Time‐Resolved Hydroxyl Radical Footprinting of RNA with X‐Rays

Sarah A. Woodson1, Michael L. Deras1, Michael Brenowitz2

1 Johns Hopkins University, Baltimore, Maryland, 2 Albert Einstein College of Medicine of Yeshiva University, Bronx, New York
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 11.6
DOI:  10.1002/0471142700.nc1106s06
Online Posting Date:  November, 2001
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Abstract

RNA tertiary structure and protein interactions can be detected by protection from hydroxyl radical cleavage. Generation of hydroxyl radicals with a synchrotron X‐ray beam provides analysis on a short timescale (50 msec to 100 sec), which enables the structures of folding intermediates or other transient conformational states to be determined. This unit provides detailed instructions on the use of the synchrotron beamline for hydroxyl radical footprinting.

     
 
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Table of Contents

  • Strategic Planning
  • Basic Protocol 1: Equilibrium X‐Ray Footprinting of RNA
  • Basic Protocol 2: Time‐Resolved X‐Ray Footprinting of RNA
  • Basic Protocol 3: Sample Work‐up and Data Analysis
  • Support Protocol 1: Preparation of Radiolabeled RNA
  • Support Protocol 2: Determine Optimal Exposure Time
  • Support Protocol 3: Set Up Rapid‐Quench Miming Apparatus
  • Support Protocol 4: Calibrate Rapid‐Quench Mixing Apparatus
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Equilibrium X‐Ray Footprinting of RNA

  Materials
  • 32P end‐labeled RNA in 6‐µCi aliquot (see protocol 4)
  • CE buffer, pH 7.5 (see recipe) or other appropriate buffer (see )
  • 1 M MgCl 2 ( appendix 2A; optional)
  • Precipitation cocktail (see recipe)
  • 100% ethanol
  • Aluminum sample holder with electronic shutter and support stand (see , Figure )
  • Support table
  • Controller cable
  • Detector for the automatic vertical alignment device
  • Refrigerated recirculating bath with attachment tubes
  • 1.5‐mL and 0.5‐mL microcentrifuge tubes with captive screw caps and O‐ring seals (Rainin)
  • Linagraph paper (Kodak)
  • Masking tape
  • Temperature‐controlled heating block or water bath
  • Lead sample box
CAUTION: Cacodylic acid is an arsenic compound and is toxic.

Basic Protocol 2: Time‐Resolved X‐Ray Footprinting of RNA

  Materials
  • CE buffer, pH 7.5 (see recipe) or other appropriate buffer (see )
  • 2 to 8 µCi 32P end‐labeled RNA, divided between two 20‐µL aliquots (see protocol 4)
  • 100% ethanol
  • Precipitation cocktail (see recipe)
  • CE20 buffer (see recipe)
  • Additional buffers or salts as desired (see )
  • 0.6 µCi RNA for prefolded controls (see protocol 4)
  • Rapid‐quench apparatus, modified for X‐ray footprinting experiments (e.g., RQF‐3, Kin‐Tek; see , Figure , and protocol 6)
  • 1‐mL and 5‐mL Luer‐lok disposable syringes
  • Temperature‐controlled heating block or water bath
  • 1.5‐mL microcentrifuge tubes with captive screw caps and O‐ring seals (Rainin)
  • 13‐G needles
  • 15‐mL sterile disposable culture tubes
  • Lead‐lined box
  • Refrigerated recirculating water bath
NOTE: It is important to become familiar with the operation of the rapid‐quench apparatus (Figure ) and the valve settings illustrated in Figure (LOAD syringes, LOAD sample, FIRE, and FLUSH). For RNA folding experiments, drive syringe B is loaded with buffer (e.g., CE or CE20 buffer), and the RNA sample is placed in the bottom right sample loop. Drive syringe A and the bottom left sample loop are loaded with CE or CE20 buffer. The third Quench syringe (C) is not used in the standard footprinting protocol. The plunger for the C syringe should remain fully depressed. CAUTION: Care must be taken to protect personnel from β radiation and prevent contamination of work area. Gloves, equipment, and work surfaces should be frequently monitored using a hand‐held survey meter. Safety procedures pertaining to use of the beamline, such as the interlock system, must be observed at all times. Caution should be used in handling the waste container, sample syringe port, and exit line, which may be contaminated with radioactive material.

Basic Protocol 3: Sample Work‐up and Data Analysis

  Materials
  • Irradiated 32P end‐labeled RNA (see protocol 1Basic Protocols 1 and protocol 22)
  • Unirradiated 32P end‐labeled RNA (see protocol 4)
  • 2× formamide loading buffer ( appendix 2A) or loading buffer containing urea
  • RNase T1 cocktail (see recipe)
  • 0.5 U/mL RNase T1 (USB)
  • CE buffer, pH 7.5 (see recipe) or TE buffer ( appendix 2A; see )
  • Prefolded control RNA (see protocol 2)
  • Imaging system with large exposure cassettes (Phosphorimager, Molecular Dynamics; or equivalent)
  • Densitometer
  • Image analysis software for personal computer (PC) or Macintosh computer (ImageQuant, Molecular Dynamics; NIH Image; or equivalent)
  • Spreadsheet software (Microsoft Excel or equivalent)
  • Graphical fitting software (KaleidaGraph, SigmaPlot, or equivalent)
  • Additional reagents and equipment for denaturing polyacrylamide gel electrophoresis ( appendix 3B)
NOTE: Samples should be stored at −20° or −70°C until they are ready to be analyzed. This should be done as soon as possible after X‐ray exposure, ideally within 1 week.

Support Protocol 1: Preparation of Radiolabeled RNA

  Materials
  • 10 pmol/µL RNA, treated with calf intestinal phosphatase (unit 6.3)
  • T4 polynucleotide kinase and 10× kinase buffer
  • 6000 Ci/mmol (10 µCi/µL)γ;‐32P]ATP
  • TE buffer, pH 7.5 (optional; appendix 2A)
  • CE buffer, pH 7.5 (see recipe)
  • Microcentrifuge tubes with captive screw caps and O‐ring seals (Rainin)
  • Additional reagents and equipment for phosphorylation reaction (unit 6.3), for preparing and running a preparative polyacrylamide sequencing gel ( appendix 3B), for phenol/chloroform extraction and ethanol precipitation ( appendix 2B and, e.g., CPMB UNIT )
NOTE: Radioactive materials must be labeled and shipped in compliance with federal and state regulations. Consult with the radiation safety officer of your home institution and the receiving institution before planning to ship radioactive materials.

Support Protocol 2: Determine Optimal Exposure Time

  • 1 to 2 µCi 5′‐32P‐labeled RNA in 10 µL CE buffer

Support Protocol 3: Set Up Rapid‐Quench Miming Apparatus

  • Detergent (e.g., Absolve, NEN)
  • Plastic‐backed absorbent bench paper
  • Diaphragm vacuum pump (details)
  • Vacuum/vent filter, 0.2 µm (Millipore Millex 50 mm, or equivalent)
  • Side‐arm flask with one‐hole stopper and Teflon tube
  • Thick‐walled soft tubing (e.g., Tygon, Nalgene) to fit Teflon tube
  • Adapter (male M6 to male Luer) to connect soft vacuum tubing with E1/16‐in.‐o.d. polypropylene tubing (exit line of rapid‐quench)
  • 5‐mL syringes

Support Protocol 4: Calibrate Rapid‐Quench Mixing Apparatus

  • 0.25% (w/v) bromphenol blue in water
  • Dental mirror
  • Small flashlight
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Figures

Videos

Literature Cited

Literature Cited
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   Breen, A.P. and Murphy, J.A. 1995. Reactions of oxyl radicals with DNA. Free Radic. Biol. Med. 18:1033‐1077.
   Brenowitz, M., Senear, D.F., Shea, M.A., and Ackers, G.K. 1986. Quantitative DNase footprint titration: A method for studying protein‐DNA interactions. Methods Enzymol. 130:132‐181.
   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.
   Celander, D.W. and Cech, T.R. 1990. Iron(II)‐ethylenediaminetetraacetic acid catalyzed cleavage of RNA and DNA oligonucleotides: Similar reactivity toward single‐ and double‐stranded forms. Biochemistry 29:1355‐1361.
   Celander, D.W. and Cech, T.R. 1991. Visualizing the higher order folding of a catalytic RNA molecule. Science 251:401‐407.
   Chaulk, S.G. and MacMillan, A.M. 2000. Kinetic footprinting of an RNA‐folding pathway using peroxynitrous acid. Angew. Chem. Int. Ed. Engl. 39:521‐523.
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   Dhavan, G.M., Chance, M.R., and Brenowitz, M. 2001. Kinetics analysis of DNA‐protein interactions by time resolved synchrotron X‐ray footprinting. Practical Applications. In press. In
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   Dizdaroglu, M. and Bergtold, D.S. 1986. Characterization of free radical‐induced base damage in DNA at biologically relevant levels. Anal. Biochem. 156:182‐188.
   Hampel, K.J. and Burke, J.M. 2001. Time‐resolved hydroxyl‐radical footprinting of RNA using Fe(II)‐EDTA. Methods 23:233‐239.
   King, P.A., Jamison, E., Strahs, D., Anderson, V.E., and Brenowitz, M. 1993. 'Footprinting' proteins on DNA with peroxynitrous acid. Nucl. Acids Res. 21:2473‐2478.
   Klassen, N.V. 1987. Primary products in radiation chemistry. Radiation Chemistry: Principles & Applications (I. Farhatazis and M.A. Rodgers eds.) pp. 29‐61. VCH Publishers, New York. In
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   Milligan, J.F. and Uhlenbeck, O.C. 1989. Synthesis of small RNAs using T7 RNA polymerase. Methods Enzymol. 180:51‐62.
   Milligan, J.F., Groebe, D.R., Witherell, G.W., and Uhlenbeck, O.C. 1987. Oligoribonucleotide synthesis using T7 RNA polymerase and synthetic DNA templates. Nucl. Acids Res. 15:8783‐8798.
   Ralston, C.Y., He, Q., Brenowitz, M., and Chance, M.R. 2000a. Stability and cooperativity of individual tertiary contacts in RNA revealed through chemical denaturation. Nat. Struct. Biol. 7:371‐374.
   Ralston, C.Y., Sclavi, B., Sullivan, M., Deras, M.L., Woodson, S.A., Chance, M.R., and Brenowitz, M. 2000b. Time‐resolved synchrotron X‐ray footprinting and its application to RNA folding. Methods Enzymol. 317:353‐368.
   Sclavi, B., Woodson, S., Sullivan, M., Chance, M.R., and Brenowitz, M. 1997. Time‐resolved synchrotron X‐ray “footprinting” a new approach to the study of nucleic acid structure and function: Application to protein‐DNA interactions and RNA folding. J. Mol. Biol. 266:144‐159.
   Sclavi, B., Sullivan, M., Chance, M.R., Brenowitz, M., and Woodson, S.A. 1998a. RNA folding at millisecond intervals by synchrotron hydroxyl radical footprinting. Science 279:1940‐1943.
   Sclavi, B., Woodson, S., Sullivan, M., Chance, M., and Brenowitz, M. 1998b. Following the folding of RNA with time‐resolved synchrotron X‐ray footprinting. Methods Enzymol. 295:379‐402.
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