Use of Chromophoric Ligands to Visually Screen Co‐Crystals of Putative Protein‐Nucleic Acid Complexes

Xiaohua Jiang1, Martin Egli1

1 Vanderbilt University School of Medicine, Nashville, Tennessee
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 7.15
DOI:  10.1002/0471142700.nc0715s46
Online Posting Date:  September, 2011
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Abstract

Distinguishing between crystals of protein‐nucleic acid complexes and those containing protein alone is a common problem in structural studies of protein‐nucleic acid interactions. Currently, there are several methods available for detecting nucleic acid in crystals, including gel electrophoresis, SYBR Gold fluorescence dye staining, and methyl violet staining. However, they require either that the crystals be sacrificed or access to a fluorescence microscope. In this protocol, we describe an approach that allows direct visualization of either the presence or absence of oligonucleotides in crystals grown from solutions containing both protein and nucleic acid—labeling with the Cy5 dye. In addition to offering the advantage of being able to distinguish between crystals of complex and protein alone with the naked eye or a light microscope, crystals of covalently Cy5‐labeled DNA can be directly used for X‐ray diffraction data collection. Curr. Protoc. Nucleic Acid Chem. 46:7.15.1‐7.15.8. © 2011 by John Wiley & Sons, Inc.

Keywords: crystallization; oligonucleotide conjugates; protein‐nucleic acid interactions; X‐ray crystallography

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Co‐Crystallization of a Duplex Composed of 5′‐Cy5‐Labeled DNA Primer and Unlabeled DNA Template with DNA Polymerase Eta from Saccharomyces cerevisiae (scPol η)
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Co‐Crystallization of a Duplex Composed of 5′‐Cy5‐Labeled DNA Primer and Unlabeled DNA Template with DNA Polymerase Eta from Saccharomyces cerevisiae (scPol η)

  Materials
  • Purified and desalted 5′‐Cy5‐labeled primer strand (5′‐Cy5‐GTGGTCAAG‐3′; Integrated DNA Technologies; http://www.idtdna.com/Home/Home.aspx)
  • Purified and desalted native DNA template strand 5′‐CTTCTTGACCAC‐3′ (IDT)
  • scPol η enzyme, catalytic core, residues 1‐513 (scPol η 1‐513) (Trincao et al., ; Alt et al., )
  • Ice
  • Magnesium chloride (Fisher Scientific)
  • 2′‐Deoxyadenosine‐5′‐[(α,β)‐methyleno]triphosphate (dAMPcPP; Jena Bioscience)
  • PEG Suite crystallization screen (Qiagen) containing reservoir solution
  • Microcentrifuge tubes
  • Various pipetman pipets and tips (Gilson)
  • Benchtop centrifuge
  • Heating block to anneal the primer and template strands
  • Vortex mixer
  • Siliconized glass circle cover slides (Hampton Research)
  • 24‐well plastic tray (Hampton Research)
  • Light microscope
  • Nylon loops for harvesting and flash‐freezing crystals (Hampton Research)
  • Liquid nitrogen
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Figures

Videos

Literature Cited

Literature Cited
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