Published online November 1, 2004
Nucleic Acids Research, 2004, Vol. 32, No. 19 e154
doi:10.1093/nar/gnh152
Optical absorption assay for strand-exchange
reactions in unlabeled nucleic acids
Besik I. Kankia*
Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN 55455, USA
Received September 14, 2004; Revised and Accepted October 15, 2004
ABSTRACT
The nucleic acid exchange reaction is a common
feature for genetic recombination, DNA replication
and transcription. Due to the fact that in the strandexchange reactions the reactant and product molecules have similar or identical nucleotide sequences,
the reaction is undetectable. As a rule, the nucleic
acids with radioactive or fluorescence labels are
used in such studies. Besides the fact that the labels
can perturb the reaction and pose a health risk to the
investigators, the assays usually involve extra experimental steps: quenching the reaction, separation,
visualization and quantification of the products.
Here, we describe a straightforward, direct and precise method to study strand-exchange reaction of
unlabeled nucleic acids by real-time measurements
of optical absorption. The method takes advantage
of the property of some guanine-rich oligonucleotides
to adopt monomolecular quadruplex conformation in
the presence of certain cations. The conformation is
characterized by significant absorption in long-wavelength range of the ultraviolet region where usually
other secondary structures are transparent. The ‘signal’ oligonucleotide is incorporated into reactant
duplex by annealing with target sequence. Adding
the replacement sequence initiates the release
of the ‘signal’ oligonucleotide into solution, which
is accompanied by ultraviolet absorption in longwavelength range.
experiments: besides the costly labeling process, attached
bulky fluorophores disturb the replacement reactions (1),
and working with radiolabel materials is undesirable since
they pose health risk and require special containment procedures. Furthermore, studies with labeled nucleic acids require
additional experimental steps: quenching the replacement
reaction at the appropriate time, separation, visualization
and quantification of the products (1,4–10). It is obvious
that there exists a need for rapid, sensitive and convenient
method to study nucleic acid replacement reaction.
In this paper, we described a method to monitor the
replacement kinetics of unmodified nucleic acids by simple
measurements of optical absorption. The assay takes advantage of the unique optical characteristics of guanine-rich
oligonucleotides folded into monomolecular quadruplex
structure [strong circular dichroism (CD) and optical density
(OD) signals in long-wavelength range of ultraviolet (UV)
region]. These oligonucleotides can be incorporated into initial
mismatched duplex by annealing with target sequence. Adding
the replacement sequence induces the release of the ‘signal’
oligonucleotide into solution, which is accompanied by an
increase in CD or OD at 300 nm.
The validity of the assay is demonstrated on DNA oligonucleotide G2T2G2TGTG2T2G2, so-called thrombin aptamer
(TBA) (11). In the presence of Sr2+, the oligonucleotide
folds into monomolecular quadruplex (Figure 1A), which is
INTRODUCTION
Displacement of a nucleic acid strand from a duplex by synchronized replacement with another equivalent or similar
strand is a common feature for many reactions in vivo. For
instance, the nucleic acid replacement occurs in genetic
recombination, DNA replication and transcription. Since the
displaced and replaced single strands, and consequently reactant and product duplexes have similar or identical sequences,
the reaction is practically invisible. The replacement process
becomes visible only after chemical modification of the
nucleic acids; for instance, attaching fluorescence (1–3) or
radioactive (4–10) labels. Obviously, this complicates the
Figure 1. (A) Sequence of the TBA and its schematic structure. (B) Scheme of
the strand-exchange reaction. Mismatched base pairs are shown in bold face.
*Tel: +1 612 624 5954; Fax: +1 612 626 7431; Email: [email protected]
Nucleic Acids Research, Vol. 32 No. 19 ª Oxford University Press 2004; all rights reserved
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characterized by strong positive band at 300 nm in both CD
and differential absorption spectra (12,13). TBA is preannealed with the complementary strand with two GA mismatches and used as a displacement strand (see Figure 1B).
After adding the replacement strand, which is fully complementary to target strand, thermodynamically favorable but
slow strand-exchange reaction takes place. The reaction
products are fully base-paired duplex and released TBA
(see Figure 1B). Released TBA binds Sr ions and folds into
quadruplex structure with strong absorption at 300 nm. Thus,
monitoring of the CD or optical absorbance signal of the
reaction allows study of strand-exchange kinetics.
MATERIALS AND METHODS
Oligonucleotides were supplied by Eppley Research Institute
at UNMC (Nebraska) and were synthesized on solid support
using standard phosphoramide chemistry. Upon completion of
synthesis, oligonucleotides were HPLC purified and desalted
by dialysis at 4 C against water using Spectrum dialysis tubing
with a molecular weight cutoff of 500 Da. The concentration
of the oligomer solutions was determined at 260 nm and
80 C using the following molar extinction coefficients,
in mM1cm1 of strands: 145 for G2T2G2TGTG2T2G2, 141
for C2A2C2ACAC2A2C2, 140 for GT3G2TGTG2T3G and 149
for CA3C2ACAC2A3C. These values were calculated by extrapolation of the tabulated values of the dimers and monomer
bases (14) at 25 C to high temperatures using procedures
reported earlier (15).
UV absorption experiments were conducted on a GBC 918
spectrophotometer equipped with thermoelectrically controlled cell holder. Absorbance versus temperature profiles
(melting curves) were measured at either 260 or 300 nm.
The temperature was scanned at a heating rate of 0.5 C per
minute. CD spectra were obtained with a JASCO J710 spectropolarimeter equipped with a water-jacketed cell holder. Quartz
cells with 10 mm path lengths were used in all studies. In a
typical strand exchange experiment, the initial DNA duplex
was incubated at the given temperature in the optical cell
placed in the spectrophotometer cell holder. The exchange
reaction was started when the displacement strand was
added into the cell.
RESULTS
Spectral properties of the oligonucleotides
Here, we present detailed optical studies of the oligonucleotides involved in the reaction. In addition to UV absorption, we
used CD technique because it is more sensitive to the sequence
and secondary structure of the nucleic acids.
Figure 2 shows CD spectra of reactant (mismatched) and
product (perfect) duplexes in 2 mM SrCl2, 100 mM NaCl,
5 mM Na-HEPES, pH 7.5. The CD spectra of perfect duplex
(solid line) and its version with two GA mismatches (dashed
line) do not differ qualitatively from each other. Both spectra
correspond to canonical B-DNA with the positive band
at 275 nm and the negative band at 250 nm (16). UV spectra
of the same duplexes did not reveal any measurable difference
Figure 2. CD spectra of the final (perfect) duplex (straight line) and initial
(mismatched) duplex (dashed line) in 100 mM NaCl, 2 mM SrCl2, 5 mM
Na-HEPES, pH 7.5. Concentration per duplex: 5 mM.
(data not shown). Thus, the duplexes on both sides of the
reaction (Figure 1) contribute into UV absorbance identically.
Figure 3 shows circular dichroism (CD), absorbance (A) and
absorbance difference (DA) spectra of displacement (left
panel) and replacement (right panel) strands in 2 mM
SrCl2, 100 mM NaCl, 5 mM Na-HEPES, pH 7.5 at 20 C
(solid lines) and in the same buffer without Sr ions (dashed
lines). In the absence of Sr ions, both strands show similar CD
and absorbance spectra (dashed lines). Adding of Sr ions into
displacement strand or TBA solution reveals dramatic effects
(left panel), while the spectra of replacement strand stays
essentially the same (right panel). CD spectrum of TBA in
the presence of Sr ions is characterized by strong positive
bands at 250 and 300 nm, and negative band 275 nm,
which is in excellent agreement with earlier measurements
under similar conditions (12). Similarly, adding of Sr2+
induces significant effects in absorbance spectrum of the
displacement strand.
Figure 3 visualizes two important aspects. First, importance
of Sr ions in quadruplex formation, and subsequently in the
strand-exchange reaction. As one can see from Figure 3, in the
absence of Sr ions, reactant and product oligonucleotides have
similar optical properties, and the strand-exchange reaction,
shown in Figure 1B, is invisible. Second, two single mutations,
exchange of guanines for thymines at positions 2 and 14 (see
Figure 1), is completely enough to inhibit Sr2+-induced
G-quartet formation.
UV difference spectrum of displacement strand in the presence of Sr2+ shows three peaks: 240, 275 and 300 nm. It
is clear that the best optical window for the tracking of the
strand-exchange reaction is at 300 nm: (i) the effect is the
largest; and (ii) this optical window is almost transparent for
all other reactants.
Thermodynamic properties
In order to choose the reaction temperatures properly, we have
studied unfolding of all reactant and product molecules.
Figure 4A shows UV melting curves of the duplexes and
displacement single-strand, TBA, in 100 mM NaCl, 2 mM
SrCl2, 5 mM Na-HEPES at pH 7.5 and at 4 mM strand
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Figure 3. Circular dichroism (CD), absorbance (A) and absorbance difference spectra (DA) of displacement (left column) and the replacement (right column) without
(dashed lines) and with 2 mM Sr ions (straight lines). Spectra are recorded in 100 mM NaCl, 5 mM Na-HEPES, pH 7.5 at 20 C. Concentration per strand: 5 mM.
Table 1. Thermodynamic parameters for duplex formationa
Duplex
Tmb ( C)
DG20 (kcal/mol)
DH (kcal/mol)
DHpred (kcal/mol)
DS (su)
DSpred (su)
Perfect (product)
Mismatched (reactant)
55.1
47.0
20.3
14.8
108.7
77.9
115.0
80.1
301.6
215.4
314.6
219.1
a
The parameters determined in 100 mM NaCl, 2 mM SrCl2, 5 mM Na-HEPES, pH 7.5.
The Tm values refer to a total strand concentration of 3.5 mM.
b
concentration. All melting curves are sigmoidal indicating on
typical two-state transition. In the case of TBA, the process is
hypochromic, which is opposite to the hyperchromic effects of
the duplexes, and typical for quadruplex melting 300 nm
(12,17). Due to the fact that our duplexes are bimolecular, their
melting temperature, Tm, depends on the strand concentration.
Therefore the melting curves of the duplexes were recorded at
different strand concentrations and the results are shown on
Figure 4B. We used van’t Hoff analysis to determine the
unfolding parameters from the dependence of Tm on strand
concentration (Figure 4B). The data are collected in Table 1
and are in excellent agreement with the parameters calculated
from perfect (18) and mismatched (19) DNA nearest-neighbor
analysis. The Tm value of TBA is 57 C, which is similar to
earlier measurements in 10 mM SrCl2, 10 mM
Cs-HEPES, pH 7.5 (12). The TBA quadruplex is monomolecular and therefore its Tm does not depend on strand
concentration (12).
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Strand-exchange reactions
Figure 5 demonstrates kinetics of strand-exchange reactions
measured at 4 mM of the reactant molecules and at temperatures between 28 and 37 C. The experimental temperatures are
significantly lower than Tm of the molecules involved in the
reaction (see Figure 4). The rate constant, k, was determined by
fitting the experimental data to a first-order reaction equation:
ðAt A0 Þ = ðA¥ A0 Þð1 expðktÞÞ
where t is time, and At, A0 and A¥ are the optical absorbance at
time t, zero time and infinite time, respectively.
The rate constants are collected in Table 2. The rate constant
increase with increasing the temperature, which is in good
agreement with earlier measurements of strand-displacement
rates on perfect duplexes using electrophoresis assay of
labeled oligonucleotides (1). As it was expected, the present
rate constants, obtained for mismatched reactant duplexes, are
significantly higher than the parameters reported for perfect
duplexes (1).
The strand-exchange kinetic is not affected by
folding of the displaced strand
We remind that in the present assay, the role of ‘signal’ molecule is played by displacement strand, which upon release
folds into the monomolecular quadruplex structure. It is
known that monomolecular quadruplexes are formed readily
(12). However, it is important to see that our strand-exchange
kinetic is not affected by kinetics of the quadruplex formation.
We performed additional experiments in which the unfolded
TBA molecule was titrated into the buffer (100 mM NaCl,
Figure 4. (A) UV melting curves of the oligonucleotides in 100 mM NaCl, 2
mM SrCl2, 5 mM Na-HEPES, pH 7.5. (B) Tm dependence on strand
concentration.
Time (s)
Figure 5. Kinetics of the strand-exchange reaction shown in Figure 2B at different temperatures. The concentration of both reactants, mismatched duplex and
replacement strand is 4 mM, or concentration ratio of displacement to replacement strand is one. Buffer: 100 mM NaCl, 2 mM SrCl2, 5 mM Na-HEPES, pH 7.5.
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Table 2. Rate constants of the strand-exchange reactions obtained from
exponential fits and the correlation coefficients, R
t ( C)
k (104 s1)
28
31
34
37
4.12
12.61
36.68
100.86
–
–
–
–
0.02
0.08
0.10
0.43
R
0.99966
0.99962
0.99979
0.99956
2 mM SrCl2, 5 mM Na-HEPES, pH 7.5) at different temperatures. In all cases, we observed immediate increase of absorbance signal at 300 nm, corresponding to folding of the strand.
Waiting periods of 15 min did not reveal any further changes
in the measured parameter.
DISCUSSION
The main point of the study is that the process of the nucleic
acid strand-exchange can be studied by the real-time measurements of optical absorbance without any modification of the
strands. The foundation of the assay is formed by following
two facts. First, in the presence of Sr ions single-stranded TBA
folds into intramolecular quadruplex structure, which strongly
absorbs light at 300 nm where all other secondary structures
are transparent, including TBA incorporated into the duplex.
Second, exchange of a few guanines for thymidines in TBA
molecule completely inhibits quadruplex formation. Thus,
both reactants (left side of Figure 1B), mismatched duplex
with TBA and mutated version of TBA, GT3G2TGTG2T3G
do not absorb the light at 300 nm. Upon their mixing, TBA is
replaced in the duplex by perfectly base-pairing
GT3G2TGTG2T3G. The release of TBA into solution is
accompanied by immediate quadruplex formation and
increase of optical absorbance at 300 nm.
It is obvious that the reaction described here can be used as a
model system for fundamental studies on the mechanism of the
nucleic acid strand-exchange reactions. In addition, the assay
can be used to study the role of non-specific binding proteins in
the strand-exchange reactions, such as HIV-1 nucleocapsid
protein (2,3).
The assay is not limited only to TBA molecule. There are
other sequences forming monomolecular quadruplexes with
light absorbance in near-UV region: e.g. human telomeric
repeat (G3TTA)3G3 [(17), B.I.Kankia, unpublished data]
and oxytria telomeric repeat (G4T4)3G4 (17). It is also
known that exchange of the nucleotides in loop regions
does not inhibit quadruplex structure [(17,20); B.I.Kankia,
unpublished data]. Keeping in mind that we also need to
introduce some mutations in G-track regions to inhibit quadruplex formation of displacement strand, it became clear that
one can design almost any nucleotide sequence in fully basepaired product duplex (for instance, a protein-binding site). In
addition, one can link the signal oligonucleotide with the
desired sequence.
The assay is easily adaptable to desired reaction rate and
experimental conditions. In addition to the ionic strength,
temperature and the duplex length, one can vary the type of
cation used for quadruplex formation. It was shown earlier that
the stability of TBA is strongly dependent on the type and size
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of cations (12,21). For instance, Tm of TBA in the presence of
Sr2+ is 60 C, while in the case of Rb+ it drops to 33 C (12).
Thus, to slow down the reaction kinetics of an unstable reactant duplex, one can use a small amount of Rb ions in combination with lower temperature. Another factor strongly
influencing the reaction rate is type and number of mismatches
in the reactant duplex. There are eight possible mismatches or
mispairs and depending on their neighboring nucleotides all of
them have different stabilities (19,22). For instance, by choosing the most stable mismatches, i.e. GA, one can increase the
number of the mismatches in the reactant duplex, or shift the
reaction into slower rates.
In the present assay, the replaced strand forms thermodynamically stable Sr2+-quadruplex which facilitates initial strandexchange reaction and as a result complicates the reverse
reaction. While beneficial in some cases (for instance, to accelerate very slow reaction), this can prevent multiple rounds of
the experiment. Since (i) TBA quadruplex easily integrates
into duplex upon addition of the complementary structure
(B.I.Kankia, unpublished results) and (ii) quadruplex stability
is sensitive to the type of cations used (12,21), one can choose
the experimental conditions favorable for reversible reactions.
For instance, one can perform the reactions in the presence of
cations which form unstable quadruplexes (i.e. Rb+ or NH4+)
(12), and by selecting proper temperature and ionic strength to
make the strand-exchange reaction reversible.
ACKNOWLEDGEMENTS
I thank Professors Victor Bloomfield, Ioulia Rouzina and
Karin Musier-Forsyth for helpful and stimulating discussions.
This investigation was supported by NIH grant GM28093 to
Prof. Victor Bloomfield.
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