Probing RNA Structure and Function by Nucleotide Analog Interference Mapping

Jesse C. Cochrane1, Scott A. Strobel1

1 Yale University, New Haven
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 6.9
DOI:  10.1002/0471142700.nc0609s17
Online Posting Date:  September, 2004
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Abstract

Nucleotide analog interference mapping (NAIM) can be used to simultaneously, yet individually, identify structurally or catalytically important functional groups within an RNA molecule. Phosphorothioate‐tagged nucleotides and nucleotide analogs are randomly incorporated into an RNA of interest by in vitro transcription. The phosphorothioate tag marks the site of substitution and identifies sites at which the modification affects the structure or function of the RNA molecule. This technique has been expanded to include identification of hydrogen bonding pairs (NAIS), ionizable functional groups, metal ion ligands, and the energetics of protein binding (QNAIM). The analogs, techniques, and data analysis used in NAIM are described here.

Keywords: RNAM; phoshphorothioate, nucleotide analog interference mapping

     
 
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Table of Contents

  • Strategic Planning
  • Basic Protocol 1: Selection for Ligand Binding
  • Basic Protocol 2: Activity Selection for Ribozymes
  • Basic Protocol 3: Selection for Structure or Folding
  • Support Protocol 1: Transcription and Purification of Phosphorothioate‐Tagged RNA
  • Basic Protocol 4: Identifying Positions of Interference and Controlling for the Level of Incorporation
  • Alternate Protocol 1: Nucleotide Analog Interference Supression (NAIS)
  • Alternate Protocol 2: Functional Group Ionization Determined by NAIM
  • Alternate Protocol 3: Metal Binding Functional Groups Identified by NAIM
  • Alternate Protocol 4: Quantitative Nucleotide Analog Interference Mapping (QNAIM)
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Tables
     
 
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Materials

Basic Protocol 1: Selection for Ligand Binding

  Materials
  • Wash buffer (see recipe)
  • Transcribed ribozyme RNA containing phosphorothioate‐tagged analogs (see protocol 4 and )
  • Stock of ligand
  • 500 mM Tris/HEPES buffer, pH 7.5 (see recipe)
  • 2 M KCl
  • 1 M MgCl 2
  • 0.1 M dithiothreitol (DTT)
  • 0.1% Igepal C‐680
  • 1 mg/mL tRNA
  • Filter elution buffer (see recipe)
  • Ethanol (EtOH), cold
  • Nitrocellulose membrane (Fisher Scientific)
  • Hybond‐N+ nylon filter (Amersham Pharmacia Biotech)
  • 90°C heating block
  • 1.5‐mL microcentrifuge tubes
  • Whatman paper
  • Glass plate
  • Platform rocker
  • Additional reagents and equipment for denaturing filter binding assays (see CPMB UNIT ) and denaturing gel electrophoresis (see appendix 3B)

Basic Protocol 2: Activity Selection for Ribozymes

  Materials
  • Transcribed ribozyme RNA containing phosphorothioate‐tagged analogs (see protocol 4 and )
  • 500 mM HEPES buffer, pH 7.0
  • 10 mM MgCl 2
  • 10 mM Mn(OAc) 2
  • Labeled substrate molecule (optional; see )
  • 2× formamide loading buffer (FLB; appendix 2A)
  • Gel elution buffer (see recipe)
  • Ethanol (EtOH), cold
  • 0.65‐mL microcentrifuge tubes
  • 37° and 50°C heating blocks
  • Autoradiography film
  • Platform rocker
  • Additional reagents and equipment for denaturing gel electrophoresis (see appendix 3B)

Basic Protocol 3: Selection for Structure or Folding

  Materials
  • Transcribed ribozyme RNA containing phosphorothioate‐tagged analogs (see protocol 4 and )
  • 500 mM Tris/HEPES buffer, pH 7.5 (see recipe)
  • 10 mM MgCl 2
  • 50% glycerol
  • Native gel mix (see recipe)
  • Gel elution buffer (see recipe)
  • Ethanol (EtOH), cold
  • 0.65‐mL microcentrifuge tubes
  • 70°C heating block
  • Autoradiography film
  • Platform rocker
  • Additional reagents and equipment for native gel electrophoresis (unit 11.4)

Support Protocol 1: Transcription and Purification of Phosphorothioate‐Tagged RNA

  Materials
  • 10× transcription buffer (see recipe)
  • 1 M MgCl 2 (store at 4°C)
  • 20× NTPs: 20 mM each ATP, CTP, GTP, and UTP (see Table 6.9.1)
  • 10× NTPαS: modified (δαS) or parent (NαS) phosphorothioate‐tagged nucleotide (see Table 6.9.1)
  • 1 µg/µL linearized template DNA, containing a T7 promoter upstream of the RNA gene to be transcribed
  • 0.1 mg/mL inorganic pyrophosphatase (store at −20°C)
  • 500 to 1000 U/µL T7 RNA polymerase (store at −20°C)
  • 2× formamide loading buffer (FLB; appendix 2A)
  • Gel elution buffer (see recipe)
  • 3 M sodium acetate (NaOAc), pH 5.2 ( appendix 2A)
  • 100% ethanol (EtOH), prechilled at −20°C
  • T 10E 0.1 buffer, pH 7.5 (see recipe)
  • 1.5‐mL microcentrifuge tubes
  • 37°C heating block
  • Platform rocker
  • Additional reagents and equipment for denaturing gel electrophoresis ( appendix 3B)

Basic Protocol 4: Identifying Positions of Interference and Controlling for the Level of Incorporation

  Materials
  • 2× formamide loading buffer (FLB; appendix 2A)
  • Selected RNA samples containing phosphorothioate‐tagged analogs (see Basic Protocols protocol 11, protocol 22, and protocol 33)
  • Parallel unselected RNA samples (control)
  • 50 mM I 2/EtOH: 50 mM I 2 dissolved in ethanol
  • Phosphorimager and screen (Molecular Dynamics)
  • ImageQuant software
  • Additional reagents and equipment for denaturing gel electrophoresis ( appendix 3B)

Alternate Protocol 1: Nucleotide Analog Interference Supression (NAIS)

  Materials
  • RNA molecules transcribed in the presence of sulfur‐substituted nucleotides
  • 100 mM Mn(OAc) 2
  • 100 mM TlOAc
  • Other mono‐ and divalent metals as required
  • 2% 2‐mercaptoethanol
  • Additional reagents and equipment for selection (see protocol 1) and for identification of sites of interference (see protocol 5)

Alternate Protocol 2: Functional Group Ionization Determined by NAIM

  Materials
  • K d RNA (labeled)
  • 500 mM Tris/HEPES buffer, pH 7.5 (see recipe)
  • 2 M KCl
  • 1 M MgCl 2
  • 0.1 M DTT
  • 0.1% Igepal C‐680
  • 1 mg/mL tRNA
  • 200× K d stock of ligand
  • Ligand dilution buffer (see recipe)
  • Wash buffer (see recipe)
  • Whatman paper
  • Cling film
  • Phosphorimager and screen
  • Additional reagents and equipment for dot blots (see CPMB UNIT ), for selection of active molecules based on ligand binding (see protocol 1), and for determining sites of interference (see protocol 5)
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Literature Cited

Literature Cited
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