Single-molecule observation of DNA charge transferTadao Takada, Mamoru Fujitsuka, and Tetsuro Majima*
The Institute of Scientific and Industrial Research (SANKEN), Osaka University, 8-1 Mihogaoka, Ibaraki, Osaka 567-0047, Japan
Edited by Jacqueline K. Barton, California Institute of Technology, Pasadena, CA, and approved May 21, 2007 (received for review January 29, 2007)
DNA charge transfer highly depends on the electronic interactionbetween base pairs and reflects the difference in the base com-position and sequence. For the purpose of investigating the chargetransfer process of individual DNA molecules and the opticalreadout of DNA information at the single-molecule level, weperformed single-molecule observation of the DNA charge transferprocess by using single-molecule fluorescence spectroscopy. TheDNA charge transfer process, leading to the oxidation of thefluorescent dye, was explored by monitoring the on–off signal ofthe dye after the charge injection by the excitation of a photosen-sitizer. The photobleaching efficiency of the dyes by the DNAcharge transfer specifically depended on the base sequence andmismatch base pair, demonstrating the discrimination of the indi-vidual DNA information. Based on this approach, the opticalreadout of a single-base mismatch contained in a target DNA wasperformed at the single-molecule level.
electron transfer � single-molecule fluorescence � SNP detection
The �-stacked array of base pairs in DNA double strandsmediates the charge transfer (1). The enormous interest in
DNA-mediated charge transfer has been spurred by its biologicalrole in the cellular process (2, 3) and the development ofDNA-based electrochemical devices, while the mechanism anddynamics of the charge-transfer process through DNA continueto attract much attention because of the general interest inunderstanding electron-transfer chemistry in the �-stacked arraysystem (4–10).
The positive charge generated by the one-electron oxidationof a nucleobase has been shown to migrate over long distances(more than a few tens of nanometers) through the DNA duplex(11, 12). At present, the multistep hopping mechanism as asimplified model, in which the guanine (G) and adenine (A)residues work as charge carriers behaving like a stepping stone,has been widely accepted (13, 14). In this proposed mechanism,a positive charge is localized on the G residues that have thelowest oxidation potential [the trend in oxidizablity for DNAnucleobases has been established to be G � A � T, C (15, 16)]and migrates to the neighboring G via a superexchange inter-action (G-hopping). When the next G is separated by more thanfour A/T base pairs, the charge is carried by the bridging A bases(A-hopping). These two hopping processes allow the charge tomigrate with an extremely shallow distance dependence. As analternative mechanism, gated hopping, in which the charge istransiently delocalized over several base pairs defined by thelocal structure and sequence, has been proposed (17).
The important characteristics of DNA charge transfer is thatthe efficiency and rates of the charge transfer are highly sensitiveto the structural perturbations in the sequence containing single-base mismatches or the changes in the electronic states inducedby protein binding (18–20). In this context, the biosensors basedon the principle of DNA charge transfer have potential appli-cations ranging from mutation detection to pathogen identifi-cation. Actually, an electrochemical DNA biosensor based oncharge transfer properties has been established to detect themismatches and base lesions involved in the target DNA (21, 22).
Single-molecule spectroscopy has been extensively used tocharacterize biophysical systems, including protein folding, en-zymatic dynamics, and ligand–receptor interactions, and chem-
ical reaction, because these techniques are especially well suitedfor revealing the dynamics and mechanistic behavior obscured byensemble averaging in conventional spectroscopic techniques(23–26). Particularly, single-molecule FRET is a powerful tech-nique for studying the conformational distribution and dynamicsof RNA folding or the unique structures of DNA (27–29).Another important aspect of single-molecule fluorescence spec-troscopy is that it can be applied to the single-molecule opticalreadout (30, 31).
So far, although single-molecule spectroscopy provides aunique and powerful tool for studying biophysical processes, thistechnique has not been applied to DNA charge transfer chem-istry. A highly sensitive optical readout to interrogate an indi-vidual DNA molecule is made possible through the combinationof DNA charge transfer chemistry and single-molecule spectros-copy. The key issue is transforming the DNA charge transferevents into optically detectable signals. We now describe astrategy for the observation of the charge transfer process inDNA at the single-molecule level using total internal reflectionfluorescence microscopy. The photobleaching of the fluorescentdyes attached to DNA on the glass surface was observed as theoptical response to the charge transfer process. We also foundthat the mismatch base pair significantly suppressed the photo-bleaching efficiency of the dyes, allowing one to identify themismatch from single DNA molecules.
Results and DiscussionDNA Charge Transfer Detection System. The DNA charge transferdetection system based on a fluorescence signal change from thefluorescent dyes is schematically shown in Fig. 1a. The positivecharge injection molecule, i.e., the photosensitizer, and thereporter fluorescence molecule are attached at the both ends ofthe DNA. A charge is injected into the DNA by excitation of thephotosensitizer, migrates through the DNA �-stack, and finallyarrives at the reporter molecule if the oxidation potential of thefluorophore is lower than that of the nucleobases. As a result, thereporter fluorescent dye is undergoing irreversible chemicaltransformation by the DNA charge transfer. On the contrary, inthe presence of a mismatch base pair in DNA, the charge transferis prohibited because the mismatch site causes a disruption ofbase stacking and a striking decrease in electronic interactionsthrough �-stacking. As a result, the reporters are protected fromoxidation and remain emissive. Accordingly, by monitoring thefluorescence signal response from the reporter fluorescencedyes, we can explore the charge transfer process and the presenceof mismatched base pairs in DNA.
For the purpose of observing the DNA charge transfer processusing the fluorescence signal response, we designed the DNA
Author contributions: T.T., M.F., and T.M. designed research, performed research, analyzeddata, and wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Abbreviations: NI, naphthalimide; TMR, 6-carboxytetramethylrhodamine; AF, AlexaFluor 532.
*To whom correspondence should be addressed. E-mail: [email protected].
This article contains supporting information online at www.pnas.org/cgi/content/full/0700795104/DC1.
conjugates shown in Fig. 1 and Table 1. Naphthalimide (NI),which is well known to be a strong oxidant in its singlet excitedstate (Ered � 2.4 eV) and can oxidize the neighboring bases, wasselected as the charge injection molecule (32). Fluorescent dyes,6-carboxytetramethylrhodamine (TMR) or Alexa Fluor 532(AF), were used as the reporter fluorescent dyes because theyhave high fluorescence quantum yields and a photostability thatenable us to use them for single-molecule spectroscopy. Previ-ously, we reported that NI can efficiently inject a positive chargeinto DNA by UV irradiation (33). The charge injection mech-anism (charge separation process between NI and G) afterexcitation of the NI site and the following charge transfer to thefluorescent dye are shown in Fig. 1b. NI is a strong enoughoxidant to oxidize the nearest A base (Eox � 1.46 V vs. NHE).The contact ion pair between NI and an adjacent A base isinitially generated by photoexcitation, and a part of the chargeescapes from the ion pair and then is trapped at the G to providea long-lived charge separated state between NI and G (34). Thecharge separated state persists for a few microseconds when NIand G are separated by four A bases, making it possible for thecharge to freely migrate to the fluorescent dye through DNA in
a submicrosecond time scale (5, 35). Although the binding modeof the fluorescent dyes to DNA is ambiguous, the charge transfercan occur as long as the dyes are close to the nucleobases (36).
Fluorescence Changes of Fluorescent Dyes in the Ensemble System byDNA Charge Transfer. The photobleaching of fluorescent dyes bythe DNA charge transfer upon UV irradiation was investigatedby steady-state fluorescence measurements. Fig. 2 shows thefluorescence spectral changes in the dyes after the UV irradia-tion. The fluorescence intensity for A4-1-TMR gradually de-creased along with the slight blue shift in the emission maximumas the irradiation time increased, demonstrating that dyes losttheir f luorescent properties by the DNA charge transfer. Thefluorescence spectral shifts indicate that the oxidized form ofTMR is also emissive, but weak.
NI and Fl represent naphthalimide and fluorescent dyes (TMR or AF) at-tached to the 5� end of DNA, respectively. BT (biotin) is attached at the 3� endof Fl-conjugated DNA. Single-base mismatches in the sequence are shown inbold.
Fig. 2. Observation of DNA charge transfer by steady-state fluorescencemeasurements. (a and b) Steady-state fluorescence spectra obtained for A4-1-TMR (a) and A4-1-AF (b) after irradiation by UV lamp. Sample solutionscontain 100 nM DNA in 20 mM sodium phosphate buffer (pH 7.0) and 100 mMNaCl. (c) Fluorescence intensity changes monitored at 583 nm for A4-1-TMR asa function of irradiation time for DNA modified with NI and TMR (�), DNAmodified with only TMR (E), and a mixture of DNA, one NI-conjugated DNA,and the other DNA modified with TMR but without NI (‚).
Fig. 1. DNA charge transfer detection system. (a) Photobleaching of thefluorescent dyes by DNA charge transfer. The NI and fluorescent dye (Fl) arerepresented by blue and red, respectively. The fluorescent dye is oxidizedwhen the charge can freely migrate through DNA, leading to the photo-bleaching of the dyes. In the presence of the mismatch site, the charge transferto the dyes is inhibited. (b) The positive charge injection (hole) process viaA-hopping and the following charge transfer to the reporter fluorophore.Excitation of NI by UV light generates NI in the singlet excited state, whichoxidizes the adjacent A base to give the contacted ion pair. The positive chargeon the A base escapes from the ion pair and migrates to the nearest G throughhopping between A bases to provide the charge separated state with a longlifetime. The hole injected into DNA migrates to the fluorescent dye, leadingto the irreversible reaction of the reporter fluorophore.
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Similar spectral changes were observed for the AF-conjugatedDNA (A4-1-AF) although the oxidation products of AF showeda very weak emission compared with that of TMR, indicatingthat other fluorescent dyes could be used for this system as longas they have an oxidation potential lower than G. This approachwould be applied to the convenient charge transfer detection.The apparent photobleaching efficiency by UV irradiation forA4-1-AF is somewhat higher than that for A4-1-TMR whencompared at the maximum peaks of emission (583 nm for TMRand 555 nm for AF). The charge injection efficiency in thissystem depends on the number of A bases between NI and G,and the charge transfer efficiency from DNA to the fluorescentdye is quantitative because the trapping of the charge by wateror molecular oxygen is not rapid enough to be competitive withthe charge transfer in this sequence (5). Considering these facts,the fluorescent dyes attached to the opposite end of the DNAshould quantitatively accept the generated charge. Accordingly,the differences in the apparent photobleaching efficiency wouldbe caused by the emissive properties of the oxidation products.
Control experiments were performed to confirm that we canrule out the effect of the direct excitation of the dyes by UV lightand the intermolecular oxidation process on the fluorescencechange (Fig. 2c). The dye decomposition by direct irradiationwas not observed in the DNA lacking NI. Similarly, a mixture ofDNA, one NI-conjugated DNA and the other DNA modifiedwith TMR lacking NI, showed no fluorescence spectral changes,indicating that the intermolecular charge transfer, i.e., oxidationof the fluorescent dyes, was ineffective. In addition, singletoxygen generation by energy transfer from NI in the excited stateto molecular oxygen can be excluded because the formation ofthe contact ion pair occurs in the picosecond time scale, whichis much faster than intermolecular energy transfer process (34).From these facts, we confirmed that the fluorescence spectralchanges of the dyes were induced by the DNA charge transfer[see supporting information (SI) Text and SI Fig. 6].
Observation of DNA Charge Transfer at the Single-Molecule Level.Observations of single-molecule fluorescence were carried outwith a conventional microscope equipped with total internalreflection illumination, which reduces background fluorescence.The DNA charge transfer process was observed at the single-molecule level from the optical response of the fluorescent dyesafter the charge injection by photoirradiation. A DNA-modifiedsurface was prepared through the biotin–streptavidin linkage,which is well established and used for single-molecule experi-ments. Biotinylated ssDNA probes with a fluorescent dye at the5� end of DNA were immobilized on the glass surface andhybridized with the complementary NI-conjugated DNA. Thehybridization efficiency was estimated to be �80% under ourexperimental conditions (see SI Figs. 7 and 8). Fig. 3 shows thesingle-molecule fluorescence images for A4-1-TMR before andafter the UV irradiation. Single DNA molecules appear as brightspots in the images, which correspond to the fluorescent signalfrom one TMR molecule (Fig. 3a). Upon irradiation by the UVlamp, the bright spots on the surface significantly disappeared(Fig. 3b). Control experiments showed no noticeable photo-bleaching of the fluorescent dyes by the intermolecular oxidationprocess or direct excitation of the dyes (see SI Fig. 9), confirmingthat the fluorescence from the dyes attached to the DNA wasquenched through an intramolecular process. Some bright spotsbecame weak upon UV irradiation, indicating that the dyes wereoxidized to provide the weak fluorescent products as observedin the steady-state fluorescent measurements. These resultsprovide clear evidence that the DNA charge transfer can beidentified from the optical response of the fluorescent dye at thesingle-molecule level.
The photobleaching efficiency of the fluorescent dyes for thelifetime of the charge separated state was investigated by single-
molecule spectroscopy (Fig. 3c). The value of the efficiency Fq(defined by the equation, 1 � N/N0, where N0 and N are thenumber of bright spots before and after UV irradiation, respec-tively) obtained for A4-1-TMR is almost three times higher thanthat for A3-1-TMR, which is consistent with the lifetime of theinitial charge separated state. Previously, we reported that thelifetime of the charge separated state between NI and Gseparated by A3 and A4 was 300 ns and 2.2 �s, respectively (35,37). In the A4-1 duplexes, in which NI and the nearest G areseparated by four intervening A residues, the charge recombi-nation between the NI radical anion and G radical cation isslower than the charge transfer process, leading to the efficientphotobleaching of the dyes. The relationship between the rate ofcharge recombination and charge transfer was closely related tothe photobleaching efficiency, indicating that the sequenceinformation of an individual DNA molecule can be obtainedfrom single-molecule spectroscopy.
A Single-Base Mismatch Detection by Single-Molecule FluorescenceSpectroscopy. A single-base mismatch incorporated into DNA,causing a local structural perturbation, is known to suppresscharge transfer efficiency (18). To investigate the photobleach-ing efficiency (Fq) for matched and mismatched DNA, wedesigned DNA possessing the A-C mismatch at different posi-tions (A4-2, A4-3, A4-4) and the T-C mismatch in the middle ofthe DNA strand (A4-5) (Table 1). Compared with the fullymatched DNA (A4-1), the photobleaching of the fluorescentdyes for A4-2, in which an A-C mismatch was incorporated nearNI, was drastically suppressed (Fig. 4 and SI Fig. 10). This resultclearly suggests that a single base mismatch incorporated into theregion, which is closely related to the charge injection efficiency,enhances the mismatch effect on the DNA charge transfer.Similarly, the suppression by the mismatch base pairing wasobserved when the AC (A4-3) or TC mismatches (A4-5) wereadded in the middle of DNA, but it is not as effective as that forA4-2. It should be noted that the mismatch base pair significantlyslows, but does not stop the DNA charge transfer. The value ofFq is determined by the relationship between the rate constantsof charge recombination and charge transfer. In A4-3 and A4-5,as the DNA charge transfer becomes competitive with the chargerecombination, the dye bleaching by the DNA charge transferwas not as effective as A4-2. These data emphasize that the singlebase mismatch can be clearly identified from the fluorescence
Fig. 3. Single-molecule observation of DNA charge transfer. (a and b)Single-molecule fluorescence images for A4-1-TMR immobilized on glass sur-face before (a) and after (b) UV irradiation for 2 s. (Scale bars: 10 �m.) (c)Photobleaching efficiency (Fq) for A3-1-TMR (red), A3-1-AF (blue), A4-1-TMR(black), and A4-1-AF (green) as a function of irradiation time. The photo-bleaching efficiency is defined by Fq, where Fq � 1 � N/N0, and N0 and N arethe number of bright spots before and after irradiation, respectively.
Takada et al. PNAS � July 3, 2007 � vol. 104 � no. 27 � 11181
response of the dyes at the single-molecule level. On thecontrary, the A-C mismatch near the fluorescent dye (A4-4) didnot change photobleaching efficiency. Considering that thefluorescent dye is attached to the 5� end of the DNA through thelong alkyl chain (C6), it is probable that the dye is positioned nearthe mismatch site and the direct charge transfer from G to thefluorescent dye occurred over the mismatch site. The inhibitoryeffect of the mismatch confirms that the bleaching of thefluorescent dye is induced by the DNA charge transfer.
Single-base mismatch detection of the target DNA was per-formed by using a DNA assembly composed of three strands: probe,reporter, and target DNA (Fig. 5a). The probe DNA possessing NIas the charge injector was immobilized on the glass surface throughthe biotin–streptavidin linkage, and then hybridized with the target
DNA and the TMR-labeled reporter DNA. As a model system, thesequence of BRCA1, which is related to the breast cancer gene, wasselected as the target DNA.
The effect of mismatch base-pairing on the bleaching of thefluorescent dyes was remarkable when it was incorporated intothe sequence related to the charge injection efficiency as men-tioned above. Hence, the DNA assembly was designed so that atarget site (R:G or A) was located in the position next to thecontinuous A sequence. Typical images of the surface-immobilized DNA assembly were displayed as 3D images for aclearer presentation (Fig. 5 b–e).
In a fully matched DNA (R:G), the remarkable photobleach-ing of the fluorescent dyes on the surface was observed as shownin Fig. 5, confirming that the charge injection and the followingcharge transfer to the dyes can occur even in the three-component DNA assembly containing the junction site. Thevalue of Fq after a 5-s irradiation was calculated to be 72 � 6%.In the mismatched assembly (R:A), as expected, the photo-bleaching of the dyes was significantly suppressed, and the valueof Fq was 34 � 9%, showing that the charge transfer was indeedinhibited by the mismatch base pairing. The obtained value of Fqfor R:A is somewhat higher than that for A4-2. Structuralvariation at the junction position may contribute to the bleachingof the dyes by the DNA charge transfer, but it is still sufficientto discriminate the target residues.
ConclusionsIn this article, we have demonstrated the observation of DNAcharge transfer at the single-molecule level using total internalreflection fluorescence microscopy. We found that a chargegenerated by the excitation of the photosensitizer migrates inDNA and leads to the oxidation of the fluorescent dyes, resultingin the fluorescent signal changes. The bleaching efficiency of thefluorescent dye by the DNA charge transfer depends on the basesequence. Single base mismatches strongly suppressed thebleaching efficiency although it depends on the position in thesequence. The observed mismatch effect allowed us to detect amutation of the target DNA in the BRCA1 sequence, providinga method to identify the mutation and SNPs from individualDNA molecules by applying DNA chip technology. Moreover,our approach for the single-molecule observation of the DNAcharge transfer can be applied to elucidate the function of theDNA charge transfer on the DNA–protein complex and wouldbecome a useful method for a better understanding of thebiological consequences of the DNA charge transfer.
Materials and MethodsReagents. The biotinylated DNA modified with fluorescent dyes(TMR and AF) and amino groups were obtained from Japan BioServices (Saitama, Japan). Streptavidin and biotinylated-BSAwere purchased from Molecular Probes (Carlsbad, CA) andSigma (St. Louis, MO), respectively.
DNA Synthesis. Synthesis of DNA modified with NI at the 5� endwas accomplished according to previous reports by using stan-dard �-cyanoethyl phosphoramidite chemistry on a DNA syn-thesizer (Applied Biosystems, Foster City, CA) (33). Modifica-tion of NI inside of DNA was carried out postsynthetically byreaction with the activated N-hydroxysuccinimide ester of NI(NI-NHS). To a 200-�M solution of DNA modified with NH2through C2 alkyl linker in 100 mM sodium phosphate buffer (pH8.0) was added 20 mM NI-NHS solution dissolved in DMSO andincubated at room temperature overnight. The reaction mixturewas purified by RP-HPLC, and characterized by MALDI-TOFMS; m/z 8,642.0 found for [M-H]�, calculated 8,642.8.
Synthesis of NI Carboxylic Acid N-Hydroxysuccinimide Ester. N-carboxymethyl-NI (2.0 g, 7.8 mmol) (38) and N-hydroxysuccin-
Fig. 4. Photobleaching efficiency (Fq) obtained for matched and mismatchedDNA. DNA duplexes immobilized on glass surfaces were irradiated with UVlight for 2 s. The obtained values of Fq for TMR and AF are represented byempty and filled bars, respectively.
Fig. 5. Single-base mismatch detection. (a) Three-component DNA assemblyconstructed by probe (blue), reporter (green), and target DNA (red) on a glasssurface. NI and TMR are represented by blue and green, respectively. R standsfor G or A. (b–e) Fluorescence images obtained for R � G or A before (b andd) and after (c and e) 5 s of UV irradiation. (Scale bars: 5 �m.)
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imide (0.897 g, 7.8 mmol) were suspended in acetonitrile (60 ml).To this solution, EDCI (1-ethyl-3-(3�-dimethylaminopropyl)car-bodiimide, 1.50 g, 7.8 mmol) was added and stirred for 2 h atroom temperature. The reaction mixture was evaporated, fil-tered, and washed with cold acetonitrile twice to provide thewhite solid (2.1 g, 6.0 mmol, 77%). 1H-NMR (DMSO, 270 MHz)� 8.55 (m, 4H), � 7.92 (t, 2H), � 5.21 (s, 2H), � 2.80 (s, 4H); fastatom bombardment-MS, m/e (%) found 353 [(M�H)�].
Surface Preparations. Surface modification with DNA was per-formed as described (39, 40). Cover glasses were cleaned bysonication in 25% alkaline detergents for a minimum of 3 h.Cover slips were then rinsed many times in Milli-Q water. Acover glass and a glass slide were used to make a sandwich-likechannel (volume �7–8 �l) using adhesive spacers. A biotinylatedBSA solution (1 mg/ml) in Tris buffer (10 mM, pH 7.6) was firstintroduced into the channel. After incubation for 10 min, thechannel was flushed with Tris buffer (20 �l 2), and strepta-vidin solution (0.25 mg/ml, 20 �l) was incubated for 10 min. Afterwashing with Tris buffer (20 �l 2), a biotinylated DNAsolution in hybridization buffer (20 mM sodium phosphatebuffer, pH 7.0 and 100 mM NaCl) was applied and incubated for30 min in a humid environment. After stringent washing with
hybridization buffer, cDNA (100 nM, 20 �l) was applied to thechannel and incubated for 30 min.
Single-Molecule Fluorescence Imaging. The surface-immobilizedDNA was imaged by total internal reflection fluorescencemicroscopy. The microscopy consisted of an inverted opticalmicroscope (Olympus, Melville, NY), one color laser excitationsource (532 nm), and an intensified CCD camera. A frequency-doubled Nd:YAG laser with 25 mW of 532-nm laser light wasused to provide the excitation for the TMR- and AF-labeledDNA. The sample cover glass was placed on the invertedmicroscope. A long-pass fluorescence filter was used to block theexcitation light. A 100-�m diameter area was illuminatedthrough an objective lens by a high-pressure Hg lamp fitted withan excitation filter (330–385 nm) for UV-induced photobleach-ing experiments. A data analysis of the fluorescence image wascarried out with Image-J software and a program written byMATLAB (Mathworks, Natick, MA).
We thank Prof. K. Kawai for valuable discussions and C. Lin and T.Tachikawa for experimental assistance. This work has been partlysupported by a Grant-in-Aid for Scientific Research on Priority Area(417), 21st Center of Excellence Research, and the Ministry of Educa-tion, Culture, Sports and Science, and Technology of Japan.
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