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Methods in Molecular Biology™

Series Editor
John M. Walker
School of Life Sciences
University of Hertfordshire
Hatfield, Hertfordshire, AL10 9AB, UK

For other titles published in this series, go to

www.springer.com/series/7651
Drug-DNA Interaction Protocols

Second Edition

Edited by

Keith R. Fox
School of Biological Sciences, University of Southampton, Southampton, UK
Editor
Keith R. Fox
School of Biological Sciences
University of Southampton
Southampton
UK
[email protected]

ISSN 1064-3745 e-ISSN 1940-6029

ISBN 978-1-60327-417-3 e-ISBN 978-1-60327-418-0
DOI 10.1007/978-1-60327-418-0
Springer New York Dordrecht Heidelberg London

Library of Congress Control Number: 2009940801

© Humana Press, a part of Springer Science+Business Media, LLC 1998, 2010

All rights reserved. This work may not be translated or copied in whole or in part without the written permission of
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Preface

DNA has been known to be the cellular target for many cytotoxic anticancer agents for
several decades. The knowledge of its structure in atomic detail and the ease with which
DNA fragments (both synthetic oligonucleotides and natural sequences) can be prepared
and manipulated has aided the design of compounds that bind to it with improved selec-
tivity. On the basis of this information, new generations of sequence reading compounds
(including triplex forming oligonucleotides and minor groove binding ligands) have been
prepared, which have the potential for targeting specific DNA sequences as anti-gene
agents. Within the last 10 years, it has also become apparent that the familiar DNA duplex
is not the only structure that can be targeted by DNA-binding ligands and there has been
increased interest in triplex and quadruplex structures as drug targets, as well as protein-
DNA complexes, such as those with nucleosomes or topoisomerases.
Each of these advances has required the availability and development of an arsenal of
techniques for probing the interactions in both qualitative and quantitative terms. This vol-
ume of Methods in Molecular Biology brings together several techniques that are currently
useful for examining these interactions. Some of these are updates on ones that were included
in the earlier volume (Methods in Molecular Biology 90), published 12 years ago, while others
are new. Molecular science is a multidisciplinary enterprise, and while individuals and labo-
ratories may become experts in a few techniques, a detailed description of DNA-ligand
interactions requires a combination of approaches. This volume should therefore be useful
for established workers who wish to broaden their experimental repertoire, as well as for
those who are new to the field and need expert advice and guidance.
The chapters have all been written by scientists who are experts in their own fields.
They will obviously reflect their local preferences in experimental protocols, which can be
modified to suit the requirements of the individual researcher. Each chapter begins with a
short introduction, which outlines the background to the technique, the principles of its
application, and the importance of the particular method. The most important part of
each chapter is the methods section. These set out the experimental protocols in a step-
by-step fashion and are accompanied by Notes sections which provide technical tips, based
on experience, giving valuable information about potential problems and pitfalls and
emphasizing the points at which special care is required. This volume should therefore be
useful for post-graduates, post-doctoral workers, and established scientists, working in
drug-DNA interactions.
The chapters in this volume combine a wide range of approaches, from the cellular to
the structural. The first nine chapters describe various biophysical techniques for quantify-
ing drug-DNA interactions and for describing these in molecular and atomic detail, while
the later chapters describe molecular and cellular approaches. Together these provide
methods for assessing the strength and mode of binding, the sequence selectivity, and
their effect on biological systems.

Southampton, UK Keith R. Fox

v
Contents

Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix

1 Quantitative Analysis of Small Molecule–Nucleic Acid Interactions

with a Biosensor Surface and Surface Plasmon Resonance Detection . . . . . . . . . . 1
Yang Liu and W. David Wilson
2 Thermal Melting Studies of Ligand DNA Interactions . . . . . . . . . . . . . . . . . . . . . 25
Aurore Guédin, Laurent Lacroix, and Jean-Louis Mergny
3 Circular and Linear Dichroism of Drug-DNA Systems . . . . . . . . . . . . . . . . . . . . . 37
Alison Rodger
4 Drug Binding to DNA⋅RNA Hybrid Structures . . . . . . . . . . . . . . . . . . . . . . . . . . 55
Richard T. Wheelhouse and Jonathan B. Chaires
5 Quantification of Binding Data Using Capillary Electrophoresis . . . . . . . . . . . . . . 71
Fitsumbirhan Araya, Graham G. Skellern, and Roger D. Waigh
6 Determination of Equilibrium Association Constants of Ligand–DNA
Complexes by Electrospray Mass Spectrometry . . . . . . . . . . . . . . . . . . . . . . . . . . 89
Valérie Gabelica
7 Detection of Adriamycin-DNA Adducts by Accelerator Mass Spectrometry . . . . . 103
Kate Coldwell, Suzanne M. Cutts, Ted J. Ognibene, Paul T. Henderson,
and Don R. Phillips
8 Molecular Modelling Methods to Quantitate Drug-DNA Interactions . . . . . . . . . 119
Hao Wang and Charles A. Laughton
9 Application of Anomalous Diffraction Methods to the Study of DNA
and DNA-Complexes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133
Derrick Watkins, Tinoush Moulaei, Seiji Komeda,
and Loren Dean Williams
10 DNase I Footprinting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153
Antonia S. Cardew and Keith R. Fox
11 Methods to Characterize the Effect of DNA-Modifying Compounds
on Nucleosomal DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173
Vidya Subramanian, Robert M. Williams, Dale L. Boger,
and Karolin Luger
12 REPSA: Combinatorial Approach for Identifying Preferred
Drug–DNA Binding Sequences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 193
Michael W. Van Dyke
13 In vitro Transcription Assay for Resolution of Drug-DNA Interactions
at Defined DNA Sequences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207
Benny J. Evison, Don R. Phillips, and Suzanne M. Cutts
14 In vitro Footprinting of Promoter Regions Within Supercoiled Plasmid DNA . . . 223
Daekyu Sun

vii
viii Contents

15 Topoisomerase I-Mediated DNA Relaxation as a Tool to Study

Intercalation of Small Molecules into Supercoiled DNA . . . . . . . . . . . . . . . . . . . . 235
Paul Peixoto, Christian Bailly, and Marie-Hélène David-Cordonnier
16 A High-Throughput Assay for DNA Topoisomerases and Other Enzymes,
Based on DNA Triplex Formation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 257
Matthew R. Burrell, Nicolas P. Burton, and Anthony Maxwell
17 Measurement of DNA Interstrand Crosslinking in Individual Cells
Using the Single Cell Gel Electrophoresis (Comet) Assay . . . . . . . . . . . . . . . . . . . 267
Victoria J. Spanswick, Janet M. Hartley, and John A. Hartley
18 Measurement of DNA Interstrand Crosslinking in Naked DNA
Using Gel-Based Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 283
Konstantinos Kiakos, Janet M. Hartley, and John A. Hartley
19 An Evaluation Cascade for G-Quadruplex Telomere Targeting
Agents in Human Cancer Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 303
Mekala Gunaratnam and Stephen Neidle
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 315
Contributors

Fitsumbirhan Araya • Institute of Pharmacy and Biomedical Sciences,

University of Strathclyde, Glasgow, UK
Christian Bailly • Jean-Pierre Aubert Research Center (JPARC),
Institut de Recherches sur le Cancer de Lille, Lille, France
IMPRT-IFR114, Lille, France
Pierre Fabre Research Institute, Toulouse, France
Dale L. Boger • Department of Chemistry, The Scripps Research Institute,
La Jolla, CA, USA
Matthew R. Burrell • Dept Biological Chemistry, John Innes Centre,
Colney, Norwich, UK
Nicolas P. Burton • Inspiralis Ltd., Norwich Bioincubator,
Norwich Research Park, Norwich, UK
Antonia S. Cardew • School of Biological Sciences, University of Southampton,
Southampton, UK
Jonathan B. Chaires • James Graham Brown Cancer Center,
University of Louisville, Louisville, KY, USA
Kate Coldwell • Department of Biochemistry, La Trobe University,
Bundoora, VIC, Australia
Suzanne M. Cutts • Department of Biochemistry, La Trobe University,
Bundoora, VIC, Australia
Marie-Helene David-Cordonnier • INSERM U-837, Jean-Pierre Aubert Research
Center (JPARC), Institut de Recherches sur le Cancer de Lille, Lille, France
IMPRT-IFR114, Lille, France
Benny J. Evison • Department of Biochemistry, La Trobe University,
Bundoora, VIC, Australia
Keith R. Fox • School of Biological Sciences, University of Southampton,
Southampton, UK
Valérie Gabelica • Physical Chemistry and Mass Spectrometry Laboratory,
Department of Chemistry, University of Liège, Belgium
GIGA-Systems Biology and Chemical Biology, University of Liège, Belgium
Aurore Guédin • Equipe Santé, Laboratoire ‘Régulation et Dynamique des
Génomes’, Muséum National d’Histoire Naturelle USM 503, INSERM UR 565,
CNRS UMR 5153, Paris, France
Mekala Gunaratnam • The Cancer Research UK Biomolecular Structure Group,
The School of Pharmacy, University of London, London, UK
Janet M. Hartley • Cancer Research UK Drug-DNA Interactions Research Group,
UCL Cancer Institute, University College London, London, UK
John A. Hartley • Cancer Research UK Drug-DNA Interactions Research Group,
UCL Cancer Institute, University College London, London, UK

ix
x Contributors

Paul T. Henderson • Division of Hematology and Oncology, Department of Internal

Medicine, UC Davis Cancer Center, University of California Davis Medical
Center, Sacramento, CA, USA
Konstantinos Kiakos • Cancer Research UK Drug-DNA Interactions Research
Group, UCL Cancer Institute, Paul O’Gorman Building, University College
London, London, UK
Seiji Komeda • School of Chemistry and Biochemistry, Georgia Institute of Technology,
Atlanta, GA, USA
Laurent Lacroix • Equipe Santé, Laboratoire ‘Régulation et Dynamique des
Génomes’, Muséum National d’Histoire Naturelle USM 503, INSERM UR 565,
CNRS UMR 5153, Paris, France
Charles A. Laughton • Centre for Biomolecular Sciences, School of Pharmacy,
University of Nottingham, Nottingham, UK
Yang Liu • Department of Chemistry, Georgia State University, Atlanta, GA, USA
Karolin Luger • Department of Biochemistry and Molecular Biology, Colorado State
University, Fort Collins, CO, USA
Anthony Maxwell • Department of Biological Chemistry, John Innes Centre,
Colney, Norwich, UK
Jean-Louis Mergny • Equipe Santé, Laboratoire ‘Régulation et Dynamique des
Génomes’, Muséum National d’Histoire Naturelle USM 503, INSERM UR 565,
CNRS UMR 5153, Paris, France
Tinoush Moulaei • School of Chemistry and Biochemistry, Georgia Institute of
Technology, Atlanta, GA, USA
Stephen Neidle • The Cancer Research UK Biomolecular Structure Group,
The School of Pharmacy, University of London, London, UK
Ted J. Ognibene • Chemistry, Materials, Earth and Life Sciences, Center for
Accelerator Mass Spectrometry, Lawrence Livermore National Laboratory,
7000 East Avenue, L-452, Livermore, CA 94551, USA
Paul Peixoto • INSERM U-837, Jean-Pierre Aubert Research Center (JPARC),
Institut de Recherches sur le Cancer de Lille, Lille, France
IMPRT-IFR114, Lille, France
Don R. Phillips • Department of Biochemistry, La Trobe University,
Bundoora, VIC, Australia
Alison Rodger • Department of Chemistry, University of Warwick, Coventry, UK
Graham G. Skellern • Institute of Pharmacy and Biomedical Sciences,
University of Strathclyde, Glasgow, UK
Victoria J. Spanswick • Cancer Research UK Drug-DNA Interactions
Research Group, UCL Cancer Institute, Paul O’Gorman Building,
University College London, London, UK
Vidya Subramanian • Department of Biochemistry and Molecular Biology, Colorado
State University, Fort Collins, CO, USA
Daekyu Sun • Department of Pharmacology and Toxicology, College of Pharmacy,
University of Arizona, BIO5 Institute, Tucson, AZ, USA
Michael Van Dyke • Molecular & Cellular Oncology, M.D. Anderson Cancer
Center, University of Texas, Houston, TX, USA
 Contributors xi

Roger D. Waigh • Institute of Pharmacy and Biomedical Sciences,

University of Strathclyde, Glasgow G4 0NR, UK
Hao Wang • Department of Oral and Dental Science, University of Bristol, UK
Derrick Watkins • School of Chemistry and Biochemistry,
Georgia Institute of Technology, Atlanta, GA, USA
Richard T. Wheelhouse • School of Pharmacy, University of Bradford,
Bradford, West Yorkshire, UK
Loren D. Williams • School of Chemistry and Biochemistry,
Georgia Institute of Technology, Atlanta, GA, USA
Robert M. Williams • Department of Chemistry, Colorado State University,
Fort Collins, CO, USA
The University of Colorado Cancer Center, Aurora, CO, USA
W. David Wilson • Department of Chemistry, Georgia State University,
Atlanta, GA, USA
 Chapter 1

Quantitative Analysis of Small Molecule–Nucleic Acid

Interactions with a Biosensor Surface and Surface
Plasmon Resonance Detection
Yang Liu and W. David Wilson

Abstract
Surface plasmon resonance (SPR) technology with biosensor surfaces has become a widely-used tool for
the study of nucleic acid interactions without any labeling requirements. The method provides
simultaneous kinetic and equilibrium characterization of the interactions of biomolecules as well as small
molecule-biopolymer binding. SPR monitors molecular interactions in real time and provides significant
advantages over optical or calorimetic methods for systems with strong binding coupled to small
spectroscopic signals and/or reaction heats. A detailed and practical guide for nucleic acid interaction
analysis using SPR-biosensor methods is presented. Details of the SPR technology and basic fundamentals
are described with recommendations on the preparation of the SPR instrument, sensor chips, and samples,
as well as extensive information on experimental design, quantitative and qualitative data analysis and
presentation. A specific example of the interaction of a minor-groove-binding agent with DNA is
evaluated by both kinetic and steady-state SPR methods to illustrate the technique. Since the molecules
that bind cooperatively to specific DNA sequences are attractive for many applications, a cooperative
small molecule–DNA interaction is also presented.

Key words: Biosensor, Surface plasmon resonance, Small molecule–nucleic acid interaction, Kinetics,
Steady-state analysis, Cooperativity, Biacore, Minor groove

1. Introduction

Biological systems function on a platform of complex and

integrated biomolecular interactions (1–3). Signaling, transcription
control of gene expression, and a host of other complex processes,
frequently, are built on sets of sequential biomolecular interactions
and subsequent reactions (4–8). These sequential processes that
control and direct cellular functions can generally be understood

K.R. Fox (ed.), Drug-DNA Interaction Protocols, Methods in Molecular Biology, vol. 613,
DOI 10.1007/978-1-60327-418-0_1, © Humana Press, a part of Springer Science + Business Media, LLC 1998, 2010

1
2 Liu and Wilson

in terms of the interaction of a small number of molecules in each

step. Appropriate assembly of the entire sequential array gives the
beautifully intricate mechanism of cell function. The entire process
can be understood and explained on the basis of thermodynamic
theory that has been firmly established for over 100 years.
The biomolecular associations involve macromolecular complexes
as well as small molecule-macromolecule interactions that are
essential for the control of cell processes. Most drugs, for example,
are small molecules that must interact with a macromolecular
receptor in order to affect the target cell function in a selective
manner (5–12). The field of chemical biology is built around
design and use of small molecules to control specific aspects of
cell function through biopolymer interactions (13–15). In order to
understand this intricate array of interactions and control systems,
it is very informative to evaluate all of the specific sequential steps
that can be isolated. This quantitative interaction information is
essential to put available structural results into the appropriate
context of cell function.
To establish a basic quantitative characterization of the
interactions, it is essential to determine a set of basic thermody-
namic quantities (16–21). Two questions must be answered at
the start of the investigations: (1) what do we want to know
about the interactions and (2) how can we accurately and with a
reasonable effort determine the desired information? In the
optimum case, the binding affinity (the equilibrium constant, K,
and Gibbs energy of binding, DG), stoichiometry (n, the number
of compounds bound to the biopolymer), cooperative effects
in binding, and binding kinetics (the rate constants, k, that
define the dynamics of the interaction) should be determined.
These fundamental parameters are the keys to a molecular under-
standing of the interactions and how they affect cellular functions.
To determine these parameters, an accurate method of determining
the concentration of each component that is not bound and the
concentration of their complex is required. The information must
be determined as a function of concentration at equilibrium for
accurate K and n, and as a function of time for k. For each system
then, the question becomes one of how to accurately determine
the necessary concentrations as a function of time, reactant total
concentrations, solution conditions, temperature, etc. The inter-
acting systems can range from as little as two molecules to as
many as required to form the final complex. As described above,
a more complete understanding of the biomolecular complexes
requires determination of additional thermodynamic quantities
that characterize complex formation in detail.
Because biological molecules have widely different molecular
characteristics that can and generally do change on complex
formation, it can be difficult to find methods to evaluate the
full array of interactions under an appropriate variety of conditions.
Quantitative Analysis of Small Molecule–Nucleic Acid Interactions 3

This is the “reasonable effort” part of question (2) above.

For many biologically important interactions, binding is very
strong, and experiments must be conducted at very low
concentrations, down to the nanomolar or lower levels. In a
binding experiment, the compound concentration should vary
from below to above the Kd in order to obtain accurate binding
results, and this requires significant concentrations of both free
and complexed molecules (16, 22–24). In many methods, for
example, those that involve some separation of free and bound
forms such as dialysis, the unbound concentration has to be
determined, and if this is below around 100 nM, as expected for
the large K values generally observed in biological systems and as
generally required for effective drug molecules, measurement can
be difficult or impossible. These concentrations fall below the
detection limit for many systems and may require special methods,
such as radiolabels or fluorescent probes, for added sensitivity in
detection. The labels may significantly increase the sensitivity of
detection; however, they may also perturb the interaction that
is being investigated. An attractive alternative method, which is
operational down to very low concentrations, is the use of
biosensors with surface plasmon resonance (SPR) detection
(17, 25–30). SPR responds to the refractive index or mass
changes at the biospecific sensor surface on complex formation
(27–32). Since the SPR signal responds directly to the amount
of bound compound in real time, as versus indirect signals at
equilibrium that are obtained for many physical measurements, it
provides a very powerful method to study biomolecular interaction
thermodynamics and kinetics. Use of the SPR signal and direct
mass response to monitor biomolecular reactions also removes
many difficulties with labeling or characterizing the diverse
properties of biomolecules (25–31).
To illustrate the power of the SPR method, small organic
cation interactions with specific DNA sequences will be used as
examples. Small molecule targeting of DNA has applications in
therapeutics, from cancer treatment to antiparasitic applications,
and in biotechnology, and development of such molecules for
control of nucleic acid function is at the heart of chemical biology
(5–10, 16, 25, 29, 32–47). The interaction of nucleic acids with
small molecules has been a primary area of interest and research
since before the discovery of the double helical structure of DNA.
There are many successful anticancer agents that intercalate or
alkylate DNA, and they can have quite varied structure and
properties (34–37). Dicationic minor groove binding heterocycles
which target eukaryotic organisms that cause parasitic diseases,
such as malaria and sleeping sickness, have also been known for
many years and are used in humans and animals (9, 10, 38–40).
Therapeutic targeting of nucleic acids recently entered a new and
very exciting area with the discovery that four-stranded, quadruplex
4 Liu and Wilson

DNA structures can occur in important cellular DNA regions from

chromosomal telomeres to oncogene promoters (34, 35, 41–46).
Several studies have clearly shown that interactions of a range
of small molecules with quadruplexes can yield anticancer
activity. Although this discussion focuses on DNA, essentially
all of the methods can be applied to RNA interactions.
Additional treatment of the biosensor surfaces may be required
when using RNA, however, due to possible hydrolysis of RNA
by a number of agents.
Applications with small molecules can be very challenging, in
terms of signal obtained on binding in a biosensor-SPR experi-
ment. The larger the bound molecule, the larger the SPR signal
and macromolecule binding can give large signal to noise ratios
(27, 29, 31). We will show, however, that the biosensor-SPR
method with a macromolecular sensor surface can be used under
appropriate conditions to investigate small molecule binding
with current state-of-the-art instruments. Because of the varied
properties of the compounds, it is difficult and time-consuming
to find other suitable methods that can quantitatively define
their interactions with DNA (or RNA) under a variety of
conditions.
As described above, in biosensor-SPR instruments and exper-
iments, it is the bound compound on the surface that is detected
by the change in plasmon resonance angle, and this can be deter-
mined very accurately from a low to a high fraction of surface
binding sites covered. The unbound or free solution compound
concentration is not measured but is simply prepared by dilution
and is in a constant concentration in the solution that flows over
the sensor surface. Since the SPR signal responds to the refractive
index or mass changes at the biospecific sensor surface on
complex formation, no specific labeling of detection ability for
the compounds is required (25–27, 31). For compounds that
have molecular weights of approximately 300 or more, the SPR
technology provides a very attractive method to study interactions
with nucleic acids or proteins immobilized to form a biospecific
target surface.

1.1. Fundamentals For a single compound (C), binding to n equivalent sites on DNA
of Molecular (or other biopolymer) to give a complex (DNA·nCbound), the
Interactions by interaction process is described by kinetic and equilibrium
Biosensor-SPR equations as follows:
Methods
DNA.nCbound Ka =ka/kd = 1/ Kd (1)
ka
DNA (site) + nCfree kd

More complex models with nonequivalent sites are also

well-known and are treated in a similar manner with more complex
functions (27) (and described below under Subheading 3.2).
Although a discussion of complex data fitting is beyond the
Quantitative Analysis of Small Molecule–Nucleic Acid Interactions 5

scope of this article, the topic is covered in most biophysical

chemistry texts, and more complex models are included in
commercial SPR instrument software packages as well as in
software available from Myszka and coworkers (http://www.
cores.utah.edu/interaction/software.html).
In the most common case, it is of interest to study a variety of
compounds for binding to a limited number of different DNAs.
For this case, however, it is more convenient and efficient to
immobilize, the DNAs on the sensor surface to monitor complex
formation. Biacore T100 and 2000/3000 instruments have
sensor chips with four channels such that three DNAs can be
immobilized, with one flow cell left blank as a control for bulk
refractive index subtraction. With a common sensor surface that
has covalently attached streptavidin, a nucleic acid strand with
biotin linked to either the 5¢ or 3¢ terminus can be captured to
create the biospecific surface. The terminal attachment of biotin,
through a flexible linker, leaves the nucleic acid binding sites open
for complex formation. If streptavidin or the biotin linker creates
problems with the interaction or nonspecific compound interac-
tions, the nucleic acid can be synthesized with a terminal alkyl
amine and the amine can be used to form a direct covalent amide
bond with the surface through activation of carboxyl groups on
the sensor surface. In Biacore instruments, which have been most
widely used to date in the area of biosensor-SPR experiments, any
molecule with free amino groups can be immobilized through
amide bonds with activated carboxyl groups from sensor chip
surface-linked carboxymethyl (CM) dextran. A range of other
sensor chips surfaces and immobilization chemistries are also
available and it is generally possible to find an appropriate surface
for any biological interaction application (for Biacore instruments,
see the web http://www.biacore.com/lifesciences/products/
sensor_chips/guide/index.html).
The results from a biosensor-SPR experiment are typically
presented as a set of sensorgrams, which plot a function that is
directly related to the SPR angle versus time as shown in Fig. 1.
The angle change is reported in Biacore technology as resonance
units (RU) where a 1,000 RU response is equivalent to a change
in surface concentration of about 1 ng/mm2 of protein or DNA
(the relationship between RU and ng of material bound will vary
with the refractive index of the bound molecule) (27, 29, 31).
With a DNA sequence immobilized on the chip surface, a
compound solution is injected and as the solution flows over the
surface, compound binding to the DNA is monitored by a change
in SPR angle (as RU). After a selected time, buffer flow is restarted
and dissociation of the complex can be monitored for an
additional selected time period (Fig. 1). Note that when the
association phase collection time is long enough, a steady state
plateau is reached such that the rate of binding of the small
6 Liu and Wilson

Fig. 1. Representative SPR sensorgrams for the interaction of DB293 with AATT and ATGA oligomer hairpin duplexes.
The DB293 concentrations from bottom to top are 0–1 mM

molecule equals the rate of dissociation of the complex and no

change of signal with time is observed. The response of interest is
the difference between the response in cells with immobilized
DNA minus the response of the blank flow cell without DNA. If
the added molecule does not bind to a target/receptor at the
surface, the SPR angle change in the sample and reference flow
cells will be the same in a properly functioning instrument, and
the signals, after subtraction, give a zero net RU response that is
indicative of no binding. In the case in which binding does
occur, an extra amount, relative to the blank surface, of the
added molecule is bound at the sensor surface, and an addi-
tional SPR angle change is generated in the sample flow cell.
Again, the amount of unbound compound in the flow solution is
the same in the sample and reference flow cells, and is subtracted
so that only the bound molecule generates a positive SPR
signal. The concentration of the unbound molecule is constant
and is fixed by the concentration in the flow solution.
Both the association and dissociation phases of the sensor-
gram can be simultaneously fit to a desired binding model in
several sensorgrams at different concentrations with a global fit
routine (27–32). Global fitting allows the most accurate determi-
nation of the kinetics constants as well as calculation of the
equilibrium constant, Ka, from the ratio of kinetic constants (Eq. 1).
It is also possible to determine Ka independently of rate constants
by fitting the steady-state response versus the concentration of
the binding molecule in the flow solution over a range of concen-
trations. For the binding process in Eq. (1) the steady-state RU
Quantitative Analysis of Small Molecule–Nucleic Acid Interactions 7

at each compound concentration is determined and Ka can be

obtained by fitting to the following equation:
RU = (RUmax • n • Ka • Cf)/(1 + Ka • Cf) (2)
where RUmax is the maximum change in RU for binding to a
single DNA site. It can be calculated, determined experimentally
at high compound concentrations or used in the above equation
as a fitting parameter such that Ka, n and RUmax are determined
by fitting RU versus C f. Note that the common term “r”,
the moles of compound bound per mole of DNA, is equal to
RU/RU max. At high C f,where all binding sites are filled with
compound, RU = RUmax • n.
The refractive index change in SPR experiments generates
essentially the same response for each bound molecule and can
provide a direct determination of the stoichiometry, n, in Eqs. 1
and 2, provided that the amount of immobilized nucleic acid is
known (29, 31). If the complex dissociates slowly, the surface can
be regenerated before complete dissociation has occurred by a
solution that causes rapid dissociation of the complex without
irreversible damage to the immobilized DNA (29, 47). For example,
a solution at low or high pH can unfold DNA and cause complex
dissociation. It should be noted, however, that accurate fitting of
the dissociation part of the curve requires collection of as much
of the total dissociation signal as possible. Additional injections of
buffer at pH near 7 allow the DNA to refold for the next binding
experiment. If the immobilized DNA is composed of separate
strands, the duplex must be reformed by hybridization.
After the dissociation/regeneration phase is over and a stable
baseline is reestablished, a second concentration sample can be
injected to generate a second sensorgram. This process can be
repeated with as many concentrations as needed to obtain a broad
coverage of the fraction of compound bound to the nucleic acid
site or sites (see Fig. 1). The kinetic and equilibrium constants
that describe the reaction in Eq. 1.1 are obtained by global fitting
the sensorgrams with equations from a kinetic model or by fitting
steady-state RU versus concentration plots to an appropriate
binding model. The models are the same for all types of binding
experiments and are not unique to SPR methods. It should be
emphasized that to obtain accurate kinetic information for a
binding reaction, it is essential that the kinetics for transfer of
the binding molecule to the surface immobilized nucleic acid
(mass transfer) be faster than the binding reaction. Equilibrium
information can be obtained, however, by fitting sensorgrams
even when mass transfer limitations prevent accurate determi-
nation of kinetics (48). It should be emphasized that, when
properly conducted, biosensor-SPR kinetic and equilibrium
results are in excellent agreement with other methods (26, 27,
29, 30, 48–50).
8 Liu and Wilson

2. Materials

2.1. Required These materials are for the Biacore T100, 3000 and 2000
Materials for research instruments but similar materials are required for other
Instrument Cleaning instruments.
1. Maintenance chip with a glass flow cell surface.
2. 0.5% SDS (Biacore desorb solution 1).
3. 50 mM glycine pH 9.5 (Biacore desorb solution 2) (see Note 1).
4. 1% (v/v) acetic acid solution.
5. 0.2 M sodium bicarbonate solution.
6. 6 M guanidine HCl solution.
7. 10 mM HCl solution. (see Note 2)

2.2. Running Buffer 1. HBS-EP buffer: 10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM

for Immobilization EDTA, 0.005%, v/v polysorbate 20. (GE Healthcare Inc.)
of DNA 2. HBS-N buffer: 10 mM HEPES pH 7.4, 150 mM NaCl.
(GE Healthcare Inc.)
3. Filter and degas all solution quite thoroughly.
4. It should be emphasized that the internal flow system of
the instrument has microcapillaries that can be damaged by
particulate matter in any solution.

2.3. Sensor chip 1. A CM5 or CM4 sensor chip that has been at room temperature
Preparation for DNA for at least 30 min (all sensor chips are available from GE
Immobilization: CM5 Healthcare Inc.).
or CM4 Chip 2. 100 mM N-hydroxysuccinimide (NHS) freshly prepared in water.
3. 400 mM N-ethyl-N¢-(dimethylaminopropyl) carbodiimide
(EDC) freshly prepared in water.
4. 10 mM acetate buffer pH 4.5 (immobilization buffer).
5. 200–400 mg/ml streptavidin in immobilization buffer.
6. Amino-labeled nucleic acid solutions (~25 nM of single strand
or hairpin dissolved in HBS-EP buffer). (5¢- end modified DNA
obtained from Integrated DNA Technologies, Coralville, IA)
7. 1 M ethanolamine hydrochloride in water pH 8.5 (deactivation
solution).
8. Dock the CM4 or CM5 chip, Prime with running buffer. Start
a sensorgram in all flow cells with a flow rate of 5 µl/min.
“Dock” and “Prime” are Biacore software commands that
instruct the instruments to carry out specific operations. The
commands and operations are listed in Table 1.
9. With NHS (100 mM) in one vial and EDC (400 mM) in other, use
the “Dilute” command to make a 1:1 mixture of NHS/EDC.
 Quantitative Analysis of Small Molecule–Nucleic Acid Interactions 9

Table 1
Biacore instrument commands

Software commands Function

Dock Docks the sensor chip

Undock Undocks the sensor chip
Prime Flushes the flow system with running buffer

Dilute Diluting samples with buffer or for preparing a defined mixture of two
samples
Manual inject Manually controlled injection
Desorb Removes adsorbed samples from the autosampler and IFC using SDS and
glycine
Sanitize Cleans pumps, IFC and autosampler from micro-organisms using BIA
disinfectant solution

10. Inject NHS/EDC for 10 min (50 µl) to activate the car-
boxymethyl surface to reactive esters.
11. Using “Manual Inject” with a flow rate of 5 µl/min, load the
loop with ~100 µl of streptavidin in the appropriate buffer
and inject streptavidin over all flow cells. Track the number of
RUs immobilized, which is available in real time readout, and
stop the injection after the desired level is reached (typically
2,500–3,000 RU for CM5 chip and 1,000–1,500 RU for
CM4 chip).
12. Inject ethanolamine hydrochloride for 10 min (50 µl) to
deactivate any remaining reactive esters.
13. Prime several times to ensure surface stability.
14. DNA is immobilized as described under Subheading 2.4.
15. To reduce the nonspecific binding to immobilized streptavi-
din by small molecules, covalent immobilization using amino-
labeled DNA could also be used. In this way, DNA can be
directly captured on the EDC/NHS activated carboxymethyl
dextran surface of the sensor chip (from step 16 to step 19).
16. Start from step 8 to step 10 to activate the sensor chip sur-
face. Then, start a new sensorgram with a flow rate of 2 µl/
min and select one desired flow cell on which to immobilize
the nucleic acid.
17. Use Manual Inject, load the injection loop with ~100 µl of a
25 nM nucleic acid solution and inject over the low cell. Track
the number of RUs immobilized and stop the injection after
a desired level is reached (see Note 3).
10 Liu and Wilson

18. Inject ethanolamine hydrochloride for 10 min (50 µl) to

deactivate any remaining reactive esters.
19. Prime several times to ensure surface stability.

2.4. Sensor chip 1. A streptavidin-coated sensor chip (SA chip or prepared as out-
Preparation for DNA lined above) that has been at room temperature for at least
Immobilization: SA 30 min.
Chip 2. HBS-EP buffer is used as running buffer.
3. Activation buffer: 1 M NaCl, 50 mM NaOH.
4. Biotin-labeled nucleic acid solutions (~25 nM of single-strand
or hairpin dissolved in HBS-EP buffer).
5. Dock a streptavidin-coated chip and start a sensorgram with
a 20 µl/min flow rate.
6. Inject activation buffer: 1 M NaCl, 50 mM NaOH for 1 min
(20 µl) five to seven times to remove any unbound streptavi-
din from the sensor chip.
7. Allow buffer to flow at least 5 min before immobilizing the
nucleic acids.
8. Start a new sensorgram with a flow rate of 2 µl/min and select
one desired flow cell on which to immobilize the nucleic acid.
Take care not to immobilize nucleic acid on the flow cell cho-
sen as the control flow cell. Generally, flow cell 1 (fc1) is used
as a control and is left blank for subtraction. It is often desir-
able to immobilize different nucleic acids on the remaining
three flow cells (see Note 4).
9. Wait for the baseline to stabilize which usually takes a few
minutes. Use Manual Inject, load the injection loop with
~100 µl of a 25 nM nucleic acid solution and inject over the
low cell. Track the number of RUs immobilized and stop the
injection after a desired level is reached (see Note 3).
10. At the end of the injection and after the baseline has stabilized,
use the instrument crosshair to determine the RUs of nucleic
acid immobilized and record this amount. The amount of
nucleic acid immobilized is required to determine the theo-
retical moles of small molecule binding sites for the flow cell.
11. Repeat steps 4–6 for another flow cell (e.g., fc3 or fc4)
(see Note 5).

2.5. Sensor chip 1. A HPA sensor chip that has been at room temperature for at
Preparation for DNA least 30 min (see Note 6).
Immobilization: HPA 2. HBS-N buffer: 10 mM HEPES pH 7.4, 150 mM NaCl is
Chip used as running buffer (see Note 7).
3. 40 mM n-octyl glucoside (Sigma Chemical Co.) water
solution.
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