DNA-block copolymer conjugates [9]

Full text of DOI:10.1021/ja0156845

5592
J. Am. Chem. Soc. 2001, 123, 5592-5593
Scheme 1
DNA-Block Copolymer Conjugates
Keith J. Watson, So-Jung Park, Jung-Hyuk Im,
SonBinh T. Nguyen,* and Chad A. Mirkin*
Department of Chemistry, Northwestern UniVersity
2145 Sheridan Road, EVanston, Illinois 60208
ReceiVed February 16, 2001
ReVised Manuscript ReceiVed April 18, 2001
The use of DNA as an interconnect for the synthesis of new
materials with preconceived architectural parameters and proper-
ties is a field of research that has seen considerable growth over
the past several years.1 The unique and reversible recognition
properties of these biomolecules are the key elements through
which their utility is derived. Exploring DNA for this purpose
already has led to the development of new detection strategies,2
novel nanostructures,3-8 and the construction of nanoelectronic
structures.9 In recent years, the coupling of synthetic oligonucle-
otides to organic polymers has emerged as a promising research
area where the combination of properties associated with both
the polymer backbone and the attached DNA can be simulta-
neously addressed, manipulated, and optimized to achieve a
particular function. For example, the attachment of DNA to
polypyrrole10-14 and other conducting polymers,15 either through
post-polymerization modification or direct copolymerization, has
led to the development of polymer-based amperometric detection
methods. While interesting, these DNA/polymer hybrids are
limited with respect to their degree of tailorability, ill-defined
compositions, and poor solubilities and dispersities, as well as
function. The synthesis of well-defined block copolymer hybrids
which can overcome these limitations would be an important
contribution to this developing technology.
Herein, we report the covalent attachment of DNA to the
backbone of a well-defined organic polymer derived from ring-
opening metathesis polymerization (ROMP). Given the thorough
exploration and optimization of ROMP during the past decade,16
its use as a template for the construction of DNA/polymer hybrid
materials offers several distinct advantages over other polymeric
systems. The commercially available catalyst Cl2Ru(PPh3)2d
CHPh (1) has been shown to initiate the polymerization of ring-
strained olefins (such as norbornene) in a living manner and to
be exceptionally tolerant to a large number of diverse functional
groups. These properties have led to the isolation of heretofore
unattainable polymers and block copolymers with virtually any
functional group covalently attached to the polymer chain, making
ROMP an ideal tool for the isolation of novel and useful
materials.17 The combination of such wide ranging functionalities
with the unique recognition properties of DNA could lead to the
development of new materials with easily programmable param-
eters.
In our attempts to incorporate DNA into ROMP polymers, we
pursued post-polymerization modification of preformed polymers
with DNA. For this task, the norbornenyl-modified alcohol 2,
which is characterized by a diphenylacetylene spacer which
separates the alcohol from the polymerizable norbornene, proved
to be extremely useful (vide infra). Starting from 5-exo-norbornen-
2-ol, 2 is isolated in five high-yielding steps. With its strong
absorption maximum at 304 nm (ꢀ ) 26 000 in MeOH), the
diphenylacetylene component serves as a convenient UV-tag
which can be used to monitor reactivity.
The synthesis of poly2 and reaction of this polymer with the
chlorophosphoramidite 3 resulted in a material with a single
resonance in the 31P NMR spectra at 149.2. This result is
consistent with that observed for the monomeric analogue.
Subsequent coupling to CPG-supported DNA using the syringe
technique, followed by deprotection of the DNA and cleavage
from the solid support in aqueous ammonia at 60 °C, yielded the
desired hybrid product. The DNA is connected to the polymer at
the 5′-end. Purification of the hybrid product from failure strands
was achieved using ultrafiltration (see Supporting Information).
As a first demonstration, we used this general experimental
strategy to isolate two polymers modified with complementary
12-mers of DNA with A10 spacers (Scheme 1, Hybrid-I and
Hybrid-II). The UV-spectra of the purified DNA/polymer hybrids
in water provide strong evidence that the DNA is attached to the
polymer backbone (Figure 1A). The absorption maximum at 310
nm demonstrates that the diphenylacetylene backbone is present,
which suggests that the water-soluble oligonucleotides are co-
valently linked to the hydrophobic polymer structure. Using
experimental and calculated extinction coefficients for the polymer
and the oligonucleotides (Supporting Information), and assuming
a repeat unit of the polymer consistent with the stoichiometry of
its synthesis, we estimate that there are, on average, five DNA
strands attached to each polymer chain. This translates to 30%
occupation of the potential polymer attachment sites by DNA
strands.
* To whom correspondence should be addressed. Email: stn@chem.nwu.edu.
(1) Storhoff, J. J.; Mirkin, C. A. Chem. ReV. 1999, 99, 1849-1862.
(2) Elghanian, R.; Storhoff, J. J.; Mucic, R. C.; Letsinger, R. L.; Mirkin,
C. A. Science 1997, 277, 1078-1080.
(3) Mirkin, C. A.; Letsinger, R. L.; Mucic, R. C.; Storhoff, J. J. Nature
1996, 382, 607-609.
(4) Alivisatos, A. P.; Johnsson, K. P.; Peng, X.; Wilson, T. E.; Loweth, C.
J.; Bruchez, M. P., Jr.; Schultz, P. G. Nature 1996, 382, 609-611.
(5) Cassell, A. M.; Scrivens, W. A.; Tour, J. M. Angew. Chem., Int. Ed.
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(10) Bazin, H.; Livache, T. Nucleosides Nucleotides 1999, 18, 1309-1310.
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When solutions of Hybrid-I and Hybrid-II are mixed in a
PBS buffer solution (PBS ) 0.3 M NaCl, 10 mM phosphate, pH
7), hybridization triggers the formation of an extended network
(13) Livache, T.; Roget, A.; Dejean, E.; Barthet, C.; Bidan, G.; Teoule, R.
Nucleic Acids Res. 1994, 22, 2915-2921.
(14) Livache, T.; Fouque, B.; Roget, A.; Marchand, J.; Bidan, G.; Teoule,
R.; Mathis, G. Anal. Biochem. 1998, 255, 188-194.
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10.1021/ja0156845 CCC: $20.00 © 2001 American Chemical Society
Published on Web 05/18/2001

Communications to the Editor
J. Am. Chem. Soc., Vol. 123, No. 23, 2001 5593
Scheme 2
Figure 1. (A) UV-vis spectrum of Hybrid-I. (B) UV-vis spectra before
and after melting of Hybrid-I/Hybrid-II mixture (melting curve inset)
(C) UV-vis spectra of DNA-modified 13-nm Au nanoparticles and
aggregates of Hybrid-I and complementary DNA-modified 13-nm Au
nanoparticles. (D) TEM image of the aggregates from C.
to block copolymers of 2 with a number of norbornene monomers,
thereby imparting tailorable functionality to branched DNA
structures. Our strategy for the synthesis of block copolymer
branched DNA is outlined in Scheme 2. In these experiments,
monomer 2 is mixed with a catalytic amount of 1 in dry THF.
After 1 h, the polymerization of the first block was determined
1
to be complete by H NMR spectroscopy. Subsequent addition
and polymerization of a second norbornene monomer, followed
by the injection of ethyl vinyl ether to terminate the reaction,
yielded block copolymers with the desired structure. When the
norbornenyl-modified ferrocene 4 was used as a second block,
post-polymerization modification with 3 and solid-phase DNA
synthesis led to the isolation of Hybrid III and Hybrid IV.
Electrochemical measurements confirm the presence of the
ferrocenes (Fc) in these hybrids (E1/2 ) 33 mV vs Fc/Fc+, Figure
2A). When PBS-bufferred solutions of these hybrids were mixed,
thermally reversible aggregate formation was again observed.
Also, a sharp melting transition was observed, consistent with
our proposed structure (Figure 2B).
Figure 2. (A) The cyclic voltammogram of Hybrid IV in 0.2 M [(n-
Bu)4N]PF6 in CH2Cl2. (B) The melting curve for Hybrid-III/Hybrid-
IV in a PBS buffer (first derivative inset).
In conclusion, we have demonstrated that post-polymerization
modification of ROMP polymers and block copolymers with DNA
can lead to DNA/polymer hybrid materials with a number of
interesting properties associated with the hybrid structure. The
initial experiments described herein reveal that the recognition
properties of the DNA strands are not adversely affected by
attachment to the polymer. These new structures can be prepared
with properties and function that depend on the choice of ROMP
monomer and DNA branch sites. Since the synthesis of block
copolymers of 2 with other norbornenyl-modified compounds is
a facile process, the isolation of other novel and potentially useful
macromolecular hybrid materials should be readily accomplished
by utilizing variations of the strategy presented herein.
aggregate of linked polymers, which is signaled by the immediate
formation of a white precipitate. Presumably, this occurs because
each polymer is modified with more than one strand of DNA,
which leads to cooperative binding between many DNA func-
tionalized complementary polymer strands, as evidenced by the
sharp melting transition (inset of Figure 1B). As expected, this
hybridization process is thermally reversible. These studies
demonstrate that attachment to the polymer does not hinder the
recognition properties of the oligonucleotides.
The DNA/polymer conjugate was utilized to form nanoparticle
assemblies. When a PBS buffer solution of Hybrid-I (12 µL of
8.3 µM in DNA) is mixed with a PBS buffer solution of 13 nm
Au nanoparticles (260 µL of 9.7 nM in nanoparticle) modified
with complementary DNA strands,2 three-dimensional nanopar-
ticle aggregates are formed. Formation of such aggregates is
signaled by the diagnostic shift in the surface plasmon resonance
of the nanoparticles (from 520 to 570 nm, Figure 1C) and a
corresponding change in color (from red to purple).2,3 Transmis-
sion electron microscopy studies reveal a highly networked
aggregate (Figure 1D). Control experiments in which a solution
of the same nanoparticles was mixed with a buffered solution of
Hybrid-II (which is noncomplementary) resulted in no aggregate
formation under nearly identical conditions.
Acknowledgment. Financial support from the AFOSR (Grants
F49620-99-1-0071 and F49620-01-1-0303 (PECASE)), ARO (MURI), NSF
(DMR-CAREER Grant 0094347), Packard Foundation, and Dreyfus
Foundation are appreciated. S.T.N. acknowledges the Afred P. Sloan
Foundation for a Research Fellowship. K.J.W. acknowledges NSERC
Canada for a predoctoral fellowship.
Supporting Information Available: Details of the synthesis and
characterization of 2, 4, poly2, poly2-block-poly4 and other experimental
procedures are available. This material is available free of charge via the
Internet at http://pubs.acs.org.
Given the exceptional functional group tolerance of catalyst 1
in ROMP, we hypothesized that this chemistry could be extended
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