CHAPTER 7Biophysical Analysis of Nucleic Acids

INTRODUCTION

T he opening commentary (UNIT 7.1) by Tinoco defines the limitations of the currentstate of the art in the biophysical analysis of nucleic acids and sets the stage for the

units that follow. In UNIT 7.2, James explains the steps to be followed in determining anNMR structure. He also discusses the care that is necessary to be sure that the structuredetermined is in fact the correct structure. The unit on optical methods (UNIT 7.3) by Plumis likely to be one of the most heavily used units in this volume and will be of particularimportance to the novice. The section on determination of oligonucleotide extinctioncoefficients, in particular, is essential to anyone needing to know solution concentrations,and the section on analysis of equilibrium melting curves contains all of the relevantequations and a discussion of which are most appropriate to a given situation.

Calorimetric methods (UNIT 7.4) are becoming much more commonly used, and, whilequite powerful, there are numerous sources of error. Pilch gives a thorough explanationof the care that is necessary to obtain meaningful results, as well as the relevant methodsfor analysis of the data produced.

In UNIT 7.5, Cheatham, Brooks, and Kollman give an introduction to computer simulationof nucleic acids. This topic is continued in UNITS 7.8-7.10, which provide more in-depthcoverage of energy and sampling, electrostatics and solvation, and setup and analysis.

In UNIT 7.6, Doudna and Ferre-D’Amare include crystallization conditions for twentyRNA and RNA-protein complexes. The overall goal of the unit, however, is to providegeneral guidance, rather than to attempt to give a detailed protocol or set of protocols.The RNA theme continues in UNIT 7.7, which deals with NMR determination of RNAstructure. Williamson focuses on the assignment of resonances and the calculation ofstructures. For preparation of the isotopically labeled RNA, references are given forpublished procedures.

UNIT 7.11 discusses analysis of DNA structure by circular dichroism (CD). For nucleicacids, CD is typically used to monitor electronic transitions in nucleobases. It is extremelysensitive to changes in environmental conditions such as pH, temperature, and ionicstrength. This unit provides both procedural guidelines and representative spectra toillustrate the usefulness of the method.

The antigene approach requires that a single-stranded oligonucleotide recognize double-stranded DNA to form a triplex structure. The two most common methods for evaluating,in vitro, the potency of modified oligonucleotides to associate with dsDNA are gel-shiftelectrophoresis and triplex melting. These methods, although conceptually simple, canbe tricky to perform. They are described in detail in UNIT 7.12.

Starting with the structure determination of the DNA double helix by X-ray fiber diffrac-tion, diffraction approaches have had a major impact on our understanding of nucleicacid structure and function. UNIT 7.13 provides an overview of diffraction techniques instructural biology. Following brief reviews of X-ray and electron and neutron diffraction,as well as electron microscopy, the unit gives a detailed account of the various steps

Current Protocols in Nucleic Acid Chemistry 7.0.1-7.0.2, September 2011Published online September 2011 in Wiley Online Library (wileyonlinelibrary.com).DOI: 10.1002/0471142700.nc0700s46Copyright C© 2011 John Wiley & Sons, Inc.

BiophysicalAnalysis ofNucleic Acids

7.0.1

Supplement 46

Introduction

7.0.2

Supplement 46 Current Protocols in Nucleic Acid Chemistry

involved in a single-crystal X-ray diffraction experiment, from sample preparation andcrystallization to model refinement and quality control.

X-ray crystallography is a key approach in the analysis of protein-nucleic acid inter-actions. Crystals grown from droplets with the protein-DNA or protein-RNA complexmay occasionally contain just one of the components (i.e., the protein), thus prevent-ing a structural investigation of the complex. UNIT 7.15 describes a facile approach usingoligonucleotides labeled with the Cy5 or Cy3 dyes to directly visualize the presence ofcomplex in crystals.

Water plays an important role in many biological processes and constitutes an integralpart of nucleic acid structure. Although crystal structures at high resolution can pinpointthe positions of first and second shell water molecules, the energetic contributions ofhydration to oligonucleotide pairing affinity, single-molecule conformational changes,and the formation of macromolecular assemblies are harder to assess. UNIT 7.14 introducesthe osmotic stress technique that probes the changes in hydration that accompany bio-logically relevant equilibria by way of the dependence of the equilibrium constant onthe water activity. The protocol contains a step-by-step description of the experimentalprocedures to determine the number of water molecules released upon melting of anoligonucleotide duplex.

Roger Jones, Piet Herdewijn, and Martin Egli