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-f...

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 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.

Author Spotlight: Efficient Nucleosome Reconstitution for Single-Molecule Techniques

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

Single molecule techniques are powerful tools to study the mechanics, coordination, and composition of chromatin systems. And therefore, researchers are always looking for better methods to generate nucleosome substrates. Here we describe a protocol to form nucleosomes across DNA in C2 in a single molecule correlative force and fluorescence microscope.Typically, nucleosome substrates are made by assembling nucleosomes on DNA containing strong positioning sequences via salt dialysis. Although this has advantages, it generates artificially stable nucleosomes and is heavy handed with reagents. Our protocol prepares nucleosome substrates for single molecule correlated force and fluorescence microscopy without specific DNA sequences and with much less reagents all within minutes.This protocol enables nucleosome assembly on native DNA sequences, easy adjustment of nucleosome density, as well as less preparation time and use of reagents. Additionally, the formation of nucleosomal DNA tethers in C2 enable simpler experimental workflow and the convenience of built-in single molecule visualization and manipulation. When uniform and specific nucleosome positioning is not an essential part of the experiment, our findings can help scientists study chromatin and its mining proteins at the single molecule level more efficiently.With the time and resources saved, more experiments can be done to further investigate additional variables and conditions. Applicable research areas that we're currently thinking about include chromatin mechanics that are regulated by histone variants and other post-translational modifications. We're also thinking about the biophysical engagements and kinetics of other chromatin binding proteins.And finally, we're also thinking about the nucleosome-driven higher order assembly processes such as biomolecular condensation.

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...

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.

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

Nucleosome Reconstitution for Single-Molecule Chromatin Studies

To begin, add 500 microliters of protein-free image buffer to channel 3 of the flow cell. Mix two nanomolar of Saccharomyces cerevisiae NAP1 and two to five nanomolar of LD655-labeled H4 histone octamer with 500 microliters of 1X HR buffer. Add this mixture to channel 4 of the flow cell.Flow all the channels at 1.0 bar for 30 seconds to flush them with samples. Set the flow to 0.2 bar, and move the optical traps to channel 1 to catch a suitable pair of streptavidin-coated beads. Next, move the optical traps to channel 3.Turn off the flow, and conduct a forced calibration for the bead pair. Turn on the red confocal laser, and calibrate the imaging area. After setting the flow to 0.2 bar, move the optical traps to channel 2 to catch biotinylated DNA.Then move the optical traps to channel 3, and stop the flow. Increase the distance between the optical traps to stretch the DNA, and monitor the associated forced distance curve to confirm the presence of a single DNA tether. Adjust the distance between the optical traps so that the DNA tension reads approximately one piconewton.Turn on the line scanning to start collecting a kymograph with the red laser on. Then move the optical traps to channel 4 with the flow off to form nucleosomes along the DNA tether. For unwrapping nucleosomes, move the optical traps to channel 3.Setting the force reading to 0 and speed to 0.1 micrometers per second, move the optical trap 1 away from the optical trap 2. Mix 10 nanomolar of Cy3-labeled linker histone H1.4 to 500 microliters of image buffer and add to channel 5. After forming a DNA tether containing nucleosomes, turn on the green laser, in addition to the red laser, and move optical traps to channel 5 for realtime imaging of H1 binding to chromatin.Nucleosome formation along the DNA tether was visualized as stationary red fluorescent foci on a 2D scan and kymograph. Photobleaching analysis showed one-step or two-step photo bleaching events, indicating the presence of mononucleosomes. In the absence of NAP1, histone octamers bound DNA, but remained mostly unwrapped, indicated by the constant tension.Using Cy3-labeled H2A, similar nucleosome assembly results were obtained, as with LD655-labeled H4.Nucleosome unwrapping increased tether distance over time, visible on the kymograph, and generated forced distance curves with characteristic rips that denote the unwrapping of individual nucleosomes. Linker histone H1 binding to chromatin was indicated by green fluorescent foci with dual color stationary trajectories representing the binding of H1 to nucleosomes, and green jagged trajectories representing diffusing H1 trajectories.

533 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.

In Situ Nucleosome Reconstitution for Single-Molecule Studies

Summary