1. Molecular recognition of single-stranded RNA: Neomycin binding to poly(A)
Hongjuan Xi, David Gray, Sunil Kumar, 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
Article history:
Received 19 March 2009
Revised 1 June 2009
Accepted 3 June 2009
Available online 9 June 2009
Edited by Hans Eklund
Keywords:
RNA recognition
Aminoglycoside
Neomycin
Poly(A)
DNA
RNA
a b s t r a c t
Poly(A) is a relevant sequence in cell biology due to its importance in mRNA stability and translation
initiation. Neomycin is an aminoglycoside antibiotic that is well known for its ability to target var-
ious nucleic acid structures. Here it is reported that neomycin is capable of binding tightly to a sin-
gle-stranded oligonucleotide (A30) with a Kd in the micromolar range. CD melting experiments
support complex formation and indicate a melting temperature of 47 °C. The poly(A) duplex, which
melts at 44 °C (pH 5.5), was observed to melt at 61 °C in the presence of neomycin, suggesting a
strong stabilization of the duplex by the neomycin.
Ó 2009 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
1. Introduction
Aminoglycoside antibiotics are established groove-binding li-
gands that are effective in targeting various nucleic acid structures
(Fig. 1) [1,2]. Aminoglycosides have been found to target the 16S
rRNA subunit of the bacterial ribosome, thereby eliciting bacterici-
dal effects by interfering with the translation of amino acids [3]
(see [1,3] and references therein). There is extensive evidence
describing the ability of aminoglycosides (e.g. neomycin) to bind
both duplex and triplex forms of DNA, in addition to quadruplex
structures, DNA:RNA hybrids, and to some extent, single-stranded
nucleic acids [1–20].
Most studies on aminoglycosides have focused on targeting
RNA and DNA duplex and triplex structures. To our knowledge
there are no reports detailing the ability of aminoglycosides to bind
poly(A) or any other single-stranded nucleic acid polymers. This is
most likely due to a lack of interest, since aminoglycosides bind
more tightly to multi-stranded nucleic acid structures than to sin-
gle-stranded polymers; however, single-stranded nucleic acids
such as poly(A) have critical roles in cell biology [21–28], so it is
important to understand how nucleic acid-targeting antibiotics af-
fect such structures. Poly(A) (Fig. 2) in particular is a very relevant
sequence in cell biology due to its important roles in mRNA stabil-
ization and translation initiation [20]. This is a ubiquitous se-
quence in eukaryotes and prokaryotes, so it needs to be
characterized as a target for nucleic acid-binding drugs like
neomycin.
It has also been shown that certain viruses actually target fac-
tors for cleavage that either bind to the poly(A) tail of mRNA or
help connect the 30
poly(A) tail with the 50
guanosine cap of mRNA
[25]. Since such action by viruses is believed to contribute to their
infecting ability, the binding of poly(A) by aminoglycosides might
be an unconsidered source of toxicity within the human cell. Pre-
vious work with poly(A) polymerase inhibition by aminoglycosides
has similarly been suggested to be a possible cause of aminoglyco-
side toxicity [29,30].
Furthermore, studies have shown that poly(A)-synthesizing
polymerases tend to be highly active in S phase of the cell cycle
and are overexpressed in certain forms of cancer [31]. The implica-
tions could mean that there is potential for greater levels of poly-
adenylated mRNA in rapidly dividing cells, or simply that more
mRNA is being produced by the cell during S phase. Either way,
more cellular poly(A) is present in S phase and the single-stranded
structure is a potential target for rapidly dividing cells that are
found in aggressive cancers.
Interestingly, isoquinoline alkaloids have been found to bind
poly(A) with low micromolar affinity [32–35], supposedly through
partial intercalation. It has also been proposed that these alkaloids
may stabilize a regular secondary structure within the poly(A)
0014-5793/$36.00 Ó 2009 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.febslet.2009.06.007
Abbreviations: RNA, Ribonucleic acid; DNA, Deoxy-ribonucleic acid; PABP,
poly(A)-binding protein; mRNA, messenger ribonucleic acid; CD, circular dichro-
ism; UV, UV absorption spectroscopy; ITC, Isothermal titration calorimetry; DSC,
Differential scanning calorimetry
* Corresponding author. Fax: +1 8646566613.
E-mail address: dparya@clemson.edu (D.P. Arya).
FEBS Letters 583 (2009) 2269–2275
journal homepage: www.FEBSLetters.org

2. OH
OH
HO
OH
OH
NH3
O
O
H3N
O
OHO
O
OH
O
R1
HO
R2
NH3
O
O
NH2R4
OH
R3
HN
OH H H
Lividomycin A OH H H H
Monomycin B OH H COCH3 H
Paromomycin I OH OH H H
Neomycin B NH3 OH H H
Ribostamycin NH3
Neamine NH3 OH H H
pKa = 8.8pKa = 7.6
pKa = 8.1
pKa = 7.6 pKa = 5.7
pKa = 8.6
+
+
+
+
1''''
4'''
2'''
1'''
3''
1''
1
5
4
2'
1'
R4
R3
R2
R1
OH H H COCH3
V
IV
III
II
I
Fig. 1. Structures/pKas of aminoglycosides with a central ribose.
-60
-40
-20
0
20
40
200 220 240 260 280 300
CD(mdeg)
λ (nm)
-35
-30
-25
-20
-15
-10
-5
0 5 10 15 20 25
CD(248nm)
r
bd
~10.0
-80
-60
-40
-20
0
20
40
60
200 220 240 260 280 300
CD(mdeg)
λ (nm)
poly(rA) alone
poly(rA)-neo complex
(b)
(c)
(a)
Fig. 2. (a) CD titration of neomycin into poly(A) (75 lM/base) at neutral pH. (b) Binding stoichiometry plots for neomycin. A binding site size of one neomycin to 10 adenine
bases was determined based on the plot of CD intensity at 257 nm against the ratio of [neomycin] to [adenine bases] (rdb). Buffer: 10 mM sodium cacodylate, 0.5 mM EDTA,
100 mM NaCl, pH 7.0.
2270 H. Xi et al. / FEBS Letters 583 (2009) 2269–2275

3. strand, which may resemble the poly(A)Ápoly(A) duplex that forms
at acidic pH. The induced formation of such a structure could pre-
vent the binding of poly(A)-binding protein to its inherent target,
but binding affinities for the drug to duplex would need to surpass
nanomolar range (the binding affinity of PABP to poly(A)). Such
affinities could be possible with a conjugate that incorporates at
least two pharmacophores with independent binding sites that
are capable of binding to poly(A) with near micromolar affinity.
While the alkaloids have been shown through viscosity studies
to interact with poly(A) duplex by partial intercalation, neomycin
would likely interact with the same target through a groove-bind-
ing mechanism. The aminoglycoside lacks planar groups capable of
intercalation, so a completely different mechanism for binding to
poly(A) should be expected. Due to the relevance of poly(A) to
mRNA stability, protein synthesis, and virology, as well as the po-
tential importance of poly(A) to cancer biology, we chose to assess
the ability of neomycin to target single-stranded poly(A). Here we
report the results of spectroscopic (UV–vis spectroscopy, circular
dichroism spectropolarimetry, and fluorescence spectroscopy)
and calorimetric (isothermal titration calorimetry) studies that
characterize the binding of neomycin to poly(A) and oligo(A)30.
2. Materials and methods
Neomycin sulfate was purchased from ICN Biomedicals Inc., and
was used without further purification. Berberine was purchased
from Acros Organics. The poly(A) was purchased from GE Health-
care Amersham Bioscience. The concentrations of the polymer
solutions were determined spectrophotometrically using the fol-
lowing extinction coefficient (in units of mol of nucleotide or bp/
LÀ1
cmÀ1
): e258 = 9800 for poly(A). Oligo(A30) was purchased from
Dharmacon RNA technologies, and further deprotected before
use, e260 = 363400 L/(mol cm) in strand. For detailed experimental
information, please see Supplementary data.
-40
-20
0
20
40
200 220 240 260 280 300
CD(mdeg)
λ(nm)
0
5
10
15
20
25
30
0 2 4 6 8 10 12 14
CD(mdeg)
rbd
∼6
(a) (b)
Fig. 3. (a) CD titration of paromomycin into poly(A) (50 lM/base) at neutral pH. (b) Binding stoichiometry plots for paromomycin. Buffer: 10 mM sodium cacodylate, 0.5 mM
EDTA, 100 mM NaCl, pH 7.0.
0
1
2
3
4
5
6
7
8
10 20 30 40 50 60 70 80
CD Melting of Poly(A)
with Neomycin, pH 6.8
0
1
2
3
4
5
6
7
8
CD(mdeg)
Temp
0
1
2
3
4
5
6
7
8
10 20 30 40 50 60 70 80
CD Melting of Poly(A)
pH 6.8
0
1
2
3
4
5
6
7
8
CD(mdeg)
Temp
Fig. 4. Two-dimensional CD melting of poly(A) (20 lM) in the presence (top) and
absence (bottom) of neomycin (5 lM). The samples were heated from 10 °C to 80 °C
while CD intensity was monitored at 257 nm at 2° intervals. Buffer conditions:
10 mM sodium cacodylate, 0.5 mM EDTA, 100 mM NaCl, pH 7.0.
0 1 2 3 4 5 6 7
-12
-10
-8
-6
-4
-2
-2.0
-1.5
-1.0
-0.5
0.0
0 25 50 75 100 125
Time (min)
µcal/sec
Molar Ratio
Ka1=(5.3+0.9)X106M-1 n1=
1.8+0.0
Kcal/molofinjectant
Fig. 5. ITC titration of neomycin into oligo(A)30. Concentrated neomycin (625 lM)
was titrated into a solution containing the oligomer at 20 lM. The experiment
consisted of 29 total titrations of 10 lL each. Binding constants and binding
enthalpies were determined by fitting the corresponding data for heats of injection
with a two-site model on Origin 7.0 software. Buffer conditions: 10 mM sodium
cacodylate, 0.5 mM EDTA, 100 mM NaCl, pH 7.0.
H. Xi et al. / FEBS Letters 583 (2009) 2269–2275 2271

4. 3. Results
3.1. Determination of binding site size and evidence of complex
formation by CD titration
Figs. 2a and 3a depicts a CD titration experiment in which neo-
mycin and paromomycin were serially titrated into a solution of
poly(A) and monitored by CD spectropolarimetry from 300 nm to
200 nm. Upon each titration, the solution was stirred for 1 min
and allowed to equilibrate for 4 min before scanning. An analysis
of the data indicated a binding site size of one neomycin to 10 ade-
nine bases. Binding site size was determined by plotting CD inten-
sity at 257 nm against the ratio of drug (neomycin/paromomycin)
to adenine bases at each successive titration (Figs. 2c and 3b). The
CD spectrum also suggests the formation of a binding complex
upon the titration of neomycin and paromomycin into the solution
of poly(A): the peak at 257 nm, in the absence of drug, shows a red-
shift with each titration of ligand. Similar results have been re-
ported for CD titrations involving the addition of berberine and
coralyne into poly(A) [32–35].
3.2. CD melting of poly(A) in the presence and absence of neomycin/
paromomycin
Fig. 4 depicts CD melting plots for poly(A) in the presence and
absence of neomycin (see Supplementary data for a 3D CD, Tm
and wavelength plot). While poly(A) in the absence of ligand
shows a gradual decrease in CD intensity, the sample of poly(A)
in the presence of ligand has a melting temperature around
47 °C. Beyond that melting temperature, the gradual decrease in
CD intensity resembles the transition observed in the absence of li-
gand. This is further evidence for the formation of a binding com-
plex between neomycin and poly(A), since the poly(A)-neomycin
complex is stable up to 47 °C, at which point the plot resembles
0.4
0.45
0.5
0.55
10 20 30 40 50 60 70 80 90
Abs
260
T (
o
C)
a b c d
a: pH5.8, 28.4
o
C
b: pH5.5, 44.0
o
C
c: pH5.0, 62.8
o
C
d: pH4.5, 76.6
o
C
(a) -2000
-1000
0
1000
2000
3000
20 40 60 80 100
C
p
(cal/mol.K)
T (
o
C)
T
m
:78.0
o
C
ΔH
m
:5.9 kcal/mol
T
m
:63.1
o
C
ΔH
m
:5.4 kcal/molT
m
:43.3
o
C
ΔH
m
:4.05 kcal/mol
T
m
:25.8
o
C
ΔH
m
:2.0 kcal/mol
(b)
1: pH 4.5
2: pH 5.0
3: pH 5.5
4: pH 5.8
4
3
2
1
-0.2
-0.1
0.0
0.1
3020
5
o
C
Time (min)
µcal/sec
0.02
-6
-4
-2
0
(c)
ΔH=-1.8+ 0.1 kcal/mol
Molar Ratio
kcal/moleofinjectant
-0.3
-0.2
-0.1
0.0
0.1
10 20
10
o
C
Time (min)
µcal/sec
0.200.00
-10
-8
-6
-4
-2
0
2
4
(d)
ΔH=-2.1 + 0.1 kcal/mol
Molar Ratio
kcal/moleofinjectant
Fig. 6. (a) UV melting profiles of a poly(A) duplex at varied pHs. (b) DSC melting profiles of poly(A) duplex at varied pHs. (c,d) ITC titration of neomycin into poly(A) duplex at
5 °C (c) and 10 °C (d). Buffer: 10 mM sodium cacodylate, 100 mM NaCl, 0.5 mM EDTA, pH 5.5.
0.3
0.35
0.4
0.45
0.5
20 30 40 50 60 70 80 90
Abs
260
T (
o
C)
44.0 61.5
1
2
Fig. 7. UV melting profiles of RNA duplex in the absence (1) and presence (2) of
neomycin at rbd of 6.5. Buffer: 10 mM sodium cacodylate, 100 mM NaCl, 0.5 mM
EDTA, pH 5.5.
2272 H. Xi et al. / FEBS Letters 583 (2009) 2269–2275

5. poly(A) in the absence of neomycin. When the same experiment
was repeated using paromomycin instead of neomycin, a much
broader melting profile without a clear transition was observed
(see Supplementary data) suggesting a weaker binding. An in-
crease in solution pH from 6.8 to 7.5 or the addition of 5 mM MgCl2
does not alter the melting profile of neomycin or paromomycin
(Supplementary data).
3.3. ITC titration of neomycin into oligo(A)30 and determination of
thermodynamics of the binding interaction
The binding affinity for neomycin to oligo(A)30 was determined
using ITC; the results of the experiment are shown in Fig. 5. A con-
centrated neomycin solution was titrated into a solution of oli-
go(A)30, and binding constants was determined by fitting the
data with a two-site binding model using Origin 7.0 software. Both
binding events are favored by high enthalpy values; however,
entropically, the first event is favored and the second event is
disfavored.
3.4. UV melting of poly(A)Ápoly(A) duplex in the presence and absence
of neomycin at acidic pH
The interaction of neomycin with the poly(A)Ápoly(A) duplex
that forms at acidic pH was then studied in order to assess the
potential for neomycin to stabilize a similar form of secondary
structure in the single-stranded poly(A) near neutral pH. By
pre-forming the duplex under acidic conditions (as evident from
our UV and DSC melting studies at various pHs, Fig. 6a and b),
melting temperatures for the duplex in the presence and absence
of ligand could be measured and the ability of neomycin to sta-
bilize a duplex structure in poly(A)Ápoly(A) determined. Results
indicate that neomycin considerably stabilizes the poly
(A)Ápoly(A) duplex at pH 5.5, as demonstrated by a increase in
melting temperature in the presence of neomycin (Fig. 7).
Fig. 6a shows the melting curve for the poly(A)Ápoly(A) duplex
at various pH values. The duplex has an approximate melting
temperature of 47 °C in the absence of ligand. In the presence
of 5 lM neomycin, the poly(A)Ápoly(A) duplex has a melting tem-
perature of 61.5 °C under the same salt conditions (Fig. 7). It was
originally reported by Davies et al. that the poly(A)Ápoly(A) du-
plex is stabilized by low pH and decreasing salt concentrations
– an observation that can most likely be attributed to the forma-
tion of hydrogen bonds between the phosphate oxygen and the
hydrogen attached to N7 of protonated adenine. Neomycin, with
its five amino groups (all but one of which are substantially pro-
tonated at pH 5.5) could potentially stabilize the inter-strand
interactions with H-bonds or ion–dipole interactions. The proton-
ated amines are well known to be involved in electrostatic inter-
actions during nucleic acid binding, so it is quite plausible that
neomycin stabilizes the adenine-to-adenine duplex upon forming
electrostatic interactions in the grooves of the poly(A)Ápoly(A)
duplex. When these experiments were repeated in the presence
of increasing concentrations of MgCl2, multiphasic melting pro-
files were observed (see Supplementary data) suggesting the for-
mation of alternate secondary structures.
3.5. CD titration of neomycin and paromomycin into a solution of
poly(A)Ápoly(A) duplex at pH 5.5
Neomycin and paromomycin were titrated into a solution of
poly(A)Ápoly(A) at pH 5.5, and the binding site size was determined
from the data depicted in Fig. 8a–d. The same method was used for
determining binding site size as described above (2.1).
-40
-20
0
20
40
60
200 220 240 260 280 300
CD(mdeg)
λ(nm)
(a)
Increase neomycin
0
50
200 220 240 260 280 300
30
35
40
45
50
55
4 5 6 7 8 9 10
CD
263
(mdeg)
r
bd
~6.5
(b)
(c) (d)
-40
-20
0
20
40
200 220 240 260 280 300
CD(mdeg)
λ(nm)
0
5
10
15
20
25
30
35
0 2 4 6 8 10 12 14
CD(mdeg)
Wavelength(nm)
~ 5.20
Fig. 8. (a) CD scans of neomycin (a) and paromomycin (c) titration with poly(rA)Ápoly(rA) duplex at acidic pH. The sample was scanned from 300 nm to 200 m after serial
additions of concentrated ligand. Following each titration, the solution was stirred by a stir bar, then allowed to equilibrate for at least three minutes before scanning. The
scan with solid circles represents the RNA alone. The insert CD spectra are RNA alone (continuous line) and drug-saturated complex (dashed line). Plot of CD intensity at
262 m against ratio of neomycin (b) and parmomycin (d) to [homoadenine base pairs].
H. Xi et al. / FEBS Letters 583 (2009) 2269–2275 2273

6. 3.6. Fluorescence titration of neomycin into poly(A) saturated with
berberine
A solution containing 20 lM poly(A) at neutral pH and berber-
ine at a saturating concentration was heated to 90 °C for five min-
utes and then allowed to cool slowly to room temperature. In this
way, a complex between berberine and poly(A) was formed prior
to the fluorescence experiment. Subsequently, neomycin was seri-
ally added to the solution containing single-stranded poly(A) and
berberine. The fluorescence was monitored from 400 nm to
600 nm after exciting the sample at 352 nm. The emission of ber-
berine was markedly enhanced upon the addition of neomycin,
indicating that berberine was stacking between adenine bases to
a greater extent than in the absence of neomycin (see Supplemen-
tary data).
4. Discussion
It has been reported previously that three isoquinoline alkaloids
are capable of binding poly(A) with micromolar affinities [32–35];
however, these ligands are believed to interact with the single-
stranded nucleic acid through partial intercalation, as opposed to
the groove-binding mechanism that neomycin is understood to
utilize in the targeting of various nucleic acids. Here we report that
neomycin is capable of binding a single-stranded nucleic acid se-
quence that is pervasive in cellular biology; specifically, neomycin
binds oligo(A)30 with micromolar affinity, in addition to forming a
complex with poly(A) at pH 6.8. According to CD studies, neomycin
induces the formation of a stable conformation in poly(A), which
has a melting temperature of 47 °C. Furthermore, the samples of
poly(A) in the presence and absence of drug are distinctly different
from one another according to the three-dimensional CD melting
plots.
A duplex poly(rA)Ápoly(rA) was then studied at acidic pHs (Figs.
6–8). At pH 5.5, as shown in Fig. 8, a strong positive band $260 nm
and a small negative band $240 nm are observed, characteristic of
the poly(rA)Ápoly(rA) duplex. This duplex structure only contains
one kind of groove because of the rotational symmetry of the A–
A base pair. Neomycin stabilizes this RNA duplex significantly, rais-
ing the melting temperature from 44 °C to 61 °C (Fig. 7). Titration
of neomycin into this duplex does not change its overall conforma-
tion and yields a binding site size of 6.5 base pair per drug (Fig. 8);
approximately one helical turn (eight base pair) binds one drug.
Paromomycin binding, on the other hand, yields a slightly lower
binding site of approximately 5.5 base pair per drug. This observa-
tion of different site sizes suggests that the extra amino group on
neomycin (ring I) is involved in binding and helps neomycin span
a slightly larger surface for interaction, when compared to paromo-
mycin, leading to a larger binding site.
We determined a binding constant for neomycin to oligo(A)30 at
20 °C using ITC. The results indicate two clear binding events, with
both association constants in the micromolar range. The first bind-
ing event has a Ka = 5.3 Â 106
MÀ1
; and the binding constant for the
second event has a Ka = 1.2 Â 105
MÀ1
and is characterized by a
stoichiometry of about 3.3 neomycin molecules to one oligo(A)30
strand, or one neomycin molecule to about 10 adenine bases,
which is also consistent with our apparent binding site size deter-
mination from CD titration of neomycin into poly(A).
A poly(A)-targeting drug could inhibit protein synthesis by dis-
placing PABP from poly(A) and disrupting the ‘‘closed-loop” model
(Fig. 1) of translation. Sufficient evidence suggests that some
mechanisms for controlling translation involve the inhibition of
PABP function [36–39]. In fact, in some instances PABP is displaced
from poly(A) (regulation by the factors Paip2 and Tob) [22,38,39],
and in other cases PABP is cleaved so that it cannot interact with
50
cap proteins (viral infections) [25]. A poly(A)-targeting drug
could displace PABP through an even different inhibitory mecha-
nism: competing with PABP for poly(A) instead of targeting actual
PABP. Since PABP binds to poly(A) with a dissociation constant on
the order of 109
MÀ1
, a drug would need to bind poly(A) with at
least nanomolar affinity. Such would be possible with a conjugate
consisting of neomycin and an isoquinoline alkaloid, since both
bind poly(A) with micromolar affinity and the resulting conjugate
with an appropriate linker arm might be capable of binding with
picomolar affinity. These findings opens the door for development
of such high affinity conjugates and are being explored in our
laboratories.
Acknowledgment
We thank the NIH (R15CA125724) for financial support.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.febslet.2009.06.007.
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