Ag+-Mediated Assembly of 5′-Guanosine Monophosphate
Kristine Loo, Natalya Degtyareva, Jihae Park, Bidisha Sengupta, Michaeal Reddish,
Christopher C. Rogers, Andrea Bryant, and Jeffrey T. Petty*
Department of Chemistry, Furman UniVersity, GreenVille, South Carolina 29613
ReceiVed: August 21, 2009; ReVised Manuscript ReceiVed: January 7, 2010
Polymorphic forms of nucleic acids provide platforms for new nanomaterials, and transition metal cations
give access to alternative arrangements of nucleobases by coordinating with electron-rich functional groups.
Interaction of Ag+ with 5′-guanosine monophosphate (5′-GMP) is considered in this work. Ag+ promotes
nucleotide stacking and aggregation, as indicated by the increased viscosity of 5′-GMP solutions with Ag+,
magnification of the circular dichroism response of guanine by Ag+, and exothermic reactions between Ag+
and guanine derivatives. Isothermal titration calorimetry studies show that the reaction is favored starting at
10 µM 5′-GMP. Utilizing the exothermic heat change associated with reaction of Ag+ with 5′-GMP, local
structure within the aggregate was assessed. On the basis of the salt dependence of the reaction and comparison
with the corresponding nucleoside, the dianionic phosphate of 5′-GMP is one binding site for Ag+, although
this electrostatic interaction is not a dominant contribution to the overall heat change. Another binding site
is the N7 on the nucleobase, as determined via studies with 7-deazaguanosine. Besides this binding site, Ag+
also associates with the O6, as earlier studies deduced from the shift in the carbonyl stretching frequency
associated with adduct formation. With these two binding sites on the nucleobase, the empirical stoichiometry
of ∼1 Ag+:nucleobase derived from the calorimetry studies indicates that Ag+ coordinates two nucleobases.
The proposed structural model is a Ag+-mediated guanine dimer within a base stacked aggregate.
Introduction
DNA hybridization has enabled creation of novel nanoma-
terials, such as self-assembled biomolecular electronic compo-
nents whose small size could greatly expand the information
density of electronic devices.1 Nucleobase assembly is dictated
by the directionality, strength, and interplay of noncovalent
interactions such as base stacking and hydrogen bonding.2,3 By
promoting alternative base pairing arrangements through coor-
dination with the electron-rich groups of nucleobases, transition
metal cations broaden the repertoire of base interactions while
also conferring magnetic and electronic functionality to DNA-
based nanostructures.4–6 Indirectly, these cations can change
patterns of hydrogen bond donors and acceptors by favoring
particular tautomers or by altering ionization constants of acidic
groups on the nucleobase. In addition, transition metal cations
can directly chelate electron-rich nitrogen and oxygen groups
to produce both canonical and noncanonical base pairs such as
coordination of adenine and thymine by Pt2+, adenine and
cytosine by Ag+, and two thymines by Hg2+.7–9
A new class of nanomaterials that provided the motivation
for the present studies is fluorescent silver clusters that form
on DNA templates.10–12 By reducing Ag+ that is bound with
DNA, clusters with ∼10 atoms are formed. These chromophores
are distinguished by their spectral tunability that depends on
base sequence, high fluorescence quantum yield, high photo-
stability, and limited blinking.11,13 Our goals are to better
understand how Ag+ is organized within a DNA matrix and
how this cation influences DNA structure. This paper considers
interaction of Ag+ with isolated guanines and the resulting
nucleobase organization in dilute solutions (10-500 µM). Ag+
favors σ bond formation with the electron-rich binding sites of
aromatic rings, as indicated by the 30-fold greater affinity for
pyridine relative to benzene, and site specific adducts have been
characterized by infrared spectroscopy and X-ray crystallography
studies.14–16 Beyond isolated 1:1 adducts, Ag+ facilitates higher
order interactions such as base pairing due to its propensity for
linear coordination.16–20 This paper evaluates interaction with
guanine derivatives, and three potential binding sites on the
nucleobase have been considered. Spectroscopic and crystal-
lographic studies show association with N7, which has been
attributed to the Lewis basicity at this site.21,22 Association with
N1 is possibly connected with the decrease in pH that ac-
companies Ag+ addition to guanine in slightly basic solutions.15
Alternatively, association could occur via an enolate-like
tautomer that concentrates electron density on the O6, as
supported by infrared spectroscopic studies.15,21 Consistent with
linear coordination, Ag+-mediated chelation between guanines
has been proposed to produce specific dimeric and tetrameric
nucleobase arrangements.15,18 Further higher order organization
via stacking is considered in this work.
This paper examines chelation and aggregation of 5′-guanosine
monophosphate (5-GMP) and related derivatives by Ag+ in pH 8
buffers (Figure 1). This choice of ribonucleoside and pH was
motivated by extensive studies of 5′-GMP aggregation in basic
solutions.23 For example, NMR studies have used the combination
of chemical shifts and translational diffusion coefficients to discern
aggregates of 5′-GMP.24 The quartet based aggregate represents a
distinctive nucleic acid form with alternating C2′-endo and C3′-
endo sugar puckers within the helical base arrangement. With Ag+,
three observations indicate that aggregation of 5′-GMP depends
on this transition metal cation. First, viscosities of solutions of the
nucleotide increase with Ag+. Second, circular dichroism associated
with the guanine base greatly increases in the presence of Ag+.
These changes are accompanied by a reduction in the extinction
coefficient of guanine. Third, exothermic heat changes result from
* To whom correspondence should be addressed. E-mail: jeff.petty@
furman.edu.
J. Phys. Chem. B 2010, 114, 4320–43264320
10.1021/jp908085s  2010 American Chemical Society
Published on Web 03/05/2010

association of Ag+ with the nucleobase, and the magnitude of these
changes is in the range expected for base stacking. Using the
calorimetric signature for the reaction, the local aggregate structure
was investigated using 5′-GMP and structural variants. Exothermic
heat changes are driven by association of ∼1 Ag+ with the guanine
base. When the potential binding sites for Ag+ on the nucleobase
are considered, a dimeric base arrangement within the aggregate
is considered to be most reasonable.
Experimental Section
Silver nitrate (99.9995%, Alfa Aesar), 5′-guanosine mono-
phosphate (Sigma-Aldrich, 99+%), guanosine (Acros Organics,
99%), inosine monophosphate (Acros Organics, 99+%), 5′-
cytosine monophosphate (Sigma-Aldrich, 99%), and 7-deaza-
guanosine (Berry & Associates, g97%) were used as received.
Buffers were prepared in deionized water (Elix 10 Water
Purification System, Millipore), and the desired pH was obtained
using a total concentration of 10 mM of the acid/conjugate base.
A concentration of 10 mM H3BO3/H2BO3- was used to maintain
pH 8 in the solutions, and supporting electrolytes were NaClO4
and LiClO4. Nucleotide and nucleoside concentrations were
determined using extinction coefficients of 14 230 M-1 cm-1
for 5′-GMP at 252 nm, 8860 M-1 cm-1 for 5′-cytosine
monophosphate at 271 nm, 11 700 M-1 cm-1 for inosine
monophosphate at 246 nm, 12 300 M-1 cm-1 for 7-deazagua-
nosine at 260 nm, and 12 500 M-1 cm-1 for guanosine at 260
nm.25–27 For the latter compound, its solubility at the 100 µM
concentrations used for the calorimetry studies was experimen-
tally verified.
Using previously described protocols, calorimetry studies
were conducted using a Microcal VP-ITC instrument (North-
hampton, MA) controlled by Origin 7.0 software.28 Following
degassing, a solution of AgNO3 was titrated into a nucleotide
or nucleoside solution. Heat changes associated with the titration
were determined by integrating the power required to maintain
reference and sample cells at the same temperature. Heat
changes associated with dilution of AgNO3 were subtracted after
saturation of the binding sites. The single site binding model in
the manufacture software was appropriate for fitting the binding
isotherms to determine the enthalpy and free energy changes
and the adduct stoichiometry. Entropy changes were derived
from the free energy and enthalpy changes. Uncertainties were
derived from a minimum of three experiments on separate
samples. The highest 5′-GMP concentration at which a full
binding isotherm could be acquired was 500 µM. Absorption
(Cary 50, Varian) and circular dichroism (J-710, Jasco) spectra
were acquired using quartz cuvettes with 1 or 0.2 cm path-
lengths. Viscosity studies followed the protocol from prior
experiments.29 Measurements were made with a Cannon-
Ubbelohde semimicro viscometer with a capillary diameter of
0.54 mm (model 75, Cannon Instrument Company), and the
viscometer was submerged in a water bath to maintain a constant
temperature of 25.00 ( 0.05 °C. Flow times between the
viscometer timing marks were 117.92 ( 0.03 s for the buffer
and 118.41 ( 0.02 s for a 1 mM solution of 5′-GMP. Flow
times were measured at least in triplicate; error was propagated
using standard procedures to give uncertainties of (1σ.30 Low
nucleotide concentrations allow intrinsic viscosities to be
calculated from differences between the flow times for the DNA
solutions and for the buffer using31,32
where [η] and [η]0 are intrinsic viscosities of the 5′-GMP
solutions with and without Ag+, respectively. Flow times of
the buffer, 5′-GMP, and 5′-GMP:Ag+ solutions are tb, t0, and t,
respectively. Nucleoside solubility at the high concentrations
needed for viscosity precluded studies with other derivatives.
Figure 1. Structures of the nucleotides and nucleosides used for these
studies. For 5′-guanosine monophosphate, the functional groups that
were considered as binding sites for Ag+ are emphasized in red. The
proposed structure for the dimer of guanine with Ag+ coordination
between N7 and O6 is shown in part F.
Figure 2. Graph of the relative viscosity (η/η0) of a 1 mM 5′-GMP
solution with increasing relative amounts of Ag+ (closed circles). For
control experiments, Ag+ was added to the buffer without 5′-GMP (open
circles). Trend lines are provided to connect the data, and the error
bars are (1σ.
[η]
[η]0
)
(t - tb)
(t0 - tb)
Guanosine Assembly Using Ag+ J. Phys. Chem. B, Vol. 114, No. 12, 2010 4321