Financial support from the Swiss National Science Foundation [200020_192153 to RKOS], the UZH Forschungskredit [FK-20-081 to EA], the UZH Stiftung f&#252;r wissenschaftliche Forschung [to RKOS and SZP], Graduate Research Campus (GRC) Short Grant [2024__SG_092 to EA], the Graduate School of Chemical and Molecular Sciences Zurich (CMSZH) and the University of Zurich is gratefully acknowledged.

Dual-Color End-Labeling and Vesicle Encapsulation of RNAs for smFRET-TIRF Studies

<ol>
	<li>Eitan, 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=FRET-based+dynamic+structural+biology:+Challenges,+perspectives+and+an+appeal+for+open-science+practices.">FRET-based dynamic structural biology: Challenges, perspectives and an appeal for open-science practices.</a> Elife. 10, e60416 (2021).</li><li>Taekjip, 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=Fluorescence+resonance+energy+transfer+at+the+single-molecule+level.">Fluorescence resonance energy transfer at the single-molecule level.</a> Nat Rev Methods Primers. 4, 22 (2024).</li><li>Ahunbay, E., Steffen, F. D., Zelger-Paulus, S., Sigel, R. K. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+dual+end-labeling+of+large+ribozymes.">Chemical dual end-labeling of large ribozymes.</a> Methods Mol Biol. 2439, 191-204 (2022).</li><li>Ahunbay, E., Zelger-Paulus, S., Sigel, R. K. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Group+II+Introns:+Highly+structured+yet+dynamic.">Group II Introns: Highly structured yet dynamic.</a> Chimia (Aarau). 77 (4), 235-241 (2023).</li><li>Steiner, M., Karunatilaka, K. S., Sigel, R. K. O., Rueda, D. <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+studies+of+group+II+intron+ribozymes.">Single-molecule studies of group II intron ribozymes.</a> Proc Natl Acad Sci USA. 105 (37), 13853-13858 (2008).</li><li>Karunatilaka, K. S., Solem, A., Pyle, A. M., Rueda, D. <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+analysis+of+Mss116-mediated+group+II+intron+folding.">Single-molecule analysis of Mss116-mediated group II intron folding.</a> Nature. 467 (7318), 935-939 (2010).</li><li>Paudel, B. P., Fiorini, E., B&#246;rner, R., Sigel, R. K. O., Rueda, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimal+molecular+crowding+accelerates+group+II+intron+folding+and+maximizes+catalysis.">Optimal molecular crowding accelerates group II intron folding and maximizes catalysis.</a> Proc Natl Acad Sci USA. 115 (47), 11917-11922 (2018).</li><li>Saha, R., Verbanic, S., Chen, I. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipid+vesicles+chaperone+an+encapsulated+RNA+aptamer.">Lipid vesicles chaperone an encapsulated RNA aptamer.</a> Nat Commun. 9 (1), 2313 (2018).</li><li>Boukobza, E., Sonnenfeld, A., Haran, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immobilization+in+surface-tethered+lipid+vesicles+as+a+new+tool+for+single+biomolecule+spectroscopy.">Immobilization in surface-tethered lipid vesicles as a new tool for single biomolecule spectroscopy.</a> J Phys Chem B. 105 (48), 12165-12170 (2001).</li><li>Cisse, I., Okumus, B., Joo, C., 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=Fueling+protein+DNA+interactions+inside+porous+nanocontainers.">Fueling protein DNA interactions inside porous nanocontainers.</a> Proc Natl Acad Sci USA. 104 (31), 12646-12650 (2007).</li><li>Zelger-Paulus, S., Hadzic, M. C. A. S., Sigel, R. K. O., B&#246;rner, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Encapsulation+of+fluorescently+labeled+RNAs+into+surface-tethered+vesicles+for+single-molecule+FRET+studies+in+TIRF+microscopy.">Encapsulation of fluorescently labeled RNAs into surface-tethered vesicles for single-molecule FRET studies in TIRF microscopy.</a> Methods Mol Biol. 2331, 1-16 (2020).</li><li>Okumus, B., Wilson, T. J., Lilley, D. M. J., 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=Vesicle+encapsulation+studies+reveal+that+single+molecule+ribozyme+heterogeneities+are+intrinsic.">Vesicle encapsulation studies reveal that single molecule ribozyme heterogeneities are intrinsic.</a> Biophys J. 87 (4), 2798-2806 (2004).</li><li>Ishitsuka, Y., Okumus, B., Arslan, S., Chen, K. H., 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=Temperature-independent+porous+nanocontainers+for+single-molecule+fluorescence+studies.">Temperature-independent porous nanocontainers for single-molecule fluorescence studies.</a> Anal Chem. 82 (23), 9694-9701 (2010).</li><li>Liu, B., Mazouchi, A., Gradinaru, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trapping+single+molecules+in+liposomes:+surface+interactions+and+freeze-thaw+effects.">Trapping single molecules in liposomes: surface interactions and freeze-thaw effects.</a> J Phys Chem B. 114 (46), 15191-15198 (2010).</li><li>Chandradoss, S. 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=Surface+passivation+for+single-molecule+protein+studies.">Surface passivation for single-molecule protein studies.</a> J Vis Exp. (86), e50549 (2014).</li><li>Paul, T., Myong, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protocol+for+generation+and+regeneration+of+PEG-passivated+slides+for+single-molecule+measurements.">Protocol for generation and regeneration of PEG-passivated slides for single-molecule measurements.</a> STAR Protoc. 3 (1), 101152 (2022).</li><li>Hadzic, M. C. A. S., Kowerko, D., B&#246;rner, R., Zelger-Paulus, S., Sigel, R. K. O., Farkas, D. L., Nicolau, D. V., Leif, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detailed+analysis+of+complex+single+molecule+FRET+data+with+the+software+MASH.">Detailed analysis of complex single molecule FRET data with the software MASH.</a> Imaging, Manipulation, and Analysis of Biomolecules, Cells, and Tissues IX. SPIE 971119, (2016).</li><li>Chu, B. C., Wahl, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Le+Orgel.+Derivatization+of+unprotected+polynucleotides.">Le Orgel. Derivatization of unprotected polynucleotides.</a> Nucleic Acids Res. 11 (18), 6513-6529 (1983).</li><li>Qin, P. Z., Pyle, 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=Site-specific+labeling+of+RNA+with+fluorophores+and+other+structural+probes.">Site-specific labeling of RNA with fluorophores and other structural probes.</a> Methods. 18 (1), 60-70 (1999).</li><li>Whitfeld, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+the+determination+of+nucleotide+sequence+in+polyribonucleotides.">A method for the determination of nucleotide sequence in polyribonucleotides.</a> Biochem J. 58 (3), 390-396 (1954).</li><li>Zamecnik, P. C., Stephenson, M. L., Scott, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Partial+purification+of+soluble+RNA.">Partial purification of soluble RNA.</a> Proc Natl Acad Sci USA. 46 (6), 811-822 (1960).</li><li>Proudnikov, D., Mirzabekov, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+methods+of+DNA+and+RNA+fluorescent+labeling.">Chemical methods of DNA and RNA fluorescent labeling.</a> Nucleic Acids Res. 24 (22), 4535-4542 (1996).</li><li>Wang, P. Y., Sexton, A. N., Culligan, W. J., Simon, 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=Carbodiimide+reagents+for+the+chemical+probing+of+RNA+structure+in+cells.">Carbodiimide reagents for the chemical probing of RNA structure in cells.</a> RNA. 25 (1), 135-146 (2019).</li><li>Mitchell, 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=In+vivo+RNA+structural+probing+of+uracil+and+guanine+base-pairing+by+1-ethyl-3-(3-dimethylaminopropyl)carbodiimide+(EDC).">In vivo RNA structural probing of uracil and guanine base-pairing by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC).</a> RNA. 25 (1), 147-157 (2019).</li><li>Rinaldi, A. J., Suddala, K. C., Walter, N. 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The authors declare no conflicts of interest.

FRET at the single-molecule level is unique because it allows the observation and analysis of individual molecules, revealing sample heterogeneity and capturing transient states that can be obscured in ensemble measurements1,2. Observing individual RNA molecules using smFRET provides high-resolution insights into their folding pathways and dynamics. This protocol describes the chemical dual-end labeling of RNA and its surface immobilization via phospholipid vesicle encapsulation, which together enable following dynamic conformational changes via smFRET-TIRF microscopy.
Studying RNA dynamics is a constantly growing field with the need for new site-specific fluorescent labeling strategies. We label the RNA ends by targeting the 5&#39;-phosphate with carbodiimides and the 3&#39;-ribose sugar with periodate. These approaches have been described earlier (5&#39;-end18,19 and 3&#39;-end19,20,21,22) but have not previously been applied to an RNA of similar size to the wild-type Sc.ai5&#947; group II intron ribozyme, which required optimization. Carbodiimide (e.g., EDC) activation of the 5&#39;-phosphate is reversible. Therefore, imidazole was used to irreversibly react with the O-acylisourea intermediate to form a highly reactive phosphorimidazolide19,20. However, it is now known that at higher pH, carbodiimides can modify nucleobases, specifically guanines and uracils, which has recently led to their use as structural probing agents23,24.
To avoid cross-reactivity, purifying the activated RNA from the EDC prior to raising the pH to 7.5 for the dye coupling step is crucial. However, when we introduced a purification step between the activation and the dye attachment25, we obtained very low yields. Analogously, in protein labeling, surface-accessible lysine residues can be activated with carbodiimides. However, instead of imidazole, which prevents the activation reversal, NHS is routinely used. We adopted this strategy too, thus replacing the phosphorimidazolide intermediate with an NHS-phosphate intermediate. This way we achieved pH control as well as an increased labeling density at lower temperatures and shorter incubation times, i.e., 25 &#176;C for 4 h compared to 37 &#176;C for 16 h. Developed for RNA, this 5&#39;-labeling strategy can be applied to any other single-stranded nucleic acid with a 5&#39;-phosphate.
The 5&#39;-phosphate activation and 3&#39;-ribose oxidation were mutually exclusive, because the chemistries are not orthogonal. To overcome this challenge and avoid cross-labeling, we started with the 5&#39;-end, followed by a blocking step to inhibit the activated but not labeled sites before proceeding with the 3&#39;-end labeling. While oxidizing the 3&#39;-diol, excess sodium meta-periodate (NaIO4) could quench the already attached fluorophore at the 5&#39;-end. Therefore, we reduced the concentration of NaIO4 used for single labeling from 20 mM to 10 mM.
We recommend working with multiple aliquots in parallel instead of scaling up the reactions. This protocol requires multiple ethanol (EtOH) precipitation steps. When working with several aliquots in parallel, prepare a precipitation mixture (30 mL of 100% absolute EtOH and 1 mL of 3 M NaOAc, pH 5.2). NaCl is not used due to its low solubility in EtOH. Precipitate the RNA with 3.1 vol. of this mixture by overnight incubation at -20 &#176;C, followed by centrifugation. Wash the RNA pellet twice with 500 &#181;L ice-cold 70% EtOH, spin down at 4 &#176;C after each time, and dry under vacuum. EtOH precipitation takes advantage of the RNA&#39;s insolubility and the free dyes&#39; solubility in 70% ethanol. Centrifugal filtration effectively removes free dyes due to their significant size difference from RNA and facilitates buffer exchange, eliminating salts. In addition to EtOH precipitation and centrifugal filtration methods, free dyes can also be removed using gel extraction and/or chromatography techniques (e.g., HPLC); however, the scale should be adapted accordingly. Do not vortex long RNAs to resuspend, as this may cause mechanical shearing26. The optimal time to pause the protocol is when the RNA is pelleted. We use DNA low-binding tubes to improve nucleic acid recovery. Although the final reaction volume is 100 &#181;L, 1.5 mL (and not lower volume) tubes are preferred for better purification by EtOH precipitation.
Once the unreacted free dyes were removed by precipitation and centrifugal filtration, we confirmed the labeling by fluorescent gel electrophoresis (Figure 4A), UV-Vis spectroscopy, analytical HPLC3. However, it is important to note that these methods cannot distinguish between an RNA molecule carrying both fluorophores and a mixture of RNAs, each labeled with a single color. Similarly, they cannot be used to determine if an RNA molecule carries multiple fluorophores of the same color. Mass spectrometry cannot be used due to size limitations. Ensemble3 and single-molecule FRET spectroscopies corroborate dual-fluorescent labeling, as shown in Figures 4B and 5B. The stoichiometry of 0.5 (sCy3 to Cy5 ratio of 1:1) in smFRET experiments confirms the equal conjugation of the two fluorophores. One concern was the double labeling at the 3&#39;-end by labeling both aldehydes instead of the proposed cyclization. The lack of species with a stoichiometry of 0.25 (sCy3 to Cy5 ratio of 1:2) in smFRET experiments suggests that the dye attachment sterically hinders and prevents the attachment of a second dye.
With this dual-fluorescent labeling, FRET signal changes can be attributed to structural rearrangements throughout RNA folding and catalysis. To maintain fluorescently labeled RNA within the evanescent field for single-molecule TIRF imaging, encapsulation is preferred over direct surface tethering. This approach involves trapping individual RNA molecules within lipid bilayers of vesicles, creating a controlled environment conducive to observing their dynamic behavior. The described protocol enriches mono-encapsulation, as photobleaching in a single step demonstrates11. To understand RNA folding and function, bridging the in vitro and in vivo gap is essential27. Molecular crowding agents can mimic the conditions inside cells to enhance RNA catalysis by group II introns7,28. Alternatively, encapsulation creates constrained microenvironments that promote RNA folding29, bringing our understanding of RNA structure and dynamics closer to a realistic cellular context.

F&#246;rster Resonance Energy Transfer (FRET) is a powerful and sensitive technique for studying inter- and intramolecular interactions of biomolecules at the nanoscale. It is based on the non-radiative energy transfer from an excited donor molecule to a nearby acceptor molecule, which occurs over distances typically between 1 nm and 10 nm. The distance between the donor and acceptor dyes determines the efficiency of this energy transfer, making FRET an invaluable tool for studying molecular dynamics, conformational changes, and interactions in a wide range of biological systems1,2, including RNAs. Total Internal Reflection Fluorescence (TIRF) microscopy has proven to be a powerful technique for smFRET investigations, as it selectively illuminates molecules only near the surface, allowing FRET dynamics of individual molecules with high spatial and temporal resolution. However, before performing smFRET-TIRF experiments, the molecule of interest must first be fluorescently labeled with an appropriate FRET pair and then immobilized on the microscopy surface. The smFRET-TIRF protocol described here was validated using the wild-type group II intron Sc.ai5&#947; from Saccharomyces cerevisiae mitochondria, flanked by its two exonic sequences (915 nucleotides)3. For a more detailed view of fluorescent labeling group II introns and their immobilization for smFRET TIRF microscopy, refer to our review4.
An ideal site-specific RNA labeling strategy would allow for the precise incorporation of donor and acceptor dyes at predetermined positions without altering the RNA&#39;s structure or function, ensuring accurate and efficient FRET measurements. This is challenging due to the chemical similarities among the four nucleobases,&#160;which complicates selective labeling. End-labeling attaches donor and acceptor dyes to the RNA ends by targeting the 5&#39;-phosphate and the 3&#39;-ribose. This approach offers a minimally invasive approach while still providing valuable insights into structural dynamics and interactions. Group II intron&#39;s ability to self-splice in the presence of Mg2+ restricts the use of metal ion-dependent enzymes. Here, we present an approach to dual end-label long and catalytically active RNAs (ribozymes) that bypass the need for enzymes or the synthesis of specialized probes.
A common approach to tether RNA molecules to the surface for TIRF microscopy is to covalently link a biotin moiety directly to the RNA or to hybridize a biotin-carrying antisense oligonucleotide (ASO)5,6,7. However, this direct immobilization method can introduce artifacts due to RNA-surface interactions, potentially resulting in misfolded RNAs8. An elegant solution to mitigate these immobilization artifacts is to encapsulate RNA in surface-attached nanoscale phospholipid vesicles9,10,11. These vesicles, which are approximately 100 nm in diameter, are anchored to the surface through a biotin-streptavidin linkage12,13,14, allowing the RNA to diffuse freely inside while permitting the exchange of ions across the lipid membrane10. After covalently labeling a large functional RNA3, we present an approach to encapsulate such RNAs in phospholipid vesicles by combining established protocols for surface passivation and vesicle encapsulation, adapted to preserve RNA functionality10,11,14. This dual-end labeling and encapsulation approach achieves a high rate of mono-encapsulation of functional RNAs for smFRET TIRF microscopy.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>RNA labeling</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1.5&#160;mL DNA LoBind tubes&#160;</td>
			<td>Eppendorf&#160;</td>
			<td>30108035</td>
			<td></td>
		</tr>
		<tr>
			<td>1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC-HCl or EDC)</td>
			<td>Thermo Scientific</td>
			<td>11844071</td>
			<td>Pierce EDC, No-Weigh Format. Store at&#160; -20 &#176;C</td>
		</tr>
		<tr>
			<td>3-morpholinopropane-1-sulfonic acid (MOPS)</td>
			<td>Sigma-Aldrich</td>
			<td>69947</td>
			<td>BioUltra, for molecular biology, &#8805;99.5% (titration)</td>
		</tr>
		<tr>
			<td>Acetic acid (glacial)</td>
			<td>Sigma-Aldrich</td>
			<td>1.00063</td>
			<td>100%, anhydrous for analysis EMSURE ACS,ISO,Reag. Ph Eur</td>
		</tr>
		<tr>
			<td>Centrifugal filtration unit&#160;</td>
			<td>Sartorius</td>
			<td>VS0132</td>
			<td>Vivaspin 500, MWCO 50.000, PES, 500 &#956;L</td>
		</tr>
		<tr>
			<td>Cyanine5 hydrazide (Cy5-hydrazide)</td>
			<td>Lumiprobe</td>
			<td>23070</td>
			<td>5 mg, CAS number 1427705-31-4&#160;</td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>New England Biolabs</td>
			<td>12611P</td>
			<td>Molecular biology grade</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>VWR Chemicals</td>
			<td>20821.296P</td>
			<td>Absolute &#8805;99,8%</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sigma-Aldrich</td>
			<td>G5516</td>
			<td>for molecular biology, &#8805;99.0%</td>
		</tr>
		<tr>
			<td>Hydrochloric acid (HCl)</td>
			<td>Sigma-Aldrich</td>
			<td>1.00317</td>
			<td>fuming 37%, for analysis EMSURE ACS,ISO,Reag. Ph Eur</td>
		</tr>
		<tr>
			<td>N-hydroxysuccinimide (NHS)</td>
			<td>Sigma-Aldrich</td>
			<td>130672</td>
			<td>98%</td>
		</tr>
		<tr>
			<td>Sodium acetate (NaOAc)&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>S8750</td>
			<td>anhydrous, ReagentPlus, &#8805;99.0%</td>
		</tr>
		<tr>
			<td>Sodium meta-periodate (NaIO4)</td>
			<td>Thermo Fisher Scientific, Life Technologies</td>
			<td>20504</td>
			<td>Pierce product line</td>
		</tr>
		<tr>
			<td>Sulfo-Cyanine3 amine (sCy3-amine)</td>
			<td>Lumiprobe</td>
			<td>213C0</td>
			<td>5 mg, CAS number 2183440-43-7</td>
		</tr>
		<tr>
			<td>Tris(hydroxymethyl)aminomethane (Tris)</td>
			<td>Biosolve</td>
			<td>200923</td>
			<td>Molecular biology grade</td>
		</tr>
		<tr>
			<td>Chamber preparation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>3-aminopropyl)triethoxysilane (APTES)&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>440140</td>
			<td>99%</td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>Sigma-Aldrich</td>
			<td>1.00014</td>
			<td>for analysis EMSURE ACS,ISO,Reag. Ph Eur</td>
		</tr>
		<tr>
			<td>Biotin-Polyethylene glycol-Succinimidyl valerate (biotin-PEG-SVA, bPEG) and Methoxy polyethylene glycol-Succinimidyl valerate (mPEG-SVA)</td>
			<td>Laysan Bio, Inc.</td>
			<td></td>
			<td>BIO-PEG-SVA, MW 5,000 and MPEG-SVA, MW 5,000 - Combo Kit</td>
		</tr>
		<tr>
			<td>Coverslips</td>
			<td>Carl Roth</td>
			<td>H876.2</td>
			<td>24 x 24 mm, 0.13-0.16&#160;mm thickness</td>
		</tr>
		<tr>
			<td>Deconex 11 universal</td>
			<td>Borer Chemie AG</td>
			<td>17416492</td>
			<td>Laboratory glassware cleaning solution</td>
		</tr>
		<tr>
			<td>Diamond coated core drill bit</td>
			<td>Crystalite corporation</td>
			<td></td>
			<td>1 mm thickness</td>
		</tr>
		<tr>
			<td>Diamond driller</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>VWR Chemicals</td>
			<td>20821.296P</td>
			<td>Absolute &#8805;99,8%</td>
		</tr>
		<tr>
			<td>Glass Coplin staining jar with cover</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Imaging spacer</td>
			<td>Grace Bio-Labs</td>
			<td>654008</td>
			<td>SecureSeal, 8 wells,&#160; 9&#160;mm &#215; 0.12&#160;mm</td>
		</tr>
		<tr>
			<td>Oxygen plasma cleaner</td>
			<td>Zepto One</td>
			<td></td>
			<td>Diener</td>
		</tr>
		<tr>
			<td>Potassium hydroxide (KOH)</td>
			<td>Sigma-Aldrich</td>
			<td>P9541</td>
			<td>for molecular biology, &#8805;99.0%</td>
		</tr>
		<tr>
			<td>Quartz slides</td>
			<td>G. Finkenbeiner, Inc.</td>
			<td></td>
			<td>7.5&#160;x&#160;2.5&#160;cm, 1 mm thickness</td>
		</tr>
		<tr>
			<td>Sodium bicarbonate (NaHCO3)&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>S6297</td>
			<td>BioXtra, 99.5-100.5%</td>
		</tr>
		<tr>
			<td>Lipid cake preparation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>16:0 Biotinyl Cap PE, bPE (1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(cap biotinyl) (sodium salt), C53H98N4O11PNaS)</td>
			<td>Avanti Polar Lipids</td>
			<td>870277P</td>
			<td>Stable for 1 year at -20 &#176;C</td>
		</tr>
		<tr>
			<td>14:0 PC, DMPC (1,2-ditetradecanoyl-sn-glycero-3-phosphocholine or 1,2-dimyristoyl-sn-glycero-3-phosphocholine, C36H72NO8P)</td>
			<td>Avanti Polar Lipids</td>
			<td>850345P</td>
			<td></td>
		</tr>
		<tr>
			<td>2.0 mL microcentrifuge tubes, Safe-Lock</td>
			<td>Eppendorf</td>
			<td>0030120094</td>
			<td>Autoclave to sterilize</td>
		</tr>
		<tr>
			<td>500&#160;mL Schlenk flask</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Merck</td>
			<td>1.02445</td>
			<td>for analysis EMSURE ACS, ISO, Reag. Ph Eur</td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Surface immobilization via encapsulation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>(&#177;)-6-Hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox&#174;)</td>
			<td>Thermo Fischer</td>
			<td>53188-07-1</td>
			<td>97%</td>
		</tr>
		<tr>
			<td>3-morpholinopropane-1-sulfonic acid (MOPS)</td>
			<td>Sigma-Aldrich</td>
			<td>69947</td>
			<td>BioUltra, for molecular biology, &#8805;99.5% (titration). Store at +4 &#176;C in the dark.</td>
		</tr>
		<tr>
			<td>Adhesive seal tabs</td>
			<td>Grace Bio-Labs&#160;</td>
			<td>629200</td>
			<td></td>
		</tr>
		<tr>
			<td>Catalase</td>
			<td>Sigma-Aldrich</td>
			<td>9001-05-2, C30</td>
			<td>from bovine liver, aqueous suspension, 10,000-40,000 units/mg protein</td>
		</tr>
		<tr>
			<td>D-glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G7528</td>
			<td>&#8805;99.5% (GC), BioXtra</td>
		</tr>
		<tr>
			<td>Extruder polycarbonate (PC) membrane</td>
			<td>Avanti Polar Lipids</td>
			<td>610005-1EA</td>
			<td>0.1 &#956;m, 19 mm</td>
		</tr>
		<tr>
			<td>Extruder set with heating block</td>
			<td>Avanti Polar Lipids</td>
			<td>610000</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose oxidase</td>
			<td>Sigma-Aldrich</td>
			<td>9001-37-0</td>
			<td>from Aspergillus niger, Type VII,&#160; lyophilized powder, &#8805;100,000 units/g solid (without added oxygen)</td>
		</tr>
		<tr>
			<td>Magnesium chloride (MgCl2)</td>
			<td>Sigma-Aldrich</td>
			<td>7786-30-3, M1028</td>
			<td>for molecular biology, 1.00 M &#177; 0.01 M solution</td>
		</tr>
		<tr>
			<td>Polyester (PE) drain disc, membrane</td>
			<td>Whatman, Cytiva</td>
			<td>230300</td>
			<td>10 mm</td>
		</tr>
		<tr>
			<td>Potassium chloride (KCl)</td>
			<td>Sigma-Aldrich</td>
			<td>60128</td>
			<td>BioUltra, for molecular biology, &#8805;99.5% (AT)</td>
		</tr>
		<tr>
			<td>Potassium hydroxide (KOH)</td>
			<td>Sigma-Aldrich</td>
			<td>P9541</td>
			<td>for molecular biology, &#8805;99.0%</td>
		</tr>
		<tr>
			<td>Streptavidin</td>
			<td>Thermo Fischer</td>
			<td>434301</td>
			<td></td>
		</tr>
	</tbody>
</table>

fluorescent end-labeling and encapsulation of long rnas for single-molecule fret-tirf microscopy

Single-molecule F&#246;rster Resonance Energy Transfer (smFRET) excels in studying dynamic biomolecules by allowing precise observation of their conformational changes over time. To monitor RNA dynamics with smFRET, we developed a method to covalently label RNAs at their termini with a FRET pair of fluorophores. This direct end-labeling strategy targets the 5&#39;-phosphate by carbodiimide (EDC)/N-hydroxysuccinimide (NHS) activation and the 3&#39;-ribose by periodate oxidation, which can be adapted to other RNAs regardless of their size and sequence to study them independently of artificial modifications. Furthermore, the 5&#39;-EDC/NHS activation is of general interest to all nucleic acids with a 5&#39;-phosphate. The use of commercially available chemicals eliminates the need to synthesize RNA-specific probes.
Total Internal Reflection Fluorescence (TIRF) microscopy requires the surface-immobilized molecules of interest to be within the evanescent field to be illuminated. A sophisticated way of keeping the RNA molecules within the evanescent field is to encapsulate them in phospholipid vesicles. Encapsulation benefits from the best of both worlds, tethering the molecule to the surface while enabling free diffusion of the molecule. We ensure that each vesicle contains only a single RNA molecule, enabling single-molecule imaging. Upon dual-end labeling and encapsulation of the RNA of interest, smFRET measurements offer a dynamic and detailed view of RNA behavior.

We present the site-specific single- and dual-fluorescent labeling of the 915-nt RNA of interest, the yeast mitochondrial Sc.ai5&#947; group II intron, flanked by exonic sequences. The FRET fluorophore pair is positioned at the RNA ends via EDC/NHS activation of the 5&#39;-phosphate and periodate oxidation of the 3&#39;-ribose, followed by respective dye attachments. We then verified the RNA-dye conjugates by fluorescent gel electrophoresis, as presented in Figure 4A. The co-migration of RNA and the fluorophores on the agarose gel confirms the successful labeling. Next, as shown in Figure 4B, ensemble fluorescence spectroscopy was used to characterize the dual-labeled group II intron. Energy transfer, that is, FRET, was observed upon excitation of the donor dye proving the dual labeling of the RNA. Notably, consistent with the distance-dependent nature of FRET, the folding of the group II intron RNA in the presence of metal ions led to an increase in FRET efficiency, as evidenced by the decrease in donor emission (green arrow) and a corresponding increase in acceptor (red arrow) emission. This indicates that this FRET labeling tracks the conformational changes of the ribozyme.
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/67391/67391fig04.jpg" /> 
Figure 4: Characterization of the RNA-dye conjugates. (A) Analytical gel-based analysis of single- and dual-fluorescently labeled RNA shows the co-migration of the dyes with the RNA on a 2% agarose gel. Co-localization of the fluorophores in the dual-labeled sample is indicated by the yellow band in the merged image (bottom) of the Cy3 (top, green) and Cy5 (middle, red) channels, visualized under 532 nm and 635 nm illumination, respectively. Lane 1: 5&#39;-sCy3 only labeled RNA, lane 2: 3&#39;-Cy5 only labeled RNA, and lane 3: dual-end (5&#39;-sCy3 and 3&#39;-Cy5) labeled RNA. (B) Ensemble fluorescence spectroscopy confirms dual-labeling. Energy transfer upon donor excitation (&#955;ex = 515 nm, &#955;em = 670 nm) verifies that both dyes are successfully coupled to the RNA. The gray curve represents the emission profile of the pre-catalytic RNA, while the black curve demonstrates increased FRET efficiency in the folded group II intron RNA (incubated with 500 mM KCl at 70 &#176;C for 3 min, cooled to 42 &#176;C for 5 min, followed by the addition of 100 mM MgCl2). This figure was adapted from Ahunbay et al.3 <a href="https://www.jove.com/files/ftp_upload/67391/67391fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
With the fluorescently dual-labeled wild-type group II intron in hand, we are now positioned to explore its dynamics at the single-molecule level. Once encapsulated in phospholipid vesicles, the labeled RNA is immobilized on a microscopy surface at very low surface densities to achieve single-molecule resolution for smFRET-TIRF. As seen in Figure 5A, several individual molecules can be tracked simultaneously. TIRF microscopy enables the real-time monitoring of FRET efficiency and its changes over time. Figure 5B exemplifies the static and dynamic single-molecule FRET traces of the labeled and encapsulated RNA. A typical dynamic trace exhibits anticorrelation between donor and acceptor signals that fluctuate the FRET efficiency. When the acceptor emission upon donor excitation increases, the donor emission correspondingly decreases, indicating a dynamic change in the inter-dye distance. This anticorrelation suggests conformational changes in the RNA molecule.
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/67391/67391fig05.jpg" /> 
Figure 5: Highly dynamic behavior of group II intron RNA revealed by smFRET. The dual-end labeled group II intron RNA is encapsulated in a phospholipid vesicle and immobilized on the surface for imaging with an objective-based TIRF microscope. (A) Merged image of individual labeled RNA molecules exhibiting donor emission (sCy3, green) and acceptor emission (Cy5, red) upon 532 nm excitation. (B) The typical smFRET trajectories of (left) a static RNA molecule where the donor and acceptor intensities do not fluctuate over time and (right) a dynamic RNA molecule, where the donor and acceptor intensities anticorrelate, with FRET efficiencies in yellow. Single-molecules are localized and analyzed using MASH-FRET17. Direct excitation, bleed-through, and &#947;-factor corrections are applied. Abbreviations: smFRET = single-molecule F&#246;rster Resonance Energy Transfer. <a href="https://www.jove.com/files/ftp_upload/67391/67391fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Fluorescent End-Labeling and Encapsulation of Long RNAs for Single-Molecule FRET-TIRF Microscopy

This article describes the dual-color labeling of long RNAs at termini positions and their surface immobilization via encapsulation in phospholipid vesicles for single-molecule FRET TIRF microscopy applications. Combining these techniques enables precise visualization and analysis of RNA dynamics at the single-molecule level.

Single-molecule F&#246;rster Resonance Energy Transfer (smFRET) excels in studying dynamic biomolecules by allowing precise observation of their ...

fluorescent-end-labeling-encapsulation-long-rnas-for-single-molecule

Research

JoVE Journal

Biochemistry

419 Views.  University of Zurich. This article describes the dual-color labeling of long RNAs at termini positions and their surface immobilization via encapsulation in phospholipid vesicles for single-molecule FRET TIRF microscopy applications. Combining these techniques enables precise visualization and analysis of RNA dynamics at the single-molecule level.

Video: Fluorescent End-Labeling and Encapsulation of Long RNAs for Single-Molecule FRET-TIRF Microscopy

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 ...

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

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

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

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

Single-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 ...

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 Diffusion and Assembly on Polymer-Crowded Lipid Membranes

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

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

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

Use of Dual Optical Tweezers and Microfluidics for Single-Molecule Studies

Visual biochemistry is a powerful technique for observing the stochastic properties of single enzymes or enzyme complexes that are obscured in the averaging that takes place in bulk-phase studies. To achieve visualization, dual optical tweezers, where one trap is fixed and the other is mobile, are focused into one channel of a multi-stream microfluidic chamber positioned on the stage of an inverted fluorescence microscope. The optical tweezers trap single molecules of fluorescently labeled DNA and fluid flow through the chamber and past the trapped beads, stretches the DNA to B-form (under minimal force, i.e., 0 pN) with the nucleic acid being observed as a white string against a black background. DNA molecules are moved from one stream to the next, by translating the stage perpendicular to the flow to enable the initiation of reactions in a controlled manner. To achieve success, microfluidic devices with optically clear channels are mated to glass syringes held in place in a syringe pump. Optimal results use connectors permanently bonded to the flow cell, tubing that is mechanically rigid and chemically resistant, and which is connected to switching valves that eliminate bubbles that prohibit laminar flow.

Visual, single-molecule biochemistry studied through microfluidic chambers is greatly facilitated using glass barrel, gas-tight syringes, stable connections of tubing to flow cells, and elimination of bubbles by placing switching valves between the syringes and tubing. The protocol describes dual optical traps that enable visualization of DNA transactions and intermolecular interactions.

Visual biochemistry is a powerful technique for observing the stochastic properties of single enzymes or enzyme complexes that are obscured in the ...

In Situ Nucleosome Reconstitution for Single-Molecule Studies

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

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 ...

CMG-Mediated DNA Unwinding Observed by ssDNA-RPA Tract Growth

Single-Molecule Real-Time Visualization of DNA Unwinding by CMG Helicase

Faithful genome duplication is essential for preserving the genetic stability of dividing cells. DNA replication is carried out during the S phase by a dynamic complex of proteins termed the replisome. At the heart of the replisome is the&#160;CDC45-MCM2-7-GINS (CMG) helicase, which separates the two strands of the DNA double helix such that DNA polymerases can copy each strand. During genome duplication, replisomes must overcome a plethora of obstacles and challenges. Each of these threatens genome stability, as failure to replicate DNA completely and accurately can lead to mutations, diseases, or cell death. Therefore, it is of great interest to understand how CMG functions in the replisome during both normal replication and replication stress. Here, we describe a total internal reflection fluorescence (TIRF) microscopy assay using recombinant purified proteins, which allows for real-time visualization of surface-tethered stretched DNA molecules by individual CMG complexes. This assay provides a powerful platform to investigate CMG behavior at the single-molecule level, allowing helicase dynamics to be directly observed with real-time control over reaction conditions.

Author Spotlight: Unraveling the Dynamics of Eukaryotic DNA Replication Through Single-Molecule Visualization

This protocol demonstrates performing a single-molecule assay for live visualization of DNA unwinding by CMG helicase. It describes (1) preparing a DNA substrate, (2) purifying fluorescently labeled Drosophila melanogaster CMG helicase, (3) preparing a microfluidic flow cell for total internal reflection fluorescence (TIRF) microscopy, and (4) the single-molecule DNA unwinding assay.

Faithful genome duplication is essential for preserving the genetic stability of dividing cells. DNA replication is carried out during the S phase by a ...