Complementary Base-Pair-Facilitated Electron Tunneling for Electrically Pinpointing Complementary Nucleobases

Complementary base-pair-facilitated electron tunneling for electrically pinpointing complementary nucleobases

Takahito Ohshiro and Yoshio Umezawa*

Department of Chemistry, School of Science, University of Tokyo, and Japan Science and Technology Agency, Hongo, Bunkyo-Ku, Tokyo 133-0033, Japan

Edited by Jacqueline K. Barton, California Institute of Technology, Pasadena, CA, and approved November 17, 2005 (received for review July 19, 2005) Molecular tips in scanning tunneling microscopy can directly detect Results and Discussion intermolecular electron tunneling between sample and tip mole- The nucleobase molecular tips were prepared by chemical cules and reveal the tunneling facilitation through chemical inter- modification of underlying metal tips with thiol derivatives of actions that provide overlap of respective electronic wave func- adenine, guanine, cytosine, and uracil (see Materials and Meth- tions, that is, hydrogen-bond, metal-coordination-bond, and ods) (for their chemical structures, see Fig. 1b; for their prepa- charge-transfer interactions. Nucleobase molecular tips were pre- rations, see Supporting Text, which is published as supporting pared by chemical modification of underlying metal tips with thiol information on the PNAS web site), and the outmost single derivatives of adenine, guanine, cytosine, and uracil and the nucleobase adsorbate probes intermolecular electron tunneling outmost single nucleobase adsorbate probes intermolecular elec- to or from a sample nucleobase molecule. Importantly, the tron tunneling to or from a sample nucleobase molecule. We found tunneling current increases when sample and tip molecules form that the electron tunneling between a sample nucleobase and its a chemical interaction that provides overlap of electronic wave complementary nucleobase molecular tip was much facilitated functions between them. The current increase is ascribed to the compared with its noncomplementary counterpart. The comple- facilitated electron tunneling through the overlapped electronic mentary nucleobase tip was thereby capable of electrically pin- wave functions. Electron tunneling observed here occurs without pointing each nucleobase. Chemically selective imaging using mo- any net chemical oxidation͞reduction of the involved bases. Fig. lecular tips may be coined ‘‘intermolecular tunneling microscopy’’ 2 a–c shows typical STM images of guanine SAMs observed with as its principle goes and is of general significance for novel complementary cytosine tips, noncomplementary adenine, and molecular imaging of chemical identities at the membrane and unmodified tips, respectively (see Materials and Methods). Cross- solid surfaces. sectional profiles of the images are shown in Fig. 2d, which represents the extent of electron tunneling between the tip and DNA ͉ nanobioscience ͉ scanning tunneling microscopy nucleobase. The complementary cytosine tip exhibited the most facilitated electron tunneling and therefore the brightest gua- lectron transfer through DNA double strands has attracted nine images among the three tips. Similarly, for adenine, cyto- Emuch interest (1–4) since Barton and colleagues (5) reported sine, and uracil, their complementary nucleobase tips gave the it in the 1990s. The processes of DNA-mediated electron transfer brightest images of their counterparts, the results of which are have been explored by spectroscopic methods for detecting shown in Fig. 2e together with those using irrelevant tips for photo-induced electron transfer through the DNA strands that validation. We have differentiated the complementary nucleo- are labeled with redox-active probes through intercalation bases from the noncomplementary ones by the tip heights for the and͞or covalent linkages (6, 7). These experiments have revealed sample nucleobases in absolute terms. The height is a quanti- that a DNA strand is capable of mediating electron transfer tative measure of the current, because the tunneling current I is through the DNA base stacking in the strand (intrastrand related to the tip height h by the relation as I ϰ exp(Ϫ2kh), where pathway) (8–13). On the other hand, the contribution of the k ϭបϪ1(2m␸)1/2 and ␸ is the work function of the sample (24). electron transfer through the complementary base pair (inter- The tip height h is usually recorded rather than the current I for strand pathway) to the overall electron transfer in DNA has been the instrumental convenience, keeping the current I constant. investigated by several researchers (14, 15). Here, we report on For example, with the cytosine tips, the heights of the tip were electron tunneling through the complementary base pair with found to be 197 Ϯ 23 pm for the complementary guanines (Fig. nucleobase molecular tips for selectively discriminating each of 2e, black columns) and 102 Ϯ 5 pm, 98 Ϯ 9 pm, and 99 Ϯ 7pm the complementary nucleobases from the other nucleobases for the noncomplementary adenines, uracils, and cytosines, (Fig. 1a). The molecular tips are prepared by chemical modifi- respectively (Fig. 2e, yellow columns). These heights quantita- cation of underlying metal tips typically with self-assembled tively represent the tunneling currents flowing within the base monolayers (SAMs) of thiols and the outermost single adsorbate pairs. On the contrary, with unmodified tips, or with gold tips probes electron tunneling to or from a sample molecule. Im- modified with 2-mercaptobenzimidazole (MB) and thiophenol portantly, the tunneling current increases when sample and tip (TP) (chemical structures, Fig. 1b), which have a pyrimidine- and molecules form chemical interactions that provide overlap of pyridine-like structure, respectively, but no particular functional their electron wave functions, that is, hydrogen-bond interac- groups for hydrogen-bond formation with nucleobases, selective tions (16–22), metal-coordination-bond interactions (20), and facilitation of electron tunneling was not detected for any charge-transfer interactions (23). We have thus far demonstrated nucleobases (Fig. 2e, yellow columns), as shown in an STM image that this phenomenon can be used for selective observation of chemical species to overcome poor chemical selectivity in con- ventional scanning tunneling microscopy (STM). We herein Conflict of interest statement: No conflicts declared. found that the electron tunneling between a sample nucleobase This paper was submitted directly (Track II) to the PNAS office. and its complementary nucleobase tip was much facilitated Abbreviations: STM, scanning tunneling microscopy; SAM, self-assembled monolayer; PNA, compared with its noncomplementary counterpart. The com- peptide nucleic acid; MB, 2-mercaptobenzimidazole; TP, thiophenol. plementary nucleobase tip was thereby capable of electrically *To whom correspondence should be addressed. E-mail: [email protected]. pinpointing each nucleobase. © 2005 by The National Academy of Sciences of the USA

10–14 ͉ PNAS ͉ January 3, 2006 ͉ vol. 103 ͉ no. 1 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0506130103 Downloaded by guest on September 28, 2021 not show selective large facilitation of electron tunneling (Fig. 2e, yellow column), confirming that the observed difference is caused solely by the hydrogen bonds of complementary base pairs between bases on a tip and substrate. We have earlier reported on the use of hydrogen bond-based molecular tips for selective STM imaging of hydrogen-bond acceptor or donor molecules and functional groups and on the use of other chemical interaction-based molecular tips, metal coordination bond-based molecular tips for selective STM imaging of metal species in metalloporphyrins (20), and charge- transfer interaction-based molecular tips for that of electron-rich porphyrin rings (23). Upon tailor-making the molecular tips with differing extents of hydrogen-bond acidity or basicity, we have succeeded in selectively pinpointing particular functional groups in sample molecules, including hydroxy, carboxy, carboxylate, ether oxygens and their orientations, and a free-base porphyrin center (16–22). We herein added another example of hydrogen- bond-facilitated electron tunneling, i.e., complementary base- pair-facilitated electron tunneling (Fig. 1a). For example, before the cytosine tip was placed on a guanine base, the guanine base did not possess any greater electron density compared with other bases, but instead a greater electron density was induced along the hydrogen-bonding plane upon placing the cytosine tip on the guanine base. This induced increase in electron density trans- lates into a greater electronic coupling between the two bases and thus an increase in the tunneling current between them. As Fig. 1. A nucleobase tip pinpoints its complementary nucleobase based on a result, nucleobase tips gave large extents of electron tunneling CHEMISTRY base-pair-facilitated electron tunneling. (a) Formation of the complementary currents only for its complementary bases. The direction of base pairs between the nucleobase tip and the sample nucleobases leads to electron flow between bases on a tip and a substrate did not greatly facilitate electron tunneling in STM. Nucleobase tips can thus pinpoint affect the extent of electron tunneling through the same com- the corresponding complementary nucleobases. (b) The chemical structures bination of the material (Fig. 2e): for instance, cytosine and for thiol derivatives of adenine (1), guanine (2), cytosine (3), uracil (4), MB (5), guanine tips gave the same extent of electron tunneling to their and TP (6) are shown. counterpart complementary bases. Therefore, the extent of overlap of electron wave functions of the base pairs solely plays (Fig. 5, which is published as supporting information on the the requisite role. PNAS web site). Taken together, it is concluded that the The formations of the specific hydrogen bonds through com- complementary combinations of the tip and sample base pairs plementary base pairs require that coplanar configurations of facilitated the largest electron tunneling compared with the the bases be achieved on the tip and surface. Although the plane noncomplementary combinations, and particular nucleobases of bases may likely be oriented randomly in mixed monolayers (Fig. 6) and orderly in pure monolayers (Fig. 2c), the nucleobase were thus discriminated from other nucleobases in STM images tips gave the selective large facilitation of electron tunneling for by using the complementary nucleobase tips. its complementary bases in both the pure and mixed monolayers, In the mixed nucleobase SAMs (see Materials and Methods), (Figs. 2a and 3 a–d), indicating that the base–base coplanar nucleobase tips were capable of pinpointing complementary orientation was in fact achieved, and thus the specific hydrogen nucleobase images in the presence of other nucleobases. Fig. 3a ͞ bonds between the complementary base pairs were formed. The shows a typical STM image of an adenine guanine-mixed SAM base–base coplanarity probably is attained by the rotation of a observed with a cytosine tip. As shown in Figs. 3a Inset and 2f, carbon–sulfur bond in the thio base on a tip, which is well known the cytosine tip gave a high- and low-contrast image for the even in the close-packed structure of thiolate SAMs (25). complementary guanine and noncomplementary adenine, re- Therefore, the complementary bases in the SAM were exclu- spectively. The number of the high-contrast guanine images sively differentiated. Although other hydrogen bonds, such as increased in proportion to the molar ratio of [guanine] to the Hoogsteen and G:U Wobble base pairs, could be formed ϩ [guanine adenine] in the sample-mixed solution for the SAM between bases on the tip and sample substrate, they were found (compare Fig. 3 b–d), the results of which are shown in Fig. 3e. to give only a small tunneling current similar to those of Similarly, uracil tips were capable of selectively pinpointing the noncomplementary base pairs (Fig. 2 e–g) and did not thereby complementary adenine images in the presence of noncomple- interfere with the chemical differentiation based on complementary guanines in the SAM (Fig. 2g), and the number of the mentary base pairs. A branch of supramolecular chemistry to use high-contrast adenine images also increased in proportion to the paradigm of individual nucleobases exists. The thermody- molar ratio of [adenine] to [guanine ϩ adenine] in the sample namic stabilities and related characteristics of these nucleobases solutions as shown in Fig. 3e. These results indicate that in the have been extensively studied, and many researchers have re- mixed nucleobase SAMs the large electron tunneling through ported Watson-Crick-type specific interactions between mono- the complementary base-pairing allows us to pinpoint a partic- meric nucleobases (26). These reports substantiate the profound ular nucleobase in the presence of other nucleobases. On the specificity observed in the present study, including strong pref- other hand, with unmodified tips, the four bases were observed erence for Watson-Crick binding and rejection of Hoogsteen and as identical images even in the mixed SAM (Fig. 6, which is Wobble base-pairing. published as supporting information on the PNAS web site). This Hole and electron transfer in a DNA strand occur via two result indicates that the chemical differentiation of complemen- pathways, along the DNA strand (intrastrand pathway) and tary bases with the nucleobase tips was not caused by the through the base pairs (interstrand pathway). In the interstrand difference in heights of the four bases. Also, MB and TP tips did pathway, electron transfer occurs preferentially through the

Ohshiro and Umezawa PNAS ͉ January 3, 2006 ͉ vol. 103 ͉ no. 1 ͉ 11 Downloaded by guest on September 28, 2021 Fig. 2. Changes in the observed image contrast for guanines; comparison with unmodified, noncomplementary, and complementary nucleobase tips. (a)An STM image of guanines observed with a complementary cytosine tip. (b) An STM image of guanines observed with a noncomplementary adenine tip. (c)AnSTM image of guanines observed with an unmodified tip. (Insets) The magnified images (2.5 ϫ 2.5 nm2)ofa–c are shown. (d) Cross-sectional profiles along the dashed lines (␣-␣Ј, ␤-␤Ј, and ␥-␥Ј)intheInsets of a–c. The y axis of the cross-sectional profiles represents the extent of electron tunneling, as measured by the height of the tip from a given position to meet the constant current mode in the STM measurement. (e) Extents of electron tunneling between tip and sample nucleobases. The mean values (n ϭ 10) of the extents of the observed electron tunneling between nucleobase tips (i.e., adenine, guanine, cytosine, and uracil tips) and sample nucleobases (i.e., adenine, guanine, cytosine, and uracil) are represented in height of the tips (pm) (see d legend). Those with irrelevant tips (i.e., unmodified, MP, and TP tips) were also obtained, for comparison, under otherwise identical conditions. (f and g) Extents of electron tunneling for adenine (red columns) and guanine (blue columns) images in pure monolayers and mixed monolayers. The results were obtained with cytosine (f) and uracil (g) tips.

hydrogen bonds of complementary base pairs (14). Kelly and The molecular tips directly detected intermolecular electron Barton (14) constructed the DNA double strands that were tunneling between sample and tip molecules and revealed the linked to a donor and acceptor located on the different strand tunneling facilitation through chemical interactions that provide and found that larger electron transfer occurred through the overlap of respective electronic wave functions, that is, hydro- interstrand connection of the complementary double strands gen-bond interactions (16–19, 21, 22), metal-coordination-bond relative to the other double strands containing mispairs. On the interactions (20), and charge-transfer interactions (23). We have other hand, in the intrastrand pathway, electron hopping is extensively studied chemical selectivity toward various func- known to occur through a pi-pi stacking interaction of base pairs tional groups based on hydrogen-bond interactions. The chem- (8–13). Of the two pathways, only the interstrand pathway is to ical selectivity can be tailored by controlling the extent of the be compared with the present results. hydrogen bond acidity or basicity of the molecular tips (22).

12 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0506130103 Ohshiro and Umezawa Downloaded by guest on September 28, 2021 CHEMISTRY

Fig. 3. Selective illumination of complementary nucleobases in guanine͞adenine-mixed SAMs. (a–c) STM images (10 ϫ 10 nm2) of adenine͞guanine-mixed SAMs prepared from the sample solutions, of which molar ratio of guanine to adenine are 10.0:1.0 (a), 1.0:1.0 (b), and 1.0:10.0 (c). (a, Inset) The magniﬁed area of a (1.5 ϫ 1.5 nm2) is shown, where low-contrast (␣) and high-contrast (␤) images exhibit guanine and adenine, respectively. (d) An STM image (10 ϫ 10 nm2) of a neat guanine SAM. a–d were obtained with cytosine tips. (e) The number of guanine images (■) increased and adenine images (E) decreased in proportion to the molar ratio of [guanine] to [guanine ϩ adenine] in the sample nucleobase-mixed solutions. The number of adenines and guanines were counted in an image (10 ϫ 10 nm2) of an adenine͞guanine-mixed SAM, and the procedure was repeated for another nine images. The results were averaged (n ϭ 10).

Larger facilitation of electron tunneling was observed at ether tions, which resulted in pinpointing complementary nucleobases oxygens in a favorable orientation than those in unfavorable in the present study. Hydrogen-bond-mediated electron-transfer orientations, allowing us to discriminate between these differ- process has been of great interest and studied by several groups ently oriented functional groups. These results substantiate the using photo-induced electron transfer with acceptor͞donor facilitated electron tunneling through hydrogen-bond interac- markers (15) because of its fundamental importance in chemical

Fig. 4. Single-nucleotide polymorphism typing in 18-mer single-stranded PNAs. (a) An STM image (10 ϫ 10 nm2) with an unmodified tip of single-stranded 18-mer PNAs, the sequence of which is TTTTTTTGGTTTTTTTTT. (Inset) A cross-sectional profile (3.5 ϫ 10 nm2) along the strand circled by white dots is also shown. White arrow drawn in the image pinpoints one end of a PNA strand. (b–d) STM images (15 ϫ 15 nm2) with cytosine tips of three kinds of PNA strands: TTTTTTTTGTTTTTTTTT (b), TTTTTTTGGTTTTTTTTT (c), and TTTTTTTTTTTTTTTTTT (d) are shown. (Insets) The magnified images (2.0 ϫ 5.0 nm2)ofb–d are shown. (e) Cross-sectional profiles of a row of base images along the PNA strand circled by white dots in Insets bЈ–dЈ are shown. The y axis of the cross-sectional profiles represents the extent of electron tunneling along the strands.

Ohshiro and Umezawa PNAS ͉ January 3, 2006 ͉ vol. 103 ͉ no. 1 ͉ 13 Downloaded by guest on September 28, 2021 reaction processes and crucial roles in biological electron- Preparation of Nucleobase-Modified Tip (Nucleobase Tip). STM transfer processes. metal tips were prepared from a gold wire (0.25 mm diameter; An example of the detection of particular nucleobases was Nilaco, Tokyo) by electrochemical etching in 3 M NaCl with ac demonstrated here with the present method in an 18-mer strand 10 V and then washed in an ultrasonic bath or cleaned in piranha of a peptide nucleic acid (PNA), an analogue of DNA (27). A solution. For constructing nucleobase molecular tips, the gold typical STM image with an unmodified tip of a PNA strand is tips were cleaned in piranha solution, and then immersed for 3 h shown in Fig. 4a, showing that bases in the strand were observed in 10 mM ethanolic solution of thiol derivatives of nucleobases. as rows of bright spots and the components of the strand, The tips were then rinsed with ethanol and dried in a stream of guanines and thymines, were not discriminated. On the contrary, argon or nitrogen. cytosine tips pinpointed the complementary guanines among the ͞ noncomplementary thymines in the strands (Fig. 4 b–d and STM Measurements of SAMs of Neat Mixed Nucleobases. STM measurements were carried out on a Nanoscope E (Digital Instru- Insets). The extent of electron tunneling along the strands shows ments, Santa Barbara, CA) at room temperature in a constant that a single- and double-base substitution in the strands was current mode. A drop (5 ␮l) of 1,2,4-trichlorobenzene deposited distinguished with the cytosine tip (Fig. 4e). on sample thio-base SAMs on Au(111) before the measure- In conclusion, we found that hydrogen-bond-mediated elec- ments. STM measurements were performed at the solution͞gold tron tunneling occurs with the complementarity between the tip interface under ambient condition at a bias voltage of Ϫ500 mV nucleobase and sample nucleobase. The electron tunneling (sample negative), and a tunneling current of 1,200 pA. It was increase is capable of electrically pinpointing each nucleobase. confirmed that no polarity dependence was observed by applying STM observations of nucleobases (28–30) and DNA oligomers the reversed potential. In the STM observation, Ϸ45% of Ͼ30 (31–33) had been reported, but those studies failed to identify nucleobase tips exhibited the facilitated electron tunneling in the chemical species of nucleobases because of the poor chemical each combination of nucleobases on tip and substrate, and the selectivity of the STM images. The present approach made it others exhibited the same STM images as those observed with possible to pinpoint particular nucleobases. Enhancement of unmodified gold tips. The lack of the facilitation is most probably electron tunneling occurred at specific functional groups and caused by the absence of a nucleobase molecule at the very apex chemical species on the basis of hydrogen-bond, metal- of the underlying gold tip at the atomic level. coordination-bond, and charge-transfer interactions, and, as a result, allowed us to identify the location of the specific chemical Preparation and STM Measurements of Single-Stranded PNA Oli- species and functional groups. This technique may be coined gomers. Three kinds of single-stranded, 18-mer PNA purified by ‘‘intermolecular tunneling microscopy’’ as its principle goes and HPLC, (i)H2N-TTTTTTTTGTTTTTTTTT-CONH2 (con- is of general significance for novel molecular imaging of chemical taining 1 guanine and 17 thymines), (ii)H2N-TTTTTTTG- identities at the membrane and solid surfaces. GTTTTTTTTT-CONH2 (containing 2 guanines and 16 thymines), and (iii)H2N-TTTTTTTTTTTTTTTTTT-CONH2 Materials and Methods (containing 18 thymines), were purchased (Fasmac, Kanagawa, Preparation of SAMs of Neat͞Mixed Nucleobases. For preparing the Japan) and used for STM measurements without further puri- sample SAMs of nucleobases (i.e., thiol derivatives of adenine, fication. Sample substrates were prepared by depositing a drop (5 ␮l) of a 1,2,4-trichlorobenzene solution containing PNAs guanine, cytosine, and uracil), gold substrates were soaked into (concentration, 0.5–1.0 mM) onto a Au(111). STM measure- 10 mM sample ethanolic solutions (HPLC-grade ethanol, Wako ments were performed at the solution͞gold interface by immer- Pure Chemical, Osaka) for 30 min, 45 min, or 1 h. After being sion, under ambient condition at a bias voltage of Ϫ500 mV taken out of the solution, the gold substrates were rinsed with (sample negative) and a tunneling current of 1,200 pA. ethanol to remove excess sample nucleobases physisorbed on the ͞ SAMs and dried in vacuum. The sample adenine guanine-mixed This work was supported by the Core Research for Evolutional Science SAMs were prepared from the aqueous 10 mM mixed solutions and Technology of the Japan Science and Technology Agency and the of adenine and guanine with their differing molar ratios. Japan Society for the Promotion of Science.

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