NANO LETTERS
A Single-Molecule Sensitive DNA Hairpin System Based on Intramolecular Electron Transfer
2003 Vol. 3, No. 7 979-982
Oliver Piestert,† Hannes Barsch,† Volker Buschmann,† Thomas Heinlein,† Jens-Peter Knemeyer,† Kenneth D. Weston,‡ and Markus Sauer*,† Physikalisch-Chemisches Institut, UniVersita¨t Heidelberg, Im Neuenheimer Feld 253, 69120 Heidelberg, Germany, and Department of Chemistry and Biochemistry, Florida State UniVersity, Tallahassee, Florida 32306 Received April 4, 2003; Revised Manuscript Received April 25, 2003
ABSTRACT We describe a method for detection of sub-picomolar concentrations of DNA or RNA sequences using novel surface-immobilized DNA hairpins. Within the DNA hairpins a fluorophore is specifically quenched by guanosine residues in the complementary stem sequence via photoinduced intramolecular electron transfer. Upon hybridization to the target sequence, fluorescence is restored due to a conformational reorganization that forces the stem apart. Proper immobilization of the DNA hairpins using biotin/streptavidin binding with minimal perturbation of the surface is required to ensure efficient quenching in the closed state.
Numerous methods for the detection and quantification of DNA and RNA based on fluorescence have been developed.1-5 Recently, molecular beacons have been used to detect the presence of unlabeled target sequences in homogeneous solution.2 Molecular beacons are single-stranded oligonucleotide probes that possess a stem-and-loop structure. A fluorescent dye and a quencher, e.g., an energy transfer acceptor dye, are linked to the two ends of the oligonucleotide forming the stem. Upon hybridization of the loop sequence to the complementary target sequence, the molecular beacon undergoes a spontaneous conformational reorganization that forces the stem apart and causes the fluorescent dye and quencher to move away from each other, leading to an increase in fluorescence intensity. Since molecular beacons are remarkably effective at detecting single base mismatches, they hold great promise for studies in genetics, disease mechanisms, and molecular interactions.2 So far, molecular beacons have been used in homogeneous and heterogeneous formats to detect target concentrations in the range of 10-8 to 10-9 M.2,3 To enable large-scale parallel analysis with increased sensitivity, the DNA hairpins have to be attached to a surface. In this letter, we describe a method for detection of target sequences down to concentrations of 10-13 M using novel surface-immobilized DNA hairpins. The method takes advantage of specific properties of naturally occurring nucle* Corresponding author. Phone: +49-6221-548460. Fax: +49-6221544255. E-mail:
[email protected]. † Universita ¨ t Heidelberg. ‡ Florida State University. 10.1021/nl0341988 CCC: $25.00 Published on Web 05/15/2003
© 2003 American Chemical Society
Figure 1. Schematic of operation of a 3′-biotinylated DNA hairpin immobilized on a solid surface. The oxazine derivative MR121 (λabs) 661, λem) 673 nm) is attached to the cytosine-containing 5′ end. Upon hybridization to the target sequence (complementary to the loop sequence), the fluorescence is restored due to a conformational reorganization that forces the stem apart.
otides, in particular, the low oxidation potential of the DNA base guanosine of 1.25 V vs SCE.6 Thus, depending on the reduction potential of the fluorescent dye used, efficient quenching via photoinduced electron transfer occurs upon contact formation with guanosine in the first excited singlet state.5 Within the novel DNA hairpins an oxazine derivative (MR121) labeled at the 5′ end is quenched by guanosine residues in the complementary stem (Figure 1). These DNA hairpins increase fluorescence upon hybridization to target DNA 6-fold in solution,5 which may provide a basis for a cost-effective sequence-specific DNA/RNA detection method. In addition, attachment of these DNA hairpins to a solid
Figure 2. Single-molecule fluorescence scanning confocal images (20 × 20 µm) of DNA hairpins immobilized on glass cover slips in PBS, pH 7.4 (50 nm/pixel, 3 ms integration time). For excitation, a pulsed diode laser (635 nm, 80 MHz, ∼2 kW/cm2) was used. Scanning was performed from top left to bottom down using modulated excitation. To visualize the difference in fluorescence quantum yield, different intensity scales were used for the closed and opened hairpins. (A) Intensity scale: 0-4 counts/3 ms. (B) Same surface at 0-16 counts/3 ms. The arrow indicates the addition of an excess of target sequence (oligo(dA)30). (C) After a washing step with PBS (0-16 counts/3 ms). Fluorescent spots of a freely rotating (D) and a DNA hairpin molecule stuck on the surface (E). The modulation stripes indicate a rotationally stationary absorption dipole of the fluorophore.
support via biotin/streptavidin binding does not require the incorporation of a modification in the loop or stem for an additional quencher, as in the case of molecular beacons.3 In addition to the fluorophore, only a simple 3′-biotinylation is needed for surface attachment. The DNA hairpin was synthesized by coupling the oxazine derivative MR121 to the 5′ end of the oligonucleotide 5′CCCCT (T)20 AGGGG TTT-Biotin-3′ using 5′ amino modifier C6.5 The synthesized oligonucleotide was purified by gel filtration chromatography and reverse phase HPLC. Five base pairs at the two ends are complementary to each other, forming the stem with a 3-base single strand overhang at the 3′ end. The stem keeps the fluorophore and the guanosine residues in close proximity to each other, causing the fluorescence of the fluorophore to be quenched. Quenching of oxazine dyes by guanine but not cytosine, thymine, or adenine nucleotides is consistent with an electron-transfer mechanism for fluorescence quenching in which the excited fluorophore serves as an electron acceptor and guanine as an ground-state electron donor. To evaluate the efficiency of photoinduced charge separation, the free energy change for charge separation, ∆Gcs, can be estimated by using Weller’s equation.7 With a reduction potential, Ered, of -0.5 V vs SCE8,9 and a zero-zero transition energy, E0,0, of ∼1.9 eV for the oxazine derivative MR121, the free energy change for charge transfer, ∆Gcs, from the ground state guanine to the excited oxazine chromophore at infinite separation can be estimated to approximately -0.15 eV. This indicates that the electron transfer reaction between MR121 and guanine is only slightly exergonic. Furthermore, intra- and intermolecular fluorescence quenching experiments with MR121 and guanosine monophosphate suggest that contact formation, i.e., van der Waals contact, is required for efficient fluorescence quenching.8 Altogether, the data indicate that the fluorophore adopts an end-capped conformation with nearly coplanar conformation with respect to the last G/C base pair (Figure 1). The formation of these ground state complexes between guanine and MR121 is the primary prerequisite for efficient electron transfer. This implies that, depending on the relative conformations and resulting interaction geometries, the complexes can be considered as essentially nonfluorescent. Nevertheless, due to the conformational flexibility of the C6 linker used, MR121 experiences various 980
interaction geometries with different quenching efficiencies. Therefore, the relative fluorescence quantum yield of MR121 is reduced to ∼0.1-0.2 with respect to the free dye.8 Molecular systems in which the excited state of a fluorophore is quenched by an electron donor form an important class of chemosensory materials.10 While in chemosensory materials the electron transfer can be turned on and off by reactions of the nonbonding electron pair of the donor with the environment,11 in our DNA hairpin the fluorescence is restored upon hybridization to the target sequence, i.e., upon a conformational reorganization that forces the stem apart.12 Within recent years fluorescence-based single-molecule spectroscopy has evolved as an important tool to study the behavior of single molecules under ambient conditions.13-15 Detecting the hybridization of a DNA hairpin to its target sequence at the single-molecule level represents the ultimate sensitivity of sensing. To explore the potentials of the novel DNA hairpins, we immobilized 3′-biotinylated DNA hairpins onto streptavidin-coated silica surfaces. Since the conformational state of a DNA hairpin determines the effectiveness of the probe, immobilization with minimal perturbation by the surface is necessary. Otherwise, nonspecific adsorption of the fluorophore and/or the oligonucleotide prevents efficient quenching and hybridization, respectively. To immobilize the 3′-biotinylated hairpin onto a cover slide, the surface first binds bovine serum albumin (BSA) by physical adsorption (5 mg/mL, 10% of the BSA carries a biotin label). After an incubation time of 4 h and a washing step, streptavidin (1 mg/mL) was allowed to bind in PBS for 5 min. Finally, DNA hairpins (10-10 M) were added to bind to the immobilized streptavidin. All measurements were carried out at ambient conditions in PBS, pH 7.4. Strong surface interaction of the fluorophore with the surface will cause the molecules to stick to the surface and become rotationally stationary (Figure 2E). As a result of deteriorated fluorescence quenching by the guanosine residues in the complementary stem, the fluorescence intensity of the fluorophore increases substantially. To determine whether such interactions were successfully avoided by the BSA surface treatment, a polarization modulated excitation method was used.16 The method is based on the fact that a single fluorophore has a well-defined absorption dipole, and the emitted fluorescence intensity depends on the angle of Nano Lett., Vol. 3, No. 7, 2003
the linear polarization of the excitation beam relative to the absorption dipole angle. For a freely rotating fluorophore, the fluorescence will be demodulated regardless of the polarization angle of excitation. The linear polarization angle of the excitation beam is rotated in time by passing the linearly polarized laser beam through an electrooptic modulator (EOM) and 1/4-wave retarder. The resulting beam remains polarized but oriented at an angle proportional to the voltage applied at the EOM. By rotating the polarization angle in time as the sample is scanned, modulation stripes in the fluorescence image can be observed if the absorption dipole of the fluorophore is fixed (or significantly hindered). In our images of immobilized DNA hairpins, such stripes are not observed, confirming that the attached fluorophore is freely rotating (Figure 2).16 As can be seen in Figure 2A, the fluorescence intensity of immobilized DNA hairpins is very low at an excitation power of ∼2 kW/cm2. Typically, average count rates of ∼0.5 kHz were detected for closed DNA hairpins. Upon addition of 10 µL of the complementary oligonulceotide oligo(dA)30 to a final concentration of 1 µM, fluorescence is restored within seconds (Figure 2B). For hybridized hairpins, average count rates >2 kHz were detected, i.e., the fluorescence intensity increases ∼4-fold upon hybridization to the target. This increase is similar to the increase observed from immobilized molecular beacons.3 The demodulation of the fluorescence intensity demonstrates that the immobilization technique ensures free rotation of the DNA hairpin. Maintaining the native end-capped conformation of the fluorophore within the hairpin is the most crucial step for the development of a single-molecule sensitive DNA chip. Any nonspecific adsorption of the fluorophore on the surface destroys the hairpin conformation required for efficient fluorescence quenching. Since the fraction of hairpin molecules that exhibit reduced quenching determines the detection sensitivity, maintaining the proper conformation in a surface-immobilized DNA hairpin is crucial for measuring the presence and quantity of target sequence.3,4 To determine the detection limit, DNA hairpins were immobilized on cover slips in PBS, pH 7.4, with areal densities ∼100 hairpins/400 µ2. After incubation with a range of concentration of target sequence for 5 min, all fluorescent spots exhibiting count rates above and below a threshold of 1 kHz within a 400 µm2 area were counted (Figure 3). In addition, only those fluorescent spots were counted that exhibit demodulated fluorescence intensity, confirming that the fluorophore is freely rotating. Fluorescence spots caused by impurity molecules stuck to the surface show modulation stripes. Therefore, our technique is ideally suited to discriminate freely rotating DNA hairpins from impurity molecules. This represents a prerequisite for highly sensitive detection of specific target sequences at the single-molecule level. At a final concentration of the complementary oligonucleotide (oligo(dA)30)) of 5 × 10-11 M, more than 50% of all hairpins restored their fluorescence (Figure 3A). Experiments more dilute in complementary sequence demonstrate that even at a final concentration of 10-13 M, 39% of the Nano Lett., Vol. 3, No. 7, 2003
Figure 3. Single-molecule fluorescence scanning confocal images (20 × 10 µm) of DNA hairpins immobilized on glass cover slips in PBS, pH 7.4 (50 nm/pixel, 3 ms integration time) incubated with different concentrations of target sequence (oligo(dA)30) for 5 min. For excitation a pulsed diode laser (635 nm, 80 MHz, ∼2 kW/ cm2) was used. Scanning was performed from top left to bottom down using modulated excitation. Intensity scale: 0-16 counts/3 ms. (A) 5 × 10-11 M target sequence, (B) 1 × 10-11 M target sequence, and (C) 1 × 10-13 M target sequence.
immobilized DNA hairpins undergo a conformational reorganization (Figure 3C). A solution with noncomplementary DNA (10-10 M) shows no fluorescent spots with count rates >1 kHz under the same conditions. For the first time, we demonstrated that immobilized DNA hairpins based on photoinduced intramolecular electron transfer from guanosine residues to excited fluorophores can be used to efficiently detect the presence of single target DNA or RNA molecules, even in the sub-picomolar concentration range within a reasonable time. This has been achieved by immobilization of the DNA hairpins on coated cover slips while maintaining their native conformation. Therefore, the method is ideally suitable for the search for specific sequences using extremely low concentrations of target sequence. It is anticipated that optimization of reaction conditions (salt, temperature, etc.) and variations of the fluorophore as well as the use of modified nucleotides will lead to even higher sensitivities. Very recently,8 we showed that the quenching efficiency can be increased substantially by using 7-deazaguanosine, which exhibits an oxidation potential lower than that of guanosine.17 In these experiments we achieved a 20-fold increase in fluorescence intensity upon specific binding to the target sequence. Acknowledgment. We thank Prof. K. H. Drexhage (Universita¨t-Gesamthochschule Siegen) for providing the oxazine derivative MR121. This work was supported by the BMBF (Grants 311864 and 13N8349). 981
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