatin-protein complexes7,8.
In smCFFM that specifically employs &#34;optical tweezers&#34; as the force manipulation method, a tightly focused laser is used to trap a dielectric object, typically a micron-sized plastic bead, in three dimensions9. A biopolymer such as a piece of DNA can be conjugated to the bead (via, e.g., streptavidin-biotin, digoxigenin-anti-digoxigenin antibody linkage), and the user can both apply a calibrated force and measure the force/displacement generated by the system10. The added fluorescence module enables simultaneous multicolor fluorescence imaging to track protein composition and behavior.
The mainstream method for reconstituting nucleosome templates for smCFFM involves assembling nucleosomes on custom DNA templates by salt dialysis11,12,13. In this procedure, purified histone octamers are incubated with DNA templates in a high-salt buffer (~1 M sodium chloride), and as the salt is slowly dialyzed away, nucleosomes spontaneously assemble on the DNA in a sequence-dependent positional manner14,15. As such, it is customary that strong nucleosome positioning sequences (e.g., Widom 60116, X. laevis 5S rDNA17) are incorporated within the DNA templates to generate custom nucleosome arrays. Although this well-established method offers precise control over the placement and number of nucleosomes across DNA, it suffers from several disadvantages: (1) incorporation of strong nucleosome positioning DNA sequences may generate artificially stable nucleosomes that bias the activity of chromatin-binding proteins, (2) the process of salt dialysis for nucleosome formation requires high amounts of DNA and octamers, and (3) the procedure takes one to several days of work as well as a considerable effort to titrate the correct DNA to octamer ratio for optimal nucleosome array density.
In this manuscript, we describe an alternative method to form nucleosomes along DNA in situ within a single-molecule correlative force and fluorescence microscope using recombinant histone chaperone Nap1, which resembles the physiological pathway of nucleosome formation in vivo18,19,20,21,22. This method allows users to assemble nucleosomes on non-specific DNA sequences, easily adjust nucleosome density, and utilize significantly fewer amounts of reagents, all within minutes. Additionally, the formation of nucleosomal DNA tethers in situ enables simpler experimental workflow and the convenience of built-in single-molecule visualization and manipulation. Thus, when uniform and specific nucleosome positioning is not a necessity, this protocol offers a useful option for the investigation of nucleosome mechanics or protein behavior on chromatin, for which we also describe example assays.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1x HR buffer</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>30 mM&#160;tris&#160;acetate pH 7.5, 20 mM magnesium acetate, 50 mM potassium chloride, 0.1 mg/mL BSA&#160;</td>
		</tr>
		<tr>
			<td>1x PBS (Phosphate-buffered saline)</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>137 mM sodium chloride, 2.7 mM potassium chloride, 10 mM sodium phosphate dibasic, 1.8 mM potassium phophate monobasic&#160;</td>
		</tr>
		<tr>
			<td>Acetic acid, glacial</td>
			<td>Millipore Sigma</td>
			<td>AX0074-6</td>
			<td>Use to make tris acetate</td>
		</tr>
		<tr>
			<td>Biotin-11-dUTP</td>
			<td>Jena Bioscience</td>
			<td>NU-803-BIOX-S</td>
			<td></td>
		</tr>
		<tr>
			<td>Biotin-14-dATP</td>
			<td>Jena Bioscience</td>
			<td>NU-835-BIO14-S</td>
			<td></td>
		</tr>
		<tr>
			<td>Biotin-14-dCTP</td>
			<td>Jena Bioscience</td>
			<td>NU-956-BIO14-S</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Millipore Sigma</td>
			<td>A9418-50G</td>
			<td>Dissolve in H2O and run through 0.22 um filter</td>
		</tr>
		<tr>
			<td>Cy3 Maleimide Mono-Reactive Dye</td>
			<td>Cytiva</td>
			<td>PA23031</td>
			<td>Maleimide functionalized Cy3 fluorophore</td>
		</tr>
		<tr>
			<td>dGTP</td>
			<td>New England Biolabs</td>
			<td>N0442S</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf Centrifuge 5425 R</td>
			<td>Fisher Scientific</td>
			<td>05-414-051</td>
			<td>Benchtop centrifuge with cooling</td>
		</tr>
		<tr>
			<td>Ethyl alcohol, Pure</td>
			<td>Millipore Sigma</td>
			<td>459844</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid (EDTA)</td>
			<td>Millipore Sigma</td>
			<td>E9884</td>
			<td>Dissolve in H2O to 0.5 M</td>
		</tr>
		<tr>
			<td>Human histone octamer (H4, L50C; H2A, K119C)&#160;</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Recombinant histone proteins and those harboring labeling mutations were purified in-house as described previously (see refs. 33, 34, 35, 36). Briefly, recombinant histones were expressed in BL21 (De3) pLySS cells (Promega). Inclusion bodies were isolated after sonication, and histones were extracted under denaturing conditions. Histones were dialyzed into buffer A (7 M urea, 10 mM tris hydrochloride pH 8.0, 100 mM sodium chloride, 1 mM EDTA, and 5 mM 2-mercaptoethanol), and the solution was added to a gravity column loaded with Q Sepharose Fast Flow (Cytiva). The flow through was then added to a gravity column loaded SP Sepharose Fast Flow (Cytiva), and the histones were eluted from the column by adding buffer A supplemented with 600 mM sodium chloride. Histones harboring labeling mutations (H4, L50C; H2A, K119C) were purified and then conjugated to the desired fluorophore via maleimide-functionalized dyes (Cytiva, Lumidyne) using a 20:1 dye-to-protein molar ratio (see refs. 33, 34). Histone octamers were assembled by adding an equal molar ratio of each wild-type or fluorophore-labeled histone under denaturing conditions, dialyzed into a high-salt buffer containing 2 M sodium chloride, and then purified by size exclusion chromatography as described previously (see refs. 36, 37). Alternatively, individual histone proteins and ready-made histone octamers can be purchased commercially (e.g., Epicypher).&#160;</td>
		</tr>
		<tr>
			<td>Image buffer</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>20 mM tris hydrochloride pH 8.0, 100 mM sodium chloride</td>
		</tr>
		<tr>
			<td>Klenow Fragment (3&#39; to 5&#39; exo-)</td>
			<td>New England Biolabs</td>
			<td>M0212S</td>
			<td></td>
		</tr>
		<tr>
			<td>Lambda DNA (dam-, dcm-)</td>
			<td>Thermo Fisher Scientific</td>
			<td>SD0021</td>
			<td>Methylation-free &#955; DNA</td>
		</tr>
		<tr>
			<td>LD655-MAL</td>
			<td>Lumidyne Technologies</td>
			<td>9</td>
			<td>Maleimide functionalized LD655 fluorophore</td>
		</tr>
		<tr>
			<td>Linker histone H1.4 (A4C)</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Purified recombinant protein made in-house (see ref. 38)</td>
		</tr>
		<tr>
			<td>LUMICKS C-Trap Dymo</td>
			<td>LUMICKS</td>
			<td>N/A</td>
			<td>Dual-trap configuration; standard materials for instrument provided by manufacturer</td>
		</tr>
		<tr>
			<td>Magnesium acetate solution</td>
			<td>Millipore Sigma</td>
			<td>63052-100ML</td>
			<td>Use to make HR buffer</td>
		</tr>
		<tr>
			<td>NEBuffer 2</td>
			<td>New England Biolabs</td>
			<td>B7002S</td>
			<td>Included with Klenow Fragment kit</td>
		</tr>
		<tr>
			<td>Pluronic F-127</td>
			<td>Millipore Sigma</td>
			<td>P2443-250G</td>
			<td>Dissolve in H2O and run through 0.22 um filter</td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>Millipore Sigma</td>
			<td>P3911</td>
			<td>Use to make PBS and HR buffer</td>
		</tr>
		<tr>
			<td>Potassium phosphate monobasic</td>
			<td>Millipore Sigma</td>
			<td>P0662</td>
			<td>Use to make PBS</td>
		</tr>
		<tr>
			<td>S. cerevisiae Nap1</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Nap1 was purified in-house as previously described (see refs. 33, 34). Alternatively, Nap1 can be purchased commercially (e.g., Active Motif).</td>
		</tr>
		<tr>
			<td>Sodium acetate</td>
			<td>Millipore Sigma</td>
			<td>241245</td>
			<td>Dissolve in H2O to 3 M</td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Millipore Sigma</td>
			<td>S9888</td>
			<td>Use to make PBS and image buffer</td>
		</tr>
		<tr>
			<td>Sodium phosphate dibasic</td>
			<td>Millipore Sigma</td>
			<td>S9763</td>
			<td>Use to make PBS</td>
		</tr>
		<tr>
			<td>SPHERO Biotin Coated Particles (3.0-3.4 &#181;m)</td>
			<td>Spherotech</td>
			<td>TP-30-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermo Scientific NanoDrop 2000/2000c Spectrophotometer</td>
			<td>Fisher Scientific</td>
			<td>ND2000</td>
			<td>NanoDrop Spectrophotometer</td>
		</tr>
		<tr>
			<td>Tris Base</td>
			<td>Fisher Scientific</td>
			<td>BP152-500</td>
			<td>Dissolve in H2O and adjust to appropriate pH; use to make image buffer and tris acetate</td>
		</tr>
	</tbody>
</table>

in situ nucleosome assembly for single-molecule correlative force and fluorescence microscopy

Nucleosomes constitute the primary unit of eukaryotic chromatin and have been the focus of numerous informative single-molecule investigations regarding their biophysical properties and interactions with chromatin-binding proteins. Nucleosome reconstitution on DNA for these studies typically involves a salt dialysis procedure that provides precise control over the placement and number of nucleosomes formed along a DNA tether. However, this protocol is time-consuming and requires a substantial amount of DNA and histone octamers as inputs. To offer an alternative strategy, an in situ nucleosome reconstitution method for single-molecule force and fluorescence microscopy that utilizes the histone chaperone Nap1 is described. This method enables users to assemble nucleosomes on any DNA template without the need for strong nucleosome positioning sequences, adjust nucleosome density on demand, and use fewer reagents. In situ nucleosome formation occurs within seconds, offering a simpler experimental workflow and a convenient transition into single-molecule measurements. Examples of two downstream assays for probing nucleosome mechanics and visualizing the behavior of individual proteins on chromatin are further described.

Using the setup described in step 3 (Figure 1A), nucleosome formation along the DNA tether was visualized as the appearance of red fluorescent foci on a 2D scan (Figures 1B, left) or trajectories over time on a kymograph (Figure 1B, right). Properly wrapped nucleosomes yielded fluorescence trajectories that were stationary over time within the diffraction limit of confocal detection (~300 nm). Notably, multiple nucleosomes formed ne...

Author Spotlight: Efficient Nucleosome Reconstitution for Single-Molecule Techniques

This article presents a detailed experimental procedure for reconstituting nucleosome-containing DNA tethers for single-molecule correlative force and fluorescence microscopy. It further describes several downstream experiments that can be conducted to visualize the binding behavior of chromatin-interacting proteins and analyze changes in the physical properties of nucleosomes.

In Situ Nucleosome Assembly for Single-Molecule Correlative Force and Fluorescence Microscopy

Nucleosomes constitute the primary unit of eukaryotic chromatin and have been the focus of numerous informative single-molecule investigations regarding ...

in-situ-nucleosome-assembly-for-single-molecule-correlative-force

Research

JoVE Journal

Biochemistry

918 Views.  The Rockefeller University. This article presents a detailed experimental procedure for reconstituting nucleosome-containing DNA tethers for single-molecule correlative force and fluorescence microscopy. It further describes several downstream experiments that can be conducted to visualize the binding behavior of chromatin-interacting proteins and analyze changes in the physical properties of nucleosomes.

Video: Author Spotlight: Efficient Nucleosome Reconstitution for Single-Molecule Techniques

Single-molecule Manipulation of G-quadruplexes by Magnetic Tweezers

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

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

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

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 ...

Imaging of Extracellular Vesicles by Atomic Force Microscopy

Exosomes and other extracellular vesicles (EVs) are molecular complexes consisting of a lipid membrane vesicle, its surface decoration by membrane proteins and other molecules, and diverse luminal content inherited from a parent cell, which includes RNAs, proteins, and DNAs. The characterization of the hydrodynamic sizes of EVs, which depends on the size of the vesicle and its coronal layer formed by surface decorations, has become routine. For exosomes, the smallest of EVs, the relative difference between the hydrodynamic and vesicles sizes is significant. The characterization of vesicles sizes by the cryogenic transmission electron microscopy (cryo-TEM) imaging, a gold standard technique, remains a challenge due to the cost of the instrument, the expertise required to perform the sample preparation, imaging and data analysis, and a small number of particles often observed in images. A widely available and accessible alternative is the atomic force microscopy (AFM), which can produce versatile data on three-dimensional geometry, size, and other biophysical properties of extracellular vesicles. The developed protocol guides the users in utilizing this analytical tool and outlines the workflow for the analysis of EVs by the AFM, which includes the sample preparation for imaging EVs in hydrated or desiccated form, the electrostatic immobilization of vesicles on a substrate, data acquisition, its analysis, and interpretation. The representative results demonstrate that the fixation of EVs on the modified mica surface is predictable, customizable, and allows the user to obtain sizing results for a large number of vesicles. The vesicle sizing based on the AFM data was found to be consistent with the cryo-TEM imaging.&#160;

A step-by-step procedure is described for label-free immobilization of exosomes and extracellular vesicles from liquid samples and their imaging by atomic force microscopy (AFM). The AFM images are used to estimate the size of the vesicles in the solution and characterize other biophysical properties.&#160;

Exosomes and other extracellular vesicles (EVs) are molecular complexes consisting of a lipid membrane vesicle, its surface decoration by membrane ...

Single-Molecule Tracking Microscopy - A Tool for Determining the Diffusive States of Cytosolic Molecules

Single-molecule localization microscopy probes the position and motions of individual molecules in living cells with tens of nanometer spatial and millisecond temporal resolution. These capabilities make single-molecule localization microscopy ideally suited to study molecular level biological functions in physiologically relevant environments. Here, we demonstrate an integrated protocol for both acquisition and processing/analysis of single-molecule tracking data to extract the different diffusive states a protein of interest may exhibit. This information can be used to quantify molecular complex formation in living cells. We provide a detailed description of a camera-based 3D single-molecule localization experiment, as well as the subsequent data processing steps that yield the trajectories of individual molecules. These trajectories are then analyzed using a numerical analysis framework to extract the prevalent diffusive states of the fluorescently labeled molecules and the relative abundance of these states. The analysis framework is based on stochastic simulations of intracellular Brownian diffusion trajectories that are spatially confined by an arbitrary cell geometry. Based on the simulated trajectories, raw single-molecule images are generated and analyzed in the same way as experimental images. In this way, experimental precision and accuracy limitations, which are difficult to calibrate experimentally, are explicitly incorporated into the analysis workflow. The diffusion coefficient and relative population fractions of the prevalent diffusive states are determined by fitting the distributions of experimental values using linear combinations of simulated distributions. We demonstrate the utility of our protocol by resolving the diffusive states of a protein that exhibits different diffusive states upon forming homo- and hetero-oligomeric complexes in the cytosol of a bacterial pathogen.

3D single-molecule localization microscopy is utilized to probe the spatial positions and motion trajectories of fluorescently labeled proteins in living bacterial cells. The experimental and data analysis protocol described herein determines the prevalent diffusive behaviors of cytosolic proteins based on pooled single-molecule trajectories.

Single-molecule localization microscopy probes the position and motions of individual molecules in living cells with tens of nanometer spatial and ...

Production of Dynein and Kinesin Motor Ensembles on DNA Origami Nanostructures for Single Molecule Observation

Cytoskeletal motors are responsible for a wide variety of functions in eukaryotic cells, including mitosis, cargo transport, cellular motility, and others. Many of these functions require motors to operate in ensembles. Despite a wealth of knowledge about the mechanisms of individual cytoskeletal motors, comparatively less is known about the mechanisms and emergent behaviors of motor ensembles, examples of which include changes to ensemble processivity and velocity with changing motor number, location, and configuration. Structural DNA nanotechnology, and the specific technique of DNA origami, enables the molecular construction of well-defined architectures of motor ensembles. The shape of cargo structures as well as the type, number and placement of motors on the structure can all be controlled. Here, we provide detailed protocols for producing these ensembles and observing them using total internal reflection fluorescence microscopy. Although these techniques have been specifically applied for cytoskeletal motors, the methods are generalizable to other proteins that assemble in complexes to accomplish their tasks. Overall, the DNA origami method for creating well-defined ensembles of motor proteins provides a powerful tool for dissecting the mechanisms that lead to emergent motile behavior.

The goal of this protocol is to form ensembles of molecular motors on DNA origami nanostructures and observe the ensemble motility using total internal reflection fluorescence microscopy.

Cytoskeletal motors are responsible for a wide variety of functions in eukaryotic cells, including mitosis, cargo transport, cellular motility, and ...

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 ...

Conventional BODIPY Conjugates for Live-Cell Super-Resolution Microscopy and Single-Molecule Tracking

Single molecule localization microscopy (SMLM) techniques overcome the optical diffraction limit of conventional fluorescence microscopy and can resolve intracellular structures and the dynamics of biomolecules with ~20 nm precision. A prerequisite for SMLM are fluorophores that transition from a dark to a fluorescent state in order to avoid spatio-temporal overlap of their point spread functions in each of the thousands of data acquisition frames. BODIPYs are well-established dyes with numerous conjugates used in conventional microscopy. The transient formation of red-shifted BODIPY ground-state dimers (DII) results in bright single molecule emission enabling single molecule localization microscopy (SMLM). Here we present a simple but versatile protocol for SMLM with conventional BODIPY conjugates in living yeast and mammalian cells. This procedure can be used to acquire super-resolution images and to track single BODIPY-DII states to extract spatio-temporal information of BODIPY conjugates. We apply this procedure to resolve lipid droplets (LDs), fatty acids, and lysosomes in living yeast and mammalian cells at the nanoscopic length scale. Furthermore, we demonstrate the multi-color imaging capability with BODIPY dyes when used in conjunction with other fluorescent&#160;probes. Our representative results show the differential spatial distribution and mobility of BODIPY-fatty acids and neutral lipids in yeast under fed and fasted conditions. This optimized protocol for SMLM can be used with hundreds of commercially available BODIPY conjugates and is a useful resource to study biological processes at the nanoscale far beyond the applications of this work.&#160;

Conventional BODIPY conjugates can be used for live-cell single-molecule localization microscopy (SMLM) through exploitation of their transiently forming, red-shifted ground state dimers. We present an optimized SMLM protocol to track and resolve subcellular neutral lipids and fatty acids in living mammalian and yeast cells at the nanoscopic length scale.

Single molecule localization microscopy (SMLM) techniques overcome the optical diffraction limit of conventional fluorescence microscopy and can resolve ...

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 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 ...