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.

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

Our research focuses on the dynamics of catalytic active RNAs and how these dynamics change under different environmental conditions. We use single-molecule FRET to study the influence of factors such as monovalent and divalent midlines, chaperone proteins, and crowding agents on RNA dynamics. Precisely incorporating donor and acceptor fluorophores without disrupting RNA structure and function is challenging.Additionally, tracking the folding of a large dynamic RNA is difficult. However, the established labeling and encapsulation protocol allows us to monitor the folding dynamics of the functional RNA over time. Our protocol addresses the challenge of observing large, catalytically active RNAs in real time and under neophysiological conditions.By covalently labeling and encapsulating the RNA and vesicles, which are then surface immobilized, we can monitor RNA function with single-Molecule TIRF. We focus on covalent FRET labeling of long catalytic RNAs and particularly group II introns. This is a challenge due to their structural complexity.When encapsulated in vesicles, we get to keep the RNA within the evanescent field for TIRF microscopy, and yet the RNA can freely float. In this way, smFRET can reveal the dynamic behavior of RNA without hindrance. We will continue trying to understand how nature really works, focusing on the structure-function relationship and dynamics of ribosomes, riboswitches, and other regulatory RNAs using a multi-technique approach.So moving towards investigations under physiological and cellular conditions will be a major emphasis of our work in the future. To begin, aliquot the RNA of interest, having 50 to 75 micrograms of RNA per tube in 55 microliters of double-distilled water, then add 45 microliters of the EDC/NHS sodium acetate pH 6 mixture to the RNA. Incubate the mixture for four hours at 25 degrees Celsius while shaking at 500 RPM.After incubation, resuspend the activated RNA pellet in 95 microliters of 100-millimolar MOPS buffer pH 7.5. Then add five microliters of two-millimolar sulfonated cyanine-3 amine to the activated RNA, and incubate at 25 degrees Celsius for 16 hours at 500 RPM to couple the fluorophore to the activated 5-prime phosphate. The next day, add 200 microliters of double-distilled water to improve the separation.Then purify the 5-prime phosphate labeled RNA by ethanol precipitation. After purification, resuspend the labeled RNA in 100 microliters of 100-millimolar tris HCl, and incubate for two hours at 25 degrees Celsius. Then to remove the free dyes, wash the labeled RNA in double-distilled water with centrifugal filtration.For dual-end-labeling, aliquot the 5-prime end-labeled RNA, having approximately 50 to 75 micrograms of RNA per tube in 90 microliters of solution. Add five microliters of one-molar sodium acetate buffer pH 5.5. Then add four microliters of freshly prepared 500-millimolar sodium metaperiodate stock solution.For 3-prime end single-labeling, add eight microliters of metaperiodate stock solution. After incubating the solutions in the dark for two hours, pipette 30 microliters of 50%glycerol to stop the reaction. After 30 minutes of incubation, add 400 microliters of ice cold ethanol sodium acetate precipitation mixture.Resuspend the oxidized RNA pellet in 95 microliters of 50-millimolar sodium acetate buffer pH six. Purify the dual-end-labeled RNA by ethanol precipitation and centrifugal filtration, as demonstrated previously. Elute the purified RNA in double-distilled water.Visually compare the supernatant and pellet colors obtained after each step. Calculate the RNA and conjugate dye concentrations on a UV-visible spectrophotometer. Fluorescent gel electrophoresis confirmed the successful single and dual labeling of RNA, as shown by the comigration of the RNA with the fluorophores.FRET efficiency increased in the presence of metal ions, indicating RNA folding, which led to a decrease in donor emission and an increase in acceptor emission. To prepare a microfluidic chamber, use a diamond driller to drill four holes in the quartz slides to form two channels. Place the drilled quartz slides and approximately twice as many coverslips into a glass Coplin staining jar with a cover.Sonicate the slides and coverslips in double-distilled water for five minutes. Next, sonicate the jar in a 10%laboratory glassware cleaning solution for 30 minutes at 50 degrees Celsius. After rinsing the jar with double-distilled water, sonicate in one-molar potassium hydroxide solution for 30 minutes and leave it in the solution overnight.After repeated rinsing and sonication with distilled water, dry the slides and coverslips using nitrogen gas flow. Treat the dried slides and coverslips with an oxygen plasma cleaner for 30 minutes. Place the clean slides and coverslips in a Coplin staining jar containing 3%AP-test ethanol solution Sonicate for one minute and then incubate for 30 minutes.Rinse the slides and coverslips three times each with absolute ethanol and double-distilled water, then dry them under nitrogen gas flow. Next, prepare a humid box by filling an empty pipet tip box halfway with double-distilled water. Place the slides inside the box with the side to be treated facing up.Add 30-microliter droplets of freshly prepared bPEG/mPEG mixture to the center of the slide to cover both channels. Cover the droplet with a clean coverslip and close the humid box. Incubate the box overnight in the dark for PEGylation.The next day, rinse the PEG-passivated and biotinylated slides and coverslips with double-distilled water and observe the change in hydrophobicity of the treated surfaces. Attach the double-sided sticker to the slide, ensuring the sticker covers the area of interest. Then carefully place the coverslip on top of the slide, aligning the PEGylated surfaces to face each other.Transfer the assembled chamber to a 50-milliliter centrifuge tube, and fill the tube with nitrogen gas. To begin, use a sterile needle to poke a hole from the inside out in the lid of a sterile clean two-milliliter tube. Add 109 microliters of bPE-DMPC mixture into the tube.Place a cardboard cell partition insert from a microtube storage box into a 500-milliliter Schlenk flask to serve as a tube holder. Using long tweezers, carefully place the tubes containing the lipid mixture into the tube holder. Evaporate the chloroform under a low flow of nitrogen gas overnight.After obtaining the dried lipid layer, seal the tubes with parafilm to cover the holes. Assemble the extruder using a 100-nanometer polycarbonate membrane and a 10-millimeter polyester drain disc. Equilibrate the syringe and membranes with anti-blinking buffer.After mixing dual-end-labeled RNA with anti-blinking buffer, hydrate the lipid cake with RNA anti-blinking buffer mixture at 30 degrees Celsius. Shake for five minutes at 1, 400 RPM, followed by 15 minutes at 700 RPM. Centrifuge the mixture for two minutes at 13, 000 G, and dilute the supernatant with 250 microliters of anti-blinking buffer.Fill the syringe with the RNA lipid suspension, and extrude 35 times at 30 degrees Celsius on a heating block to encapsulate the dual-end-labeled RNA in phospholipid vesicles of 100-nanometer diameter. Flush the chamber two times with 200 microliters of 5X standard buffer at room temperature. Fill the chamber with 50 microliters of 20 microliters per milliliter streptavidin solution, and incubate for five minutes.Then flush the chamber with 200 microliters of standard buffer and 100 microliters of anti-blinking buffer. Add 75 microliters of vesicle suspension and incubate for 10 minutes for immobilization of the encapsulated RNA on the surface. After immobilization, flush the chamber with 200 microliters of freshly prepared imaging buffer.For data acquisition, mount the chamber on the TIRF microscope and acquire smFRET data with alternating laser excitation. Single-molecule FRET imaging using TIRF microscopy tracked individual RNA molecules and detected real-time changes in FRET efficiency. Dynamic Single-Molecule traces showed anti-correlated fluctuations between donor and acceptor signals, indicating confirmational changes in RNA.

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>

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

282 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.

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

Summary