Site‐Specific Fluorescent Labeling of Large RNAs with Pyrene

Mary K. Smalley1, Scott K. Silverman1

1 University of Illinois at Urbana‐Champaign, Urbana, Illinois
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 11.11
DOI:  10.1002/0471142700.nc1111s19
Online Posting Date:  December, 2004
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Abstract

Pyrene is a useful chromophore for monitoring the tertiary structure and folding of large RNAs. This unit describes the general preparation of a large RNA (>80 nucleotides in length) that has been site‐specifically modified with pyrene at the 2′‐position of an individual internal nucleotide. A protocol is provided for derivatizing a 2′‐amino‐RNA oligonucleotide with a suitably activated pyrene reagent. This pyrene‐labeled oligonucleotide is then assembled with other RNA(s) either by covalent ligation or by noncovalent hybridization to form a full‐length structured RNA, which may then be studied by equilibrium and stopped‐flow fluorescence spectroscopy.

Keywords: RNA folding; fluorescence; pyrene; ligation; annealing

     
 
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Table of Contents

  • Strategic Planning
  • Basic Protocol 1: Derivatization of A 2′‐Amino RNA Oligonucleotide with Pyrene
  • Basic Protocol 2: Assembly of the Large RNA by Ligation
  • Basic Protocol 3: Assembly of the Large RNA by Annealing
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Derivatization of A 2′‐Amino RNA Oligonucleotide with Pyrene

  Materials
  • RNA oligonucleotide with a 2′‐amino group
  • 500 mM sodium phosphate buffer, pH 8.0 ( appendix 2A)
  • 10 mM EDTA, pH 8.0 ( appendix 2A)
  • 50 mM pyrene 4‐sulfotetrafluorophenyl ester (pyr3‐STP; Molecular Probes or Gee et al., ) in N,N‐dimethylformamide (DMF)
  • 3 M NaCl (prepared directly or diluted from 5 M NaCl; see appendix 2A)
  • Absolute ethanol (e.g., Fisher)
  • 80% formamide gel loading buffer with dye (see recipe)
  • TEN buffer (see recipe)
  • 1.7‐mL RNase‐free microcentrifuge tubes (e.g., Eppendorf or Fisher)
  • Speedvac evaporator (Savant)
  • Glass rod
  • 50‐mL plastic tube (e.g., Fisher)
  • Platform rocker (Clay‐Adams Nutator or equivalent)
  • 0.45‐µm syringe filter (e.g., Fisher)
  • 40‐mL polypropylene tube (e.g., Oak Ridge tube from Fisher)
  • Tabletop centrifuge (e.g., IEC HN‐SII), chilled to 4°C
  • Refrigerated centrifuge (e.g., Beckman J2‐HS with Beckman JS‐13.1 rotor)
  • Additional reagents and equipment for denaturing polyacrylamide gel electrophoresis (PAGE; appendix 3B)

Basic Protocol 2: Assembly of the Large RNA by Ligation

  Materials
  • PAGE‐purified pyrene‐derivatized RNA oligonucleotide (see protocol 1)
  • DNA splint oligonucleotide (e.g., Integrated DNA Technologies or other commercial supplier)
  • Polynucleotide that constitutes the remaining portion of the large RNA (e.g., transcribed from a plasmid DNA template, as described in Silverman and Cech, )
  • 10× annealing buffer (see recipe)
  • 10× ligation buffer (see recipe)
  • T4 DNA ligase (e.g., USB; see )
  • 80% formamide gel loading buffer with dye (see recipe)
  • 1.7‐mL RNase‐free microcentrifuge tubes (e.g., Eppendorf or Fisher)
  • Dry heating block
  • Additional reagents and equipment for denaturing polyacrylamide gel electrophoresis (PAGE; appendix 3B)

Basic Protocol 3: Assembly of the Large RNA by Annealing

  Materials
  • PAGE‐purified pyrene‐derivatized RNA oligonucleotide (see protocol 1)
  • Polynucleotide that constitutes the remaining portion of the large RNA (e.g., transcribed from a plasmid DNA template, as described in Silverman and Cech, )
  • 10× TB buffer (equivalent to 10× TBE but omitting EDTA; see appendix 2A)
  • 1.7‐mL RNase‐free microcentrifuge tubes (e.g., Eppendorf or Fisher)
  • Dry heating block
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Figures

Videos

Literature Cited

   Bevilacqua, P.C., Kierzek, R., Johnson, K.A., and Turner, D.H. 1992. Dynamics of ribozyme binding of substrate revealed by fluorescence‐detected stopped‐flow methods. Science 258:1355‐1358.
   Blount, K.F. and Tor, Y. 2003. Using pyrene‐labeled HIV‐1 TAR to measure RNA‐small molecule binding. Nucl. Acids Res. 31:5490‐5500.
   Cate, J.H., Gooding, A.R., Podell, E., Zhou, K., Golden, B.L., Kundrot, C.E., Cech, T.R., and Doudna, J.A. 1996. Crystal structure of a group I ribozyme domain: Principles of RNA packing. Science 273:1678‐1685.
   Flynn‐Charlebois, A., Wang, Y., Prior, T.K., Rashid, I., Hoadley, K.A., Coppins, R.L., Wolf, A.C., and Silverman, S.K. 2003. Deoxyribozymes with 2′‐5′ RNA ligase activity. J. Am. Chem. Soc. 125:2444‐2454.
   Gee, K.R., Archer, E.A., and Kang, H.C. 1999. 4‐Sulfotetrafluorophenyl (STP) esters: New water‐soluble amine‐reactive reagents for labeling biomolecules. Tetrahedron Lett. 40:1471‐1474.
   Golden, B.L., Gooding, A.R., Podell, E.R., and Cech, T.R. 1996. X‐ray crystallography of large RNAs: Heavy‐atom derivatives by RNA engineering. RNA 2:1295‐1305.
   Kierzek, R., Li, Y., Turner, D.H., and Bevilacqua, P.C. 1993. 5′‐Amino pyrene provides a sensitive, nonperturbing fluorescent probe of RNA secondary and tertiary structure formation. J. Am. Chem. Soc. 115:4985‐4992.
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   Moore, M.J. and Query, C.C. 1998. Use of site‐specifically modified RNAs constructed by RNA ligation. In RNA‐Protein Interactions: A Practical Approach (C.W.J. Smith, ed.) pp. 75‐108. Oxford University Press, Oxford.
   Moore, M.J. and Sharp, P.A. 1992. Site‐specific modification of pre‐mRNA: The 2′‐hydroxyl groups at the splice site. Science 256:992‐997.
   Pan, T. 2000. Probing RNA structure and function by circular permutation. Methods Enzymol. 317:313‐330.
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   Preuss, R., Dapprich, J., and Walter, N.G. 1997. Probing RNA‐protein interactions using pyrene‐labeled oligodeoxynucleotides: Qβ replicase efficiently binds small RNAs by recognizing pyrimidine residues. J. Mol. Biol. 273:600‐613.
   Silverman, S.K. and Cech, T.R. 1999a. Energetics and cooperativity of tertiary hydrogen bonds in RNA structure. Biochemistry 38:8691‐8702.
   Silverman, S.K. and Cech, T.R. 1999b. RNA tertiary folding monitored by fluorescence of covalently attached pyrene. Biochemistry 38:14224‐14237.
   Silverman, S.K. and Cech, T.R. 2001. An early transition state for folding of the P4‐P6 RNA domain. RNA 7:161‐166.
   Silverman, S.K., Deras, M.L., Woodson, S.A., Scaringe, S.A., and Cech, T.R. 2000. Multiple folding pathways for the P4‐P6 RNA domain. Biochemistry 39:12465‐12475.
   Sontheimer, E.J., Gordon, P.M., and Piccirilli, J.A. 1999. Metal ion catalysis during group II intron self‐splicing: Parallels with the spliceosome. Genes & Dev. 13:1729‐1741.
   Strobel, S.A. and Ortoleva‐Donnelly, L. 1999. A hydrogen‐bonding triad stabilizes the chemical transition state of a group I ribozyme. Chem. Biol. 6:153‐165.
   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.
   Whitaker, M. 2000. Fluorescent tags of protein function in living cells. BioEssays 22:180‐187.
   Young, B.T. and Silverman, S.K. 2002. The GAAA tetraloop‐receptor interaction contributes differentially to folding thermodynamics and kinetics for the P4‐P6 RNA domain. Biochemistry 41:12271‐12276.
Key Reference
   Silverman and Cech, 1999b. See above.
  Presents the derivatization and ligation approaches to synthesizing pyrene‐labeled P4‐P6 RNA.
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