Molecular recognition of a DNA:RNA hybrid: Sub-nanomolar binding by a neomycin-methidium conjugate

Article abstract of DOI:10.1016/j.bmcl.2008.05.090

A novel neomycin-methidium conjugate was synthesized. The covalent linkage of the aminoglycoside to an intercalator, a derivative of ethidium bromide, results in a new conjugate capable of selective recognition of the DNA:RNA hybrid duplex. Spectroscopic methods: UV, CD, fluorescence, and calorimetric techniques: DSC and ITC were used to characterize the sub-nanomolar binding displayed by the conjugate for the DNA:RNA hybrid duplex, poly(dA):poly(rU).

Full text of DOI:10.1016/j.bmcl.2008.05.090

Bioorganic & Medicinal Chemistry Letters 18 (2008) 4142–4145
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Molecular recognition of a DNA:RNA hybrid: Sub-nanomolar binding
by a neomycin–methidium conjugate
*
Nicholas N. Shaw, Hongjuan Xi, Dev P. Arya
Laboratory of Medicinal Chemistry, Department of Chemistry, Clemson University, Clemson, SC 29634, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel neomycin–methidium conjugate was synthesized. The covalent linkage of the aminoglycoside to
an intercalator, a derivative of ethidium bromide, results in a new conjugate capable of selective recog-
nition of the DNA:RNA hybrid duplex. Spectroscopic methods: UV, CD, fluorescence, and calorimetric
techniques: DSC and ITC were used to characterize the sub-nanomolar binding displayed by the conju-
gate for the DNA:RNA hybrid duplex, poly(dA):poly(rU).
Received 13 February 2008
Revised 21 May 2008
Accepted 21 May 2008
Available online 28 May 2008
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
DNA
Aminosugar
Ethidium bromide
Intercalator
Neomycin
Aminoglycoside
RNA
DNA:RNA hybrid
Telomerase
DNA:RNA hybrid structures have attracted recent attention due
to the key roles they play in a number of biological processes.
DNA:RNA hybrids can be found in transcription,1 reverse transcrip-
tion2,3 and in the priming of DNA prior to replication.4,5 DNA:RNA
hybrids play crucial roles in different types of enzymatic activity,
notably telomerases6 and HIV RNase. However, when one attempts
to identify small molecule binders of DNA:RNA hybrids, fewer than
10 pharmacophores can be found.7–9 In fact, few reported attempts
have been made to target the unique structure of DNA:RNA hybrids
and only a handful of molecules have been identified.8 For compar-
ison, the number of molecules studied that target duplex DNA is in
the thousands. A few years ago, we identified aminoglycosides as
lead candidates in the groove-based recognition of A-form nucleic
acid structures.2 DNA:RNA hybrids normally adopt A-form struc-
tures. These major groove binders were subsequently shown to
bind DNA:RNA hybrids in the sub-nanomolar range and inhibit
RNA processing enzymes, RNase H and RNase A, by binding to
RNA:DNA hybrids.10 Two other laboratories, using two different as-
says, have shown the preference of ethidium bromide for DNA:RNA
new class of molecules with high DNA:RNA hybrid affinity can be
developed. A neomycin–methidium conjugate (NM, Scheme 1,
compound 3) has therefore been synthesized by formation of an
amide bond linkage between neomycin and methidium carboxylic
acid, 2. We report that 3 is more potent in stabilizing DNA:RNA hy-
brids than neomycin, ethidium bromide, or a non-covalent combi-
nation of both. In fact, the affinity of the conjugate parallels the
highest affinity aminoglycoside ligands ever identified for nucleic
acid recognition.
The following assumptions were made in the design of the con-
jugate: the amino groups on rings I, II, and IV of neomycin are nec-
essary in stabilizing and recognizing the nucleic acid grooves
(aminoglycosides without any of these amines do not stabilize
nucleic acids as efficiently).7,12–14 The 500 –OH on ring III was thus
chosen to provide linkage to the intercalating unit. The intercalator
6-(4-carboxyphenyl)-3,8-diamino-5-methylphenanthridinium chlo-
ride (2) was linked to neomycin amine, 1, (prepared in three steps
from neomycin)15–17 using DCC-mediated amide bond formation.
TFA deprotection yielded the conjugate, 3, in good yields.
CD and UV melting experiments were carried out to confirm hy-
brid duplex stabilization in the presence of the ligands. Addition of
hybrids,9,11 which binds in the low
lM range.
We hypothesized that by conjugating neomycin with a specific
DNA:RNA binding intercalator, ethidium bromide, high selectivity
and binding of DNA:RNA hybrids can be achieved. Furthermore, a
2.2 lM of neomycin {ratio of poly(dA):poly(rU) base pairs to drug
(rbd) = 9} and ethidium bromide leads to a Tm increase of 12 °C,
whereas addition of 3 at the same rbd leads to a Tm increase of
20 °C (Fig. 1a). Increased concentration of 3 to the DNA:RNA hybrid
resulted in a gradual increase in the melting temperature of the
* Corresponding author.
E-mail address: dparya@clemson.edu (D.P. Arya).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.05.090

N. N. Shaw et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4142–4145
4143
M)
Table 1
NHBoc
O
I
Melting temperatures of nucleic acid structures (20 lM/bp) with NM, 3, (2.2 l
O
HO
HO
Tm0 (°C)
Tm (°C)
D
Tm (°C)
OH
HN
II
Boc
N
BocH
H2N
NHBoc
O
A-site RNA
Calf thymus DNA
Poly(dA):2poly(dT)
71.7
86.9
23.1
68.2
68.4
67.9
93.6
57.1
43.6
62.3
79.8
>95
—
75.9
76.1
75.3
>95
91.4
90.5
82.4
8.1
>8.1
—
7.7
7.7
7.4
—
34.3
46.9
20.1
S
O
O
OH
III
+
N+
NHBoc
Poly(dA):poly(dT)
Poly(dA–dT):poly(dA–dT)
Poly(dG):poly(dC)
Poly(rA):poly(rU)
Poly(dA):poly(rU)
Poly(rA):poly(dT)
O
OH
IV
H2N
NH2
O
OH
Boc HN
OH
2
1
a, b
Buffer: 10 mM sodium cacodylate, 0.5mM EDTA, pH 5.5 and 100 mM NaCl.
NH2
O
HO
HO
0.2
O
H2N
H
N
(4)
H2N
NH2
(2)
O
0.15
S
O
(5)
O
OH
0.1
(1)
(3)
NH2
N+
O
OH
0.05
O
OH
0
H2N
H2N
NH2
OH
32
40
48
56
64
72
80
88
T (oC)
3
Scheme 1. Reagents and conditions: (a) DCC, DMAP, DMF, rt, 28 h, 80%; (b) TFA/
CH2Cl2, HSCH2CH2SH, rt, 30 min, 90%.
Figure 2. Mixed melting profile of various nucleic acids. The panel shows
derivative plot for the mixed melting of poly(dG):poly(dC); peak 5, poly(dA):po-
ly(dT); peak 4, poly(rA):poly(dT); peak 3, poly(rA):poly(rU); peak 2, poly(dA):po-
a
ly(rU); peak 1. Individual polynucleotide concentration was 10 lM/bp, NM, 3,
(rbd = 20). In the panel, the solid line reflects native melting and the dashed line
represents the melting with NM. Buffer: 1.5 mM Na2HPO4, 0.5 mM NaH2PO4,
0.25 mM Na2EDTA and 46.25 mM NaCl at pH 7.0.
hybrid. As depicted in Figure 1b, as the concentration of 3 is
increased from 0 to 3 lM the melting temperature of poly(dA):
poly(rU) increases from 48.9 to 70.1 °C, a 21.2 °C increase overall.
In order to ascertain the selectivity of this novel conjugate,
nucleic acid structures known to bind ethidium bromide and neo-
mycin were thermally denatured in the presence of equimolar
amounts of 3, affording a thermal melting profile for the conjugate,
Table 1. Additionally, a mixture composed of various equimolar
nucleic acids was thermally denatured in the presence of 3 to fur-
ther illustrate selectivity of the conjugate, Figure 2. A relationship
between binding preference and thermal stabilization afforded by
3 was obtained using UV melting experiments. Table 1 shows those
nucleic acids which 3 displays a high preference for and shows
significant thermal stabilization. Poly(dA):poly(rU) stability is
increased by 46.9 °C, 12.6 °C higher than any other nucleic acid
structure. Not only does 3 show a high preference for DNA:RNA
hybrids, poly(dA):poly(rU) thermal stabilization afforded by 3 is
significantly higher than any other nucleic acid structure.
Most recently, an assay using thermal denaturation of mixtures
containing various nucleic acids in the presence of ligand was
developed to determine structural and sequence selectivity.18 As
seen in Figure 2, when assayed against a solution containing
poly(dA):poly(rU), poly(rA):poly(dT), poly(dA):poly(dT), poly(rA):
poly(rU), and poly(dG):poly(dC), at rbd = 20, only the melting tem-
perature of poly(dA):poly(rU) is increased, while all other duplexes
remain unchanged. In fact, increasing the concentration to
rbd = 10 affords stabilization to poly(dA):poly(rU) (2.0 °C) and
1.2
0.62
a
b
0.6
0.58
0.56
0.54
0.52
0.5
1
0.8
0.6
2
0
1
3
A
B
C
D
E
0.4
0.2
0.48
0.46
30
40
50
60
70
80
40
45
50
55
60
65
70
75
80
T (oC)
T (oC)
Figure 1. (a) CD melting profile of poly(dA):poly(rU) in the presence of ligands. DNA:RNA hybrid (A) (20.0
neomycin, and ethidium bromide (D) and NM, 3, (E). All ligands were at a concentration of 2.2 M. (b) UV melting profile of poly(dA):poly(rU) in the presence of increasing 3.
DNA:RNA hybrid (20.0 M/bp) was mixed with NM 3 at increasing concentrations (0, 1, 2, and 3 M) before slow heating to 90 °C at a rate of 10 °/h. Buffer conditions: 10 mM
sodium cacodylate, 0.5 mM EDTA, 100 mM NaCl, and pH 6.8.
lM/bp) was mixed with ethidium bromide (B), neomycin (C),
l
l
l

4144
N. N. Shaw et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4142–4145
Enthalpy values were obtained by subtraction of the heat of dilution
of ligand into buffer, see Supporting Information, from the sample
titration of ligand into duplex. We have calculated KT for neomycin,
ethidium bromide, and 3 using this approach. The data shown in
Table 2 illustrate the power of conjugating two ligands that bind
to a receptor at different sites. Neomycin binds to the hybrid duplex
with a 106 Mꢀ1 affinity, while ethidium bromide binds the hybrid
duplex with a similar affinity at 106 Mꢀ1. The conjugate shows a
10,000-fold improvement over neomycin, with an affinity of
poly(rA):poly(dT) (0.9 °C), see Supporting Information. Convenient
in this strategy, is the combination of both ligand selectivity and
thermal stabilization afforded by 3. The smaller changes in Tm, as
observed in the mixed melting experiments, are consistent with
the previously reported data.18 Provided, is irrefutable evidence
of NM preference for poly(dA):poly(rU). The difference in stabiliza-
tion of the two hybrids by 3 can be attributed to the conforma-
tional differences adopted by the two hybrids of which
poly(dA):poly(rU) adopts
a more A-like conformation than
4.8 ꢁ 1010 Mꢀ1
.
poly(rA):poly(dT).19–21
In an effort to compare the affinity of 3 to other nucleic
acid structures, KT values were calculated for both the RNA
and DNA duplexes, Table 2. The conjugate binds the DNA duplex
poly(dA–dT)2 with an affinity of 1.0 ꢁ 106 Mꢀ1 and the RNA
duplex poly(rA):poly(rU) with an affinity of 2.1 ꢁ 109 Mꢀ1. The
affinity for the RNA duplex, to 3, is higher than its affinity for
both neomycin (107 Mꢀ1) and ethidium bromide (106 Mꢀ1). The
conjugate, when binding the duplex DNA, displays a moderate
increase in affinity, when compared to the DNA duplex affinity
for neomycin (104 Mꢀ1), and ethidium bromide (105 Mꢀ1). More
significant is the conjugates selectivity for the hybrid duplex
over the RNA duplex and DNA duplex. Compound 3 binds to
the hybrid poly(dA):poly(rU) with 20-fold higher affinity than
the RNA:RNA duplex, and almost 40,000 higher affinity than
the DNA:DNA duplex. Compound 3 clearly displays the power
of conjugation and expansion of aminoglycoside based binding
to non-traditional targets. In fact, not only does the conjugate
bind non-traditional target, poly(dA):poly(rU), the affinity for
this duplex is much higher than the reported neomycin binding
to the 16S A-site RNA (9.1 ꢁ 108 Mꢀ1).26
Theoretically, if one looks at the product of the Ka values for
methidium and neomycin, one expects a Ka in the 1012 Mꢀ1 range
(Table 2). There are very few methods which can accurately deter-
mine such large binding constants. Fortunately, a calorimetric
method has previously been used to determine Ka in this range.22
The apparent ligand–DNA:RNA hybrid association constants
were estimated using the following equations. The melting tem-
perature difference in the absence and presence of drug was used
to estimate the association constant, KT , using the following
m
equation23
1
1
R
ꢀ
¼
lnð1 þ KT LÞ:
ð1Þ
m
Tm0 Tm
n
D
Hwc
In Eq. (1), terms Tm0 and Tm represent the melting temperatures of
the native hybrid without and with ligand, as determined with ther-
mal denaturation, UV and CD. The number of drug molecules bound
per duplex, n, was determined using titrations of drug into duplex
(fluorescence for ethidium bromide and 3 and CD for neomycin).
DHwc, Watson–Crick duplex melting enthalpy, was determined
using DSC. L is the free drug concentration at Tm, estimated at
one-half the total drug concentration. The binding constant at the
melting temperature, Tm, was extrapolated to a reference tempera-
ture (T) of 20 °C using the integrated van’t Hoff equation24
The experiments were conducted at pH 5.5 to eliminate bind-
ing induced drug protonation effects and to determine intrinsic
enthalpies of interaction between drug and polymer. Protonation
effects on aminoglycoside binding have been previously stud-
ied22 and binding-linked protonation leads to an increase in
observed binding enthalpy. In fact, it has been shown that a
decrease in pH leads to a moderate increase in affinities of
aminoglycosides to their nucleic acid targets (3- to 5-fold
increase from pH 7.0 to pH 6.0 in aminoglycoside binding to
the DNA:RNA hybrid and rRNA A-site).10,22 Therefore, the affini-
ties described here are only slightly higher than the affinities
that would be observed under physiological conditions. Further-
more, low salt concentration, 100 mM NaCl, was used to isolate
the primary, high affinity, binding site.
KT
m
KT
¼
;
C =R
p
ð2Þ
D
T
m
DCpT=Rð1=Tmꢀ1=TÞ
ð T
Þ
DHT =Rð1=Tm
eꢀ
ꢀ1=TÞe
where
constant, and
D
HT was determined experimentally using ITC, R is the gas
D
Cp was determined using the following equation25
D
D
H
T
D
Cp
¼
:
ð3Þ
Enthalpy values (
D
H) were determined using binding enthalpies
from excess site ITC titrations (used to identify the heat of interac-
tion of the primary high affinity site), at various temperatures.
Table 2
Thermodynamic profile of nucleic acid interactions with NM, 3, neomycin and ethidium bromide
Poly(dA):poly(rU)
Neomycin
Poly(rA):poly(rU)
Poly(dA–dT):poly(dA–dT)
NM (3)
EtBr
NM (3)
Neomycin
EtBr
NM (3)
Neomycin
EtBr
a
D
Tm0
Hwc
3.84
43.0
90.5
9.7
4.4 0.1
1.9 0.1
1.1 0.1
ꢀ339 16
3.84
43.0
65.8
6.5
3.6 0.1
2.7 0.1
2.1 0.1
ꢀ150 13
3.84
43.0
61.8
4.6
6.34
53.4
77.0
9.6
3.3 0.1
1.9 0.1
0.8 0.1
ꢀ256
6.34
53.4
64.7
8.0
1.8 0.1
0.7 0.1
ꢀ0.5 0.1
ꢀ223 11
6.34
53.4
64.2
2.5
3.90
62.4
66.2
9.1
1.2 0.1
0.8 0.1
0.2 0.1
ꢀ104 12
3.90
62.4
62.9
7.3
ꢀ1.1 0.1
ꢀ1.1 0.1
ꢀ1.3 0.1g
ꢀ29 16
3.90
62.4
63.2
5.3
b
b
Tm
nc
d
d
d
ð15 ꢂCÞ
ð20 ꢂCÞ
ð25 ꢂCÞ
D
D
D
D
H
H
H
ꢀ5.6 0.1
ꢀ5.9 0.1
ꢀ6.5 0.1
ꢀ5.8 0.1
ꢀ6.2 0.1
ꢀ6.5 0.1
ꢀ3.7 0.1
ꢀ4.5 0.1
ꢀ5.1 0.1
e
Cp
ꢀ93
7
9
ꢀ90
9
ꢀ139
6
f
KTð20 ꢂ
(4.8 0.1) ꢁ 1010 (9.9 0.1) ꢁ 106 (9.3 0.1) ꢁ 106 (2.1 0.2) ꢁ 109 (2.6 0.1) ꢁ 107 (1.8 0.1) ꢁ 106 (1.0 0.1) ꢁ 106 (7.0 0.5) ꢁ 104 (2.0 0.1) ꢁ 105
CÞ
a
Data obtained from DSC melting profiles (kcal/mol).
Data obtained from CD and UV melting profiles (°C).
Data obtained from titrations, as outlined in text.
b
c
d
D
H is corrected binding heat (kcal/mol) derived by integration of heat burst curve from the sample titration, followed by subtraction of the dilution head from the control
titration.
e
D
Cp calculated from Eq. (3) (cal/mol K).
f
Binding affinities calculated from Eqs. 1–3 (Mꢀ1).
DH values were recorded at 15, 20, and 25 °C (for the titration of neomycin into poly(dA–dT)2, DH values were also
recorded at 30 °C, see Supporting Information). Buffer: 10 mM sodium cacodylate, 0.5mM EDTA, 100mM NaCl, and pH 5.5.

N. N. Shaw et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4142–4145
4145
Conjugation leads to a much more negative
(ꢀ339 cal/mol K) than neomycin or ethidium bromide binding to
the polymer. Cp values can be impacted by a number of factors
including the release of constrained water molecules from the
hydration shell,27 binding induced changes in internal vibrational
modes,28 electrostatic interactions29 and the temperature depen-
dent equilibrium, such as protonation, upon drug uptake.30 It has
DCp of interaction
Supplementary data
D
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2008.05.090.
References and notes
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3. Varmus, H. E. Nature 1982, 299, 204.
previously been shown that groove binders exhibit negative
DCp
values, a result attributed to the displacement of large amounts
of non-polar surface area.31,32 However, Barbieri, et al. suggest that
aminoglycosides do not disrupt the spine of hydration,33 even at
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pH 5.5, and the observed negative
DCp value is unrelated to change
in solvent accessible areas. Perturbations to helical structure
through disruption of adenine base stacking has been observed
upon aminoglycoside binding to the A-site and is the main contrib-
utor to observed negative
D
Cp values.30,33 Further work will help
assign the exact contribution of these effects in the
observed here.
DCp values
Previous studies have shown the preference of aminoglycosides
to RNA:RNA duplex over the DNA:RNA hybrid.10 Our work shows
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sides. Even though the conjugate does not display the energetic
additivity estimated from the individual binding moieties, the
sub-nanomolar binding affinity remains very significant and far
exceeds the affinities of any known DNA:RNA binding drugs.
Further studies using different linker modifications should allow
us to optimize the affinity of the conjugate.
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acid targets. A recent report in C&E News cites a critical need for
development of such high affinity DNA:RNA binders.34 This work
should allow one to translate the recognition event to therapeuti-
cally relevant DNA:RNA hybrids sequences such as telomerase and
RNase H inhibitors.
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Acknowledgment
33. Barbieri, C. M.; Srinivasan, A. R.; Pilch, D. S. J. Am. Chem. Soc. 2004, 126, 14380.
34. Borman, S. C&E News 2006, 84, 32.
The author is grateful for financial support from NSF-CAREER
(CHE/MCB-0134972).