RNA Intramolecular Dynamics by Single‐Molecule FRET

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Abstract
Table of Contents
Materials
Figures
Literature Cited

Abstract

Investigation of single RNA molecules using fluorescence resonance energy transfer (FRET) is a powerful approach to investigate dynamic and thermodynamic aspects of the folding process of a given RNA. Its application requires interdisciplinary work from the fields of chemistry, biochemistry, and physics. The present work gives detailed instructions on the synthesis of RNA molecules labeled with two fluorescent dyes interacting by FRET, as well as on their investigation by single?molecule fluorescence spectroscopy. Curr. Protoc. Nucleic Acid Chem. 34:11.12.1?11.12.22. © 2008 by John Wiley & Sons, Inc.

Keywords: fluorescence resonance energy transfer (FRET); single molecule; splint ligation; dynamics; fluorescent dyes; nucleotide modifications; RNA folding

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Introduction
Strategic Planning
Basic Protocol 1: Preparation of RNA by Multiple Enzymatic Splinted Ligation
Basic Protocol 2: Initial Spectroscopic Characterization by Bulk FRET Measurements
Basic Protocol 3: Single‐Molecule FRET Measurements
Reagents and Solutions
Commentary
Literature Cited
Figures
Tables

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Materials

Basic Protocol 1: Preparation of RNA by Multiple Enzymatic Splinted Ligation

Materials

RNA oligonucleotides for ligation; preferably lyophilized, or in aqueous solutions, concentration >100 µM (Dharmacon or IBA GmbH, http://www.iba‐go.com)
T4 polynucleotide kinase (PNK; Fermentas)
5× KL buffer (see recipe )
100 µM DNA template (splint; IBA GmbH, http://www.iba‐go.com; for more information, see Kurschat et al., )
30 U/µL T4 DNA ligase HC (Fermentas)
DNase I (Fermentas)
Water‐saturated phenol (Carl Roth GmbH)
Diethyl ether, water‐saturated (Carl Roth GmbH)
5 M ammonium acetate (Sigma)
Ethanol, absolute (VWR)
80% (v/v) ethanol
Acrylamide gel casting solutions (e.g., Rotiphorese Sequencing Gel system, Carl Roth GmbH)
- Sequencing gel concentrate solution: 25% 19:1 acrylamide/bisacrylamide/8.3 M urea
- Sequencing gel buffer concentrate: 8.3 M urea/10× TBE
- Sequencing gel diluter solution: 8.3 M urea
Repellant solution: 2% dichlorodimethlysilane (Fluka) in CHCl₃
10% (w/v) ammonium persulfate (Fluka)
N ,N ,N ′,N ′‐Tetramethylethan‐1,2‐diamine (TEMED; Fluka)
10× TBE buffer (Invitrogen)
Loading buffer (see recipe )
0.5 M ammonium acetate (Sigma)

Thermoshaker (e.g., Eppendorf)
400 × 280 × 5–mm gel casting plates with 1‐ to 2‐mm spacers (Biometra, http://www.biometra.de/)
Gel comb with 80 × 40–mm teeth (Biometra, http://www.biometra.de/)
Polyacrylamide gel electrophoresis apparatus with power source (also see appendix 3B )
Instrument for visualization of gel bands, one of the following:
- UV handlamp or UV‐transilluminator
- Fluorescence light table (Mobitec, http://www.mobitec‐us.com)
- Typhoon Variable Mode Imager, equipped with suitable lasers (GE Healthcare)
0.22‐µM spin filter columns (Pall Corp.)
UV spectrometer (e.g., Nanodrop 1000, Thermo Fisher)

Additional reagents and equipment for polyacrylamide gel electrophoresis ( appendix 3B )

Basic Protocol 2: Initial Spectroscopic Characterization by Bulk FRET Measurements

Materials

Ultrapure H₂ O
Ethanol
∼ 2 to 10 pmol RNA sample (depending on spectrometer sensitivity)
Appropriate folding buffer for the RNA under investigation

Blackened fluorescence quartz cuvette (Hellma, Suprasil series), suitable for the fluorescence spectrometer used
Thermoshaker (e.g., Eppendorf)
Emission‐calibrated fluorescence spectrometer (e.g., JASCO FP6500)

Basic Protocol 3: Single‐Molecule FRET Measurements

Materials

Appropriate buffer (see protocol 2 )
1 pmol RNA sample
1 mg/mL BSA‐biotin (Sigma‐Aldrich) in 100 mM sodium phosphate buffer, pH 7.4 (Fluka)
10 µg/mL streptavidin (Sigma‐Aldrich) in 100 mM sodium phosphate buffer, pH 7.4 (Fluka)
100 mM sodium phosphate buffer, pH 7.4 (Fluka)
Epoxy glue (optional)

Untreated glass cover slips (24 × 32 mm and 20 × 20 mm, Menzel‐Glaser)
Double‐sided adhesive tape
Bunsen burner

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Figures

Figure 11.12.1 Fluorescence emission spectra of a Cy3 and Cy5 dye pair with high and low FRET coupling efficiency. I _A and I _D are fluorescence intensities emitted by acceptor and donor at their respective λ_max .

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Figure 11.12.2 FRET efficiency versus distance in R ₀ units. R is distance between donor and acceptor; R ₀ is distance at which transfer efficiency is 50% (Förster radius).

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Figure 11.12.3 Examples for attachment sites of internal labels: C ⁶ ‐8‐amino‐dG (left) and Cy5‐ C ⁶ ‐rT (right).

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Figure 11.12.4 Schematic depiction of scanning confocal fluorescence microscopy setup.

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Figure 11.12.5 Intramolecular tRNA dynamics revealed by smFRET. (A ) Histograms of FRET efficiency values, E , taken from freely diffusing human mitochondrial tRNA^Lys molecules. (B ) FRET trajectories of immobilized single molecules and the corresponding histograms of FRET efficiencies, obtained with 100‐msec dwell time binning. Figure reprinted with permission from Voigts‐Hoffmann et al. (). Copyright (2007) American Chemical Society. Kwt, wild‐type; Km¹ A, modified with 1‐methyladenosine at position 9.

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Figure 11.12.6 ALEX‐based data analysis. (A ) The stoichiometry map is calculated from fluorescence bursts due to freely diffusing tRNA molecules. (B ) FRET efficiency histograms are compiled from those bursts with 0.35 < S < 0.75, so that “donor‐only” molecules are efficiently suppressed.

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Videos

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Internet Resources
	http://probes.invitrogen.com/handbook/boxes/0422.html
	Molecular Probes Note 1.2—Technical Focus: Fluorescence Resonance Energy Transfer (FRET).

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RNA Intramolecular Dynamics by Single‐Molecule FRET

Abstract

Table of Contents

Materials

Basic Protocol 1: Preparation of RNA by Multiple Enzymatic Splinted Ligation

Basic Protocol 2: Initial Spectroscopic Characterization by Bulk FRET Measurements

Basic Protocol 3: Single‐Molecule FRET Measurements

Figures

Videos

Literature Cited