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Immobilization of Platinated and Iodinated Oligonucleotides on Carbon Nanotubes.

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Atigcw. C/icm. In!.
€d. EngI. 1997. 36. No. 20
,c WILEY-VCH Verlag GmbH. D-69451 Weinheim. 1997
0570-0833,97 3620-2197 S 17.50+.5010
Immobilization of Platinated and Iodinated
Oligonucleotides on Carbon Nanotubes**
technique. For this purpose a self-complementary 14-mer (I)
d(SA-T-G-G-T-A-G-'C-T-A-'C-'C-A-T3') I
Shik Chi Tsang, Zijian Guo, Yao Kuan Chen,
Malcolm L. H. Green, H. Allen 0. Hill,
Trevor W. Hambley, and Peter J. Sadler*
Rapid advances in biotechnology are making major contributions to our understanding of health and disease, stimulating
much current interest in designing novel systems for detecting
abnormalities and for delivering (bio)chemicals to target diseases with limited side effects. In this context, the development
of techniques for the study of molecular recognition is particularly important in the area of biochemical sensing, drug delivery, and gene therapy. For example, there has been recent interest in anchoring DNA onto a solid surface so that it may
function as a chemical recognition agent for complementary
DNA or for specific proteins.[']
Carbon is the most common support material for the immobilization of biomolecules because of its strength, stability, and
electrical conductivity.['] However, only a limited amount of
work on the structure and morphology of biomolecules adsorbed onto carbon materials has been reported. Recently discovered nanotubes have fullerene-type structures of concentric
tubular carbon layers and a prominent central cavity 30-60 A
in diameter. This novel form of carbon has a higher electrical
conductivity than graphitef31and can be prepared on a gramscale by high-temperature arc vaporization of graphite elect r o d e ~ . [ ~We
* report here the synthesis and immobilization of
small, platinated and iodated DNA oligomers and their immobilization on carbon nanotubes. These heavy-atom-labeled
oligomers readily scatter electrons, which allows indirect visualization by high-resolution transmission electron microscopy
Carbon nanotubes were prepared as previously reported.I6]
TEM examination of the nanotube-rich distillate showed it to
consist of fiberlike nanotubes (about 30%) and graphitic particles (about 70%). The TEM images of the tip region showed
that the carbon tubes invariably had closed ends. In order to
investigate whether DNA could be trapped inside nanotubes,
opened nanotubes were prepared by treatment with HNO,, and
most of the surface acidic groups were removed by heat treatment.['] TEM examination of the sample showed that more than
80% of the tubes were selectively opened in the cap region[*]
and that the internal cavities were clean and empty.
The nanotubes were then treated with a DNA duplex labeled
with a heavy atom, which could be clearly seen by the TEM
[*I Prof. Dr. P. J. Sadler, Dr. Z. Guo, Dr. T. W. Hambley[+'
Department of Chemistry
University of Edinburgh
West Mains Road, Edinburgh EH9 3JJ (UK).
Fax: Int. code +(131)650-6452
e-mail: p.
Permanent address: School of Chemistry, University of Sydney (Australia)
Dr. S. C. Tsang
The Catalysis Research Centre, Department of Chemistry
University of Reading (UK)
Y. K. Chen, Prof. Dr. M. L. H. Green, Prof. Dr. H. A. 0. Hill
Inorganic Chemistry Laboratory, University of Oxford (UK)
This work was supported by the Association for International Cancer Research
and BBSRC. PJS thanks the EC and EPSRC for support of HCM and COST
programs, and SCT The Royal Society for a University Fellowship. We thank
the MRC NMR Centre, Mill Hill, for provision of NMR facilities, and Dr. J. L.
Hutchinson, Department of Materlals, University of Oxford, for use of the
JEOL 4000 EX microscope and for helpful discussions, and Dr R. Henderson
(MRC Laboratory of Molecular Biology, Cambridge) for critlcal comments on
the script.
0 WILEY-VCH Verlag GmbH, D-69451 Weinheim, 1997
containing 5-iodocytosine ('C) and a G G pair, which could
be labeled with cheIated eis-[Pt(NH,),]'+ was chosen. The
'H NMR spectrum of this oligonucleotide over the pH range of
4.4 to 7 (H,O/D,O (9/l), 0.1 M NaClO,) showed 5 to 11 peaks
in the region 6 = 11.5- 13.5, typical of imino NH protons in
Watson-Crick base pairs.1g' The duplex (11) was platinated
specifically at G(3).G(4) by treatment with 0.9 equivalents (i.e.,
ca. one Pt per duplex) of a solution containing equimolar
amounts of cis-[Pt(H,O),(' 5NH,),J2 and cis-[PtCl(H,O)(15NH,),]f, the reactive hydrolysis products of the anticancer
drug cisplatin.['O1The reaction was monitored by ['H,' 5N]
HSQC 2D NMR spectroscopy.[",'21 The diaqua complex reacted very rapidly to give the G G chelate, and no monofunctional aqua intermediates were seen, whereas the monoaquamonochloro complex gave rise to two monofunctional intermediates cis-[PtC1(NH3),(G3-N7)] and cis-[PtC1(NH3),(G4-N7)]
(Figure 1). One of the monofunctional adducts predominated,
c- &('H)
Figure 1. Two-dimensional ['H,I5N] NMR spectrum recorded 12 h after mixand
ing duplex I1 with 0.9equiv of a solution of c~~-[P~(H,O),('~NH,),]~+
At the end of the reaction (about 72 h) only peaks a and
a' were present, which are assignable to the nonequivalent PI-NH, ligands in the
G G chelate Pt-11. Peaks b, ,'b and c, c' are assignable to the monofunctional intermediates platinated at G(3) or G(4) (two peaks in each case: NH, trans to N7
and Cl).
an effect which has been observed recently for a 14-mer DNA
oligonucleotide duplex." After three days the G G chelate was
the only final product detected by ['H,''N] NMR spectroscopy.
The presence of about ten resonances in the imino region of the
'H NMR spectrum suggested that the duplex structure of I1 was
retained after platination, as is usual for such adducts.["] The
model of Pt-I1 shown in Figure 2 serves to indicate the position
of the heavy atoms in this duplex.
Figure 2. B-DNA model of Pt-I1 showing the positions of the heavy atoms (artificially enlarged; unlabeled heavy atoms are iodine atoms).
0570-0833/97/3620-2198$17.50+ .50/0
Angew. Chem. Int. Ed. Engl. 1997,36, No. 20
Both closed and opened nanotubes were treated with Pt-11.
Figure 3 shows closed nanotubes (with graphitic layers) entirely
embedded beneath a layer of amorphous material. A small
amount of the amorphous material could be due to contamination of the sample during preparation and transfer, and local
electrical charges induced by the electron beam could lead to the
attraction of amorphous material inside the microscope chamber. However, contamination by such a large quantity of amorphous material on nanotube surfaces such as those shown in
Figure 3 has never been observed. Nanotubes treated similarly
was still observable on open nanotubes (2 mg) after treatment
with Pt-I1 (1 mL of a 3 p solution)
for 24 h and followed by
incubation in water for 24 h. The adsorption of duplex I1 was
studied by UV spectroscopy (see Experimental Section). Even
after washing, about 25pg of I1 (46% of added DNA) was
adsorbed onto 2 mg of closed tubes and 12 pg of I1 (22% of
added DNA) onto open tubes. The material washed off the
tubes was shown to be double helical DNA by melting experiments. The greater adsorption of DNA onto closed nanotubes
is consistent with the introduction of negative charges on the
surface after oxidation of the tubes.
No clear image of the surface adsorbed DNA was obtainable,
perhaps because of radiation damage. Blurring of images due to
beam-induced specimen movement and charging has been reported previ~usly.['~]
Nevertheless the fairly uniform thickness
of adsorbed molecules (7- 13 A) indicates excellent surface
recognition properties. The large major groove of B-DNA results in it having a regular cross-section and is consistent with
the observed two-dimensional width variation of 7- 13 A, being
dependent on the projection angle and position relative to the
electron beam. Thus, the surface layer coating the nanotubes is
likely to be double-stranded helical DNA. DNA on some inert
surfaces (for example, glass) can have a similar morphology as
in bulk aqueous solution."'' Some TEM pictures appeared to
show the presence of highly coiled Pt-I1 trapped inside open
nanotubes with filling along the entire length of the tube but in
a discontinuous manner. Presumably when the tubes were dried
and water was removed, the DNA anchored to the internal
surface in a regular fashion. The included materials appeared to
form long aggregates inside the nanotubes.
We have previously reportedC6]immobilization of proteins
and enzymes in and on the nanotubes and now anticipate that
carbon nanotubes with their well-defined structures, defect-free
surfaces and tubular morphology may be of general utility in
the study of molecular recognition processes including biomolecule-carbon interactions of importance to the design of
Experimental Section
Figure 3. TEM images of closed nanotubes after treatment with Pt-11. Top: side
view; bottom: an end. The arrows indicate adsorbed layers of amorphous material
(DNA) on the surface.
but without the presence of Pt-I1 showed clear surfaces free of
contamination. X-ray dispersive analysis (EDX) indicated that
adsorbed material contained Pt, P, I, CI, Na, and some Ca. We
therefore infer that the amorphous surface layer is due to Pt-I1
(and NaClO,). The immobilized material gave IR absorption peaks at 1630cm-' (NH, bending mode), 1097cm-'
(phosphate backbone), 1000-1400 cm-' (nucleobases), and
800 cm- ' (deoxyribose), typical of DNA.['31
Good contrast from the amorphous material was obtained in
some TEM image:;. This may arise from the heavy elements Pt
and I, which scai.ter electrons more readily than the background. The amorphous material tended to cover the nanotube
surface very evenly with an average thickness of 7-13 A, although some larger aggregation patches were also observed
(Figure 3, top). This indicates that the material interacts strongly with the carbon surface and has good surfact-wetting properties. The strong binding was illustrated by the fact that material
Angiw. Clwm. In!.
Ed Kngl. 1997, 36, No. 20
Chemicals: The iodinated oligonucleotide I was supplied by OSWEL DNA Service
(University of Southampton). cis-[PtCI,(lSNH,),] was synthesized by a standard
and cis-[Ptmethod [36], and the solution of C~~-[P~(H,O),('~NH,)~]~+
CI(H,0)(15NH,),]+ was prepared by treating cis-[PtCI,("NH,),] with 1.5 equiv of
AgNO, for 24 h followed by removal ofAgCI. Carbon nanotubes were prepared by
the arc vaporization method [6]. Open tubes were obtained by suspending closed
tubes in HNO, and heating at reflux for 4 h [S]. The open tubes were washed
dried in air at 100 "C, and heated at 700 ' C for 4 h to remove
thoroughly with H20,
most of the surface acidic groups [7]. Immobilization was carried out by mixing
0.5 mL of a 0 S m solution
of Pt-I1 (as used for NMR) with 2 mg of closed or
opened tubes. To avoid DNA damage, no sonication was used. Typically the
sample was preconcentrated under vacuum, and a droplet ofthe suspension was
placed on a copper grid (400 mesh) covered with perforated carbon film Tubes for
the control experiments were treated in the same way except for the absence of
added Pt-11.
Instruments: DNA samples were characterized by UV, IR, NMR, and HRTEM.
U V spectra were recorded on a Perkin Elmer Lambda 16 spectrometer in 1 cm cells
and a PTP-1 Peltier temperature programmer. The NMR spectra were recorded on
Varian UNITY plus-SO0 and UNITY-600 spectrometers fitted with pulsed-fieldgradient modules. 'H chemical shifts were internally referenced to TSP
in 1 M HCI. The 2D ['H,
(Me,Si(CD,),CO,Na), and the 15Nshifts to 1 . 5 NH,CI
"N] HSQC spectra (optimized for J(N,H) = 72 Hz) were recorded as previously
reported [11,12,17]with the sequence of Stonehouse et a]. [18]. The electron micrographs were recorded on JEOL 4000EX and 2000FX high resolution microscopes.
The accelerating voltages were adjusted to 200 kV in all cases. The microscopes were
operated at their optimum defocusingconditions.In order to reducecontamination,
short illumination times (less than one minute), low beam Intensity, and improved
vacuum by use of liquid nitrogen traps were employed. Energy X-ray analysis
(EDX) was used to confirm the presence of Pt and I on nanotubes treated with Pt-11.
UV studies of DNA adsorption: Open or closed nanotubes (2 mg) were incubated
with 1 mL of a 7 PM solution of duplex 11 in 0.1 M NaCIO, for 24 h at ambient
0 WILEY-VCH Verlag GmbH, D-69451 Weinheim, 1997
0570-0833/97/3620-2199$ 17.50+ .5OiO
temperature with occasional agitation. The supernatant was separated by ultrafiltration (Centricon, Amicon, cut-off 30 kDa, 6 h at 15 "C). The nanotubes were
then washed with water (0.6 mL), and the separation was repeated. In a control
experiment (to check for adsorption of DNA onto the membrane filter), DNA
solution was added to the ultrafiltration tube in the absence of nanotuhes, and the
separation and washing steps carried out as before. DNA concentrations were
determined from the absorption at 260 nm. The recovery of DNA in the control
experiment was >87%. A second set of experiments was carried out with 0.1 M
NaCIO, to wash the tubes, and then melting curves for the desorbed DNA were
determined by UV.
Molecular rnodelmg: A bifunctional Pt adduct was constructed on one strand of a
B-DNA model with the HyperChem program [19] and restrained energy minimization, followed by docking of the ammine ligands in an appropriate geometry. This
produced a model of the bifunctional adduct similar to those obtained with Newton- Raphson energy minimization and a force field developed for PtiDNA interactions [20]. Iodine atoms were then added to the 5-positions of each cytidine base.
Beltlike Aromatic Hydrocarbons by Metathesis
Reaction with Tetradehydrodianthracene""
Stefan Kammermeier, Peter G. Jones, and
Rainer Herges*
One of the main motivations for the synthesis of cyclophanes
is the preparation and investigation of compounds in which the
n-electron system is spherically deformed by the introduction of
bridging ligands (Scheme 1, left) .[IJ Fully conjugated belt- and
Received: December 3, 1996
Revised version: May 23, 1997 [Z9850IE]
German version: Angen. Chem. 1997,109, 2291 -2294
- immobilization
- nanotubes - oligonucleotides
Keywords: electron microscopy
Scheme 1. Spherically deformed
conjugated system (right)
[l] K. M. Millan, A. J. Spurmanis, S. R. Mikkelsen, Electroanalysis 1992,4, 929932.
[2] P. Pantano, W. G. Kuhr, Electroanalysis 1995, 7, 405-416.
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[4] S. Iijima, Nature 1991, 354, 56-58.
[5] T. W Ebbesen, P. M. Ajayan, Nature 1992, 358, 220-222.
[6] S. C. Tsang, J. J. Davis, M. L. H. Green, H. A. 0. Hill, Y C. Leung, P. J. Sadler
J: Chem. SOC.Chem. Commun. 1995, 1803-1804,
[7] R. M Lago, S . C. Tsang, K. L. Lu, Y K. Chen, M. L. H. Green, J: Chem. SOC.
Chem Comm. 1995, 135551356,
[XI S. C. Tsang, Y K. Chen, P. J. F. Harris, M. L. H. Green, Nature 1994, 372,
[9] G. C. K. Roberts, N M R of Macromolecules. a Practical Approach, Oxford
University Press, Oxford, 1993.
[lo] J. Reedijk, Chem. Commun. 1996, 801-806.
[ l l ] K. J. Barnham, S. J. Berners-Price, T. A. Frenkiel, U. Frey, P. J. Sadler,
Angew Chem. 1995, 107, 2040-2043; Angew. Chem. I n t . Ed. Engl. 1995, 34,
1874- 1877.
[12] S. J. Berners-Price, K. J. Barnham, U. Frey, P. J. Sadler, Chem. Eur. J: 1996,
2. 1283-1291.
[13] R. J. H. Clark, E. E. Hester, Sperrroscopy of Biological Systems, Wiley,
Chichester, 1986.
[14] R. Henderson, Q.Rev. Eiophys. 1995,28, 171-193.
[15] Y S Melnikova, N. Kumazawa, K. Yoshikawa, Biochem. Biophys. Res. Commun. 1995,214, 1040-1044.
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[17] S. 1. Berners-Price, U. Frey, J. D.Ranford, P. J. Sadler, J. Am. Chem. Soc.
1993,115, 8649-8659.
[18] J. Stonehouse, G. L. Shaw, J. Keeler, E. D. Laue, J. Magn. Reson. Ser. A 1994,
[19] HyperChem, Release 2 for Windows, Autodesk, Sausalito, California, USA.
[20] T. W. Hambley, Inorg. Chem. 1991, 30, 937-942.
systems in a cyclophane (left) and in a beltlike
tubelike structures can be viewed as an extreme example of such
a deformation (Scheme 1, right)."] The p orbitals in these structures are perpendicular to the surface of a cylinder and their
inner lobes point towards the axis of the cylinder. To date,
three such systems have been prepared by conventional synthesis and isolated as stable compound^.^^
Formally, the nanotubes generated by vaporization of carbon[@and chemical
vapor deposition (CVD)['] also belong to this class of compounds.
Our approach to the synthesis of molecular belts and tubes is
based on the ring enlargement metathesis[*] of tetradehydrodianthracene 1 (TDDA).[91Scheme 2 illustrates the molecular unit
construction system employed. a) Bianthraquinodimethanes
are obtained starting from TDDA 1 and noncyclic a l k e n e ~ ; ~ ~ ]
b) cyclophanelike, bridged bianthraquinodimethanes are available from TDDA and cyclic a l k e n e ~ ; [c)~ ]tubelike anthracene9,10-bisylidenes, which are connected by quinoid double
bonds, can be constructed by dimerizing metathe~is;'~]
d) bianthraquinodimethanes bridged by conjugated chains are
formed from [ n l a n n ~ l e n e sThe
. ~ ~double
bonds in the bridge of
these beltlike conjugated systems should again be able to undergo metathesis with TDDA. This should provide access to larger
conjugated systems composed of anthracenylidene units connected by quinoid double bonds and diene units in an alternating sequence. We now report on the synthesis of the smallest of
these beltlike, fully conjugated systems which has 20 carbon
atoms in its perimeter (n = 0, Scheme 2d).
In principle, cyclobutadiene, the smallest [nlannulene, would
be suitable for the synthesis of the conjugated, bridged bianthraquinodimethane. Cyclobutadiene can be generated in
situ["J and also undergoes [2 + 2]~ycloadditions;~' however,
[*I Prof. Dr. R. Herges, Dr. S. Kammermeier
Institut fur Organische Chemie der Technischen Universitat
Hagenring 30, D-38106 Braunschweig (Germany)
Fax: Int. code +(531)391-5266
e-mail : r.
Prof. Dr. P. G. Jones
Institut fur Anorganische und Analytische Chemie der Technischen Universit& Braunschweig
[**I This work was supported by the Deutsche Forschungsgemeinschaft and by the
Fonds der Chemischen Industrie (scholarship for S. K.).
0 WILEY-VCH Verlag GmbH, D-69451 Weinheim, 1997
Angew. Chem. I n t . Ed. Engl. 1997,36, No. 20
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iodinated, immobilization, oligonucleotide, platinated, nanotubes, carbon
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