Influence of metal coordination on the mismatch tolerance of ligand-modified PNA duplexes

Article abstract of DOI:10.1021/ja051336h

Recent studies on metal incorporation in ligand-modified nucleic acids have focused on the effect of metal coordination on the stability of metal-containing duplexes or triplexes and on the metal binding selectivity but did not address the effect of the sequence of the nucleic acid in which the ligands are incorporated. We have introduced 8-hydroxyquinoline Q in 10-mer PNA strands with various sequences and have investigated the properties of the duplexes formed from these strands upon binding of Cu²⁺. Variable-temperature UV-vis spectroscopy shows that, in the presence of Cu ²⁺, duplexes are formed even from ligand-modified Q-PNA strands that have a large number of mismatches. Spectrophotometric titrations demonstrate that at any temperature, one Cu²⁺ ion binds a pair of Q-PNA strands that each contain one 8-hydroxyquinoline, but below the melting temperature, the PNA duplex exerts a supramolecular chelate effect, which prevents the transformation in the presence of excess Cu²⁺ of the 1:2 Cu ²⁺:Q-PNA complexes into 1:1 complexes. EPR spectroscopy gives further support for the existence in the duplexes of [CuQ₂] moieties that are similar to the corresponding square planar synthetic complex formed between Cu²⁺ and 8-hydroxyquinoline. As PNA duplexes show a preferred handedness due to the chiral induction effect of a C-terminal L-lysine, which is transmitted through stacking interactions within the duplex, only if the metal-containing duplex has complementary strands, does it show a chiral excess measured by CD spectroscopy. The strong effect of the metal-ligand moiety is suggestive of an increased correlation length in PNA duplexes that contain such moieties. These results indicate that strong metal-ligand alternative base pairs significantly diminish the importance of Watson-Crick base pairing for the formation of a stable PNA duplex and lead to high mismatch tolerance, a principle that can be used in the construction of hybrid inorganic-nucleic acid nanostructures.

Full text of DOI:10.1021/ja051336h

Published on Web 09/29/2005
Influence of Metal Coordination on the Mismatch Tolerance of
Ligand-Modified PNA Duplexes
Richard M. Watson, Yury A. Skorik, Goutam K. Patra, and Catalina Achim*
Contribution from the Department of Chemistry, Carnegie Mellon UniVersity, 4400 5th AVenue,
Pittsburgh, PennsylVania 15213
Received March 2, 2005; E-mail: achim@cmu.edu
Abstract: Recent studies on metal incorporation in ligand-modified nucleic acids have focused on the effect
of metal coordination on the stability of metal-containing duplexes or triplexes and on the metal binding
selectivity but did not address the effect of the sequence of the nucleic acid in which the ligands are
incorporated. We have introduced 8-hydroxyquinoline Q in 10-mer PNA strands with various sequences
and have investigated the properties of the duplexes formed from these strands upon binding of Cu2+
.
Variable-temperature UV-vis spectroscopy shows that, in the presence of Cu2+, duplexes are formed even
from ligand-modified Q-PNA strands that have a large number of mismatches. Spectrophotometric titrations
demonstrate that at any temperature, one Cu2+ ion binds a pair of Q-PNA strands that each contain one
8-hydroxyquinoline, but below the melting temperature, the PNA duplex exerts a supramolecular chelate
effect, which prevents the transformation in the presence of excess Cu2+ of the 1:2 Cu2+:Q-PNA complexes
into 1:1 complexes. EPR spectroscopy gives further support for the existence in the duplexes of [CuQ2]
moieties that are similar to the corresponding square planar synthetic complex formed between Cu2+ and
8-hydroxyquinoline. As PNA duplexes show a preferred handedness due to the chiral induction effect of a
C-terminal L-lysine, which is transmitted through stacking interactions within the duplex, only if the metal-
containing duplex has complementary strands, does it show a chiral excess measured by CD spectroscopy.
The strong effect of the metal-ligand moiety is suggestive of an increased correlation length in PNA duplexes
that contain such moieties. These results indicate that strong metal-ligand alternative base pairs significantly
diminish the importance of Watson-Crick base pairing for the formation of a stable PNA duplex and lead
to high mismatch tolerance, a principle that can be used in the construction of hybrid inorganic-nucleic
acid nanostructures.
Introduction
Metal ions play important roles in the structure and function
Besides their biological functions, nucleic acids’ ability to
store and transmit information has been recently exploited for
the creation of nanostructures and molecular electronics
devices.8-10 Incorporation of transition metal ions into these
systems is a means to expand the range of electronic and
magnetic properties of the nanostructures. Furthermore, the
combined use of the hybridization properties of nucleic acids
and coordination properties of the metal ions offers an op-
portunity to create hybrid nucleic-acid structures with new
topologies. Transition metal ions can be incorporated into
nucleic acids by substitution of the natural nucleobases with
ligands. In the presence of metal ions, these ligand-containing
nucleic acids form supramolecular structures based on a
combination of coordination and hydrogen bonds. This strategy
has been successfully applied in the last 5 years to introduce
Ni2+, Cu2+, and Ag+ in DNA duplexes and triplexes.11-19
Recently, we have extended this approach to incorporation of
of nucleic acids, and numerous modified nucleic acids that bind
transition metal ions are presently being used for diagnostic and
therapeutic applications. As a consequence, the effects of metal
binding to backbone phosphates or nucleobases on DNA
properties have been extensively investigated.1-4 Eichhorn et
al. have studied the effect of transition metal ions, in particular
of Cu2+, on DNA structure and stability, and they have
determined that in solutions containing at least one Cu2+
equivalent per DNA base pair, Cu2+ binds to nucleobases at
high temperature, decreases significantly the thermal stability
of DNA duplexes, and can prevent DNA hybridization upon
cooling.5-7 In the presence of less than 0.4 equiv of Cu2+ per
DNA base pair, the thermal stability of DNA was found to
slightly increase, an effect attributed to possible Cu2+ binding
to the phosphate backbone.5
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9
14628
J. AM. CHEM. SOC. 2005, 127, 14628-14639
10.1021/ja051336h CCC: $30.25 © 2005 American Chemical Society

Mismatch Tolerance of Ligand-Modified PNA Duplexes
A R T I C L E S
Ni2+ into peptide nucleic acid (PNA) duplexes by replacing two
complementary nucleobases with two bipyridine moieties.20,21
The properties of these metal-containing supramolecular
assemblies depend on both the nucleic acid part and the metal-
ligand moiety but are not simply the sum of their properties.
For example, replacement of two complementary bases with
two ligands that cannot form hydrogen bonds leads to desta-
bilization of the nucleic acid duplexes, with the destabilization
being more pronounced for PNA than for DNA duplexes, in a
manner similar to the better mismatch discrimination of PNA
when compared to DNA. When metal ions are present, the
duplex stabilization is partly or completely restored, as expected
because coordination bonds are formed in these structures. As
these bonds are stronger than hydrogen bonds, the fact that in
some cases the thermal stability of the metal-containing duplexes
is lower than that of the nonmodified duplexes indicates that
other factors, such as steric interactions that weaken hydrogen
bonds in, and π-π stacking interactions between, nucleobase
pairs adjacent to the metal-bridged ligand pair, play an important
role in determining the properties of the duplexes.
Predictions of the duplex properties based on coordination
chemistry concepts do not hold in a consistent fashion. For
example, Schultz et al. have introduced in DNA duplexes the
monodentate ligand pyridine and either pyridine-2,6-dicarboxy-
late to create a [1+3] binding site for Cu2+ or 2,6-bis-
(ethylthiomethyl)pyridine to create a [1+3] binding site for the
softer Ag+.13,16 Cu2+ incorporation occurred and had a predict-
able effect on a DNA duplex containing the former ligand
combination, but surprisingly, in the presence of Ag+, DNA
duplexes with the latter ligand combination had thermal stability
slightly lower than that of nonmodified duplexes. Also, signifi-
cantly more stable duplexes were formed when two tridentate
2,6-bis(ethylthiomethyl)pyridine ligands were present in comple-
mentary positions in the DNA duplex, suggesting a six-
coordinate Ag+ complex, which is unusual. Furthermore,
comparison between results obtained for pyridine-modified DNA
duplexes that have different sequences indicates a context
dependent effect of the metal-ligand moiety on the duplex
properties.16,22
with the formation of hydrogen bonds within the base pairs
adjacent to the metal-ligand pair and reduce π-stacking, and
thus have a destabilization effect on the duplex.
To test this hypothesis, 8-hydroxyquinoline (Q) has been
introduced into PNA oligomers (Q-PNA), and the properties
of duplexes formed by these oligomers in the presence of Cu2+
have been investigated. As a synthetic complex [CuQ2] is known
and its square planar geometry is well documented,24-27 we
anticipated that the Cu2+-containing PNA duplexes would have
higher thermal stability than the nonmodified PNA. This paper
presents the results of the study of 10-mer Q-PNA oligomers
in the presence of Cu2+ using UV-vis, CD, and EPR spec-
troscopy. While this work was in progress, Zhang et al. have
shown that the formation of a 15 base pair DNA duplex that
contains in the middle of the duplex, in complementary
positions, two Q ligands leads in the presence of Cu2+ to
formation of very stable DNA duplexes and makes possible
modifications of the DNA backbone that would otherwise
prevent the duplex assembly.14 In the present study, we show
that strong metal binding to 8-hydroxyquinoline ligands within
two PNA oligomers promotes the formation of a PNA duplex
even when several mismatches exist between the two strands,
a result which suggests an important role of metal coordination
in the stabilization of peptide nucleic acid structures.
Experimental Section
Materials. tert-Butyl N-(2-Boc-aminoethyl)-glycinate 2 was prepared
according to a published procedure.28 The 5-carboxymethyl-8-hydroxy-
quinoline 3 was prepared as shown in Scheme 1, using literature
procedures for steps a-c.29,30
Scheme 1 a
a (a) HCl, formaldehyde; (b) NaCN, DMSO, 90 °C; (c) HCl; (d) 2, DCC,
DhbtOH; (e) NaOH, MeOH; (f) HCl.
During the study of metal incorporation in PNA duplexes,
we have found that the stability of a 2,2′-bipyridine-modified
PNA duplex in the presence of Ni2+ is lower than that of a
duplex that has an AT base pair.20 This observation may be
attributed to the fact that in a four-coordinate bis(bipyridine)
metal complex, the two ligands cannot be coplanar due to steric
interactions between protons situated in the 6 and 6′ positions.
As a consequence, square planar complexes undergo a tetra-
hedral distortion, with the two ligands being twisted, or adopt
a bow-step conformation in which the two ligands are situated
in off-set parallel planes.23 These distortions potentially interfere
All other reagents were obtained from commercially available
sources, were of analytical grade quality, and were used without further
purification. Silica gel for column chromatography was 200-400 mesh
size. All reactions were monitored by TLC on SiO2 (UV detection).
Melting points were measured using a Mel-Temp II instrument and
are uncorrected. Elemental analysis was done by Prevalere Life
Sciences, Inc. H NMR spectra were recorded on a Bruker Cryospec
WM 300. A Finnegan Mattson instrument was used for electrospray
mass spectrometry (ES-MS).
tert-Butyl-2-(N-(tert-butyloxycarbonyl-2-aminoethyl)-2-(8-hydroxy-
quinoline-5-yl)acetamido) Acetate (4). 0.82 g (3 mmol) of 2 was
dissolved in 15 mL of DCM. To this solution were added 0.49 g (3
mmol) of DhbtOH and 0.72 g (3 mmol) of 3‚HCl dissolved in 20 mL
of DMF (by slight warming). The reaction mixture was cooled to
1
(16) Zimmermann, N.; Meggers, E.; Schultz, P. G. J. Am. Chem. Soc. 2002,
124, 13684-13685.
(17) Weizman, H.; Tor, Y. J. Am. Chem. Soc. 2001, 123, 3375-3376.
(18) Switzer, C.; Shin, D. Chem. Commun. 2005, 1342-1344.
(19) Switzer, C.; Sinha, S.; Kim, P. H.; Heuberger, B. D. Angew. Chem., Int.
Ed. 2005, 44, 1529-1532.
(24) Ammor, S.; Coquerel, G.; Perez, G.; Robert, F. Eur. J. Solid State Inorg.
Chem. 1992, 29, 445.
(25) Bevan, J. A.; Graddon, D. P.; McConnell, J. F. Nature 1963, 199, 373.
(26) Hoy, R. C.; Morriss, R. H. Acta Crystallogr. 1967, 22, 476-482.
(27) Murray-Rust, P.; Wright, J. D. J. Chem. Soc. A 1968, 247-253.
(28) Dueholm, K. L.; Egholm, M.; Behrens, C.; Christensen, L.; Hansen, H. F.;
Vulpius, T.; Petersen, K. H.; Berg, R. H.; Nielsen, P. E.; Buchardt, O. J.
Org. Chem. 1994, 59, 5767-5773.
(20) Popescu, D.-L.; Parolin, T. J.; Achim, C. J. Am. Chem. Soc. 2003, 125,
6354-6355.
(21) Franzini, R.; Watson, R. M.; Popescu, D.-L.; Patra, G. K.; Achim, C. Polym.
Prepr. 2004, 45, 337-338.
(22) Tanaka, K.; Yamada, Y.; Shionoya, M. J. Am. Chem. Soc. 2002, 124, 8802-
8803.
(29) Burckhalter, J. H.; Leib, R. I. J. Org. Chem. 1961, 26, 4078-4083.
(30) Warner, V. D.; Sane, J. N.; Mirth, D. B.; Turesky, S. S.; Soloway, B. J.
Med. Chem. 1976, 19, 167-169.
(23) Geremia, S.; Randaccio, L.; Mestroni, G.; Milani, B. J. Chem. Soc., Dalton
Trans. 1992, 2117-2118 and references therein.
9
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A R T I C L E S
Watson et al.
0 °C, and 0.62 g (3 mmol) of solid DCC was added. The solution was
stirred vigorously at 0 °C for 2 h. The reaction mixture was then allowed
to reach room temperature and was stirred for another 2 h. The finely
powdered white precipitate of dicyclohexyl urea (DCU) was removed
by filtration. 75 mL of DCM was added to the filtrate. The resulting
DCM solution was washed successively with dilute aqueous NaHCO3
(3 × 25 mL), dilute aqueous KHSO4 (2 × 25 mL), and finally with
brine (2 × 30 mL). The organic phase was dried over anhydrous
Na2SO4, filtered, and the solvent was evaporated. The solid product
obtained after evaporation was redissolved in 10 mL of ethyl acetate.
A small amount of white solid DCU, which remained undissolved, was
filtered out. The ethyl acetate solution was evaporated to obtain 4 as
nanopure water and were stored at -18 °C. The concentration of PNA
oligomers was determined by UV absorption at 95 °C using the sum
of the extinction coefficients of the constituent nucleosides ꢀ260 taken
from the literature.32 The extinction coefficient for 8-hydroxyquinoline
ꢀ260 ) 2570 ( 110 M-1 cm-1 (pH ) 7.0) was determined from the
slope of the calibration curve A260 versus concentration. PNA solutions
for melting curves and titrations had concentrations in the micromolar
range (10-50) and were prepared in pH 7.0 10 mM phosphate buffer.
UV-vis titrations were carried out by addition of standard 0.5-2 mM
CuCl2 solutions in water to PNA solutions. The absorbance A after
each addition was corrected (Acorr) for dilution and for the contribution
of CuCl2 and PNA.
1
yellow solid. Yield, 0.798 g (58%). H NMR (300 MHz, in CDCl3):
UV melting curves were recorded in the temperature range
5-95 °C for both cooling and heating modes, at the rate of 1 °C/min.
Prior to the measurement of the melting profiles, the solutions were
kept at 95 °C for at least 10 min. The difference between the high-
and low-temperature spectra of Q-modified PNA oligomers in the
absence of Cu2+ indicates that the maximum absorbance changes
induced by temperature occur at 240-244 nm and at 266-270 nm. In
the presence of Cu2+, the maximum absorbance change occurs at
262-267 nm. Therefore, melting curves were measured at wavelengths
within these intervals. Tm is the inflection point of a sigmoidal function
used to fit the melting curve.
δ, 8.76 (d, 1H, H2); 8.36 (d, 1H, H4); 7.48 (dd, 1H, H3); 7.35 (d, 1H,
H6); 7.10 (d, 1H, H7); 5.47 (m br, NH, 1H); 4.05 (s, 2H, N-CH2-
CO); 3.90 (s, 2H, Q-CH2); 3.55 (t, 2H, CH2-Nd); 3.28 (t, 2H, CH2-
NHBoc); 1.40 (m, 18H, Boc + OtBu). ES-MS for (M + H)+
(CH3CN): m/z ) 459.93 observed, 460.24 calculated.
2-(N-(tert-Butyloxycarbonyl-2-aminoethyl)-2-(8-hydroxyquinolin-
5-yl)acetamido) Acetic Acid (1). Ester 4 (0.918 g, 2 mmol) was
dissolved in 10 mL of ethanol. 20 mL water was added to this solution,
followed by dropwise addition of 1.5 mL of 12 M aqueous NaOH
solution with stirring. The reaction mixture was stirred at room
temperature for 4 h. The resulting solution was then filtered, acidified
to pH 3 by addition of 4 M HCl, and extracted with 3 × 50 mL ethyl
acetate. The ethyl acetate fractions were combined, dried over anhydrous
Na2SO4, filtered, and the solvent was evaporated under vacuum. The
hydrolyzed monomer was obtained as a light green solid after being
dried under vacuum overnight. Yield, 0.61 g (76%); mp 114-118 °C.
Anal. Calcd for C20H25N3O6: C, 59.54; H, 6.25%. Found: C, 59.17;
EPR Spectroscopy. EPR spectra were recorded on an X-band
(9 GHz) Bruker ESP 300 spectrometer equipped with an Oxford ESR
910 cryostat. The microwave frequency was calibrated with a frequency
counter and the magnetic field with a NMR gaussmeter. The temper-
ature was calibrated using devices from Lake Shore Cryonics. Spectra
were collected under nonsaturating conditions. Samples were prepared
in pH 7.0 10 mM sodium phosphate buffer with 25% glycerol as
glassing agent. Samples containing PNA and Cu2+ in appropriate molar
ratio were heated at 95 °C for 10 min, slowly cooled to room
temperature, and then transferred into EPR tubes and frozen. EPR
spectra were simulated using the program SpinCount written by Prof.
Michael P. Hendrich.33 Spin quantitation was done relative to a 0.499
mM Na2[Cu(edta)] standard, the copper concentration of which was
determined by plasma emission spectroscopy.
1
H, 6.47%. H NMR (300 MHz, in CDCl3): δ, 8.84 (d, 1H, H2); 8.29
(d, 1H, H4); 7.56 (dd, 1H, H3); 7.30 (d, 1H, H6); 7.01 (d, 1H, H7); 4.28
(s, 2H, N-CH2-CO); 3.97 (s, 2H, Q-CH2); 3.53 (t, 2H, CH2-Nd);
3.11(t, 2H, CH2-NHBoc); 1.37 (m, 9H, Boc). ES-MS for (M - H)-
(CH3CN): m/z ) 402.07 observed, 402.17 calculated.
Solid-Phase PNA Synthesis. PNA oligomers were synthesized using
the Boc-protection strategy.31 PNA monomers were purchased from
Applied Biosystems and used without further purification. After
cleavage, PNA was precipitated using ethyl ether and was purified by
reversed-phase HPLC using a C18 silica column on a Waters 600
Controller and Pump. Absorbance was measured with a Waters 2996
Photodiode Array Detector. Characterization of the oligomers was
performed by MALDI-ToF mass spectrometry on an Applied Biosys-
tems Voyager Biospectrometry Workstation with Delayed Extraction
and an R-cyano-4-hydroxycinnamic acid matrix (10 mg/mL in 1:1
water:acetonitrile, 0.1% TFA). MALDI-TOF calcd/found for (Xi + H)+
m/z XA 2855.8/2855.3, XB 2855.8/2855.2, Q-XA 2865.8/2866.0, Q-XB
2874.8/2875.0, Q-XC 2865.8/2866.3, Q-XD 2865.8/2866.4, Q-XE
2812.9/2813.0, G-XA 2871.8/2872.0.
Physical Methods. CD Spectroscopy. CD spectra were measured
for 20 µM total PNA concentration in pH 7.0 10 mM sodium phosphate
buffer solutions. In the case where complementary PNA strands were
used, solutions were equimolar in the two strands. CD measurements
were conducted on a JASCO J-715 spectropolarimeter equipped with
a thermoelectrically controlled, single-cell holder. CD thermal dena-
turation curves were measured using response time 16 s, sensitivity
20 mdeg, and bandwidth 5.0 nm. The temperature was changed at a
rate of 1 °C/min, and data were collected every 0.5 °C. CD spectra
were collected using bandwidth 1 nm, response time 1 s, speed
50 nm/min, sensitivity 20 mdeg, and scan accumulation 16.
Results
Synthesis of 8-Hydroxyquinoline-PNA. The 8-hydroxy-
quinoline PNA monomer was prepared by coupling the acetic
acid derivative of 8-hydroxyquinoline 3 activated using DCC
and DhbtOH to the Boc-protected, tert-butyl aminoethyl gly-
cinate 2. In contrast to typical PNA monomer synthesis in which
an excess of the acetic acid derivative is used in this coupling
step, we have used a stoichiometric amount of 3 to minimize
the risk that the phenolic group of 3 reacts with the secondary
amino group of 2. Next, the tert-butyl ester 4 obtained from
the coupling was hydrolyzed using a dilute sodium hydroxide
solution in water/ethanol. The 8-hydroxyquinoline PNA mono-
mer 1 was incorporated into PNA oligomers using solid-phase
peptide synthesis based on a Boc-protecting group strategy.31
PNA Sequences. The antiparallel PNA duplex XA:XB (a in
Chart 1) formed by complementary Watson-Crick base pairing
of all of its 20 bases has been extensively investigated since
PNA’s discovery in 1991.34,35 One A and T nucleobase in the
(32) Nielsen, P. E., Egholm, M., Eds. Peptide Nucleic Acid: Protocols and
Applications; Horizon Scientific Press: Wymondham, UK, 1999.
(33) Hendrich, M. P.; Petasis, D.; Arciero, D. M.; Hooper, A. B. J. Am. Chem.
Soc. 2001, 123, 2997-3005.
UV-Vis Spectroscopy. UV-vis experiments were performed on a
Varian Cary 3 spectrophotometer with programmable temperature block,
in 10 mm quartz cells. PNA stock solutions were prepared with
(34) Wittung, P.; Nielsen, P. E.; Buchardt, O.; Egholm, M.; Norden, B. Nature
1994, 368, 561-563.
(31) Christensen, L.; Fitzpatrick, R.; Gildea, B.; Petersen, K. H.; Hansen, H.
F.; Koch, T.; Egholm, M.; Buchardt, O.; Nielsen, P. E. J. Pept. Sci. 1995,
1, 175-183.
(35) Egholm, M.; Buchardt, O.; Christensen, L.; Behrens, C.; Freier, S. M.;
Driver, D. A.; Berg, R. H.; Kim, S. K.; Norden, B.; Nielsen, P. E. Nature
1993, 365, 566-568.
9
14630 J. AM. CHEM. SOC. VOL. 127, NO. 42, 2005

Mismatch Tolerance of Ligand-Modified PNA Duplexes
A R T I C L E S
Chart 1. PNA Sequences and Duplexesa
that cannot form hydrogen bonds generally leads to destabiliza-
tion of nucleic acid duplexes, while metal binding to these
ligand-modified duplexes can partly or completely restore the
thermal stability. Variable-temperature UV-vis spectroscopy
was used to evaluate changes in the stability of our PNA
duplexes upon 8-hydroxyquinoline incorporation and Cu2+
binding.
Analysis of the melting curve for the complementary duplex
Q-XA:Q-XB in the absence of Cu2+ (Figure 1a), which
displays a sigmoidal shape indicative of cooperativity, gives
Tm ) 46 °C, which is 21 °C less than the Tm of the nonmodified
XA:XB.34 In the presence of less than 1 equiv of Cu2+ per duplex,
two transitions are clearly observable in the melting curves for
Q-XA:Q-XB (Figure 1a): one at ∼46 °C, attributed to melting
of the metal-free duplex, and one at >79 °C, attributed to
melting of the metal-containing duplex. The latter melting
temperature represents a lower limit as the transition is not
complete up to 95 °C. The relative contribution of the two
transitions correlates with the amount of Cu2+ present in
solution, and at ratios Cu2+:(Q-XA:Q-XB) g 1:1, the lower
temperature transition disappears and only the transition with
Tm > 79 °C is observed. The large stabilization of the
Q-containing duplex upon Cu2+ addition (∆Tm > 33 °C) is in
contrast to the nonmodified XA:XB duplex for which Cu2+
addition leads to a very small decrease in Tm (∆Tm ) 2 °C).
Surprisingly, in the presence of Cu2+, homoduplexes formed
from Q-PNA strands that are only partly complementary show
a cooperative transition with considerable hyperchromicity
(Figure 1b and Table 1). The melting temperatures for homo-
duplexes based on Q-XA, Q-XB, Q-XC, and Q-XD are 72,
>79, 77, and 66 °C, respectively. To determine whether the
change in absorption with temperature is due to the [CuQ2]
moiety situated within the duplex, we have monitored the
absorption of a solution containing 5 µM Cu2+ and 10 µM
8-hydroxyquinoline PNA monomer 1 (Figure S1). We have
found that the changes in absorption of this solution with the
temperature are linear and significantly smaller (∆A267 ) 0.03)
than those measured for Q-PNA in the presence of Cu2+ (∆A267
) 0.20). This experiment established that the changes in
absorbance at 267 nm for Q-PNA solutions in the presence of
Cu2+ are indeed due to PNA duplex formation rather than to
the CuQ2 moiety.
a Watson-Crick and wobble base pairs are represented in blue and green,
respectively. Alignment of the strands within the duplexes is such that either
the Q ligands are in complementary position or the number of Watson-
Crick base pairs is maxim.
middle of each of the PNA 10-mers XA and XB, respectively,
has been replaced by 8-hydroxyquinoline ligands such that in
the duplex formed by the modified strands Q-XA and Q-XB,
the two ligands are situated in complementary positions (b in
Chart 1).
To investigate the synergy between hydrogen and coordina-
tion bonds on PNA duplex formation and stability, we have
also studied the properties of Q-modified PNA 10-mer strands
that are partly or non-self-complementary. Q-XA and Q-XB
can form antiparallel homoduplexes (Q-Xi:Q-Xi)-Cu2+ sta-
bilized by four Watson-Crick base pairs and two GT wobble
base pairs (c and d in Chart 1). Q-XC and Q-XD can also
form homoduplexes (Q-Xi:Q-Xi)-Cu2+ that contain up to six
and four Watson-Crick base pairs, respectively (Chart 2). To
investigate the effect of copper on completely noncomplemen-
tary Q-PNA strands, we have also synthesized the 10-mer
Q-XE, which contains alternating A and C bases that are size
complementary but cannot form Watson-Crick base pairs (g
in Chart 1).
Having observed the formation of duplexes even from partly
complementary strands, we have measured the temperature
dependence of the absorption at 267 nm for PNA oligomer
Q-XE, which cannot form a homoduplex containing Watson-
Crick base pairs. Changes in absorbance for this oligomer in
the presence of Cu2+ are noncooperative, indicating that a duplex
is not formed (Figure 1b).
Variable-Temperature UV-Vis Spectroscopy. As men-
tioned above, substitution of GC or AT base pairs with ligands
These results indicate that strong binding of Cu2+ to the Q
ligands in the PNA oligomers leads to high mismatch tolerance,
Chart 2. Alternative Alignments for (Q-XC:Q-XC)-Cu2+ and (Q-XD:Q-XD)-Cu2+, Which Make Possible the Formation of Watson-Crick
Base Pairsa
a Watson-Crick base pairs are represented in blue.
9
J. AM. CHEM. SOC. VOL. 127, NO. 42, 2005 14631

A R T I C L E S
Watson et al.
Figure 2. Electronic absorption spectra of Q-XA:Q-XB solution at
25 °C (purple) and 95 °C (red) and of those in the presence of an equimolar
amount of CuCl2 at 25 °C (green) and 95 °C (blue). Solutions were prepared
in pH ) 7.0 10 mM phosphate buffer.
nucleobases and of the Q ligand, respectively.36-38 A shoulder
at 320 nm (inset in Figure 2) is due to π-π* transitions of Q.
At both 25 and 95 °C, the effect of Cu2+ addition to Q-Xi
solutions is the expected bathochromic shift of the ligand
absorption band from 247 to 260, the hypochromicity of the
ligand π-π* band at 320 nm, and the appearance of a metal-
to-ligand charge-transfer band at ∼400 nm,39 demonstrating that
Cu2+ binds indeed to Q at both temperatures. To determine the
stoichiometry of Cu2+ binding to Q-PNA, we have conducted
spectrophotometric titrations of Q-PNA with Cu2+ by monitor-
ing changes in absorbance at the above wavelengths. Q-PNA
solutions with total concentrations of 10 µM (Figures 3 and 4)
and 50 µM (Figures 5 and 6) were titrated with CuCl2 solutions
at 25 °C (Figures 3, 5, S4, S5, S7, S9) and at 95 °C (Figures 4,
6, S2, S3, S6, S8, S10, S11). The results of spectrophotometric
titrations for Q-Xi single strands where i ) A-D and for
equimolar mixtures of Q-XA and Q-XB are similar to each
other at both temperatures (Figures 3-6 and S2-S9).
At 95 °C, the UV-vis titration curves at 247, 260, 320, and
400 nm of all Q-PNA strands show a first inflection point
at Cu2+:Q-Xi of 1:2 (part b of Figures 4, 6, S2, S3, S6, S8,
S10, and S11). Two isosbestic points are observed at 253
and 349 nm, indicating a direct conversion of Q-Xi to
[Cu(Q-Xi)2] (part a of Figures 4, 6, S2, S3, S6, S8, S10, and
S11). Further addition of Cu2+ leads to a second inflection point
corresponding to the formation of 1:1 [Cu(Q-Xi)] complexes.
The transformation of the 1:2 complex into a 1:1 complex is
also accompanied by a hypsochromic shift (∆λ ) 7 nm) of the
metal-to-ligand charge-transfer transition from 400 to 393 nm.
The small hyperchromicity observed at 260 nm after the 1:1
complex has formed can be attributed to weak interaction of
copper ions with unpaired PNA nucleobases (Figures 4b, S2b,
Figure 1. (a) Absorbance change at 267 nm for 5 µM Q-XA:Q-XB
solutions in the absence and presence of CuC12. Concentration of CuCl2:
0, 1, 2, 3, 5 µM; (b) Absorbance change at 267 nm for 10 µM Q-PNA pH
) 7.0 10 mM phosphate buffer solutions in the presence of 5 µM Cu2+
.
Q-XA (green); Q-XB (blue); Q-XC (black); Q-XD (red); Q-XE (cyan);
Q-XA:Q-XB (purple).
that is, to the formation of PNA duplexes from strands that
contain a relatively small number of Watson-Crick base pairs.
However, strong metal coordination to the ligands within the
PNA strands cannot be a high enough driving force for the
formation of duplexes in the absence of any Watson-Crick
complementarity.
UV-Vis Titrations. To evaluate the influence of hydrogen-
bonding association of Q-PNA strands on metal binding, we
have conducted titrations at 25 and 95 °C, which are temper-
atures above and below Tm for the duplexes formed from these
strands. At both temperatures, the electronic absorption spectra
of Q-PNA oligomers in pH 7.0 sodium phosphate buffer
(Figure 2) are dominated by two strong absorption bands at 260
and 247 nm, which correspond to π-π* transitions of the
Table 1. Melting Temperatures Tm and Interactions That Affect Tm
+
+
+
+
+
(Q
−XA:Q
−XB)−
Cu2
(Q
−XB:Q
−XB)−
Cu2
(Q
−XC:Q
−XC)−
Cu2
(Q
−XA:Q
−XA)−
Cu2
(Q
−XD:Q
−XD)−
Cu2
Cu2+(Q
−XE)2
Tm
>79
>79
77
6
0
72
4
2
66
4
0
Watson-Crick bp
wobble bp
overhangs
9
0
0
4
2
0
0
2 A
2 A
0 or 2 CT
2 T
0 or 2 CT
9
14632 J. AM. CHEM. SOC. VOL. 127, NO. 42, 2005

Mismatch Tolerance of Ligand-Modified PNA Duplexes
A R T I C L E S
Figure 3. Spectrophotometric titration of a 5 µM Q-XA and 5 µM Q-XB
solution in pH ) 7.0 10 mM phosphate buffer with 0.500 mM CuCl2
solution, T ) 25 °C.
Figure 4. Spectrophotometric titration of a 5 µM Q-XA and 5 µM Q-XB
solution in pH ) 7.0 10 mM phosphate buffer with 0.500 mM CuCl2
solution, T ) 95 °C.
that occurs at 376 nm for the synthetic CuQ2 is shifted to 412
nm for the (Q-Xi:Q-Xi)-Cu2+ duplexes formed at 25 °C,
which is likely due to steric influence exerted by the PNA duplex
on the geometry of the CuQ2 moiety. Similar UV-vis titrations
of Q-XE with Cu2+ show that addition of Cu2+ leads to
transformation of [Cu(Q-XE)2] into [Cu(Q-XE)] not only at
95 °C (Figures S10 and S11) but also at 25 °C (Figures 7 and
8), as expected because Q-XE cannot form a homoduplex based
on Watson-Crick base pairing. The transformation of
[Cu(Q-XE)2] to [Cu(Q-XE)] is supported not only by the
observation of two inflection points in titration curves (Figures
7b and 8b) but also by the hypsochromic shift of the 400 nm
band characteristic for [Cu(Q-XE)2] to 393 nm characteristic
for the [Cu(Q-XE)]complex (Figure 8a).
Additionally, the transformation of [Cu(Q-PNA)2] to
[Cu(Q-PNA)] upon addition of excess Cu2+ has been con-
firmed by titrations with 5,5′-dimethyl-2,2′-bipyridine (Me-bipy),
monitored at ∼400 nm, which is a charge-transfer transition of
CuQ2, and at 318 nm, which is an absorption band of
coordinated Me-bipy. Upon Me-bipy addition to solutions that
are 40 µM in Cu2+ and 40 µM in Q-XB, besides the appearance
of bands characteristic for the Cu2+-coordinated Me-bipy at 318
S8b, S15b). This hypothesis is supported by the titration of the
nonmodified PNA duplex XA:XB with Cu2+, which shows
indeed a very weak increase in absorbance at 260 nm with
increasing Cu2+ concentration (Figure S12). Furthermore, the
increase in absorbance after quantitative Cu2+ binding to Q
ligands in the Q-PNA duplexes is significantly lower at
25 °C, where all nucleobases are involved in hydrogen bonding.
The same two complexes [Cu(Q-XB)2] and [Cu(Q-XB)] form
in the opposite order in the reverse titration of Cu2+ solutions
with Q-XB at 95 °C (Figures S13).
UV-vis titrations at 25 °C show two isosbestic points at 254
and 353 nm, but in contrast to the observations made at 95 °C,
the changes in absorbance show a single inflection point at
2:1 Q-PNA:Cu2+ molar ratio, indicating that once the
(Q-Xi:Q-Xi)-Cu2+ (i ) A-D) duplexes form, they cannot
be converted by excess Cu2+ to Cu(Q-Xi) complexes (Figures
3, 5, S4, S5, S7, S9, S14). Notably the charge-transfer transition
(36) Philips, J. P.; Huber, W. H.; Chunga, J. W.; Merritt, L. L. J. Am. Chem.
Soc. 1951, 73, 630-632.
(37) Stone, K. G.; Friedman, L. J. Am. Chem. Soc. 1947, 69, 209-211.
(38) Sone, K. J. Am. Chem. Soc. 1953, 75, 5207-5211.
(39) Amundsen, A. R.; Whelan, J.; Bosnich, B. J. Am. Chem. Soc. 1977, 99,
6730-6739.
9
J. AM. CHEM. SOC. VOL. 127, NO. 42, 2005 14633

A R T I C L E S
Watson et al.
Figure 5. Spectrophotometric titration of a 25 µM Q-XA and 25 µM
Q-XB solution in pH ) 7.0 10 mM phosphate buffer with 2.00 mM CuCl2
solution, T ) 25 °C.
Figure 6. Spectrophotometric titration of a 25 µM Q-XA and 25 µM
Q-XB solution in pH ) 7.0 10 mM phosphate buffer with 2.00 mM CuCl2,
T ) 95 °C.
mentarity of Q-Xi strands. The latter behavior is different from
that of 8-hydroxyquinoline under the same conditions, which
at 95 °C forms [CuQ2] complexes stable with respect to further
addition of Cu2+ (see Supporting Information, pages S8-S10).
This difference is attributed to the conformational entropy loss
for the complex formation, which is larger for Q-PNA strands
than for 8-hydroxyquinoline itself. Comparison of the low-
temperature behavior of the noncomplementary Q-XE strand
with the partly complementary Q-Xi, i ) A-D, strands
indicates that if the [Cu(Q-Xi)2] complexes are held together
not only by metal ion coordination bonds but also by hydrogen
bonds within Watson-Crick base pairs, the enthalpic stabiliza-
tion overcomes the entropic destabilization of 1:2 complexes
with respect to 1:1 complexes and leads to the relationship
between stepwise binding constants for Cu2+ to Q-PNA:
Κ1 , Κ2.
nm, the charge-transfer band of the Cu2+-coordinated 8-hydroxy-
quinoline moiety undergoes a bathochromic shift from 393 to
400 nm (Figure 9). The absorbance increase at 318 and at
400 nm has an inflection point at a Cu2+:Me-bipy ratio of 1:2.
The bathochromic shift and inflection point indicate that
[Cu(Q-XB)] (λmax ) 393 nm) transforms into [Cu(Q-XB)2]
complex (λmax ) 400 nm) concomitant with formation of
[Cu(Me-bipy)] (λmax ) 318 nm):
2[Cu(Q-XB)] + Me-bipy f
[Cu(Q-XB)2] + [Cu(Me-bipy)]
Titrations at 95 °C of solutions with a 1:2 Cu:Q-XB ratio
showed no change in the absorbance or position of the 400 nm
band, indicating that the [Cu(Q-XB)2] complex is stable to Me-
bipy addition (Figure 10).
These observations indicate that binding of 0.5 equiv of Cu2+
to Q ligands leads to association of Q-Xi strands into
[Cu(Q-Xi)2] complexes at any temperature. Above the melting
temperature, [Cu(Q-Xi)2] are converted to [Cu(Q-Xi)] upon
addition of Cu2+, irrespective of the degree of (self)-comple-
EPR Spectroscopy. To further investigate the coordination
of Cu2+ to Q-PNA, we have recorded EPR spectra for solutions
containing duplexes annealed in the presence of Cu2+. In the
presence of phosphate anions, Cu2+ forms EPR-silent, poly-
nuclear complexes. Thus, for our samples that are prepared in
phosphate buffer, an EPR spectrum is expected only if the
9
14634 J. AM. CHEM. SOC. VOL. 127, NO. 42, 2005

Mismatch Tolerance of Ligand-Modified PNA Duplexes
A R T I C L E S
Figure 7. Spectrophotometric titration of a 10 µM Q-XE solution in pH
) 7.0 10 mM phosphate buffer with 0.500 mM CuCl2 solution, T ) 25 °C.
Figure 8. Spectrophotometric titration of a 40 µM Q-XE solution in pH
) 7.0 10 mM phosphate buffer with 2.00 mM CuCl2 solution, T 25 °C.
phosphate complexes are destroyed by binding of Cu2+ to
stronger ligands in the modified Q-PNA. Indeed, a 100 µM
Cu2+ solution in pH 7.0 sodium phosphate buffer showed a very
weak EPR spectrum, which integrated to <5 µM Cu2+. No
spectrum was observed for the duplex XA:XB formed by
annealing in the presence of Cu2+, which confirms that at this
concentration level Cu2+ does not bind to the nucleobases
involved in hydrogen bonding within the Watson-Crick base
pairs.
[CuQ2] and for the synthetic complex may be due to weak
interactions between the adjacent base pairs and the Cu2+ ion
coordinated to two Q ligands, as previously observed by Atwell
et al.,42 to weak binding of water or phosphate molecules in
the axial positions of the complex, or to differences between
the structure of the free- and PNA-enclosed-[CuQ2] moiety.
Integration of the EPR signals for all homoduplexes and for
(Q-XA:Q-XB)-Cu2+ confirmed the quantitative 1:2 binding
of Cu2+ to the two 8-hydroxyquinoline ligands present within
the ligand-containing duplexes, in agreement with the results
of spectrophotometric titrations.
CD Spectroscopy. Having obtained from UV-vis and EPR
spectroscopy information about the Cu2+ coordination to
Q-PNA, we have used CD spectroscopy to investigate the
secondary structure of the duplexes formed upon metal binding.
In contrast to DNA, which has a chiral backbone and shows a
CD spectrum characteristic for each type of duplex (e.g., A, B,
or Z), PNA has an achiral backbone and PNA duplexes show
a preference for a given handedness only upon chiral induction
by an enantiomerically pure component, such as an L-amino
acid attached to the carboxy terminus of the PNA oligomers.34,43
EPR spectra for solutions of duplexes that contain 8-hydroxy-
quinoline ligands, (Q-XA:Q-XA)-Cu2+, (Q-XB:Q-XB)-
Cu2+, (Q-XC:Q-XC)-Cu2+, and (Q-XA:Q-XB)-Cu2+, were
similar to each other and indicative of Cu2+ coordinated to the
Q ligands (Figure 11). Simulation of the EPR spectra with an
1
S ) /2 spin Hamiltonian that includes hyperfine parameters
for 63,65Cu2+ (I ) 3/2) and 14N (I ) 1) leads to parameters (Table
2) similar to those measured for the [CuQ2] complex in toluene/
chloroform solutions or in pH 8.5 ethylene glycol-water glasses
and confirms binding of Cu2+ to two Q ligands.40,41 The small
difference between the A3 values for the PNA-incorporated
(40) Walker, F. A.; Sigel, H.; McCormick, D. B. Inorg. Chem. 1972, 11, 2756-
2763.
(41) Gersmann, H. R.; Swalen, J. D. J. Chem. Phys. 1962, 36, 3221-3233.
(42) Atwell, S.; Meggers, E.; Spraggon, G.; Schultz, P. G. J. Am. Chem. Soc.
2001, 123, 12364-12367.
9
J. AM. CHEM. SOC. VOL. 127, NO. 42, 2005 14635

A R T I C L E S
Watson et al.
Figure 11. Perpendicular mode X-band EPR spectrum of a 100 µM (Q-
XA:Q-XB)-Cu solution in pH 7.0 10 mM sodium phosphate buffer with
25% glycerol (solid line) and simulation (dotted line) using the parameters
given in Table 2. T ) 20 K, frequency 9.65 GHz, microwave power 0.02
mW, modulation amplitude 6.676 G.
Figure 9. Spectrophotometric titration of a 40 µM CuCl2 and 40 µM Q-XB
solution with a 2.00 mM Me-bipyridine solution in pH ) 7.0 10 mM
phosphate buffer, T ) 95 °C (inset: view of absorbance for 300 < λ <
500 nm).
Figure 12. CD spectra of annealed 20 µM Q-XA (red) and 20 µM Q-XB
(blue), and 10 µM Q-XA:Q-XB in the absence (black dotted line) and in
the presence (black solid line) of 10 µM Cu(NO3)2. Solutions were prepared
in pH 7.0 10 mM sodium phosphate buffer.
observation, one would predict that mismatches and coplanar
metal-coordinated ligand pairs have a negative and positive
impact, respectively, on the chirality of modified PNA duplexes,
a hypothesis supported by our experimental results.
We have measured the CD spectra of Q-modified PNA
homoduplexes [Q-XA:Q-XA] and [Q-XB:Q-XB] in the
absence and presence of Cu2+ and found that they do not
show a preferred handedness (Figure 12). The heteroduplex
Q-XA:Q-XB has a preferred left-handedness in the absence
of Cu2+, which coincides with that of the nonmodified PNA
duplex.44,45 (Q-XA:Q-XB)-Cu2+ has a significantly more
intense CD spectrum than that of the Q-XA:Q-XB duplex
Figure 10. Spectrophotometric titration of a 20 µM CuCl2 and 40 µM
Q-XB solution with a 2.00 mM Me-bipyridine solution in pH ) 7.0 10
mM phosphate buffer, T ) 95 °C.
(43) Wittung, P.; Eriksson, M.; Lyng, R.; Nielsen, P. E.; Norden, B. J. Am.
Chem. Soc. 1995, 117, 10167-10173.
The chiral induction effect depends on the type of base pair
that is adjacent to the chiral component of the PNA duplex,
with the more stable GC base pair being more efficient in
transmitting the effect than an AT pair.43 On the basis of this
(44) Sforza, S.; Haaima, G.; Marchelli, R.; Nielsen, P. E. Eur. J. Org. Chem.
1999, 197-204.
(45) Lagriffoule, P.; Wittung, P.; Eriksson, M.; Jensen, K. K.; Norden, B.;
Buchardt, O.; Nielsen, P. E. Chem.-Eur. J. 1997, 3, 912-919.
9
14636 J. AM. CHEM. SOC. VOL. 127, NO. 42, 2005

Mismatch Tolerance of Ligand-Modified PNA Duplexes
A R T I C L E S
Table 2. EPR Parameters for [CuQ2] within the PNA Context and in the Synthetic Complexa
+
Cu2
+
Cu2
+
Cu2
+
(Q
−XA:Q
−XB)−
(Q
−XA:Q
−XA)−
(Q
−XB:Q
−XB)−
[CuQ2]40
(G:G)−
Cu2
g1
g2
g3
2.06
2.05
2.22
72
80
611
39
2.06
2.05
2.22
73
80
612
39
2.06
2.05
2.23
69
79
607
39
2.06
2.06
2.24
72
72
530
39
2.07
2.07
2.32
A1 (MHz)
A2 (MHz)
A3 (MHz)
486
A
A
A
1N (MHz)
2N (MHz)
3N (MHz)
32
36
31
35
34
37
33
33
a Ai and AiN are hyperfine coupling constants for copper and nitrogen nuclei, respectively.
(Figure 12), which suggests that despite being situated far from
the chiral, C-terminal amino acid, the metal-ligand pair
significantly influences the transmission of the chiral effect
through the duplex. On the other hand, even in the presence of
Cu2+, no chiral excess is induced by the L-lysine residue in
Q-XA:Q-XA and Q-XB:Q-XB duplexes, which have a
significantly lower number of hydrogen-bonded base pairs. This
observation reinforces the idea that π-stacking interactions
throughout the duplex are an important condition for transmis-
sion of the chiral effect.34,43
Given the intense CD signal at 256 nm of Q-XA:Q-XB and
(Q-XA:Q-XA)-Cu2+, we have also performed thermal dena-
turation studies by monitoring the CD intensity at this wave-
length and found good agreement with the melting curves
measured using UV hypochromicity (Figure S19).
the binding of two extraneous small ligands (e.g., water or
phosphate) to the [CuQ2] moiety within PNA is unlikely because
it would require significant distortions of the duplex to accom-
modate the axial ligands of Cu2+. It is also notable that the
intermolecular distance between stacked [CuQ2] molecules in
single crystals is 3.36 Å, which is similar to the distance between
natural base pairs in PNA duplexes.50,51 If indeed the differences
in the EPR values are due to weak binding of axial ligands,
these ligands would have to be nucleobases adjacent to the
[CuQ2] site, as previously observed by Atwell et al.42 Addition-
ally, while all structurally characterized, four-coordinate syn-
thetic complexes of Cu2+ with two 8-hydroxyquinoline have a
trans arrangement of the ligands,24-27 we cannot rule out a cis
orientation of the two Q ligands within the PNA duplex, and
such an orientation could also lead to differences between the
EPR spectra of the Q-PNA duplexes and of the synthetic
complex. This orientation has been proposed by Zhang et al.,14
but no experimental evidence was brought forth. For the cis
isomer, the relative orientation of the C-C bonds that link the
8-hydroxyquinoline to the PNA or DNA backbone is closer to
that of the C-C bonds that link GC and AT base pairs to the
duplex backbone than the relative orientation for a trans isomer.
On the other hand, a cis orientation of the two 8-hydroxyquino-
line within a square planar complex leads to a steric clash
between the protons situated on the C2 atoms of the ligand,
which could lead to tetrahedral distortion of the complex and
weakening of the H bonds of the bases adjacent to the CuQ2
moiety. We are currently pursuing combined electronic structure
and molecular modeling calculations to evaluate these possibili-
ties.
Discussion
EPR spectroscopy and spectrophotometric titrations measure
the stoichiometry of complexes formed between Cu2+ and
Q-PNA and show that, in solutions containing
a
Cu2+:Q-PNA ratio of 1:2, [Cu(Q-Xi)2] complexes form
irrespective of the nucleobase sequence of the ligand-containing
PNA strands Q-Xi and of the temperature. The similarity
between the UV-vis and EPR spectra for the synthetic [CuQ2]
complex known to adopt a four-coordinate, square planar
coordination and for (Q-Xi:Q-Xi)-Cu2+ duplexes indicates
that the copper coordination in the duplexes is also square planar.
The small difference between the A3 values for these compounds
could be due to binding of one or two more ligands in axial
positions to Cu2+ or to differences between the geometry of
the [CuQ2] unit in the synthetic complex and that in the PNA-
bound one. Coordination of a single water molecule to Cu2+ is
unlikely because, as observed in the crystal structure of the five-
coordinate complex [CuQ2(OH2)], the planes of the aromatic
rings of the two Q ligands would form a 50° dihedral angle, an
arrangement which would have a destabilization rather than a
stabilization effect on the PNA duplex.46-48 Examination of the
crystal structure of a six-coordinate [CuQ2(OH2)2] complex
Variable-temperature UV spectroscopy shows that full Wat-
son-Crick complementarity is not necessary for the formation
of metal-bridged duplexes. Solutions of Q-PNA strands that
contain one 8-hydroxyquinoline and have as few as four
complementary base pairs form in the presence of 0.5 equiv of
Cu2+ duplexes that show a cooperative melting transition. This
transition takes place with significant hyperchromicity, and Tm
can be correlated with the degree of self-complementarity of
the Q-Xi single strands. The fully complementary strands
Q-XA and Q-XB form the duplex (Q-XA:Q-XB)-Cu2+
,
shows that the axial Cu-OH O distances are 2.45 Å and that
2
the complexes are arranged in the crystal in stacks of parallel-
oriented molecules with the distance between the planes of
adjacent molecules being 5.54 Å.49 Based on these observations,
which has the highest thermal stability, while the noncomple-
mentary strand Q-XE does not form a stable duplex despite
the fact that Cu2+ leads to association of the strands into
[Cu(Q-XE)2] complexes. In contrast, the partially complemen-
(46) Jian, F.; Wang, Y.; Lu, L.; Yang, X.; Wang, X.; Chantrapromma, S.; Fun,
H. K.; Razak, I. A. Acta Crystallogr., Sect. C 2001, 57, 714-716.
(47) Kruh, R.; Dwiggins, C. W. J. Am. Chem. Soc. 1955, 77, 806.
(48) Suito, E.; Arakawa, M.; Kobayashi, T. Kolloid Z. Z. Polym. 1966, 212,
155-161.
(50) Petit, S.; Coquerel, G.; Perez, G.; Louer, D.; Louer, M. Chem. Mater. 1994,
6, 116-121.
(51) Petersson, B.; Nielsen, B. B.; Rasmussen, H.; Larsen, I. K.; Gajhede, M.;
Nielsen, P. E.; Kastrup, J. S. J. Am. Chem. Soc. 2005, 127, 1424-1430.
(49) Okabe, N.; Saishu, H. Acta Crystallogr., Sect. E 2001, 57, m251-m252.
9
J. AM. CHEM. SOC. VOL. 127, NO. 42, 2005 14637

A R T I C L E S
Watson et al.
tary homoduplexes (Q-Xi:Q-Xi)-Cu2+ have intermediate
thermal stability (c-f in Chart 1). In Chart 1, all duplexes are
written with their strands in antiparallel alignment and with the
Q ligands placed in complementary positions. In these align-
ments, no Watson-Crick base pair would form in duplexes
Q-XC:Q-XC and Q-XD:Q-XD, and their stability would be
expected to be low. In contrast, arrangements shown in Chart
2 for the Q-XC:Q-XC and Q-XD:Q-XD duplexes lead to
six and four Watson-Crick base pairs, respectively, and place
the two Q ligands of each duplex in close proximity to each
other. The EPR spectrum of the (Q-XC:Q-XC)-Cu2+ duplex
and the spectrophotometric titration at 25 °C (Figure S9) of
Q-XD:Q-XD with Cu2+ establishes coordination of one Cu2+
to two Q ligands per duplex.52 To allow the formation of the
CuQ2 moiety in these duplexes, the bases across from the two
Q ligands can either be situated within the duplex and have
π-π interactions with the adjacent base pairs or be bulged out
of the duplex. Table 1 shows the Tm’s for all homoduplexes as
well as the maximum number of Watson-Crick and wobble
base pairs and the number and type of overhangs of each duplex.
Analysis of the data contained in the table indicates a correlation
between the number of complementary base pairs in each duplex
and its Tm. Duplex (Q-XC:Q-XC)-Cu2+, which can form six
Watson-Crick base pairs, is more stable than homoduplexes
A, B, D, which can form at most four Watson-Crick base pairs.
For duplexes (Q-Xi:Q-Xi)-Cu2+, where i ) B, A, and D,
the thermal stability increases if besides the four Watson-Crick
base pairs they can either form two additional wobble base pairs
(i.e., duplexes with i ) A or B) or contain overhang bases,
with A overhangs having a larger stabilization effect than T
overhangs as previously observed in PNA duplexes53 (com-
pare (Q-XB:Q-XB)-Cu2+ with (Q-XA:Q-XA)-Cu2+ and
(Q-XD:Q-XD)-Cu2+ in Charts and in Table 1).
duplex, as indicated by the high hypochromicity observed in
the melting curves. A related effect of zipper-like recognition
based on interstrand π-stacking interactions has been reported
by Brotschi et al.55,56 In a study of DNA duplexes containing
hydrophobic biphenyl (Bph) residues instead of nucleobases,
they have shown that an increasing number of Bph moieties
situated in the middle of a DNA duplex leads to an increase of
the Tm as measured by monitoring hypochromicity at 260 nm.55
The CD spectra of the DNA duplexes containing up to eight
Bph residues did not differ from those of the corresponding
nonmodified DNA duplexes, which suggests that the Bph
residues adopt a highly ordered, stacked arrangement within
the double helix. It is notable that in contrast to the biphenyl-
modified DNA in which complementary natural base pairs flank
the biphenyl residues situated in the middle of the PNA duplex,
in the PNA system under consideration in the present paper,
interactions of the natural base pairs at the extremities of the
duplex are triggered by strong binding between the metal ion
and the ligands situated in the middle of the duplex.
At temperatures above Tm and in the absence of complemen-
tarity of PNA strands, addition of excess Cu2+ leads to
conversion of [Cu(Q-PNA)2] complexes to [Cu(Q-PNA)].
This transformation does not occur at 25 °C if the CuQ2 moiety
is situated in a PNA duplex, because the duplex exerts a
supramolecular chelate effect, prearranging the ligands in close
proximity and making the stability of the square planar [CuQ2]
complex exceptionally high.
Chiral induction by L-Lys is observed only for the fully
complementary (Q-XA:Q-XB)-Cu2+ and Q-XA:Q-XB du-
plexes. Norden et al. determined that the correlation length in
PNA is around 10 base pairs; that is, for PNA hairpins, the CD
amplitude increases almost linearly with the number of comple-
mentary base pairs of the stem up to 10 pairs, while in the case
of intermolecular duplexes, the CD amplitude also increases
with the duplex length up to 10 base pairs, although to a smaller
extent. Notably, even if heteroduplexes (Q-XA:Q-XB)-Cu2+
and Q-XA:Q-XB differ only by the presence and absence,
respectively, of one central [CuQ2] moiety, the amplitude of
their CD spectra differs by almost a factor of 2. The significantly
larger intensity of the CD spectrum for (Q-XA:Q-XB)-Cu2+
when compared to that of Q-XA:Q-XB supports the idea that
π-interactions throughout the duplex are significantly stronger
upon metal binding to the Q-PNA duplexes, and thus the chiral
effect of the Lys residue is better transmitted in the former,
metal-containing duplex. The strong effect of the metal ion on
the CD spectra and, implicitly, on the helicity of the duplex
suggests that the correlation length may be longer for PNA
duplexes that contain metal-ligand alternative base pairs, a
possibility that will be investigated in future studies.
Formation of PNA duplexes even from partly complementary
PNA strands can be rationalized by assuming that the tight
binding of the two strands by Cu2+ coordination to the ligands
centrally located in the PNA strands overcomes the loss of
translational entropy for the duplex formation and brings the
nucleobases in sufficient proximity such that π-π interactions
and a few hydrogen bonds are a sufficient driving force for
duplex formation.54 The noncomplementary bases within the
duplexes formed from partly complementary Q-PNA strands
are likely involved in π-π stacking interactions within the
(52) For 25 °C UV titrations of Q-XD with Cu2+, an inflection point occurs in
the absorbance change of the π-π* transition of 8-hydroxyquinoline at a
Q-XD:Cu2+ ratio of 2:1. This observation rules out the possibility that
two Cu2+ ions would bind to the two mixed-ligand, 8-hydroxyquinoline-
nucleobase coordination sites that can form within the duplex in arrange-
ments 2 and 3 of Chart 2. Thus, if two Q-XD strands are aligned to form
a Q-XD:Q-XD duplex with maximum number of Watson-Crick base
pairs, which is four, one Cu2+ must bind to the two Q ligands situated in
close proximity but not in complementary positions (Chart 2). To further
verify the binding ability of a Q-G motif, we have prepared the PNA
This study demonstrates that incorporation of a strong metal-
ligand moiety within a PNA duplex has a strong stabilization
effect on duplexes and that PNA duplexes able to form strong
metal-based pairs have high tolerance to mismatches. A similar
duplex G-XA:Q-XB
) H-GTAGGTCACT-Lys-NH2:H2N-Lys-
CATCQAGTGA-H. The EPR spectrum of the (G-XA:Q-XB)-Cu2+
duplex is similar to that of the (Q-XA:Q-XB)-Cu2+ duplex, indicating
that one Cu2+ is coordinated by two Q ligands. As we have ruled out the
possibility that the presence of Cu2+ would lead to the formation of (Q-
XB:Q-XB)-Cu2+ because the thermal denaturation curves do not show a
Tm of 79 °C characteristic for this duplex, Cu2+ must bridge G-XA:Q-
(54) This hypothesis is supported by the fact that in the absence of Cu2+ all
partly complementary Q-PNA strands show very weakly cooperative
thermal denaturation curves with Tm’s < 45 °C (Figure S20), even if the
same number of Watson-Crick base pairs as in the presence of Cu2+ could
be formed.
XB duplexes to achieve a coordination of two 8-hydroxyquinoline ligands.
Finally, attempts to do 25 °C spectrophotometric titrations of G-XA:Q-
XB with Cu2+ failed because upon addition of small amounts of Cu2+ the
UV absorbance changes with time due to an increase in the background,
which is indicative of scattering due to PNA aggregation and supports the
idea that inter-duplex CuQ2 bridges form instead of Cu2+ coordinating a
G nucleobase and a Q ligand within the duplex.
(55) Brotschi, C.; Leumann, C. J. Angew. Chem., Int. Ed. 2003, 42, 1655-
1658.
(56) Brotschi, C.; Ha¨berli, A.; Leumann, C. J. Angew. Chem., Int. Ed. 2001,
40, 3012-3014.
(53) Datta, B.; Armitage, B. A. J. Am. Chem. Soc. 2001, 123, 9612-9619.
9
14638 J. AM. CHEM. SOC. VOL. 127, NO. 42, 2005

Mismatch Tolerance of Ligand-Modified PNA Duplexes
A R T I C L E S
stabilization effect of Cu2+-Q coordination has been observed
by Zhang et al. in Q-DNA duplexes, where it was exploited
to introduce drastic changes to the backbone. In the absence of
metal ions, such a backbone modification would impose an
energetic penalty too high for duplex formation.14 Interestingly,
the shape of the melting curve measured by Zhang et al. for
Q-DNA in the presence of Cu2+ is similar to that measured
for Q-PNA, which suggests that the metal-ligand moiety is
the major determinant of the properties of the nucleic acid in
which it is incorporated.
The potential for nanotechnology applications of metal-
containing nucleic acid structures has been recognized in the
past 5 years.11,16 The results presented in this paper indicate
that the presence of strongly bound metal-ligand moieties
makes possible formation of nucleic acid-based structures even
in the presence of a small number of Watson-Crick base pairs
of natural nucleobases, a principle that can be exploited in the
design and synthesis of hybrid nucleic acid-metal structures
with new topologies and properties.
Acknowledgment. We are grateful to the National Science
Foundation (CHE-0347140) and the Camille and Henry Dreyfus
Foundation for financial support of this research. We thank Dr.
Michael Hendrich for the EPR simulation software. NMR
spectra were recorded on instruments purchased with an NSF
grant (CHE-0130903). MALDI-TOF and ES mass spectrograms
were recorded in the Center for Molecular Analysis at Carnegie
Mellon University, which is supported by NSF grants CHE-
9808188 and DBI-9729351.
Supporting Information Available: Absorption spectra at 25
and 95 °C for solutions containing the 8-hydroxyquinoline PNA
monomer 1 and Cu2+, spectrophotometric titrations of single
strand Q-Xi (i ) A, B, D, E) and XA:XB with Cu2+
,
spectrophotometric titration of Cu2+ solution with Q-XB,
spectrophotometric titration of 8-hydroxyquinoline, and titrations
with Me-bipy. This material is available free of charge via the
Internet at http://pubs.acs.org.
JA051336H
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J. AM. CHEM. SOC. VOL. 127, NO. 42, 2005 14639