Directed DNA metallization

Article abstract of DOI:10.1021/ja055517v

Genes of interest can be selectively metallized via the incorporation of modified triphosphates. These triphosphates bear functions that can be further derivatized with aldehyde groups via the use of click chemistry. Treatment of the aldehyde-labeled gene mixture with the Tollens reagent, followed by a development process, results in the selective metallization of the gene of interest in the presence of natural DNA strands. Copyright

Full text of DOI:10.1021/ja055517v

Published on Web 01/12/2006
Directed DNA Metallization
#
#
#
§
§
Glenn A. Burley, Johannes Gierlich, Mohammad R. Mofid, Hadar Nir, Shay Tal,
§
,#
Yoav Eichen, and Thomas Carell*
Department of Chemistry, Technion, Haifa, Israel, and Department of Chemistry, Ludwig-Maximilians UniVersity
Munich, Butenandtstr. 5-13, D-81377 Munich, Germany
Received August 12, 2005; E-mail: thomas.carell@cup.uni-muenchen.de
DNA is an outstanding material for the preparation of nano- and
microscale assemblies,1 which are believed to have potential for
the construction of nanoelectronic devices. DNA metallization
procedures were developed in order to increase the conductivity
of subsequent DNA nanostructures, thereby enabling their use as
molecular wires. The metallization process, which involves the
-3
2
a-d
chemical reduction of DNA-complexed metal salts (e.g., Ag,
4
a
4b
4c
Pd, Pt, and Cu ), results in uniformly metallized DNA
architectures. For the construction of DNA-based electronic devices,
however, a more selective protocol that allows sequence-selective
metallization of DNA is required. In this communication, we report
a simple method to direct the metallization process to specific DNA
strands or stretches of DNA. Current protocols for nonspecific silver
deposition of DNA strands involve either photoreduction (254 nm)
2f
Figure 1. Schematic depiction of the selective metallization process.
5
of Ag(I) ions complexed to DNA or chemical reduction of Ag(I)
ions by glutaraldehyde-modified DNA. Both procedures provide
2
uniformly metallized DNA. We reasoned that it may be possible
to program the metallization process to occur only on selected DNA
strands if one could specifically label DNA with aldehyde groups.
Since the construction of nanodevices requires the preparation
of long DNA double-stranded scaffolds from several to hundreds
1
of nanometers in length, we surmised that selective aldehyde
labeling could be achieved via an enzymatic method. Subsequently,
we devised the two-step protocol depicted in Figure 1. In the first
step, DNA polymerases are used to introduce acetylene reporter
groups into selected genes via the enzymatic incorporation of
5
1
-position-modified pyrimidine nucleoside triphosphates, such as
and 2 (Figure 2). The second step involved reaction of the
6
acetylene reporter groups with aldehyde azides using the Cu(I)-
7
catalyzed Huisgen 1,3-cycloaddition “click reaction”. As a con-
sequence of this derivatization process, the selected genes are now
adorned with aldehyde functions. Although the direct incorporation
of an aldehyde-modified triphosphate analogue provides a more
expeditious route toward aldehyde-modified DNA, their enzymatic
incorporation provided mixed results.
To evaluate how efficiently both compounds were accepted by
various polymerases, we used the polη and polH genes from yeast
8
and human cDNA as suitable template DNA strands. These genes
were initially isolated, then cloned into a plasmid, and subsequently
used for the PCR. Amplification of both the polη and polH genes
9
Figure 2. Depiction of molecules 1-5 used for this study.
using standard PCR conditions with a mixture of triphosphates
used. In both examples, the class B Pwo polymerase provided the
highest yields of PCR products. Enzymatic digestion of the
acetylene-decorated polη gene amplicon incorporating 2 and
subsequent HPLC and mass spectrometric analysis confirmed the
(
(
3
dATP, dCTP, dGTP, and 1 or 2) afforded, for both triphosphates
1 and 2), full-length amplicons (∼2142 bp for human polη and
18 bp for yeast polη) (Figure 3a).9 In experiments where
triphosphate 1 replaced dTTP, full-length amplicons were readily
obtained using a variety of commercially available high-fidelity
polymerases. For compound 2, however, full-length amplicons of
suitable quantity were only obtained when the Pwo polymerase was
9
complete replacement of thymidine by the nucleoside 2.
In addition, the acetylene-modified DNA strands can also be used
as template strands. PCR amplicons generated with 1 or 2 could
be used as templates for the PCR using either the four natural
triphosphates or a triphosphate mixture containing, again, 1/2 instead
of dTTP. Sequencing of the PCR products obtained with the natural
§
Technion.
#
Ludwig-Maximilians University Munich.
1398
9
J. AM. CHEM. SOC. 2006, 128, 1398-1399
10.1021/ja055517v CCC: $33.50 © 2006 American Chemical Society

C O M M U N I C A T I O N S
We finally investigated the structure of the metallized DNA using
9
AFM. Nonmodified DNA gave under our conditions no metal
deposition, in line with the gel electrophoresis results. Sugar-
modified DNA, however, exhibited Ag(0) deposition after limited
exposure to the Tollens reagent and a subsequent development
process, therefore proving that Ag(0) deposition is indeed localized
along the sugar (aldehyde)-modified DNA. Further confirmation
of the Ag(0)-templating properties of sugar-modified DNA was
demonstrated by an increase in DNA diameter as a function of the
development time.
In conclusion, we have developed an efficient and selective
method for the deposition of Ag(0) around aldehyde-modified DNA.
The modification involves incorporation of acetylene-containing
nucleotide triphosphates using DNA polymerases followed by a
click reaction that can be efficiently performed directly on a
polyacrylamide gel. Using this method, Ag(0) deposition can be
confined only to the modified DNA. The ability to insert the
acetylene labels enzymatically offers the possibility to exploit the
arsenal of molecular biological tools in order to construct conductive
DNA nanodevices. Experiments to ascertain whether these metal-
lized DNA constructs conduct electricity are currently underway.
Figure 3. PCR assays (Pwo pol.) of the polη gene from yeast (318 bp).
a) Lanes 1 and 5: DNA ladder (NEB 2-log; 0.1-10.0 kb). Lane 2: Positive
(
control (using dTTP). Lane 3: PCR using 1. Lane 4: PCR using 2. (b)
PCR assay incorporating triphosphate 2 [lane 1, 7.0 ng; lane 3, 3.5 ng DNA
loadings] and dTTP [lane 2, 7.0 ng; lane 4, 3.5 ng DNA loadings]. Gel A
corresponds to treatment with a Tollens solution followed by development,
whereas gel B corresponds to treatment with the fluorescent stain SYBR
Green II.
set of triphosphates provided the correct base sequence, reflecting
the high fidelity of the polymerases when incorporating 1 or 2.9
We then investigated the compatibility and efficiency of the click
reaction between acetylene-modified DNA prepared using 1 and 2
and the galactose azide 3, containing the aldehyde as a pro-
tected hemiacetal. Clicking onto synthetic oligodeoxyribonucleo-
tides (ODNs) comprising one or several consecutive alkyne-modi-
fied nucleosides 1 revealed incomplete conversion by MALDI-
Acknowledgment. We thank the VW Foundation, the DFG,
and the BASF AG (Ludwigshafen) for generous financial support.
J.G. thanks the Fonds of the Chemical Industry. G.A.B. thanks the
Alexander von Humboldt Foundation.
9
TOF. This is most likely attributed to the steric shielding of the
acetylene by the DNA backbone. In contrast, the yield of click
product of ODN’s comprising the more flexible alkyne with azide
3
was always quantitative even in ODN examples incorporating
Supporting Information Available: Information concerning the
preparation of 1 and 2, the PCR assays, the enzymatic digest of modified
PCR fragments, “on the gel” and ODN clicking efficiency and Ag
deposition procedures. This material is available free of charge via the
Internet at http://pubs.acs.org.
the nucleoside 2 in six consecutive positions.
We then investigated whether the Ag deposition process can be
directed to just aldehyde-modified DNA. To this end, nonacetylene-
modified DNA (318 nucleobases) and its DNA cognate prepared
with either 1 or 2 were loaded onto a 5% TBE-urea polyacrylamide
gel (Figure 3b). The click reaction was then performed directly on
the gel by agitation of the DNA-containing gel in a 1:1 MeOH:
References
(1) Seeman, N. C. Nature 2003, 421, 427-431.
H
2
O solution comprising 3, CuSO
TCEP or sodium ascorbate). Washing the gel with an aqueous
Ag(NH OH solution (Tollens reagent) and subsequent develop-
4
, and a reducing agent (e.g.,
(
2) (a) Braun, E.; Eichen, Y.; Sivan, U.; Ben-Yoseph, G. Nature 1998, 391,
7
75-778. (b) Keren, K.; Krueger, M.; Gilad, R.; Ben-Yoseph, G.; Sivan,
3
)
2
U.; Braun, E. Science 2002, 297, 72-75. (c) Keren, K.; Berman, R. S.;
Buchstab, E.; Sivan, U.; Braun, E. Science 2003, 302, 1380-1382. (d)
Deng, Z. X.; Mao, C. D. Angew. Chem., Int. Ed. 2004, 43, 4068-4070.
ment of the Ag(0) nuclei with a typical developer solution (citric
acid and formaldehyde) furnished, only in the case of the aldehyde-
modified DNA strands, yellow/brown spots on the gel, indicative
of Ag deposition (Figure 3b, Gel A). The whole process of the
Tollens reaction and development is performed in just 30 and 3
min, respectively. To prove that the unmodified DNA is indeed
present on the gel, we repeated the experiment but now treated the
gel with SYBR Green II (Figure 3b, Gel B). This time, both
aldehyde-modified and natural DNA bands were observed. The click
reaction followed by Ag deposition is highly efficient because the
(
e) Yan, H.; Park, S. H.; Finkelstein, G.; Reif, J. H.; LaBean, T. H. Science
2
003, 301, 1882-1884. (f) Keren, K.; Berman, R. S.; Braun, E. Nano
Lett. 2004, 4, 323-326.
(
3) Chworos, A.; Severcan, I.; Koyfman, A. Y.; Weinkam, P.; Oroudjev, E.;
Hansma, H. G.; Jaeger, L. Science 2004, 306, 2068-2072.
(4) (a) Richter, J.; Seidel, R.; Kirsch, R.; Mertig, M.; Pompe, W.; Plaschke,
J.; Schackert, H. K. AdV. Mater. 2000, 12, 507. (b) Mertig, M.; Ciacchi,
L. C.; Seidel, R.; Pompe, W.; De Vita, A. Nano Lett. 2002, 2, 841-844.
(c) Monson, C. F.; Woolley, A. T. Nano Lett. 2003, 3, 359-363.
(5) Berti, L.; Alessandrini, A.; Facci, P. J. Am. Chem. Soc. 2005, 127, 11216-
1217.
1
(
6) (a) Sakthivel, K.; Barbas, C. F. Angew. Chem., Int. Ed. 1998, 37, 2872-
875. (b) Held, H. A.; Benner, S. A. Nucleic Acids Res. 2002, 30, 3857-
3869. (c) Jaeger, S.; Famulok, M. Angew. Chem., Int. Ed. 2004, 43, 3337-
340.
galactose-modified 318-mer DNA was detectable by eye down to
1
2
9
.3 ng. The click reaction using 1-modified DNA is less efficient,
3
in agreement with the data from the small oligonucleotides, as
indicated by the lower detection sensitivity of only 3.5 ng. When
the aldehyde density on the DNA was significantly increased, by
the use of azide containing dendrimers 4 and 5, the detection limit
of 2-modified DNA was reduced to 0.9 and 0.5 ng, respectively,
indicative of an increase in the amount of Ag(0) deposition around
the modified DNA
(7) Rostovtsev, V. V.; Green, L. G.; Fokin, V. V.; Sharpless, K. B. Angew.
Chem., Int. Ed. 2002, 41, 2596-2598.
(
8) (a) Johnson, R. E.; Prakash, S.; Prakash, L. Science 1999, 283, 1001-
1004. (b) Hubscher, U.; Maga, G.; Spadari, S. Annu. ReV. Biochem. 2002,
71, 133-163.
(9) For further details, please consult the Supporting Information.
JA055517V
J. AM. CHEM. SOC.
9
VOL. 128, NO. 5, 2006 1399