High-affinity multivalent DNA binding by using low-molecular-weight dendrons

Article abstract of DOI:10.1002/anie.200500066

(Chemical Equation Presented) Nine spermines are better than one: Compound C2 exhibits high-affinity, salt-independent DNA binding as a consequence of the spermine array on its surface. Comparison with smaller dendrons and model compounds indicates a clea

Full text of DOI:10.1002/anie.200500066

Communications
Multivalent DNA Binding
High-Affinity Multivalent DNA Binding by Using
Low-Molecular-Weight Dendrons**
Mauri A. Kostiainen, John G. Hardy, and
David K. Smith*
High-affinity binding between nanoscale objects is an essen-
tial prerequisite for “bottom-up” fabrication.[1] In recent
years, interest has focused on the use of dendritic macro-
molecules as supramolecular nanoscale building blocks.[2] The
branched superstructure of dendrons and dendrimers offers
specific advantages, for example, enhancement of weak
binding by using multivalent arrays of recognition units on
the dendritic surface. This multivalency principle, in which
organized arrays amplify the strength of a weak binding
process, such as the binding of saccharides to proteins on cell
surfaces, is now well established.[3]
DNA constitutes a particularly interesting target for
nanotechnological exploitation.[4] High-affinity binding of
DNA is useful for protecting DNA and ultimately for
delivering genetic information into cells.[5] Noncovalent
interactions between dendritic macromolecules and DNA
are, therefore, of considerable current interest.[6] Polyamido-
amine (PAMAM) dendrimers were the first systems to be
investigated,[7] while dendritic poly(l-lysine)[8] and poly(prop-
ylene imine)[9] have also been studied recently. In general,
higher-generation, or structurally fractured, systems are
usually more effective for DNA binding and delivery. In an
important recent study, however, Diederich and co-workers
reported low-molecular-mass dendrons designed to self-
assemble with DNA, which were capable of gene therapy.[10]
The interaction between a single protonated amine and
the phosphate backbone of DNA is relatively weak and must
compete with salt binding under biological conditions.
Biology therefore uses tetraamines, such as spermine
(Scheme 1), to achieve DNA binding.[11] Synthetic spermine
derivatives are also widely used for applications in DNA
binding and delivery.[12] However, although spermine is better
than an isolated amine for binding DNA, the interaction is
still relatively weak and the complex is mobile. Consequently,
spermine struggles to compete with DNA-bound inorganic
cations.[13]
[*] M. A. Kostiainen, J. G. Hardy, Dr. D. K. Smith
Department of Chemistry
University of York
Heslington, York, YO10 5DD (UK)
Fax: (+44)1904-432-516
E-mail: dks3@york.ac.uk
[**] This work was supported through the Socrates–Erasmus European
Exchange Scheme (M.A.K.) and by the Engineering and Physical
Sciences Research Council and Syngenta through the CASE Scheme
(J.G.H.). We acknowledge Ian Ashworth and Colin Brennan
(Syngenta) for support and Meg Stark (Department of Biology,
University of York) for assistance with TEM measurements.
Supporting Information for this article is available on the WWW
under http://www.angewandte.org or from the author.
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DOI: 10.1002/anie.200500066
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Scheme 1. Spermine and target spermine derivatives G0, G1, and G2.
We became interested in optimizing DNA binding and
developing low-molecular-mass dendrons with very high
affinities for DNA—such systems would be particularly
useful for DNA encapsulation and protection.[14] We there-
fore decided to develop dendrons that express multivalent
spermine arrays on their surfaces. Some previously reported
multivalent spermine arrays comprised multiple spermine
groups grafted onto dextran polymers;[15] however, we wanted
to develop multivalent systems with well-defined molecular
structures. Such monodisperse systems enable an understand-
ing of structure–activity relationships and, in addition, have a
greater chance of being licensed for therapeutic applications
in the longer term. Herein, we report on multivalent dendritic
spermine constructs with extremely high, salt-independent
binding affinities for DNA.
We used a divergent synthetic approach to construct
target compounds G1 and G2 (Scheme 1, details of the
synthesis can be found in the Supporting Information).
Newkome-type branching[16] was used as the dendritic scaf-
fold, as such structures are readily synthesized[17] and should
be biocompatible. After dendron synthesis, the surface was
functionalized with spermine groups which had been appro-
priately protected by using the methodology of Blagbrough
and Geall.[18] Deprotection of the spermine groups with HCl
gas then yielded target compounds G1 and G2. Model
compound G0 was constructed by using a similar approach
(Scheme 1). All synthetic steps were high yielding and all
target compounds and intermediates were fully characterized
with standard methods (see the Supporting Information).
Initially, the binding of the spermine derivatives to DNA
was studied by using an ethidium bromide displacement assay.
This method is commonly used to study the binding of
polyammonium compounds to DNA.[19] The displacement of
ethidium bromide from its complex with DNA can be
monitored because it has enhanced fluorescence when
intercalated. This is
a powerful comparative method,
although the resulting data reflect a competition assay,
rather than an absolute binding strength, and furthermore
give no information about binding stoichiometry.
Fluorescence titrations were performed in buffered water
at pH 7.2 using G0, G1, G2, and spermine itself. The resultant
titration profiles are shown in Figure 1. At this physiologically
relevant pH value, the spermine groups are largely proto-
nated whilst the DNA is largely deprotonated, so electrostatic
interactions are maximized.
The data can be presented in terms of C50 and CE50 values
(Table 1). C50 values report the concentration of polyamine
causing a 50% decrease in fluorescence intensity. CE50 values
represent the “charge excess”[20] required to achieve the same
Figure 1. Fluorescence titration profiles for the addition of spermine,
G0, G1, or G2 to a solution of calf thymus DNA and ethidium bromide
in buffered water (pH 7.2) in the presence of 150 mm NaCl.
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Table 1: Results for spermine, G0, G1, and G2 from an ethidium
bromide displacement assay.[a]
constructs G1 and G2 retarded
the electrophoretic mobility of
DNA as
a consequence of
Compound
[NaCl] [mm]
Nominal charge
C50 [mm]
CE50
charge neutralization, whilst
the spermine and G0 ana-
spermine
G0
G1
9.4
9.4
9.4
9.4
4+
3+
9+
1.33
20
0.076
0.030
5.3
60
0.68
0.81
logues
were
ineffective
(Figure 2). The CE values at
which mobility was retarded
were in agreement with the
results of the ethidium bro-
mide displacement assays.
G2
27+
spermine
G0
G1
150
150
150
150
4+
3+
9+
390
220
0.300
0.028
1560
660
2.70
0.76
G2
27+
Finally, transmission elec-
[a] Conditions: Measurements performed in buffered water at pH 7.2
(with 2 mm 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid
(HEPES) and 0.05 mm ethylenediaminetetraacetic tetraacetate
(EDTA)). The calf thymus DNA (1 mm) and ethidium bromide
(1.26 mm) concentrations were kept constant. The total added polyamine
solution did not exceed 5% of the total volume; therefore, corrections
were not made for sample dilution. Results are an average of at least
three titrations. Experimental errors ꢀ10%.
tron microscopy (TEM) was
used to image the complexes
(Figure 3). The addition of
spermine at CE = 1.8 to plas-
mid DNA in buffered water
Figure 2. Gel electrophoresis
of plasmid DNA (250 ng per
lane). Lane 1: Plasmid DNA.
Lane 2: Plasmid DNA +
spermine (250 ng). Lane 3:
Plasmid DNA + G0
(250 ng). Lane 4: Plasmid
DNA + G1 (250 ng).
Lane 5: Plasmid DNA + G2
(250 ng).
(pH 7.1,
[NaCl] = 9.4 mm)
prior to deposition on
a
carbon-coated copper grid
gave rise to large unsymmet-
rical aggregates approximately
250 nm in diameter (Fig-
ure 3A). Compound G0, how-
ever, led to little or no com-
paction of DNA under the
same conditions. On the other hand, use of G1 or G2 (CE =
2.7) gave rise to well-defined, approximately spherical nano-
scale complexes (G1: approximately 100 nm in diameter; G2:
approximately 400 nm in diameter) with no free plasmid
being detected (Figures 3B,C). The size range of the aggre-
gates formed was relatively large. Nonetheless, these obser-
vations indicate that compounds G1 and G2 efficiently bind
DNA and condense it into approximately spherical com-
plexes.
50% reduction in fluorescence—the very best DNA binders
should have CE50 values below 1.0. Spermine binds to DNA
with moderate strength under low-salt (9.4 mm NaCl) con-
ditions (C50 = 1.33 mm, CE50 = 5.3), but a very large charge
excess would have been required at physiological salt
concentrations (150 mm NaCl) in order to achieve 50%
quenching of ethidium bromide fluorescence (C50 = 390 mm,
CE50 = 1560). These results were in good agreement with
literature data.[19a] Compound G0 showed similar, if slightly
weaker, DNA binding. This was expected, as one of the
primary amines of spermine has been converted into an
amide, which is incapable of protonation, and G0 should
therefore exhibit weaker electrostatic interactions with poly-
anionic DNA.
The dendritic systems G1 and G2 showed significantly
enhanced DNA binding. Under low-salt conditions, G1
In summary, we have reported novel dendrons that bind
DNA with remarkably high affinities. Notably, G2 showed
salt-independent DNA binding and it was a factor of ten more
effectively displaced ethidium bromide from DNA (C50
=
76 nm, CE50 = 0.68). Notably, the affinity for DNA is consid-
erably more than three times higher than that of G0. This
indicates that the organization of three spermine units on the
dendritic framework enables DNA-binding activity that is
more than the simple sum of its individual parts—the
multivalency principle[3] in operation. Compound G1 is
somewhat affected by the increase in salt concentration but
still shows reasonable binding under these conditions (C50
300 nm, CE50 = 2.70).
=
Compound G2 shows a similar binding affinity to G1
under low-salt conditions (C50 = 30 nm, CE50 = 0.81) but
demonstrates its power at physiological salt concentrations,
where the binding remains just as strong (C50 = 28 nm, CE50 =
0.76). The binding is therefore salt independent—a proactive
dendritic effect. The multivalent system can therefore com-
pete with Na+ ions for binding sites on the surface of the DNA
helix. Indeed, compound G2 exhibits one of the best binding
profiles reported by using this method, thereby proving that
the strategy of organizing spermine units into a well-defined
multivalent array has considerable power.
Figure 3. TEM images of DNA in the presence of A) spermine
(CE=1.8), B) G1 (CE=2.7), or C) G2 (CE=2.7). Samples were
deposited from buffered water (pH 7.1).
Gel electrophoresis was used to confirm the affinities of
the dendrons for DNA in a direct binding assay. The dendritic
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Chemie
examples, see: b) G. Byk, C. Dubertret, V. Escriou, M. Frederic,
effective than the G1 analogue under high-salt conditions,
whilst G1 was, in turn, significantly more effective than the
G0 analogue. It can be concluded that the expression of
multiple spermine units, natureꢀs own DNA binder, on the
surface of a dendritic scaffold offers a powerful approach for
achieving high-affinity DNA binding under physiological
conditions. These molecules have potential for further
synthetic variation, and in current and future work, we will
be investigating the effect of structural modifications on DNA
binding and nanoscale assembly, as well as looking at
applications of the novel dendritic constructs in gene protec-
tion and delivery.
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Received: January 7, 2005
Published online: March 22, 2005
Keywords: dendrimers · DNA · multivalency · nanochemistry ·
.
self-assembly
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