Insulating behavior of $\lambda$-DNA on the micron scale
Y. Zhang$^1$
R. H. Austin$^1$
J. Kraeft$^2$
E. C. Cox$^2$
and N. P. Ong$^1$
We have investigated the electrical conductivity of $\lambda$-DNA using DNA covalently bonded to Au electrodes. %%@ Thiol-modified dTTP was incorporated into the `sticky' ends of bacteriophage $\lambda$-DNA using DNA polymerase. %%@ Two-probe measurements on such molecules provide a hard lower bound for the resistivity $\rho>10^6\,\mathrm{\Omega %%@ cm}$ at bias potentials up to 20 volts, in conflict with recent claims of moderate to high conductivity. By direct %%@ imaging, we show that the molecules are present after the measurements. We stress the importance of eliminating %%@ salt residues in these measurements.
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\begin{document}

\title{Insulating behavior of $\lambda$-DNA on the micron scale}
\author{Y. Zhang$^1$, R. H. Austin$^1$, J. Kraeft$^2$, E. C. Cox$^2$, and N. P. Ong$^1$}

\affiliation{$^1$Department of Physics, and $^2$Department of Molecular Biology, Princeton University, Princeton, %%@
New Jersey 08544}

\date{\today}

\begin{abstract}
We have investigated the electrical conductivity of $\lambda$-DNA using DNA covalently bonded to Au electrodes.  %%@
Thiol-modified dTTP was incorporated into the `sticky' ends of bacteriophage $\lambda$-DNA using DNA polymerase.  %%@
Two-probe measurements on such molecules provide a hard lower bound for the resistivity $\rho>10^6\,\mathrm{\Omega %%@
cm}$ at bias potentials up to 20 volts, in conflict with recent claims of moderate to high conductivity.  By direct %%@
imaging, we show that the molecules are present after the measurements.  We stress the importance of eliminating %%@
salt residues in these measurements.
\end{abstract}
\pacs{}

\maketitle
The question whether DNA is electrically conducting has generated broad interest.  The initial spurt of interest %%@
arose in photoexcitation experiments which were interpreted in terms of long-range electron transfer~\cite{barton}.  %%@
In the past few years, there have been upwards of 20 papers reporting the results of more direct electrical %%@
measurements ranging from contactless meaurements at microwave frequencies to DC measurements.  A distressingly wide %%@
range of conductivity values -- from $\rho<10^{-4}\,\mathrm {\Omega cm}$ to $\rho>10^6\,\mathrm{\Omega cm}$ -- has %%@
been reported~\cite{fink,braun,dekker,spanish,DekkerAPL}.  Proximity-induced superconductivity in DNA has also been %%@
claimed~\cite{superconducting}.  Recently, local polarization measurements by `electrostatic force microscopy' have %%@
been used to show that $\lambda$-DNA is insulating~\cite{spanish2,Sohn}.  We note, however, that the %%@
force-microscopy experiments probe conductivity at relatively weak bias potentials.  

In many of the DC measurements, contact with the metal electrodes (usually Au) was achieved by laying down the %%@
molecules directly on the electrodes. Although expedient, this approach raises several concerns.  It is very %%@
difficult to prove that the DNA molecule is in direct physical contact with the electrodes.  Even if contact is %%@
attained, the weak physical adhesion between DNA and Au may produce an insulating contact and possibly account for %%@
the wide variation in reported resistivities~\cite{sciencecomment}.  A recent experiment on %%@
octanedithiol~\cite{science} has shown that deliberate chemical bonding between organic molecules and metal %%@
electrodes is a pre-requisite for achieving reproducible conductivity results.  Thus a better approach would be to %%@
achieve direct chemical binding between the open ends of $\lambda$-DNA and Au.  The bonds should be strong enough to %%@
withstand shear forces in a flow, and should survive the measurement process.  A second concern is the shunting %%@
effect of buffer residue.  Because of its finite conductance, the buffer salts which coat the electrodes and %%@
substrate produce a spurious conductance signal.  Hence adequate salt removal is important.  We report the results %%@
of experiments performed along these lines. Our results show that $\lambda$-DNA is a good insulator up to bias %%@
potentials of 20 volts. 

Chemical binding between organic molecules and Au is usually achieved by the Au-thiol (SH) chemical %%@
bond~\cite{mirkin}.  Commercially available oligonucleotides modified to incorporate the thiol group usually have %%@
carbon-chain spacers (C3 or C6) between the thiol group and DNA~\cite{braun,DekkerAPL,glen}, which may present %%@
barriers to electron transfer.  To avoid the spacer problem, we adopted an approach in which the DNA base-pair is %%@
bound {\it directly} to gold electrodes by a Au-thiol bond.  This approach should provide the most direct %%@
conductance channel between the gold electrode and the putative electronic ``$\pi$-way'' proposed for the DNA %%@
helix~\cite{history}. 

$\lambda$-DNA is a double-stranded DNA helix comprised of 48,502 base pairs (length $\sim 16\,\mu$m).  At the %%@
extremities, there are single-stranded 12-base 5' overhangs (`sticky ends'), with the complementary sequences 
$$\rm 5'-GGG\; CGG\; CGA\; CCT,\quad 5'-AGG\; TCG\; CCG\; CCC,$$
where A,C,G,T are the nucleotides adenine, cytosine, guanine and thymine, respectively.  Our technique relies on the %%@
incorporation of T's modified to include the desired thiol group~\cite{modT,LeziusRao}.  The `sticky' ends are %%@
filled in by a standard reaction~\cite{chemistry} using the Klenow fragment of DNA polymerase and the three %%@
deoxynucleoside triphosphates dATP, dGTP, and S$^4$-dTTP (see Fig.~\ref{fig1}-A).  Because of the preponderance of %%@
modified dTTPs (and absence of dCTP) in solution, we can incorporate a significant number of modified T's at both %%@
ends of each DNA molecule~\cite{mismatch}.  To prevent the Klenow fragment from excising T's that are not %%@
Watson-Crick matched to the template, we use a mutated form of the Klenow fragment which lacks the 3'$\rightarrow$5' %%@
proof-reading activity~\cite{Klenow}.

We tested the incorporation of the nucleotides into the DNA ends by a ligation assay~\cite{ligation}.  Unmodified %%@
$\lambda$-DNA is readily ligated by T4 DNA ligase to form multimers.  In the modified $\lambda$-DNA, however, the %%@
sticky ends -- now filled in by the incorporated bases -- are blunt, and multimer formation is strongly suppressed.  %%@
The reaction products were analyzed by pulsed-field gel electrophoresis.  As shown in Fig.~\ref{fig1}-B, unmodified %%@
$\lambda$-DNA (``natural-$\lambda$'') was efficiently ligated (lane 3).  To control for the possibility that %%@
unincorporated S$^4$-dTTP inhibited ligation, $\lambda$-DNA monomers were ligated in the presence of all 3 dNTP's %%@
minus the polymerase (``control-$\lambda$''), and the ligation was also efficient (lane 6).  The majority of the %%@
thiol-modified $\lambda$-DNA (``HS-$\lambda$''), however, remained as monomers (lane 5).  This provides strong %%@
evidence that the protocol is effective in incorporating bases into the ends of $\lambda$-DNA.

% FIG1 chemistry
\begin{figure}[h] %FIG 1
\centerline{\epsfig{figure=chemistry.eps,height=5in,width=3.3in,clip=0}}
\caption {\label{fig1} (A) The schematic of the incorporation of deoxynucleoside triphosphates into $\lambda$-DNA %%@
`sticky' ends using the Klenow fragment (3'$\rightarrow$5' exo$^-$); (B) Pulsed-field gel (PFG) electrophoresis of %%@
thiol-modified and natural $\lambda$-DNA before and after ligation~\cite{PFG}.  Lane 1: $\lambda$-DNA PFG marker %%@
(New England Biolabs); 2: unmodified ``natural-$\lambda$'' monomers; 3: ligated ``natural-$\lambda$'' multimers; 4: %%@
thiol-modified $\lambda$-DNA (``HS-$\lambda$'') monomers; 5: ``HS-$\lambda$'' after ligation, with no significant %%@
multimer formation; 6: ``control-$\lambda$'' after ligation, proving that the presence of unincorporated S$^4$-dTTP
and other dNTPs does not inhibit the ligation reaction.}
\end{figure} 

Using standard photolithography, we constructed Au electrodes on a quartz substrate in parallel strips, 4 $\mu$m %%@
wide and 5 mm long, and separated by 4 or 8 $\mu$m.  The Au surfaces were rigorously cleaned~\cite{cleangold} before %%@
depositing the modified DNA.  At several stages during these experiments, it was important to observe the molecules %%@
in an optical microscope.  To image the thiol-modified DNA molecules, we stained them with the fluorescent %%@
intercalating dye TOTO1 (Molecular Probes), and then loaded them on the chip.  After a 20-min. incubation period, %%@
many of the molecules were observed to be attached to the electrodes at one end.  The unattached molecules were %%@
carefully rinsed in $1\times$TE~\cite{TE}.  The chip was then covered with a clean coverslip, and a flow of the %%@
buffer solution was applied perpendicular to the electrodes.  We observed that DNA molecules anchored at one end %%@
were stretched by the buffer flow to bridge the space between the electrodes.  Many of these molecules subsequently %%@
attached to the second electrode by their free end.  After this occurs, the flow may be repeatedly reversed to %%@
demonstrate that the anchored DNA molecules bow out with the flow while their ends remain anchored (Panels A and B %%@
of Fig.~\ref{fig2}.  See video in Ref.~\cite{video}).  This is direct evidence that chemical binding between the %%@
ends to Au is much stronger than physical adhesion of the rest of the molecule to either quartz or Au.  (For the %%@
specific DNA samples used in the resistivity measurements, we carried out the dye-staining step {\em after} the %%@
measurements to avoid inadvertent damage from dye intercalation.)

% FIG2  NewInBuffer
\begin{figure}[h]   %FIG 2
\centerline {\epsfig{figure=NewInBuffer.eps,height=2.64in,width=3.25in,clip=0}} 
\caption {\label{fig2}  Images of HS-$\lambda$ DNA on quartz chips with Au electrodes. The DNA molecules were %%@
pre-stained with TOTO1.  Observations were through a Nikon Eclipse TE300 inverted microscope equipped with a %%@
60$\times$ oil-immersion lens (N.A.=1.4), and excited by a collimated Ar:Kr ion laser at 488 nm.  Fluorescence %%@
images were collected at 533 nm by an intensified CCD camera (RoperScientific, Princeton Instruments) and digitally %%@
enhanced. Au electrodes appear as vertical stripes.  Arrows represent the flow direction. Panel A-1 \& 2: Molecule %%@
A, anchored at both ends, and flexing with the flow.  Panel B-1 \& 2: Both ends of molecule B were attached on the %%@
same electrode, with the mid-segment moving freely in the buffer, showing that the attachment was specific to the %%@
thiol-modified ends. Panel C: Many HS-$\lambda$ DNA molecules spanning two electrodes.  The image was taken after
the Mg$^{2+}$ and NH$_4$Ac rinsing. Scale bar applies to all panels~\cite{video}.}
\end{figure}

%%%%%%%%%%%%
% 

As discussed above, a crucial step in the experiment was the removal of buffer.  In preliminary experiments, we %%@
repeatedly observed a finite, semiconductor-like, history-dependent conductance after loading DNA solution and %%@
removing the buffer solution.  The inset of Fig.~\ref{fig3} is a representative $I$-$V$ curve of the buffer salt %%@
residue, 1$\times$TE.  Such spurious signals are of particular concern when DNA is laid down on electrodes in a %%@
thick bundle, because the salts trapped between the DNA molecules can form conduction paths.  The spurious %%@
background vanished after we adopted the following procedure.  Chips containing bridging DNA molecules were %%@
carefully rinsed in 5 mM ammonium acetate (NH$_4$Ac, pH 6.6), a volatile buffer that can be completely removed in %%@
high vacuum.  A drawback of this rinsing is that a large fraction of the anchored DNA molecules are cut after %%@
rinsing and drying.  However, rinsing in 10 mM MgSO$_4$/40 mM Tris-HCl (pH 8) {\em before} the NH$_4$Ac rinsing %%@
introduces Mg$^{2+}$ ions which coat the quartz surface with weak positive charges~\cite{DekkerAPL}.  As the %%@
negatively charged DNA molecules stick to the substrate by electrostatic interaction, damage due to NH$_4$Ac
rinsing is minimized.  Panel C of Fig.~\ref{fig2} shows a typical image of anchored DNA after Mg$^{2+}$ and NH$_4$Ac %%@
rinsing.  [Rinsing with the MgSO$_4$ solution also led to binding of unmodified $\lambda$-DNA.  However, the yield %%@
of anchored molecules was much smaller.]

To perform the electrical measurements, unstained thiol-modified $\lambda$-DNA was attached to the Au electrodes as %%@
described above.  After the final NH$_4$Ac rinsing, the chip was dried in the dark to avoid possible photon-induced %%@
damage. Two-probe $I$-$V$ measurements were performed in moderately high vacuum ($<10^{-7}\mathrm{Torr}$).  

A typical room-temperature $I$-$V$ curve, measured on $\lambda$-DNA spanning electrodes 4 $\mu$m apart, is shown in %%@
the main panel of Fig.\ref{fig3}.  The voltage was swept between $\pm$20 V.  A linear fit to the data in %%@
Fig.~\ref{fig3} yields $dI/dV=(-3 \pm 9)\times 10^{-14}\mathrm{S}$.  Using a cross-section of $\sim$3 nm$^2$ per %%@
molecule, and the estimated number of bridging molecules ($\sim$1000), we obtain the bound on the resistivity of %%@
$\rho>10^6\, \mathrm{\Omega cm}$ in electric fields $E$ up to $\sim 10^4\mathrm{V/cm}$.  Measurements performed on %%@
several chips yielded consistent results.  No current was detected within the noise level of our %%@
measurement($\sim\pm$10 pA), despite sustained and deliberate efforts to improve electrical contacts between the %%@
base pair stack of $\lambda$-DNA and Au.

Immediately after the measurements, a buffer solution with an appropriate amount of TOTO1 dye dissolved in %%@
1$\times$TE was loaded on the chip.  By direct optical microscopy inspection of the post-stained DNA, we confirmed %%@
that there were $\sim$1000 DNA molecules bridging the electrodes, and thus the measurements did not destroy the DNA.

As a final check that the observed images are those of $\lambda$-DNA, we introduce DNase to digest the %%@
molecules~\cite{DNase}.  Complete deletion of all fluorescent DNA molecules was observed.  These tests leave very %%@
little room for doubt that a large number of {\em intact} DNA molecules were chemically bound to the electrodes %%@
during the electrical measurements~\cite{denature}.

% FIG 3  IVmsmt
\begin{figure}[h]  %FIG 3
\centerline {\epsfig{figure=IVmsmt.eps,height=4.07in,width=3.14in,clip=0}} 
\caption {\label{fig3} The two-probe current vs. voltage ($I$-$V$) curve for a sample of $\lambda$-DNA bridging 2 %%@
parallel Au electrodes separated by 4 $\mu$m (the sample comprises $\sim$1000 molecules).  The DNA was rinsed with %%@
NH$_4$Ac before the measurement to remove the buffer salt residue.  The dashed line is a linear fit to the data. The %%@
inset shows the two-probe $I$-$V$ curve for a test chip containing 1$\times$TE buffer solution (without DNA).  The %%@
chip was dried in vacuum but not subject to NH$_4$Ac rinsing.  The observed conductance is entirely from trace TE %%@
salt residue. Both measurements were done in vacuum ($<10^{-7}$ Torr) at 295 K. Open-circuit impedance between any %%@
two electrodes was always $\gg$10 T$\mathrm{\Omega}$.}
\end{figure}

The bound $\rho > 10^6\, \Omega$cm in our experiment and the large bias potential applied (20 V) is at odds with %%@
many recent reports of moderately high conductivity.  In some DC experiments, the DNA molecules formed bundles or %%@
networks between the microfabricated electrodes~\cite{ZnDNA,DNAnetwork,dyeDNA}.  As noted above, high conductance %%@
may arise from residual salts trapped between the DNA strands.  Contamination from other sources (C or Re) may be a %%@
problem as well in the experiment on proximity-induced superconductivity in $\lambda$-DNA~\cite{superconducting}. 
Microwave absorption experiments have been used to infer that $\rho\simeq  1\;\mathrm{\Omega cm}$ at 295 K in %%@
$\lambda$-DNA~\cite{gruner}.  The high microwave conductivity, 10$^6$ times larger than our bound, is very difficult %%@
to reconcile with our data.  If $\lambda$-DNA had such a high uniform conductivity, all the applied potential should %%@
fall across the contacts ($\sim$2-3 nm) to produce an $E$-field $\sim 10^8$ V/cm, high enough to produce a large %%@
tunneling current, if not breakdown of the contact barrier altogether.  This is not observed.  Possibly, the %%@
microwave is detecting very short dissipative regions embedded in the insulating molecule.


Two groups recently used electrostatic force microscopy to probe the electrostatic polarization of %%@
DNA~\cite{spanish2,Sohn}.  Our results are consistent with their conclusion that DNA is insulating.  However, the %%@
electrostatic force microscopy technique probes conductivity in the limit of weak bias potentials, so it does not %%@
rule out a transition to moderately large conductivity above a bias threshold of several volts (as reported in some %%@
experiments~\cite{dekker,Watanabe}).  The present experiments show that insulating behavior extends to bias %%@
potentials as high as 20 volts.

We are grateful to P. M. Chaikin for invaluable suggestions, and acknowledge fruitful discussions with Shirley S. %%@
Chan, J. Tegenfeldt, P. Silberzan, Christelle Prinz, S. Park and R. Huang.  This research is supported by a U.S. %%@
National Science Foundation MRSEC grant (DMR 98-09483) and by the National Inst. Health (NIH HG01506).


\begin{references}

\bibitem{barton} C. J. Murphy {\it et al.}, Science {\bf 262}, 1025 (1993); S. O. Kelley 
and J. K. Barton, Science {\bf 283}, 375 (1999).

\bibitem{fink} H.-W. Fink and C. Schonenberger, Nature {\bf 398}, 407 (1999).

\bibitem{braun} E. Braun {\it et al.}, Nature {\bf 391}, 775 (1998).

\bibitem{dekker} D. Porath, {\it et al.}, Nature {\bf 403}, 635 (2000).

\bibitem{spanish} P. J. de Pablo {\it et al.}, Phys. Rev. Lett. {\bf 85}, 4992 (2000).

\bibitem{DekkerAPL} A. J. Storm {\it et al.}, Appl. Phys. Lett. {\bf 79}, 3881 (2001).

\bibitem{superconducting} A. Yu. Kasumov {\it et al.}, Science {\bf 291}, 280 (2001).

\bibitem{spanish2} C. G\'omez-Navarro {\it et al.}, Proc. Natl. Acad. Sci. USA {\bf 99}, 8484 (2002).

\bibitem{Sohn} M. Bockrath {\it et al.}, Nano Lett. {\bf 2}, 187 (2002).

\bibitem{sciencecomment} K. W. Hipps, Science {\bf 294}, 536 (2001).

\bibitem{science} X. D. Cui {\it et al.}, Science {\bf 294}, 571 (2001).

\bibitem{mirkin} C. A. Mirkin {\it et al.}, Nature {\bf 382}, 607 (1996).

\bibitem{glen} User Guide to DNA Modification, Glen Research, 1999. See the refs. therein.

\bibitem{history} D. D. Eley and D. I. Spivey, Trans. Faraday Soc. {\bf 58}, 411 (1962).

\bibitem{modT} In the modified nucleotide 4-thiothymidine-5'-triphosphate (S$^4$-dTTP, Trilink Biotechnologies), a
sulfur atom is covalently bound to the C4 site of the thymine molecule.  Its close proximity to the hydrogen ion at
the N3 site results in a resonant state that forms the desired thiol group.  

\bibitem{LeziusRao} A. G. Lezius and K. H. Scheit, European J. Biochem. {\bf 3}, 85 
(1967); T. V. S. Rao {\it et al.}, Bioorg. Med. Chem. Lett. {\bf 10}, 907 (2000).

\bibitem{chemistry} The incorporation reaction was 100 $\mu$g/ml $\lambda$-DNA, 0.1 mM dATP, 0.1 mM dGTP and 0.2 mM
S$^4$-dTTP in 1$\times$ EcoPol buffer(10 mM Tris-HCl, 5 mM MgCl$_2$ and 7.5 mM dithiothreitol (DTT); pH7.5 @
25$^\circ$C; New England Biolabs), for a total volume of 100 $\mu$l. Five units of Klenow fragment
(3'$\rightarrow$5' exo$^-$) were added. The mixture was incubated at 37 $^\circ$C for 30 min. To stop the reaction,
1$\mu$l of 0.5 M EDTA was added, and the mixture heated at 75$^\circ$C for 20 min.  To exchange the buffer, the
product was dialyzed against 30 ml 0.1$\times$TE at room temperature for 45 min across a 50 nm Millipore membrane.

\bibitem{mismatch} One 5' overhang of the $\lambda$-DNA ends with an A, which is Watson-Crick matched to S$^4$-T. 
The other overhang ends with G's, which are matched to C's.  While sulfur-modified dCTP is available, the sulphur
is remote from H atoms and the resonant thiol group can not form.  To maximize the probability of modified T's at
both ends, we excluded dCTP altogether from the reaction, so that S$^4$-T:G pairing could occur in place of C:G. 
The mutated Klenow fragment used here is incapable of detecting the mis-matches.


\bibitem{Klenow} The Klenow fragment is a large fragment of DNA polymerase I lacking the error-correcting
5'$\rightarrow$3' exonuclease activity (this function damages the $\lambda$-DNA `sticky' ends).  Here, we employed
a mutated form of the Klenow fragment (3'$\rightarrow$5' exo$^-$, New England Biolabs) in which the proof-reading
3'$\rightarrow$5' exonuclease activity is deleted.  S$^4$-dTTP binds more weakly to A than unmodified
dTTP~\cite{LeziusRao}, and hence the proof-reading activity could possibly remove incorporated S$^4$-dTTP.


\bibitem{ligation} The reaction was carried out in 1$\times$ ligation buffer (50 mM Tris-HCl, 10 mM MgCl$_2$, 10 mM
DTT, 1 mM ATP and 25 $\mu$g/ml BSA; pH 7.5@25 $^\circ$C) at a $\lambda$-DNA concentration of 50 $\mu$g/ml. 400
units of T4 DNA ligase (New England Biolabs) was added to the reaction volume of 20 $\mu$l, and incubated at room
temperature for 5 h.

\bibitem{PFG} 1\% agarose gel in 0.5$\times$TBE buffer; $E$=6 V/cm; 14 $^\circ$C; 16 hours; switch time ramped from
0.1 sec to 40 sec.

\bibitem{cleangold} To clean the Au surface, freshly prepared chips were soaked in fuming HNO$_3$ for 30 min, and
further cleaned in a mixture of 18 M$\mathrm{\Omega cm}$ deionized water, ammonium hydroxide and hydrogen peroxide
(5:1:1 by volume, 70 $^\circ$C) for 20 min. The chips were thoroughly rinsed in deionized and distilled water (DD
H$_2$O), and kept in Ar-flushed DD H$_2$O for no more than 30 min before loading DNA samples. The Au surface was
never allowed to dry during the process.

\bibitem{TE} 1$\times$TE buffer contains 10 mM Tris (tris(hydroxymethyl)-aminomethane) and 1 mM EDTA (ethylene
diamine tetra acetic acid), pH 8.0 .

\bibitem{video} See videos at http://suiling.princeton.edu/research/ DNAconductance/DNAconduction.html. 

\bibitem{denature} The strong fluorescence observed after the resistivity measurements strongly argues against %%@
denaturation of DNA into single strands when affixed to quartz in vacuum.  TOTO1 displays strong fluorescence only %%@
when bound to double-strand DNA.  Moreover, AFM images of B-DNA in vacuum have recently been obtained by Takayuki %%@
Uchihashi {\em et al.}, Langmuir {\bf 16}, 1349 (2000); {\em ibid} Jpn. J. Appl. Phys. {\bf 39}, L887 (2000).


\bibitem{ZnDNA}A. Rakitin {\it et al.}, Phys. Rev. Lett. {\bf 86}, 3670 (2001).

\bibitem{DNAnetwork} L. Cai, H. Tabata, and T. Kawai, Appl. Phys. Lett. {\bf 77}, 3105 
(2000).

\bibitem{dyeDNA} J. Gu {\it et al.}, Appl. Phys. Lett. {\bf 80}, 688 (2001).

\bibitem{DNase} To digest the DNA, we used 10 units of DNase I (RNase-free, Roche Molecular Biochemicals) dissolved
in 10 $\mu$l reaction buffer (40 mM Tris-HCl, 10 mM MgSO$_4$, and 1mM CaCl$_2$).

\bibitem{gruner} P. Tran, B. Alavi, and G. Gruner, Phys. Rev. Lett. {\bf 85}, 1564 
(2000).

\bibitem{Watanabe} H. Watanabe {\it et al.}, Appl. Phys. Lett. {\bf 79}, 2462 (2001).

\end{references}

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physics0205016.extracted_bib
\bibitem{barton} C. J. Murphy {\it et al.}, Science {\bf 262}, 1025 (1993); S. O. Kelley 
and J. K. Barton, Science {\bf 283}, 375 (1999).


\bibitem{fink} H.-W. Fink and C. Schonenberger, Nature {\bf 398}, 407 (1999).


\bibitem{braun} E. Braun {\it et al.}, Nature {\bf 391}, 775 (1998).


\bibitem{dekker} D. Porath, {\it et al.}, Nature {\bf 403}, 635 (2000).


\bibitem{spanish} P. J. de Pablo {\it et al.}, Phys. Rev. Lett. {\bf 85}, 4992 (2000).


\bibitem{DekkerAPL} A. J. Storm {\it et al.}, Appl. Phys. Lett. {\bf 79}, 3881 (2001).


\bibitem{superconducting} A. Yu. Kasumov {\it et al.}, Science {\bf 291}, 280 (2001).


\bibitem{spanish2} C. G\'omez-Navarro {\it et al.}, Proc. Natl. Acad. Sci. USA {\bf 99}, 8484 (2002).


\bibitem{Sohn} M. Bockrath {\it et al.}, Nano Lett. {\bf 2}, 187 (2002).


\bibitem{sciencecomment} K. W. Hipps, Science {\bf 294}, 536 (2001).


\bibitem{science} X. D. Cui {\it et al.}, Science {\bf 294}, 571 (2001).


\bibitem{mirkin} C. A. Mirkin {\it et al.}, Nature {\bf 382}, 607 (1996).


\bibitem{glen} User Guide to DNA Modification, Glen Research, 1999. See the refs. therein.


\bibitem{history} D. D. Eley and D. I. Spivey, Trans. Faraday Soc. {\bf 58}, 411 (1962).


\bibitem{modT} In the modified nucleotide 4-thiothymidine-5'-triphosphate (S$^4$-dTTP, Trilink Biotechnologies), a
sulfur atom is covalently bound to the C4 site of the thymine molecule.  Its close proximity to the hydrogen ion at
the N3 site results in a resonant state that forms the desired thiol group.  


\bibitem{LeziusRao} A. G. Lezius and K. H. Scheit, European J. Biochem. {\bf 3}, 85 
(1967); T. V. S. Rao {\it et al.}, Bioorg. Med. Chem. Lett. {\bf 10}, 907 (2000).


\bibitem{chemistry} The incorporation reaction was 100 $\mu$g/ml $\lambda$-DNA, 0.1 mM dATP, 0.1 mM dGTP and 0.2 mM
S$^4$-dTTP in 1$\times$ EcoPol buffer(10 mM Tris-HCl, 5 mM MgCl$_2$ and 7.5 mM dithiothreitol (DTT); pH7.5 @
25$^\circ$C; New England Biolabs), for a total volume of 100 $\mu$l. Five units of Klenow fragment
(3'$\rightarrow$5' exo$^-$) were added. The mixture was incubated at 37 $^\circ$C for 30 min. To stop the reaction,
1$\mu$l of 0.5 M EDTA was added, and the mixture heated at 75$^\circ$C for 20 min.  To exchange the buffer, the
product was dialyzed against 30 ml 0.1$\times$TE at room temperature for 45 min across a 50 nm Millipore membrane.


\bibitem{mismatch} One 5' overhang of the $\lambda$-DNA ends with an A, which is Watson-Crick matched to S$^4$-T. 
The other overhang ends with G's, which are matched to C's.  While sulfur-modified dCTP is available, the sulphur
is remote from H atoms and the resonant thiol group can not form.  To maximize the probability of modified T's at
both ends, we excluded dCTP altogether from the reaction, so that S$^4$-T:G pairing could occur in place of C:G. 
The mutated Klenow fragment used here is incapable of detecting the mis-matches.



\bibitem{Klenow} The Klenow fragment is a large fragment of DNA polymerase I lacking the error-correcting
5'$\rightarrow$3' exonuclease activity (this function damages the $\lambda$-DNA `sticky' ends).  Here, we employed
a mutated form of the Klenow fragment (3'$\rightarrow$5' exo$^-$, New England Biolabs) in which the proof-reading
3'$\rightarrow$5' exonuclease activity is deleted.  S$^4$-dTTP binds more weakly to A than unmodified
dTTP~\cite{LeziusRao}, and hence the proof-reading activity could possibly remove incorporated S$^4$-dTTP.



\bibitem{ligation} The reaction was carried out in 1$\times$ ligation buffer (50 mM Tris-HCl, 10 mM MgCl$_2$, 10 mM
DTT, 1 mM ATP and 25 $\mu$g/ml BSA; pH 7.5@25 $^\circ$C) at a $\lambda$-DNA concentration of 50 $\mu$g/ml. 400
units of T4 DNA ligase (New England Biolabs) was added to the reaction volume of 20 $\mu$l, and incubated at room
temperature for 5 h.


\bibitem{PFG} 1\% agarose gel in 0.5$\times$TBE buffer; $E$=6 V/cm; 14 $^\circ$C; 16 hours; switch time ramped from
0.1 sec to 40 sec.


\bibitem{cleangold} To clean the Au surface, freshly prepared chips were soaked in fuming HNO$_3$ for 30 min, and
further cleaned in a mixture of 18 M$\mathrm{\Omega cm}$ deionized water, ammonium hydroxide and hydrogen peroxide
(5:1:1 by volume, 70 $^\circ$C) for 20 min. The chips were thoroughly rinsed in deionized and distilled water (DD
H$_2$O), and kept in Ar-flushed DD H$_2$O for no more than 30 min before loading DNA samples. The Au surface was
never allowed to dry during the process.


\bibitem{TE} 1$\times$TE buffer contains 10 mM Tris (tris(hydroxymethyl)-aminomethane) and 1 mM EDTA (ethylene
diamine tetra acetic acid), pH 8.0 .


\bibitem{video} See videos at http://suiling.princeton.edu/research/ DNAconductance/DNAconduction.html. 


\bibitem{denature} The strong fluorescence observed after the resistivity measurements strongly argues against %%@
denaturation of DNA into single strands when affixed to quartz in vacuum.  TOTO1 displays strong fluorescence only %%@
when bound to double-strand DNA.  Moreover, AFM images of B-DNA in vacuum have recently been obtained by Takayuki %%@
Uchihashi {\em et al.}, Langmuir {\bf 16}, 1349 (2000); {\em ibid} Jpn. J. Appl. Phys. {\bf 39}, L887 (2000).



\bibitem{ZnDNA}A. Rakitin {\it et al.}, Phys. Rev. Lett. {\bf 86}, 3670 (2001).


\bibitem{DNAnetwork} L. Cai, H. Tabata, and T. Kawai, Appl. Phys. Lett. {\bf 77}, 3105 
(2000).


\bibitem{dyeDNA} J. Gu {\it et al.}, Appl. Phys. Lett. {\bf 80}, 688 (2001).


\bibitem{DNase} To digest the DNA, we used 10 units of DNase I (RNase-free, Roche Molecular Biochemicals) dissolved
in 10 $\mu$l reaction buffer (40 mM Tris-HCl, 10 mM MgSO$_4$, and 1mM CaCl$_2$).


\bibitem{gruner} P. Tran, B. Alavi, and G. Gruner, Phys. Rev. Lett. {\bf 85}, 1564 
(2000).


\bibitem{Watanabe} H. Watanabe {\it et al.}, Appl. Phys. Lett. {\bf 79}, 2462 (2001).

