C3-symmetric opioid scaffolds are pH-responsive DNA condensation agents

Article abstract of DOI:10.1093/nar/gkw1097

Herein we report the synthesis of tripodal C₃-symmetric opioid scaffolds as high-affinity condensation agents of duplex DNA. Condensation was achieved on both supercoiled and canonical B-DNA structures and identified by agarose electrophoresis, viscosity, turbidity and atomic force microscopy (AFM) measurements. Structurally, the requirement of a tris-opioid scaffold for condensation is demonstrated as both di- (C₂-symmetric) and mono-substituted (C₁-symmetric) mesitylene-linked opioid derivatives poorly coordinate dsDNA. Condensation, observed by toroidal and globule AFM aggregation, arises from surface-binding ionic interactions between protonated, cationic, tertiary amine groups on the opioid skeleton and the phosphate nucleic acid backbone. Indeed, by converting the 6-hydroxyl group of C₃-morphine (MC3) to methoxy substituents in C₃-heterocodeine (HC3) and C₃-oripavine (OC3) molecules, dsDNA compaction is retained thus negating the possibility of phosphate - hydroxyl surface-binding. Tripodal opioid condensation was identified as pH dependent and strongly influenced by ionic strength with further evidence of cationic amine-phosphate backbone coordination arising from thermal melting analysis and circular dichroism spectroscopy, with compaction also witnessed on synthetic dsDNA co-polymers poly[d(A-T)₂] and poly[d(G-C)₂]. On-chip microfluidic analysis of DNA condensed by C₃-agents provided concentration-dependent protection (inhibition) to site-selective excision by type II restriction enzymes: BamHI, HindIII, SalI and EcoRI, but not to the endonuclease DNase I.

Full text of DOI:10.1093/nar/gkw1097

Published online 29 November 2016
Nucleic Acids Research, 2017, Vol. 45, No. 2 527–540
doi: 10.1093/nar/gkw1097
C 3-symmetric opioid scaffolds are pH-responsive
DNA condensation agents
Natasha McStay1,†, Zara Molphy1,†, Alan Coughlan1, Attilio Cafolla2, Vickie McKee1,
Nicholas Gathergood3,* and Andrew Kellett1,*
1School of Chemical Sciences and National Institute for Cellular Biotechnology, Dublin City University, Glasnevin,
Dublin 9, Ireland, 2School of Physical Sciences, Dublin City University, Glasnevin, Dublin 9, Ireland and 3Department
of Chemistry, Tallinn University of Technology, Akadeemia tee 15, 12618 Tallinn, Estonia
Received August 23, 2016; Revised October 11, 2016; Editorial Decision October 23, 2016; Accepted October 27, 2016
ABSTRACT
INTRODUCTION
The search for new synthetic DNA recognition agents is an
area of considerable research importance. These agents can
be categorized, broadly, into two main areas: (i) those that
covalently bind nucleic acids at electron rich sites on the
nucleobase (1–3) and (ii) those that bind in non-covalent
fashion through hydrogen (H)-bonding, ionic or ␲-stacking
interactions (4–6). Accordingly, non-covalent recognition
scaffolds may be further classified to the area of DNA where
recognition occurs thus giving rise to groove binders (7–9),
intercalators (9–12) (including threading intercalators (13))
and surface-binding compounds (14). Within this last cat-
egory, surface interacting compounds have been designed,
and compared to naturally occurring protein binding do-
mains containing, for example, cationic arginine (arginine
fork (15)) or lysine (poly-L-lysine (16)) amino acid residues
that facilitate their complexation to nucleic acids. Indeed,
a commonality between synthetic and naturally occurring
surface binders is their ability to efficiently bind electron-
rich phosphate oxygen atoms located at the nucleic acid
backbone, principally through H-bonding and electrostatic
interactions. Furthermore, electrostatic binding by polyca-
tionic molecules can directly lead to a collapse in the B-
DNA conformation with resultant condensation or aggre-
gation of tertiary DNA helical structures (17). Condensa-
tion agents have, in turn, found widespread utility in molec-
ular biology as transfection agents capable of mediating cel-
lular delivery of nucleic acids for ubiquitous therapeutic and
bioprocessing applications (18). Thus, an important con-
sideration in the development of new DNA condensation
agents is their relative inertness toward chemical reactivity
(e.g. artificial nuclease) or secondary interactions (e.g. inter-
calation) that may interrupt or block efficient transport and
incorporation of target nucleic acids into mammalian cells.
While nucleic acid condensation can be achieved through
mechanisms of neutral crowding, antisolvent precipitation
and liposomal packaging (amongst others), significant at-
Herein we report the synthesis of tripodal C3-
symmetric opioid scaffolds as high-affinity conden-
sation agents of duplex DNA. Condensation was
achieved on both supercoiled and canonical B-
DNA structures and identified by agarose elec-
trophoresis, viscosity, turbidity and atomic force
microscopy (AFM) measurements. Structurally, the
requirement of a tris-opioid scaffold for conden-
sation is demonstrated as both di- (C2-symmetric)
and mono-substituted (C1-symmetric) mesitylene-
linked opioid derivatives poorly coordinate dsDNA.
Condensation, observed by toroidal and globule
AFM aggregation, arises from surface-binding ionic
interactions between protonated, cationic, tertiary
amine groups on the opioid skeleton and the phos-
phate nucleic acid backbone. Indeed, by convert-
ing the 6-hydroxyl group of C3-morphine (MC3)
to methoxy substituents in C3-heterocodeine (HC3)
and C3-oripavine (OC3) molecules, dsDNA com-
paction is retained thus negating the possibility of
phosphate––hydroxyl surface-binding. Tripodal opi-
oid condensation was identified as pH dependent
and strongly influenced by ionic strength with further
evidence of cationic amine-phosphate backbone co-
ordination arising from thermal melting analysis and
circular dichroism spectroscopy, with compaction
also witnessed on synthetic dsDNA co-polymers
poly[d(A-T)2] and poly[d(G-C)2]. On-chip microfluidic
analysis of DNA condensed by C3-agents provided
concentration-dependent protection (inhibition) to
site-selective excision by type II restriction enzymes:
BamHI, HindIII, SalI and EcoRI, but not to the endonu-
clease DNase I.
*To whom correspondence should be addressed. Tel: +353 1 7005461; Email: andrew.kellett@dcu.ie
Correspondence may also be addressed to Nicholas Gathergood. Tel: +372 0 6204381; Email: nicholas.gathergood@ttu.ee
†These authors contributed equally to this work as the first authors.
ꢀ
C
The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which
permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

528 Nucleic Acids Research, 2017, Vol. 45, No. 2
tention focuses on the condensation properties of multiva-
lent cationic compounds including cationic polymers. Al-
though DNA condensation has been reviewed by Bloom-
field (17) and others including theoretical descriptions of
the dynamics of polymer collapse (19,20), it is worth consid-
ering some examples of multivalent cations currently acces-
sible to this field. Cationic charge density––essential for the
neutralization of anionic phosphate charge and the reorga-
nization of water dipoles adjacent to DNA surfaces––plays
a critical role in driving condensation and the majority
of cationic agents carry a formal charge of 3+ or higher.
Prominent examples here include the naturally occurring
polyamines spermine4+ and spermidine3+ that are abun-
dantly found in living organisms and aid in the packaging
of cellular DNA by lowering the free energy of transition
required for condensation (21). Within the polyamine class,
it was demonstrated that DNA collapse occurs once ∼90%
of phosphate charge neutralization has occurred (22) and
that polyamine structure, allied with charge, plays a role
within condensation transitions (21). Polyamines of varying
aliphatic linker length have recently been incorporated into
substitution inert tri-platinum(II) complexes (Triplatin) and
these highly cationic agents are capable of aggregating nu-
cleic acids (both RNA and DNA) through clamping in-
teractions along the phosphate backbone and across the
minor groove of DNA (23–25). Evidence of condensation
by Triplatin agents was identified by turbidity and atomic
force microscopy (AFM) analysis with DNA (24), particu-
larly poly[d(A-T)2] (23), undergoing a B −→ ␺ transition
during the collapse. Another substitutionally inert com-
plex cation, cobalt(III) hexammine [Co(NH3)6]3+, has un-
dergone extensive study for its ionic surface-binding, con-
densation and dehydration properties with DNA (26,27).
Although [Co(NH3)6]3+ can also induce a B −→ ␺ tran-
sition in short oligonucleotide sequences, aggregation of
long DNA polymers occurs without modification to the
secondary B-DNA conformation (28). Furthermore, while
the morphology of DNA condensates with [Co(NH3)6]3+
are commonly toroidal in structure, non-toroidal (rod-like)
structures are observed in the presence of alcohol solvents.
A further example of metal complex-promoted DNA con-
densation was recently identified in di-Fe2+ supramolec-
ular helicates; these 4+ cationic agents bind DNA non-
covalently and are capable of inhibiting DNA-processing
enzymes including RNA polymerase, DNA topoisomerase
I, deoxyribonuclease I, along with a variety of restriction
endonucleases (29). Polycations can also induce alterna-
tive changes in B-DNA configuration; a reversible B −→
A transition, regulated by dehydration, was recently evi-
denced in short G-C rich oligonucleotide sequences exposed
to a poly(allylamine)-graft-dextran synthetic cationic poly-
mer (16). Finally, although exceptions to the ≥3+ cationic
valence requirement for DNA collapse are rare, both Mn2+
and the putrescine2+ cations are known to aggregate DNA
but only at high (millimolar) concentration (30).
was the first opioid isolated from the poppy and is the most
abundant opioid interacting with three principal opioid re-
ceptors located in the central nervous system (CNS): mu
(␮, MOR), kappa (␬, KOR) and delta (␦, DOR). The in-
teraction of opioids with CNS receptors has led toward
the development of synthetic analogues (including hete-
rocodeine, buprenorphine and naltrexone) and to more
heavily modified structures containing multiple opiate sub-
stituents. Morphine congeners can also be extracted from
the poppy (e.g. codeine, thebaine and oripavane) and offer
a range of bio-renewable derivatives to support proposed
SAR studies. Our group has recently developed a high-
throughput ethidium bromide (EtBr) displacement screen
to identify potential new DNA binding molecules (32)
and, as such, identified a synthetic C3-symmetric morphine
molecule (MC3), containing a mesitylene bridging ligand,
as a lead compound that was subsequently found to in-
duce DNA condensation at low micromolar concentration.
Our motivation for developing heterocodeine (HC3) and
oripavine (OC3) analogues stemmed from structural anal-
ysis of MC3 that focused on the 6ꢁ-OH group in the mor-
phine C ring as a possible hydrogen(H)-bonding site for
phosphate coordination at the nucleic acid backbone. If this
were the case, masking this H-bond through the introduc-
tion of methyl groups in the form of methoxy substituents
present in heterocodeine (H) and oripavine (O) should ren-
der the DNA binding properties negligible. In addition, as
oripavine is a natural product, and commercially available,
a short and direct synthesis of the C3-symmetry target was
envisaged. Surprisingly, however, enhanced EtBr displace-
ment was observed for both HC3 and OC3 agents, thus
implying only a limited role for 6ꢁ-OH phosphate interac-
tion in the binding mechanism. Consequently, the DNA
binding mode and condensation properties of this class of
molecule were elucidated using an array of biophysical tech-
niques including turbidity, viscosity, thermal melting, circu-
lar dichroism (CD) spectroscopy, electrophoresis and AFM
analysis and are reported herein.
MATERIALS AND METHODS
Materials synthesis and characterization
Chemicals and reagents were sourced from Sigma-Aldrich
and were used without any further purification required.
High pressure (or high performance) liquid chromatogra-
phy (HPLC) grade chloroform, methanol and acetonitrile
were used with no further purification. All other solvents
were used as supplied. Morphine and oripavine were pro-
vided from Johnson Matthey MacFarlan Smith Ltd. Thin
layer chromatography was performed on Fluka Silica gel
(60 F254) coated on aluminium plates. The TLC plates were
˚
visualized using UV light. Davisil 60 A silica gel was used
for column chromatography. 1H and 13C NMR spectra were
obtained on a Bruker AC 400 and 600 MHz NMR spec-
trometer. The pH was monitored by a Mettler Toledo InLab
Expert Pro-ISM pH probe. Electrospray ionization mass
spectra (ESI-MS) were recorded using a Thermo Fisher Ex-
active Orbitrap mass spectrometer coupled to an Advion
TriVersa Nanomate injection system with samples being
prepared in 100% HPLC-grade acetonitrile prior to ESI-
MS analysis. CD spectrometry was conducted on an Ap-
In this study we report the synthesis of a new class
of DNA binding scaffold incorporating opioid derivatives.
Opioids are naturally derived alkaloids from the opium
poppy, Papaver somniferum, and serve as one of the ma-
jor drug classes within pharmaceutical chemistry widely
known for their potent analgesic properties (31). Morphine

Nucleic Acids Research, 2017, Vol. 45, No. 2 529
12.6, 5.1 Hz, 3H); 1.78–1.75 (m, 3H). 13C NMR (151 MHz,
CDCl3) ␦: 152.96, 146.07, 142.13, 139.65, 133.99, 132.59,
132.06, 128.73, 119.46, 117.48, 111.73, 96.09, 89.16, 89.11,
77.37, 77.16, 76.95, 67.74, 61.07, 55.04, 46.25, 46.16, 42.57,
37.11, 29.93, 16.02. IR (ATR, cm−1): 2908, 1605, 1491,
1437, 1368, 1331, 1302, 1231, 1143, 1105, 1066, 1021, 987,
914, 867, 812, 767, 748, 698. ESI-MS: [OC3]+ 1048 m/z.
[␣]D = −88◦ (c = 0.12, CHCl3, 589 nm, 25◦C).
plied Photophysics Chirascan plus qCD spectrometer with
samples being prepared in acetonitrile.
Morphine-C3 (MC3). Morphine (0.573 g, 2.01 mmol)
and potassium carbonate (1.110 g, 8.03 mmol) were sus-
pended in acetonitrile (ACN) (30 ml) and heated to reflux.
2,4,6-Tris-(bromomethyl)-mesitylene (0.268 g, 0.66 mmol)
was added in small aliquots to the reaction mixture and vig-
orously stirred overnight (18 h) under reflux conditions. Af-
ter allowing the reaction mixture to cool to room tempera-
ture (RT), the reaction solvent was removed by rotary evap-
oration and crude product dissolved in dichloromethane
(DCM) (50 ml). The organic layer was washed with d.H2O
(40 ml) and the aqueous layer extracted with DCM (3 × 20
ml). All organic layers were combined, then washed with
d.H2O (3 × 20 ml) and with a saturated brine solution
(20 ml). The organic layer was dried over magnesium sul-
phate, filtered and solvents removed by rotary evaporation.
The crude product was purified by column chromatogra-
phy (SiO2, 95:1:1 to 92:8:1 CH2Cl2:MeOH:NH4OH). The
title compound MC3 was isolated as a white solid in 50%
yield (0.338 g, 0.33 mmol). mp. 187–189◦C. 1H NMR (600
MHz, CDCl3) ␦: 6.80 (d, J = 8.2 Hz, 3H); 6.58 (d, J = 8.2
Hz, 3H); 5.69 (ddt, J = 9.9, 3.2, 1.4 Hz, 3H); 5.30–5.27
(m, 3H); 5.21 (d, J = 10.5 Hz, 3H); 5.05 (d, J = 10.4 Hz,
3H); 4.85 (dd, J = 6.5, 1.1 Hz, 3H); 4.17–4.13 (m, 3H); 3.35
(s, 3H); 3.05 (d, J = 18.7 Hz, 3H); 2.66 (s, 3H); 2.61–2.56
(m, 3H); 2.48 (s, 9H); 2.44 (s, 9H); 2.41 – 2.37 (m, 3H);
2.31 (dd, J = 18.7, 6.2 Hz, 3H); 2.05 (td, J = 12.4, 5.0
Hz, 3H); 1.88 (d, J = 11.3 Hz, 3H). 13C NMR (151 MHz,
CDCl3) ␦: 147.59, 141.43, 139.43, 133.41, 131.82, 131.44,
128.19, 119.61, 116.36, 91.27, 77.22, 77.00, 76.79, 67.32,
66.46, 58.93, 46.47, 43.09, 42.96, 40.81, 35.78, 20.64, 15.98.
IR (ATR, cm−1): 2907, 1632, 1602, 1492, 1443, 1247, 1200,
1157, 1118, 1099, 1034, 983, 940, 833, 784, 766, 731. ESI-
MS: [MC3] 1012 m/z. [␣]D = −85◦ (c = 0.154, CHCl3, 589
nm, 25◦C).
Heterocodeine (33,34). Reaction carried out on parallel
synthesizer. Potassium hydride (4.421 g, 110.23 mmol) was
prepared in the reaction vessel under nitrogen flux and
washed with dry hexane, suspended in dry tetrahydrofuran
(THF) (150 ml) over ice. A solution of morphine (2.862
g, 10.03 mmol) in THF (30 ml) was added slowly over
30 min to the reaction under a nitrogen atmosphere and
the resulting solution was allowed to stir at RT for 16 h.
Methyl iodide (1.710 g, 0.75 ml, 12.05 mmol) was added
to the reaction slowly over 15 min and reaction left stir-
ring for 4 h. The reaction was quenched slowly with a mix-
ture of THF/H2O (10:1) at 0◦C. The solution was neutral-
ized to pH 7.0 with 2 M HCl and volatiles were then re-
moved by rotary evaporation. The pH was adjusted to 8.0
by the addition of 1M NaOH and the aqueous layer ex-
tracted with chloroform/isopropanol (3:1, 3×25 ml). The
resulting organic layer was washed with H2O (4×30 ml) and
a final wash with saturated brine solution (20 ml). The or-
ganic layer was dried over magnesium sulphate, filtered and
solvents removed by rotary evaporation. The crude prod-
uct was purified by column chromatography (SiO2, 95:1:1
to 92:8:1 CH2Cl2:MeOH:NH4OH), heterocodeine was iso-
lated as a white solid in 25% yield (756 mg, 2.53 mmol). 1H
NMR (600 MHz, CDCl3) ␦: 6.57 (d, J = 8.1 Hz, 1H); 6.41
(d, J = 8.1 Hz, 1H); 5.64 (ddt, J = 9.9, 3.2, 1.5 Hz, 1H); 5.26
(dt, J = 9.8, 2.7 Hz, 2H); 4.91 (dd, J = 5.8, 1.3 Hz, 1H); 3.72
(td, J = 5.5, 2.3 Hz, 1H); 3.45 (s, 3H); 3.32 (dd, J = 6.3, 3.2
Hz, 1H); 2.97 (d, J = 18.6 Hz, 1H); 2.63 – 2.49 (m, 2H); 2.43
– 2.31 (m, 4H); 2.23 (dd, J = 18.7, 6.4 Hz, 1H); 1.99 (td, J
= 12.4, 5.1 Hz, 1H); 1.88–1.79 (m, 2H).
Oripavine-C3 (OC3). A flask was charged with oripavine
(0.595 g, 2.00 mmol), tetrabutylammonium hydroxide (40%
aqueous solution, 18 ml) and DCM (6 ml) and stirred under
nitrogen for 30 min. A solution of 2,4,6-tris-(bromomethyl)-
mesitylene (0.269 g, 0.66 mmol) in DCM (4 ml) was added
and the biphasic reaction mixture was stirred for 6 h at
RT. The reaction solution was transferred into d.H2O (150
ml) and washed with DCM (4 × 10 ml). Organic layers
were combined and washed with aqueous NaOH solution
(0.1 M, 2 × 20 ml) followed by d.H2O (3 × 20 ml) then
saturated brine solution (20 ml). The organic layer was
dried over magnesium sulphate, filtered and solvents re-
moved by rotary evaporation. The crude product was pu-
rified by column chromatography (SiO2, 95:1:1 to 92:8:1
CH2Cl2:MeOH:NH4OH). The title compound OC3 was
isolated as a golden yellow solid in 47% yield (0.329 g, 0.31
mmol). mp 174–176◦C. 1H NMR (600 MHz, CDCl3) ␦: 6.73
(d, J = 8.1 Hz, 3H); 6.58 (d, J = 8.1 Hz, 3H); 5.56 (d, J = 6.4
Hz, 3H); 5.28 (s, 3H); 5.25 (d, J = 10.7 Hz, 3H); 5.17 (d, J =
10.7 Hz, 3H); 5.03 (d, J = 6.4 Hz, 3H); 3.62 (d, J = 6.6 Hz,
3H); 3.59 (s, 9H); 3.32 (d, J = 18.0 Hz, 3H); 2.83 (td, J =
12.7, 3.3 Hz, 3H); 2.68 (dd, J = 18.1, 7.0 Hz, 3H); 2.63 (dd,
J = 12.7, 4.6 Hz, 3H); 2.47 (s, 9H); 2.46 (s, 9H); 2.20 (td, J =
Heterocodeine-C3 (HC3). A flask was charged with het-
erocodeine (0.700 g, 2.34 mmol), tetrabutylammonium hy-
droxide (40% aqueous solution, 20 ml) and DCM (8 ml) and
stirred under nitrogen for 30 min. A solution of 2,4,6-tris-
(bromomethyl)-mesitylene (0.269 g, 0.66 mmol) in DCM
(4 ml) was added and the mixture was stirred for 6 h at
RT. The reaction solution was transferred into d.H2O (150
ml) and washed with DCM (4 × 10 ml). Organic lay-
ers were combined and washed with aqueous NaOH so-
lution (0.1 M, 2 × 20 ml) followed by d.H2O (3 × 20
ml) then a saturated brine solution (20 ml). The organic
layer was dried over magnesium sulphate, filtered and sol-
vents removed by rotary evaporation. The crude product
was purified by column chromatography (SiO2, 95:1:1 to
92:8:1 CH2Cl2:MeOH:NH4OH), HC3 was isolated by col-
umn chromatography as a white solid in 13% yield (95 mg
0.09 mmol). mp. 164–166◦C. 1H NMR (400 MHz, CDCl3)
␦: 6.73 (d, J = 8.1 Hz, 3H); 6.49 (d, J = 8.1 Hz, 3H); 5.71
(d, J = 9.9 Hz, 3H); 5.32 (dt, J = 10.0, 2.7 Hz, 3H); 5.27–
5.16 (m, 6H); 5.00 (d, J = 5.1 Hz, 3H); 3.80 (dd, J = 5.4,
2.7 Hz, 3H); 3.51 (s, 9H); 3.36 (dd, J = 5.9, 3.1 Hz, 3H);

530 Nucleic Acids Research, 2017, Vol. 45, No. 2
3.04 (d, J = 18.7 Hz, 3H); 2.69–2.65 (m, 3H); 2.61–2.56
(m, 3H); 2.52 (s, 9H); 2.44 (s, 9H); 2.40 (d, J = 3.4 Hz,
3H); 2.31 (dd, J = 18.7, 6.3 Hz, 3H); 2.04 (td, J = 12.4, 5.0
Hz, 3H); 1.93 (d, J = 11.0 Hz, 3H).13C NMR (101 MHz,
CDCl3) ␦: 148.88, 141.35, 139.66, 132.09, 131.46, 131.01,
128.56, 128.18, 119.02, 118.34, 89.14, 77.48, 77.36, 77.16,
76.84, 75.67, 68.01, 59.06, 56.92, 53.58, 46.68, 43.61, 43.26,
43.19, 41.23, 36.06, 29.82, 20.66, 16.10. IR (ATR, cm−1):
2905, 2798, 1632, 1601, 1492, 1442, 1247, 1199, 1104, 984,
941, 831, 787, 768, 727, 679. [␣]D = −185◦ (c = 0.08, CHCl3,
589 nm, 25◦C).
the absorbance equilibrated and values obtained remained
constant.
DNA condensation investigation (pUC19 scDNA). The
ability of the organic compounds to condense supercoiled
plasmid DNA was determined using a method previously
published by this laboratory with minor changes being
made (23). Tripodal opiates were initially prepared in DMF
and further diluted in 80 mM HEPES buffer (Fisher). Re-
actions were carried out according to the following general
procedure: in a total volume of 20 ␮l using 80 mM HEPES
buffer (pH 7.2) with 25 mM NaCl, 400 ng pUC19 (NEB,
N3041) and varying concentrations of test compound (5,
10, 20 and 30 ␮M), samples were incubated at 37◦C for both
5 and 12 h. Reactions were quenched by adding 6x loading
buffer (Fermentas) containing 10 mM Tris-HCl, 0.03% bro-
mophenol blue, 0.03% xylene cyanole FF, 60% glycerol, 60
mM EDTA and samples were loaded onto an agarose gel
(1.2%) containing 3 ␮l EtBr. Electrophoresis was completed
at 60 V for 1 h in 1x TAE buffer.
DNA binding experiments
Competitive ethidium bromide displacement assay. The
DNA binding affinity of the tripodal series was determined
over a 5 h time period using calf-thymus DNA (ctDNA,
Ultra-Pure Invitrogen, 15633019) and synthetic alternat-
ing co-polymers poly[d(A-T)2)] (Sigma Aldrich, P0883) and
poly[d(G-C)2] (Sigma Aldrich, P9389) by ethidium bromide
fluorescence quenching in a similar manner to the high-
throughput method previously reported by Kellett et al.
(35). Each drug concentration was measured in triplicate,
on at least two separate occasions, and the apparent bind-
ing constants were calculated using Kapp = Kb × 12.6/C50
DNA condensation investigation in the presence of non-
covalently bound recognition elements (pUC19 scDNA).
This protocol was carried out as previously reported by this
group with minor changes made (37). Briefly, 400 ng pUC19
was incubated with 25 mM NaCl, and 20 ␮M of either
methyl green, netropsin or hexamine cobalt(III) chloride in
80 mM HEPES buffer (pH 7.2) for 30 min at 37◦C. Sample
tubes were then vortexed and varying concentrations of test
compound were added (5,10, 20 and 30 ␮M). The reaction
mixture was further incubated at 37◦C for 5 h. The reaction
was then quenched and subjected to gel electrophoresis as
previously described.
where Kb = 8.80 × 106 M(bp)−1 (Kapp = apparent binding
constant).
Viscosity studies. Experiments were conducted in a sim-
ilar manner to the method reported previously using DV-
II-Programmable Digital Viscometer equipped with En-
hanced Brookfield UL Adapter at room temperature by
gradually increasing the [compound/DNA] ratios from
0.02–0.20 (36).
Thermal melting studies. The thermal melting of DNA of
varying %AT content was conducted using an Agilent Cary
100 dual beam spectrophotometer equipped with a 6 × 6
Peltier multicell system with temperature controller. The
protocol was previously developed within the Kellett group
and reported elsewhere (6,7).
Investigation of DNase I inhibition by DNA condensation
(pUC19 scDNA). pUC19 DNA (400 ng) was initially ex-
posed to 75, 100, 200 and 300 ␮M of test compounds with
25 mM NaCl in a total volume of 20 ␮l using 80 mM
HEPES buffer (pH 7.2) for 5 h at 37◦C. The condensed
DNA was then treated with the endonuclease, DNase I
(NEB, M0303S), for 10 min at 37◦C and heat inactivated at
75◦C for 10 min. Samples were then loaded onto an agarose
gel (1.5%) containing 3 ␮l EtBr and electrophoresis was
completed at 50 V for 40 min in 1x TAE buffer.
DNA condensation studies (dsDNA)
Turbidity investigation (ctDNA). This method was
adapted from the Brabec laboratory (24). Absorbance was
initially measured at 260 nm in order to give a final mea-
surement of ∼0.5 units and the concentration of ctDNA
was determined using the extinction coefficient ⑀260 = 12
824 M(bp)−1 cm−1. The turbidity of the ctDNA solution
was determined spectrophotometrically by monitoring the
absorbance of DNA at both 350 nm and 260 nm at 25◦C
using an Agilent Cary 100 dual beam spectrophotome-
ter equipped with a 6 × 6 Peltier multicell system with
temperature controller and stirring mechanism. In a final
volume of 3 ml containing ∼40 ␮M ctDNA, 1 mM PBS
buffer (pH 7.0) and 25 mM NaCl, varying concentrations
of MC3, OC3 and HC3 (2.5, 5, 7.5, 10, 12.5, 15, 20, 25,
30, 35, 45, 50, 65, 75, 80, 90, 100 ␮M) were titrated into
quartz cuvettes. Between each aliquot, solutions were
mixed thoroughly and allowed to incubate at 25◦C until
CD spectroscopy. Opioid profiles (MC3 and OC3) and
opioid–DNA interactions were analysed using Starna
quartz cuvettes in 10 mM PBS solution (pH 7.0) in the pres-
ence of 25 mM NaCl. Salmon testes DNA (stDNA, Sigma
Aldrich, D1626) was initially quantified using the extinction
co-efficient ⑀260 = 12 824 M(bp)−1 cm−1 to give a working
solution with final stDNA concentration of ∼100 ␮M. The
investigation was conducted in the range of 200–400 nm and
measurements were recorded at a rate of 1 nm per second. A
total of 100 ␮M stDNA solution was incubated with MC3,
OC3 and control agents hexammine cobalt(III) chloride
and spermine at r = 0.1 (r being the ratio [drug]/[DNA])
over a 7 h period at 37◦C. The DNA free CD spectra for
MC3 and OC3 are shown in Supplementary Figure 13.

Nucleic Acids Research, 2017, Vol. 45, No. 2 531
DNA condensation studies (linear dsDNA)
controlled by a Nanoscope IIIa controller, Digital Instru-
ments), in tapping-mode, using standard unmodified silicon
cantilevers (BudgetSensors, Windsor Scientific Ltd.) with a
40 N/m force constant. Topographic images are recorded
at a scanning rate of 1–2 Hz, and a resonance frequency
of about 300 kHz (nominal value). Images were processed
using the WSxM software (38) to remove the background
slope and normalize the z-scale across all images, no addi-
tional filtering was performed.
PCR primer design. Primers were designed (Eurofins Ge-
nomics) such that it was possible to generate a 742 bp long
sequence of linear dsDNA from the pUC19 vector encom-
passing the lacZ␣ gene. The short nucleotide sequence was
generated through PCR (35 cycles) with 1 ng pUC19 plas-
mid using 2x MyTaq Red Mix (Bioline) at an annealing tem-
perature of 66◦C and the band generated was compared to
a 50 bp DNA ladder (Invitrogen, 10416014).
Forward: 5ꢁ-TCGCGCGTTTCGGTGATGACGG-3ꢁ
Reverse: 5ꢁ-CCGCTCGCCGCAGCCGAACG-3ꢁ
Influence of pH and ionic strength on condensation
Influence of pH on condensation. Absorption measure-
ments spectra were initially measured at 260 nm to give
a final absorbance of ∼0.4 units and the concentration of
ctDNA was determined using the extinction co-efficient ⑀260
= 12 824 M(bp)−1 cm−1. The condensation aggregates of
the ctDNA solution was determined spectrophotometri-
cally by monitoring the absorbance of DNA at both 350
nm and 260 nm at 25◦C using an Agilent Cary 100 dual
beam spectrophotometer equipped with a 6 × 6 Peltier mul-
ticell system with temperature controller. In a final volume
of 5 ml containing ∼30 ␮M ctDNA, nuclease free water
(pH 7) and 25 mM NaCl, containing 35 ␮M OC3, the pH
of solution was adjusted with 1 M HCl and 1 M NaOH
to basic and acidic conditions as required using a Mettler
Toledo Inlab expert Pro-ISM pH probe. One hundred mi-
croliters aliquots were titrated into quartz cuvettes and be-
tween each pH adjustment solutions were mixed thoroughly
and allowed to incubate at 25◦C until the absorbance equi-
librated and values obtained remained constant.
DNA condensation investigation. A total of 400 ng of the
742 bp transcript was then treated under identical condi-
tions as described (5 h incubation at 5, 10, 20 and 30 ␮M)
and samples were loaded onto an agarose gel (1.2%) con-
taining EtBr. Electrophoresis was completed at 70 V for 40
min in 1x TAE buffer.
Endonuclease enzyme Inhibition
Endonuclease optimization on duplex DNA. A total of 400
ng of linear dsDNA was treated with 1 ␮l of EcoRI (NEB,
R0101S), BamHI (NEB, R0136S), SalI (NEB, R0138S) and
HindIII (NEB, R0104S), respectively, at 37◦C overnight to
verify the presence of their recognition sites within the short
742 bp sequence. All enzymes induced double stranded
nicks to this sequence as evidenced by agarose gel elec-
trophoresis.
Microfluidic analysis of endonuclease inhibition using an Ag-
ilent Bioanalyzer DNA 1000 chip. This assay was con-
ducted as previously described by the Kellett Group with
small changes being made (23). A total of 400 ng of the PCR
fragment was pre-treated with 10, 25, 50, 100 and 200 ␮M
of either MC3, HC3 or OC3. Subsequent digestion exper-
iments were performed by incubating drug treated and un-
treated DNA with 1 ␮l of EcoRI, BamHI, SalI and HindIII
overnight and heat deactivated as per NEB guidelines. The
reactions of EcoRI, BamHI, SalI and HindIII, in the pres-
ence and absence of tripodal scaffolds, were then exam-
ined using the Agilent DNA 1000 microfluidic chip (Agilent
5067-1504) with data being collected on the Agilent Bioan-
alyzer 2100 platform.
DNA condensation in acidic and basic buffers. A total of
400 ng of pUC19 DNA was treated as previously stated with
slight modifications (5 h incubation at 5, 10, 20, 30 and 50
␮M). Samples were incubated in acidic and basic buffers
at pH 4.0 and 8.0, respectively. Sodium acetate (NaOAc)
and Tris buffers were prepared and adjusted with HCl and
NaOH accordingly to achieve the desired pH. Samples were
loaded onto an agarose gel (1.2%) containing 3 ␮l EtBr.
Electrophoresis was completed at 70 V for 40 min in 1x TAE
buffer.
RESULTS AND DISCUSSION
Synthesis of opioid scaffolds
Morphine scaffolds MC3, HC3 and OC3 were gener-
ated and their molecular structures are shown in Fig-
ure 1. Treating 1 equivalent of 2,4,6-tris-(bromomethyl)-
mesitylene with 3 equivalents of either morphine (M), het-
erocodeine (H) or oripavine (O) yielded the respective C3-
symmetric opioid compounds MC3, HC3 and OC3. These
were isolated as yellow or white solids after purification
by column chromatography. A satisfactory yield for both
MC3 (50%) and OC3 (48%) was obtained, however, HC3
could only be isolated in a low yield (13%). Indeed, genera-
tion of the HC3 opioid was restricted due to the poor syn-
thetic conversion of morphine to heterocodeine with yields
<25% (39) that leads to a low overall yield for HC3 from
morphine. C1 and C2 opiate analogues (MC1, MC2, OC1
and OC2) were synthesized in a similar manner to the C3
Atomic force microscopy (AFM)
AFM was used to determine the morphology of the DNA
condensates induced by a morphine C3 opiate to support
the effects of condensation at low concentrations. AFM
samples were prepared according to the following general
procedure: in a total volume of 10 ␮l final concentrations
of 3 ng/␮l of pUC19 (NEB, N3041) or linearized pUC19,
5 mM MgCl and varying concentrations of test compound,
were incubated at 37◦C for 1 h. Ten microliters of each sam-
ple was pipetted directly on to freshly cleaved mica and al-
lowed to incubate for 5 min followed by rinsing with 500 ␮l
of water. The samples were dried under compressed air for
a period of 1 h. AFM examinations were performed in am-
bient air with a commercial microscope (Dimension 3100

532 Nucleic Acids Research, 2017, Vol. 45, No. 2
Figure 1. Molecular structures of morphine, heterocodeine and oripavine along with C3-symmetic opioid molecules developed in this study: morphine
(MC3), heterocodeine (HC3) and oripavine (OC3). Left to right, morphine, heterocodeine and oripavine scaffolds. Structures (i), (ii) and (iii) show the
geometries of each scaffold (morphine, heterocodeine and oripavine respectively), with labeled ring substituents A–E, modified from X-ray structures
reported in the CSD.
congeners using ␣-2-chloroisodurene (MC1 and OC1) and
2, 4-bis-(chloromethyl)-1,3,5-trimethylbenzene (MC2 and
OC2), respectively, of each opioid derivative (Supplemen-
tary Section S-7). The series of compounds for this study
were all prepared in either a one- or two-step synthesis that
enabled rapid access to samples for evaluation in biological
investigations. As part of this work, a green chemistry met-
ric analysis (40) has been performed (Supplementary Sec-
tion S-10) with the aim of identifying aspects of the synthe-
sis that can be improved in future studies (41,42). This will
ensure that further efforts to improve the yield for the above
reactions can be in tandem with the development of greener
detected, supporting the C3 symmetry of the compounds as
a stable configuration.
Condensation of duplex DNA
C 3 opiate binding interactions with duplex DNA were first
identified using a saturated EtBr fluorescence quenching
study with calf thymus DNA (ct-DNA) (Figure 2). In our
initial high-throughput screen, which involved a series of
novel opioid scaffolds, MC3 was identified as a potentially
new DNA-binding molecule. Our motivation for develop-
ing HC3 and OC3 isomers stemmed from structural anal-
ysis on MC3 that focused on the 6ꢁ-OH group in the mor-
phine C ring as a possible H-bonding site for phosphate co-
ordination. Since this group is masked by methylation in
heterocodeine (H) and oripavine (O), we anticipated HC3
and OC3 analogues would have negligible DNA binding
properties. Unexpectedly, however, enhanced EtBr displace-
ment was observed (Table 1), particularly on poly[d(G-C)2]
and poly[d(A-T)2] co-polymers with apparent binding con-
stants (Kapp) of ∼106 M(bp−1) and ∼107 M(bp−1), respec-
tively.
1
methodologies. All compounds were characterized by H
and 13C nuclear magnetic resonance (NMR, Supplemen-
tary Section S-1) and attenuated total reflectance (ATR)
Fourier transform infrared (FTIR) spectroscopies, and by
ESI-MS. Solution-based NMR studies in deuterated chlo-
roform were performed to elucidate whether all three opiate
units were on the same face of the molecule (C3) or whether
rotation of one opiate ‘arm’ was rapidly occurring at room
temperature. To test the latter, a temperature study was con-
ducted. This confirmed a C3-symmetric opioid compound
was formed for each of the tris target compounds; a sin-
gle set of peaks in the 1H NMR was observed for the opi-
oid substructure for MC3, HC3 and OC3 in both 1D and
2D NMR studies. As the probe temperature was decreased
from 293 K to 243 K, no changes in the proton NMR were
To identify the significance of the C3 opioid scaffold sym-
metry toward duplex DNA binding, OC1, OC2, MC1 and
MC2 compounds were generated and tested using gel elec-
trophoresis experiments (24,35,37). Condensation results,
however, revealed negligible C1 and C2 activity (Supplemen-

Nucleic Acids Research, 2017, Vol. 45, No. 2 533
Figure 2. Competitive fluorescence quenching of ethidium bromide bound to calf thymus (ct-DNA) by opioid drugs MC3, HC3 and OC3, viscosity
properties of MC3, ethidium bromide and spermine (SPM) exposed to salmon testes dsDNA, and turbidity profiles of CT-DNA in the presence of titrated
C3 opioids and spermine. Data points being displayed as an average of triplicate measurement for fluorescence quenching and turbidity measurements.
Table 1. Apparent binding constants of opioid C3 compounds to dsDNA polymers. (N.D. = not detected)
Kapp M(bp−1
)
ꢀTM ◦C ( S.D.)
Poly A-T (100% A-T)
ct-DNA (58% A-T)
Poly G-C (0% A-T)
Poly A-T (100% A-T)
Poly G-C (0% A-T)
Actinomycin D (35)
Netropsin (35)
MC3
OC3
HC3
N.D.
2.92 × 107
2.5 × 106
5.35 × 107
N.D.
−0.32 0.29
12.32 0.79
0.38 0.71
−0.17 0.25
0.73 0.18
12.10 0.95
2.83 0.38
2.63 0.58
0.36 0.17
1.72 0.61
5.75 × 107
3.03 × 105
3.63 × 106
9.1 × 106
6.40 × 106
3.82 × 106
7.84 × 106
2.69 × 105
3.5 × 107
1.15 × 107
tary Figure 14 and 15) confirming the requirement of a ter-
tiary opioid substituent to facilitate efficient nucleic acid co-
ordination at low micromolar concentration. Thermal melt-
ing analysis of duplex polymers were then completed (Table
1), and, unlike classic DNA binding agents netropsin and
actinomycin D (35), examined previously under identical
conditions by this group, no significant stabilization ener-
gies were identified for the tris-opiate compounds.
DNA compaction was then followed by electrophoresis.
Samples were titrated against both supercoiled pUC19 plas-
mid DNA and a 742 bp dsDNA fragment amplified from
pUC19 encompassing the lacZ␣ gene, before incubation for
5 h prior to analysis by agarose gel electrophoresis (Figure
3). All three opioid scaffolds were found to condense both
supercoiled and linear dsDNA; MC3 and HC3 have simi-
lar profiles with the onset of aggregation at 20 ␮M, beyond
which native DNA bands become fainter in appearance or
disappear entirely from view, thus reflecting total conden-
sation. OC3, however, exhibits improved condensation ef-
fects in comparison to morphine and heterocodeine scaf-
folds. Here, the initiation of aggregation can be visualized
at 5 ␮M with the condensation process being completed at
20 ␮M and 30 ␮M on supercoiled plasmid and linear DNA,
respectively. Indeed, on comparing the gels, it is evident that
opioid-induced aggregation of supercoiled pUC19 (Figure
3A) occurs more efficiently than for linear dsDNA (Figure
3B).
To further probe the condensation mechanism, the in-
teraction of C3 opioids with pUC19 were examined in
the presence of non-covalently bound recognition elements
netropsin, which is well-characterized to bind within the mi-
nor groove (43), and methyl green – an agent that binds at
the major groove (44). In these experiments plasmid DNA
was pre-exposed to 20 ␮M of each recognition element
prior to the introduction of opioid sample (5–30 ␮M). No
differences were detected in the condensation process be-
tween the control (opioid + pUC19) and tested samples
in any case, indicating interactions within the minor and
major grooves are unlikely as prospective recognition sites
for C3 opioid binding (data not shown). Indeed, no evi-
dence of minor or major groove binding, or of intercala-
tion could be found in the CD spectra of MC3 and OC3
with st-DNA (Figure 4). It was further noted that no con-
formational change was induced upon opioid binding and
In order to probe the nature of DNA binding, viscos-
ity studies with salmon testes DNA (st-DNA) at vary-
ing drug load were evaluated (Figure 2). Classical conden-
sation behavior by C3 opioids was observed whereby all
three agents exhibited identical, downward curving, hydro-
dynamic (η/ηo) values. As shown in Figure 2, the profile
of MC3 is similar to the well-studied DNA compaction
agent spermine, which was run as a control agent along-
side the classical DNA intercalator EtBr. Since conden-
sation of dsDNA can be monitored at 350 nm, a wave-
length where nucleic acids do not normally absorb unless
condensation/aggregation has occurred (24), UV-vis ab-
sorption spectrophotometry was employed to further char-
acterize the condensation process. (Although turbidity of
dsDNA can also be monitored at 260 nm, electronic absorp-
tion of C3 opioids in this region precluded this measure-
ment.) All three opioids exhibit concentration-dependent
aggregation of dsDNA with binding isotherms of MC3 and
HC3 being sigmoidal in nature and approximately similar in
shape to spermine (Figure 2). OC3, however, has a markedly
different profile that increases almost linearly between 1–25
␮M before reaching a plateau beyond this titration point.
Overall, and in comparison to spermine, greater aggrega-
tion was observed for C3 opioids tested up to 25 ␮M, be-
yond which, OC3 and MC3 scaffolds can be described as
having the highest condensation effects in the series.
Since condensation by C3 opioids had been established
by viscosity and turbidity measurements, visualization of

534 Nucleic Acids Research, 2017, Vol. 45, No. 2
Figure 3. (A) Agarose gel electrophoresis of supercoiled (400 ng) and (B) a 742 bp dsDNA fragment of pUC19 (400 ng) exposed to increasing concentrations
of MC3, OC3 and HC3. Reactions were carried out in the presence of 25 mM NaCl for 5 h at 37◦C prior to electrophoretic analysis.
Figure 4. Circular dichroism (CD) spectra of st-DNA treated with MC3, OC3 and electrostatic DNA binding controls hexammine cobalt(III) chloride
and spermine over 7 h at r ([opioid] / [DNA]) values of 0.1.
condensation. Thus, the profiles for both C3 scaffolds do
not yield spectral shifts expected of classical DNA binding
agents but, instead, are similar to surface-binding conden-
sation agents spermine and [Co(NH3)6]Cl3 (45) when tested
with long DNA polymers. Finally, while the spectrum of
HC3 could not be examined owning to its limited solubil-
ity in CD accessible solvents, it is reasonable to assume the
binding interaction should not deviate from that observed
from MC3 or OC3.
amine within the morphine D (piperidine) ring as a possible
cationic site responsible for H-bonding and/or electrostatic
binding to the phosphate backbone. Indeed, this particular
site is well recognized to undergo protonation prior to bind-
ing with specific amino acid residues within opioid receptor
cavities (46). To examine this hypothesis, the condensation
effects of supercoiled pUC19 were initially examined under
sodium chloride titration on agarose electrophoresis. Here,
25 ␮M aliquots of MC3, OC3 and HC3 were analyzed with
increasing NaCl ionic strength (25–1000 mM) with super-
coiled pUC19 (Figure 5A). At low salt concentrations (25–
75 mM) the plasmid was condensed by all three opioids,
however, in the presence of ≥100 mM NaCl, aggregation
by MC3 and HC3 agents was inhibited as evidenced by the
fraction of DNA migrating in supercoiled form. In contrast,
Influence of pH and ionic strength on condensation
Since the biophysical evidence gathered on C3 opioid–
DNA binding points toward a surface-binding coordina-
tion mode, we considered the possibility of the tertiary

Nucleic Acids Research, 2017, Vol. 45, No. 2 535
lapse of DNA proceeds at lower C3 concentration. Indeed,
on comparing Figure 3A with Figure 5B and C it is clear
that condensation in pH 4 buffered solution occurs with the
highest efficiency across all three tested opioids, followed
thereafter by condensates at pH 7.2 and then finally pH 9.0
where very little aggregation for both MC3 and HC3. This
analysis supports proposal that the amine group within the
piperidine opioid ring is the protonation site responsible for
condensation and, based on these condensation results, is
highly probable the 3+ polycationic state of the C3 symmet-
ric opioid scaffold is required to induce aggregation.
Atomic force microscopy (AFM) analysis
AFM imaging provides a direct tool for exploring the ef-
fects of ligand binding on DNA morphology (47,48). Due
to its nanoscale resolution there are numerous examples of
AFM techniques used to determine the binding mode, affin-
ity and site-exclusion number of DNA in the presence of
suitable binding substrates. Recent AFM studies have char-
acterized the binding modes of well-studied ligands such as
doxorubicin, ethidium bromide and netropsin (49). More-
over, this technique can successfully probe morphological
changes induced in closed circular plasmid and linear forms
of DNA, along with RNA. In this work, AFM studies
were performed with closed circular and HindIII linearized
pUC19 in ambient air using a Tapping-Mode with unmod-
ified silicon cantilevers at a 40 N/m force constant. Fur-
thermore, we established that 5 mM of MgCl2 was required
for complete mica adhesion with no appreciable conforma-
tional changes to DNA morphology. MC3 was selected as
a representative compound in this series for AFM analysis.
In the presence of 8 ␮M of MC3, supercoiled pUC19 exhib-
ited small cluster formation where tight packing is present in
the cluster center with DNA strands extending from its mid-
point (Figure 6A). As the concentration of MC3 increased
to 9 ␮M, a sizeable increase in cluster formation was ob-
served, however, unbound strands of DNA were evident at
this point (Figure 6B). A gradual increase in MC3 concen-
tration to 10 and 20 ␮M led to the near disappearance of un-
bound pUC19, leaving only large and tightly packed glob-
ules of ∼800 nm and ∼3.3 ␮m spherical dimension, respec-
tively (Figure 6C and D). Data here supports our earlier gel
electrophoresis analysis where 20 ␮M of MC3 was required
to fully condense the supercoiled plasmid (see Figure 3A).
In the presence of linearized pUC19, a lower concentration
of MC3 (5 ␮M) initiated the onset of condensation (Fig-
ure 6E) with both toroidal and cluster formation evident.
Toroid condensates, however, were evident at low MC3 con-
centration only with larger aggregates, >1 ␮m, appearing
at 10 and 20 ␮M exposure with compact globules form-
ing thereafter (50 ␮M). These results agree with our elec-
trophoretic data on linear pUC19 (see Figure 3B), where 50
␮M of MC3 was established to condense this sequence.
Figure 5. (A) Influence of ionic strength on pUC19 condensation (400 ng)
by OC3 and MC3 (25 ␮M) opioid compounds. Condensation reactions on
pUC19 (400 ng) by opioid compounds in (B) acidic NaOAc buffer (80 mM,
pH = 4.0), and (C) basic Tris buffer (80 mM, pH = 9.0) in the presence of
25 mM NaCl.
OC3 maintained its condensation effects up to 750 mM of
titrated NaCl and only at the highest ionic strength exam-
ined, 1 M, could a fraction of native supercoiled pUC19
be identified. These results indicate that condensation by
C3 morphine analogues are dependent on ionic charge, and
that disruption of this interaction by the introduction of an
ionic gradient can, at least partially, reverse DNA aggrega-
tion. Furthermore, it is worth highlighting that compaction
by MC3 and HC3 is disrupted to the same degree by NaCl,
while OC3 is substantially more resistant to changes in ionic
strength.
To further probe the involvement of quaternary amine
cations in the condensation process, aggregation of pUC19
was then examined under acidic (pH 4.0) and basic (pH 9.0)
buffered conditions. As anticipated, a clear enhancement of
the condensation process at lower pH with sodium acetate
buffer (Figure 5B) was observed, while under basic condi-
tions (Tris buffer) DNA aggregation was reduced (Figure
5C). These results indicate that in an acidic environment,
the piperidine opioid D ring becomes protonated and col-
Microfluidic analysis of endonuclease inhibition
The condensation effects of the opioid series on a 742 bp
dsDNA fragment, amplified from the pUC19 vector en-
compassing the lacZ␣ gene (Supplementary Section S-6),
and subsequent access by sequence-recognition type II re-

536 Nucleic Acids Research, 2017, Vol. 45, No. 2
Figure 6. Atomic force microscopy (AFM) images of MC3-treated supercoiled and HindIII linearized pUC19 DNA; (A–D) supercoiled pUC19 with 8, 9,
10 and 20 ␮M MC3; (E–H) linear pUC19 with 5, 10, 20 and 50 ␮M MC3.
Figure 7. Experimental design for the Bioanalyzer 2100 to identify site-specific endonuclease inhibition by opioid compounds, HindIII, EcoRI, BamHI
and SalI.
striction endonucleases were initially examined by gel elec-
trophoresis. Results showed the inhibition of migration at
∼20 ␮M of C3 opioid exposure indicating condensation had
occurred. An on-chip microfluidic assay using an Aglient
2100 Bioanalyzer was then employed (Figure 7) to deter-
mine whether C3-opoid induced condensation could block
sequence recognition by type II restriction enzymes BamHI,
HindIII, SalI and EcoRI. These restriction enzymes were
selected based on their single recognition site within our
transcript with control experiments (Supplementary Figure
17) establishing no direct interaction between restriction en-
zymes and C3 opiates. A DNA 1000 microfluidic chip was
employed in this study to detect and quantify excision frag-
ments, resulting in the disappearance of the parent 742 bp
band and the emergence of daughter fragments sized be-
tween 295 and 447 bp (Figure 8A). Pre-incubation of the
transcript with 50 ␮M of both MC3 and HC3 afforded
little protection toward endonuclease accessibility (Figure
8B and C). An exception, however, was noted for BamHI
activity to HC3 exposed DNA; here, the parent fragment
at 742 bp emerged with notable reduction in the excision
fragment peak area at 325 and 417 bp. Interestingly, pre-
incubation of the transcript with low micromolar loading of
OC3 (10 ␮M) was found to inhibit endonuclease accessibil-
ity by BamHI, SalI and EcoRI. Furthermore, in the case of
the BamHI-treated transcript pre-exposed to OC3, almost
complete protection of the oligo was observed. It was noted,
however, that the peak area of OC3 exposed fragments were
diminished in comparison to MC3 and HC3 experiments,
most likely through the enhanced condensation effects of
this agent. Finally, in a complementary study using gel elec-
trophoresis, we identified that the non-specific endonuclease
DNase I degraded all C3-opioid plasmid pUC19 conden-
sates with identical efficiency (data not shown).
CONCLUSION
To our knowledge opioid compounds have not previ-
ously been shown to interact with nucleic acids. This work

Nucleic Acids Research, 2017, Vol. 45, No. 2 537
Figure 8. (A) Electrograms generated using the Bioanalyzer 2100 of 742 bp dsDNA fragment with treatment by endonucleases BamHI, HindIII, SalI and
EcoRI. Electrograms of the 742 bp fragment were pre-incubated for 5 h with either (B) MC3, (C) HC3 and (D) OC3, followed by exposure over night to
the type II restriction endonuclease.
may therefore open up new applications for these well-
established analgesic compounds as semi-synthetic natu-
ral products that facilitate nucleic acid recognition. Target
compounds were prepared via short synthetic routes, which
in most cases involved a single step (i.e. from an opioid
natural product and commercially available benzyl bromide
derivative). The high-affinity DNA binding and condensa-
tion properties of this scaffold demonstrate the requirement
for a tris-opiate, along with a polycationic charge of 3+, as
the C1 and C2 opiate congeners showed negligible conden-
sation effects by comparison. In this regard, this class of
agent falls squarely in line with the majority of established
DNA condensation agents where a ≥3+ cationic valence re-
quirement for DNA collapse is generally required (22). Bio-
physical assays on duplex DNA polymers revealed no in-
Figure 9. Proposed ionic binding by the C3 opioid scaffold to the nucleic
acid phosphate backbone.
tercalative or major/minor groove residency, which led to
the probing of C3-opioids as potential electrostatic and/or
H-bonding agents. Thus, our preliminary studies using C3-
morphine (MC3) focused on the possibility of the 6ꢁ-OH,

538 Nucleic Acids Research, 2017, Vol. 45, No. 2
Figure 10. Structures of protonated diacetylmorphine (FAZDAM left) and protonated 3, 6-dimethoxy-5, 17-dimethyl-6,7,8,14-tetradehydro-4,5-
epoxymorphinan (LOBGUG right) redrawn from the CSD data (the amine proton has been added to the structure at a calculated position for LOBGUG).
The amine proton (highlighted in cyan) is more exposed in the oripavine. Dashed lines show the interactions with neighbouring axial protons.
located in the morphine C ring, as a possible surface bind-
ing moiety. Further, while physiological conditions promote
interplay between morphine protonation (kO) and zwitteri-
onic (kN) isomers (Supplementary Section S-8), the 6ꢁ-OH
does not engage in acid/base speciation and was therefore
considered as a potential phosphate-interacting site (50,51).
To test this axiom, the development of C3-heterocodeine
(HC3) and C3-oripavine (OC3) derivatives, where the 6ꢁ-
OH site is masked by methoxy substituents, was under-
taken. Biophysical studies, however, revealed comparable
(HC3) and enhanced (OC3) high-affinity binding to DNA;
these data, coupled with condensation properties observed
at varying pH and ionic strength, implicate N-protonation
of the piperidine (E) ring––a known cationic site that under-
goes ionic bond formation with the opioid receptor (46)––as
the nucleation site responsible for nucleic acid coordination.
A proposed model for DNA binding is shown in Figure 9
where each opiate is protonated (and charged) at the piperi-
dine ring that, in turn, surface binds the vicinal nucleic acid
backbone. Based on this proposed model, the binding of C3
opioids to other nucleic acid structures can be expected and
preliminary experiments on the condensation of tRNA by
MC3 (Supplementary Figure 38) evidences this. To corrob-
orate the binding mode described in Figure 9, methylation
of each N-piperidine moiety in MC3 was achieved (Sup-
plementary Section S-7) leading to isolation of the quat-
eranized piperidium cation MC3-NMe2 as an iodide salt.
The condensation effects of MC3-NMe2 with pUC19, when
directly compared to MC3 (Supplementary Figure 16), re-
sulted in a significantly altered condensation profile. Thus,
MC3-NMe2 did not aggregate DNA at any exposure value
(1–30 ␮M) where MC3 exhibited condensation and only at
higher concentrations (>30 ␮M) was MC3-NMe2 conden-
sation observed; we propose this interaction is electrostatic
in nature and similar to NMe2-quaternarized nucleic acid
binding observed in the literature (52,53). It is highly likely,
therefore, that N-protonation of the C3 opioid scaffold is
responsible for efficient condensation and that piperidium
phosphate hydrogen bonding interactions are required for
these high-affinity interactions.
Further examination of C3 agents revealed (i) no con-
formational modification to long canonical DNA poly-
mers, (ii) improved affinity toward aggregation of su-
percoiled plasmid versus linear DNA conformation and
(iii) an overall aggregation activity profile OC3 >>
HC3 ≈ MC3. To derive some insight into the dif-
ferences in binding between the oripavine compound
and the morphine or heterocodeine analogs, we consid-
ered structural data for examples of each class taken
from the Cambridge Structural Database (CSD) (54).
Figure 10 shows the cation of diacetylmorphine hy-
drogen chloride monohydrate (FAZDAM, ccdc 242245)
and 3, 6-dimethoxy-5,17-dimethyl-6,7,8,14-tetradehydro-
4,5-epoxymorphinan (LOBGUG, ccdc 985664), for the
purpose of comparison a proton has been added to the
amine at the calculated position in the latter case. The in-
creased unsaturation in the oripavine means that the ‘C’ ring
is flattened relative to the morphine or codeine systems and

Nucleic Acids Research, 2017, Vol. 45, No. 2 539
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that one of the hydrogen atoms in an axial position relative
to the amine proton is lost. As a consequence, the amine
site is clearly less sterically crowded in the oripavine system
and this might be expected to make an interaction with the
DNA phosphate backbone easier. There are also electronic
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linear DNA structures. The combined effect of high binding
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DNA binding scaffold capable of mediating condensation
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with the phosphate backbone. Our attention now turns to
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native DNA structures by these molecules and for their abil-
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SUPPLEMENTARY DATA
Supplementary Data are available at NAR Online.
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Irish Research Council (IRC) [GOIPG/2013/826]; Marie
-
Sklodowska-Curie Innovative Training Network (ITN)
ClickGene [H2020-MSCA-ITN-2014−642023]; Equip-
ment used in this study was funded under the Programme
for Research in Third Level Institutions (PRTLI) Cycle
5; The PRTLI is co-funded through the European Re-
gional Development Fund (ERDF), part of the European
Union Structural Funds Programme 2011–2015; EU
COST Action: Biomimetic Radical Chemistry [CM1201];
European Union’s Seventh Framework Programme for
research, technological development and demonstration
[621364 (TUTIC-Green)]. Funding for open access charge:
European Union’s Seventh Framework Programme for
research, technological development and demonstration
[621364 (TUTIC-Green)].
Conflict of interest statement. None declared.
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