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
Table of Contents
- 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
Materials
Basic Protocol 1: Preparation of RNA by Multiple Enzymatic Splinted Ligation
Materials
Basic Protocol 2: Initial Spectroscopic Characterization by Bulk FRET Measurements
Materials
Basic Protocol 3: Single‐Molecule FRET Measurements
Materials
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Figures
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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 λmax . View Image -
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). View Image -
Figure 11.12.3 Examples for attachment sites of internal labels: C 6 ‐8‐amino‐dG (left) and Cy5‐ C 6 ‐rT (right). View Image -
Figure 11.12.4 Schematic depiction of scanning confocal fluorescence microscopy setup. View Image -
Figure 11.12.5 Intramolecular tRNA dynamics revealed by smFRET. (A ) Histograms of FRET efficiency values, E , taken from freely diffusing human mitochondrial tRNALys 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. View Image -
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. View Image
Videos
Literature Cited
Aitken, C.E., Marshall, R.A., and Puglisi, J.D. 2007. An oxygen scavenging system for improvement of dye stability in single‐molecule fluorescence experiments. Biophys. J. 94:1826‐1835. | |
Amirgoulova, E.V., Groll, J., Heyes, C.D., Ameringer, T., Röcker, C., Möller, M., and Nienhaus, G.U. 2004. Biofunctionalized polymer surfaces exhibiting minimal interaction towards immobilized proteins. Chemphyschem 5:552‐555. | |
Bassi, G.S., Murchie, A.I., Walter, F., Clegg, R.M., and Lilley, D.M. 1997. Ion‐induced folding of the hammerhead ribozyme: A fluorescence resonance energy transfer study. EMBO J. 16:7481‐7489. | |
Chan, B., Weidemaier, K., Yip, W.T., Barbara, P.F., and Musier‐Forsyth, K. 1999. Intra‐tRNA distance measurements for nucleocapsid protein‐dependent tRNA unwinding during priming of HIV reverse transcription. Proc. Natl. Acad. Sci. U.S.A. 96:459‐464. | |
Clegg, R.M. 1992. Fluorescence resonance energy transfer and nucleic acids. Methods Enzymol. 211:353‐388. | |
Coban, O., Lamb, D.C., Zaychikov, E., Heumann, H., and Nienhaus, G.U. 2006. Conformational heterogeneity in RNA polymerase observed by single‐pair FRET microscopy. Biophys. J. 90:4605‐4617. | |
Deniz, A.A., Dahan, M., Grunwell, J.R., Ha, T., Faulhaber, A.E., Chemla, D.S., Weiss, S., and Schultz, P.G. 1999. Single‐pair fluorescence resonance energy transfer on freely diffusing molecules: Observation of Forster distance dependence and subpopulations. Proc. Natl. Acad. Sci. U.S.A. 96:3670‐3675. | |
Dietrich, A., Buschmann, V., Müller, C., and Sauer, M. 2002. Fluorescence resonance energy transfer (FRET) and competing processes in donor‐acceptor substituted DNA strands: A comparative study of ensemble and single‐molecule data. J. Biotechnol. 82:211‐231. | |
Groll, J., Amirgoulova, E.V., Ameringer, T., Heyes, C.D., Röcker, C., Nienhaus, G.U., and Möller, M. 2004. Biofunctionalized, ultrathin coatings of cross‐linked star‐shaped poly(ethylene oxide) allow reversible folding of immobilized proteins. J. Am. Chem. Soc. 126:4234‐4239. | |
Ha, T., Enderle, T., Ogletree, D.F., Chemla, D.S., Selvin, P.R., and Weiss, S. 1996. Probing the interaction between two single molecules: Fluorescence resonance energy transfer between a single donor and a single acceptor. Proc. Natl. Acad. Sci. U.S.A. 93:6264‐6268. | |
Ha, T., Zhuang, X., Kim, H.D., Orr, J.W., Williamson, J.R., and Chu, S. 1999. Ligand‐induced conformational changes observed in single RNA molecules. Proc. Natl. Acad. Sci. U.S.A. 96:9077‐9082. | |
Heyes, C.D., Kobitski, A.Y., and Nienhaus, G.U. 2004. Biocompatible surfaces for specific tethering of individual protein molecules. J. Phys. Chem. B 108:13387‐13394. | |
Heyes, C.D., Groll, J., Möller, M., and Nienhaus, G.U. 2007. Synthesis, patterning and applications of star‐shaped poly(ethylene glycol) biofunctionalized surfaces. Mol. Biosyst. 3:419‐430. | |
Huang, Z., Ji, D., Xia, A.D., Koberling, F., Patting, M., and Erdmann, R. 2005. Direct observation of delayed fluorescence from a remarkable back‐isomerization in Cy5. J. Am. Chem. Soc. 127:8064‐8066. | |
Ishii, Y., Yoshida, T., Funatsu, T., Wazawa, T., and Yanagida, T. 1999. Fluorescence resonance energy transfer between single fluorophores attached to a coiled‐coil protein in aqueous solution. Chem. Phys. 247:163‐173. | |
Jares‐Erijman, E.A. and Jovin, T.M. 2003. FRET imaging. Nat. Biotechnol. 21:1387‐1395. | |
Kapanidis, A.N., Lee, N.K., Laurence, T.A., Doose, S., Margeat, E., and Weiss, S. 2004. Fluorescence‐aided molecule sorting: Analysis of structure and interactions by alternating‐laser excitation of single molecules. Proc. Natl. Acad. Sci. U.S.A. 101:8936‐8941. | |
Kim, H.D., Nienhaus, G.U., Ha, T., Orr, J.W., Williamson, J.R., and Chu, S. 2002. Mg2+‐dependent conformational change of RNA studied by fluorescence correlation and FRET on immobilized single molecules. Proc. Natl. Acad. Sci. U.S.A. 99:4284‐4289. | |
Klostermeier, D. and Millar, D.P. 2001. Tertiary structure stability of the hairpin ribozyme in its natural and minimal forms: Different energetic contributions from a ribose zipper motif. Biochemistry 40:11211‐11218. | |
Kobitski, A.Y., Nierth, A., Helm, M., Jäschke, A., and Nienhaus, G.U. 2007. Mg2+‐dependent folding of a Diels‐Alderase ribozyme probed by single‐molecule FRET analysis. Nucleic Acids Res. 35:2047‐2059. | |
Kurschat, W.C., Muller, J., Wombacher, R., and Helm, M. 2005. Optimizing splinted ligation of highly structured small RNAs. RNA 11:1909‐1914. | |
Kuzmenkina, E.V., Heyes, C.D., and Nienhaus, G.U. 2005. Single‐molecule Forster resonance energy transfer study of protein dynamics under denaturing conditions. Proc. Natl. Acad. Sci. U.S.A. 102:15471‐15476. | |
Lakowicz, J.R. 2006. Principles of Fluorescence Spectroscopy. Springer, New York. | |
Lee, N.K., Koh, H.R., Han, K.Y., and Kim, S.K. 2007. Folding of 8‐17 deoxyribozyme studied by three‐color alternating‐laser excitation of single molecules. J. Am. Chem. Soc. 129:15526‐15534. | |
McKinney, S.A., Declais, A.C., Lilley, D.M., and Ha, T. 2003. Structural dynamics of individual Holliday junctions. Nat. Struct. Biol. 10:93‐97. | |
Mujumdar, R.B., Ernst, L.A., Mujumdar, S.R., Lewis, C.J., and Waggoner, A.S. 1993. Cyanine dye labeling reagents: Sulfoindocyanine succinimidyl esters. Bioconjug. Chem. 4:105‐111. | |
Murchie, A.I., Thomson, J.B., Walter, F., and Lilley, D.M. 1998. Folding of the hairpin ribozyme in its natural conformation achieves close physical proximity of the loops. Mol. Cell 1:873‐881. | |
Nienhaus, G.U. 2006. Exploring protein structure and dynamics under denaturing conditions by single‐molecule FRET analysis. Macromol. Biosci. 6:907‐922. | |
Panchuk‐Voloshina, N., Haugland, R.P., Bishop‐Stewart, J., Bhalgat, M.K., Millard, P.J., Mao, F., Leung, W.Y., and Haugland, R.P. 1999. Alexa dyes, a series of new fluorescent dyes that yield exceptionally bright, photostable conjugates. J. Histochem. Cytochem. 47:1179‐1188. | |
Pinard, R., Hampel, K.J., Heckman, J.E., Lambert, D., Chan, P.A., Major, F., and Burke, J.M. 2001. Functional involvement of G8 in the hairpin ribozyme cleavage mechanism. EMBO J. 20:6434‐6442. | |
Qin, P.Z. and Pyle, A.M. 1999. Site‐specific labeling of RNA with fluorophores and other structural probes. Methods 18:60‐70. | |
Rasnik, I., McKinney, S.A., and Ha. T. 2005. Surfaces and orientations: Much to FRET about? Acc. Chem. Res. 38:542‐548. | |
Rasnik, I., McKinney, S.A., and Ha, T. 2006. Nonblinking and long‐lasting single‐molecule fluorescence imaging. Nat. Methods 3:891‐893. | |
Rothwell, P.J., Berger, S., Kensch, O., Felekyan, S., Antonik, M., Wöhrl, B.M., Restle, T., Goody, R.S., and Seidel, C.A. 2003. Multiparameter single‐molecule fluorescence spectroscopy reveals heterogeneity of HIV‐1 reverse transcriptase:primer/template complexes. Proc. Natl. Acad. Sci. U.S.A. 100:1655‐1660. | |
Sabanayagam, C.R., Eid, J.S., and Meller, A. 2005. Using fluorescence resonance energy transfer to measure distances along individual DNA molecules: Corrections due to nonideal transfer. J. Chem. Phys. 122:061103. | |
Scaringe, S.A., Wincott, F.E., and Caruthers, M.H. 1998. Novel RNA synthesis method using 5′‐Silyl‐2′‐Orthoester protecting groups. J. Am. Chem. Soc. 120:11820‐11821. | |
Selvin, P.R. 2000. The renaissance of fluorescence resonance energy transfer. Nat. Struct. Biol. 7:730‐734. | |
Singh, K.K., Parwaresch, R., and Krupp, G. 1999. Rapid kinetic characterization of hammerhead ribozymes by real‐time monitoring of fluorescence resonance energy transfer (FRET). RNA 5:1348‐1356. | |
Slatko, B.E. and Albright, L.M. 2001. Denaturing gel electrophoresis for sequencing. Curr. Protoc. Mol. Biol. 16:7.6.1‐7.6.13. | |
Tan, E., Wilson, T.J., Nahas, M.K., Clegg, R.M., Lilley, D.M.J, and Ha, T. 2003. A four‐way junction accelerates hairpin ribozyme folding via a discrete intermediate. Proc. Natl. Acad. Sci. U.S.A. 100:9308‐9313. | |
Telser, J., Cruickshank, K.A., Morrison, L.E., and Netzel, T.L. 1989. Synthesis and characterization of DNA oligomers and duplexes containing covalently attached molecular labels: Comparison of biotin, fluorescein, and pyrene labels by thermodynamic and optical spectroscopic measurements. J. Am. Chem. Soc. 111:6966. | |
Tuschl, T., Gohlke, C., Jovin, T.M., Westhof, E., and Eckstein, F. 1994. A three‐dimensional model for the hammerhead ribozyme based on fluorescence measurements. Science 266:785‐789. | |
Voigts‐Hoffmann, F., Hengesbach, M., Kobitski, A.Y., van Aerschot, A., Herdewijn, P., Nienhaus, G.U., and Helm, M. 2007. A methyl group controls conformational equilibrium in human mitochondrial tRNA(Lys). J. Am. Chem. Soc. 129:13382‐13383. | |
von Ahsen, U. and Noller, H.F. 1995. Identification of bases in 16S rRNA essential for tRNA binding at the 30S ribosomal P site. Science 267:234‐237. | |
Walter, N.G., Hampel, K.J., Brown, K.M., and Burke, J.M. 1998. Tertiary structure formation in the hairpin ribozyme monitored by fluorescence resonance energy transfer. EMBO J. 17:2378‐2391. | |
Walter, N.G., Burke, J.M., and Millar, D.P. 1999. Stability of hairpin ribozyme tertiary structure is governed by the interdomain junction. Nat. Struct. Biol. 6:544‐549. | |
Walter, N.G., Yang, N., and Burke, J.M. 2000. Probing non‐selective cation binding in the hairpin ribozyme with Tb(III). J. Mol. Biol. 298:539‐555. | |
Walter, N.G., Chan, P.A., Hampel, K.J., Millar, D.P., and Burke, J.M. 2001. A base change in the catalytic core of the hairpin ribozyme perturbs function but not domain docking. Biochemistry 40:2580‐2587. | |
Widengren, J. and Schwille, P. 2000. Characterization of photo‐induced isomerization and back‐isomerization of the cyanine dye Cy5 by fluorescence correlation spectroscopy. J. Phys. Chem. 104:6416‐6428. | |
Zhuang, X., Bartley, L.E., Babcock, H.P., Russell, R., Ha, T., Herschlag, D., and Chu S. 2000. A single‐molecule study of RNA catalysis and folding. Science 288:2048‐2051. | |
Zhuang, X., Kim, H., Pereira, M.J., Babcock, H.P., Walter, N.G., and Chu, S. 2002. Correlating structural dynamics and function in single ribozyme molecules. Science 296:1473‐1476. | |
Internet Resources | |
http://probes.invitrogen.com/handbook/boxes/0422.html | |
Molecular Probes Note 1.2—Technical Focus: Fluorescence Resonance Energy Transfer (FRET). |