Direct Detection of DNA Conformation in Hybridization Processes

Article pubs.acs.org/ac

Direct Detection of DNA Conformation in Hybridization Processes George Papadakis,† Achilleas Tsortos,† Florian Bender,†,∥ Elena E. Ferapontova,§ and Electra Gizeli*,†,‡ †

Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology Hellas, 100 N. Plastira, Vassilika Vouton, 70013 Heraklion, Greece ‡ Department of Biology, University of Crete, Vassilika Vouton, 71409, Heraklion, Greece § Danish National Research Foundation: Center for DNA Nanotechnology, and Department of Chemistry and iNANO, Aarhus University, Ny Munkegade 1521, DK-8000 Aarhus C, Denmark ∥ Department of Electrical and Computer Engineering, Marquette University, 1515 West Wisconsin Avenue, Milwaukee, Wisconsin 53233, United States ABSTRACT: DNA hybridization studies at surfaces normally rely on the detection of mass changes as a result of the addition of the complementary strand. In this work we propose a mass-independent sensing principle based on the quantitative monitoring of the conformation of the immobilized single-strand probe and of the final hybridized product. This is demonstrated by using a label-free acoustic technique, the quartz crystal microbalance (QCM-D), and oligonucleotides of specific sequences which, upon hybridization, result in DNAs of various shapes and sizes. Measurements of the acoustic ratio ΔD/ΔF in combination with a “discrete molecule binding” approach are used to confirm the formation of straight hybridized DNA molecules of specific lengths (21, 75, and 110 base pairs); acoustic results are also used to distinguish between single- and double-stranded molecules as well as between same-mass hybridized products with different shapes, i.e., straight or “Y-shaped”. Issues such as the effect of mono- and divalent cations to hybridization and the mechanism of the process (nucleation, kinetics) when it happens on a surface are carefully considered. Finally, this new sensing principle is applied to single-nucleotide polymorphism detection: a DNA hairpin probe hybridized to the p53 target gene gave products of distinct geometrical features depending on the presence or absence of the SNP, both readily distinguishable. Our results suggest that DNA conformation probing with acoustic wave sensors is a much more improved detection method over the popular mass-related, on/off techniques offering higher flexibility in the design of solid-phase hybridization assays.

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effect of the probe attachment surface chemistry,13−15 the distance of the probe DNA from the surface,16 the target and probe secondary sequence,17 the number of mismatches in the target sequence,12 the solution ionic strength,7,18 and the degree of nonspecific binding to the underlying substrate.15 Some general guidelines in relation to what parameters favor thermodynamically and kinetically DNA−surface hybridization have been derived from previous studies. Low surface density of the immobilized probe (in the range of ∼3 × 1012 molecules/ cm2) leading to a less crowded surface presenting smaller sterical hindrance,10,11 full complementarity of the target strand to the immobilized probe sequence,12 and longer distance from the solid surface defined by the diluent layer used in mixed thiol layers16 are some of the factors that result in high kinetic kon and Keq values. The above conclusions have been derived using a plethora of biophysical techniques: label-free methods include

ybridization, i.e., the annealing of two complementary pieces of DNA or RNA to form a double-stranded (ds) molecule, is a process of significance to DNA molecular devices, motors, and nanoswitches.1 Moreover, the efficient detection of such processes, in combination with microarray surfaces, is used in gene analysis, clinical diagnosis of genetic disorders, tissue matching, and forensic applications.2 DNA arrays consist of surface-immobilized single-stranded (ss) probes which can selectively bind their complementary clone from a cocktail of various ssDNA molecules. Careful control of the thermodynamic and kinetic parameters of the interaction allows for the sensitive and specific discrimination between fully complementary strands and those with one or more mismatches.2 Such parameters are normally derived by using biosensing technologies in combination with surface-immobilized DNA; in addition, biosensors are used for the study of DNA interactions with various ligands, e.g., drugs,3−6 nucleic acid mimics,7 and proteins.8 Currently, there is a vast number of studies related to DNA in situ hybridization at a solid surface. These studies investigate issues such as thermodynamics,9 the probe density,10−12 the © 2012 American Chemical Society

Received: September 22, 2011 Accepted: January 16, 2012 Published: January 16, 2012 1854

dx.doi.org/10.1021/ac202515p | Anal. Chem. 2012, 84, 1854−1861

Analytical Chemistry

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that the acoustic ratio ΔD/ΔF (or ΔA/ΔPh) is directly related to the intrinsic viscosity [η] of the surface-bound molecule.51−53 This finding becomes significant when one realizes that the intrinsic viscosity is characteristic for each molecule and depends on its hydrodynamic volume and shape. Moreover, well-established theoretical and experimentally derived mathematical formulas exist correlating [η] (and, subsequently, the acoustic ratio) to specific geometrical features of the molecule, i.e., length and shape, may that be spherical, rod, bent rod, triangle, cross, coil, etc.51−54 For a detailed description of the mathematical model, see refs 52 and 53. On the basis of the above, it was reported that for straight DNAs the acoustic ratio can be used to predict the actual length of the molecule with an accuracy of ∼10 base pairs.53 Here we show that DNA hybridization can be followed in a label-free way through acoustic ratio measurements as a result of the transition of a coiled or straight single-strand probe to an elongated doublestranded molecule. Specifically, the QCM-D system is employed for (a) characterizing the conformation of in situ and prehybridized DNA molecules of various lengths in two different buffers, (b) distinguishing between two hybridized DNA molecules of same mass but different shape (straight and Y-shaped) in two different buffers, and (c) distinguishing between two DNA hairpin structures and their conformational change upon hybridization as well as detecting a singlenucleotide polymorphism (SNP). An alternative way to monitor solid-phase hybridization and answer the posed questions on the mechanism of the hybridization process is, thus, offered.

two-color surface plasmon resonance (SPR),19 SPR-spectroscopy,20,21 imaging,3 and acoustic22−29 biosensors. Other methods employing fluorescent or electrically active labels have been reported in combination with optical30,31 and electrochemical devices,32,33 respectively. Important problems though still remain regarding the optimum design parameters required for efficient solid-phase hybridization mechanism at surfaces. In general, the double-helix formation comprises two distinct steps: (a) the nucleation step, i.e., the initial formation of 2−5 base pairs,34−39 and (b) the sequential pairing of the remaining bases (hybridization step).39 However, regarding the mechanism of hybridization at surfaces, two persisting questions are still open: (a) does the binding of the second strand result in the “final” duplex structure or is it a mere “nucleation” event16 and (b) how fast does the whole process, “nucleation” plus transition into the “final” form, take place.40 Is the first event, i.e., the “nucleation” step, the one immediately detected (by most techniques) while it takes hours to form the final product of the complete double strand? Regardless of the nature of the assay, i.e., optical, acoustic, electrochemical, and direct or label-dependent, most of the techniques utilized so far rely on the same detection principle: the detection of the quantity of the target DNA that binds to the surface-immobilized probe. This observation clearly suggests that achieving high hybridization efficiency would always depend on our ability to attract more and more molecules to the sensor surface until a 1:1 stoichiometry is achieved (100% efficiency). In addition, the previously posed questions regarding the “final” product and its kinetics are not answerable with the above techniques, e.g., a mass increase would occur and be detected by the sensor as soon as recognition and initial binding of a few bases (“nucleation”) occurs regardless of this being an initial step or the complete final product−the mass remains constant after the initial binding and for the whole process. An alternative way is clearly needed capable of following the process not simply as a “mass” evolution but also as a “shape” evolution, exploiting the dynamic, structural changes of a single-strand molecule to a double-helical one as a result of target−probe binding. Such an approach would require a detection method sensitive to the structural features of the DNA molecule. Previous studies using electrochemical devices and DNA or RNA molecules have indirectly correlated DNA curvature, folding, or conformation (open vs closed) to a change in the efficiency of the electrically active probe to reach the electrode surface.5,40−45 Acoustic studies employing a quartz crystal microbalance platform have also been used to provide structural information of surfacehybridized DNA;23,24,27,46−49 however, these were only qualitative and/or relied on a model that treats bound DNA as a film; consequently, they provided evidence related to the film structure (being less or more hydrated) and its mechanical features (shear modulus, effective viscosity, etc.) rather than the geometrical features of the DNA molecules themselves.50 Here, we exploit the capability of a recently developed approach to follow conformation changes in DNA hybridization. The method is based on an acoustic wave system, employing a quartz crystal microbalance (QCM-D) sensor that provides simultaneously two measurements: mass-related frequency (F) and viscosity-sensitive energy dissipation (D); an equally wellsuited method uses a surface acoustic wave (SAW) sensor, which records wave phase (Ph) and amplitude (A). Previous studies in our laboratory using a model based on the binding of discrete molecules (rather than formation of a film) have shown

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MATERIALS AND METHODS Acoustic Device. A QCM (Q-Sense D300, Sweden) acoustic device was used; the operating frequency for the results reported here is the 35 MHz overtone. Immediately prior to the start of each experiment, the sensor chip was subjected to plasma cleaning for 3 min using a Harrick plasma cleaner PDC-002 (“HI” setting) to ensure a clean surface. After etching, to clean off adsorbed samples, the devices were reused. Real-Time Acoustic Detection of Neutravidin and DNA Binding. A continuous flow of Tris/Mg buffer (50 mM Tris pH 7.5, 10 mM MgCl2) or PBS (2.7 mM KCl, 8.1 mM Na2HPO4, 1.76 mM KH2PO4, 137 mM NaCl, pH 7.4) was pumped over the surface of the gold-coated devices at a flow rate of 60 μL/min. The signal was allowed to equilibrate prior to the first addition, and all samples were added in the same buffer. Neutravidin, purchased from Pierce, was dissolved in double-distilled water at a concentration of 10 mg/mL and stored at 4 °C in aliquots according to the manufacturer’s protocol. Neutravidin was physically adsorbed on the gold surface by adding 3.3 μM (200 μg/mL) diluted in the appropriate buffer for 8 min, long enough to saturate the sensor surface with a protein layer. After a buffer rinse, DNA samples of various concentrations (50 nM to 2.5 μM) were added in the desired buffer solution. Design and Synthesis of DNA Sequences. The DNA oligonucleotides used in the hybridization experiments were synthesized by FRIZ Biochem (Germany) at the 200 nmol scale and purified with high-performance liquid chromatography (HPLC). For the formation of straight DNA hybridization products, the following oligos were used as surfaceimmobilized probes: oligo21, 5′-biotin-tag AGC TCC CTT CAA TCC AAA-3′; oligo75, 5′-biotin−CCA CCA AAC GTT TCG GCG AGA AGC AGG CCA TTA TCG CCG GCA 1855



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TGG CGG CCG ACG CGC TGG GCT ACG TCT TGC TGG-3′; oligo110, 5′-biotin−CCA CCA AAC GTT TCG GCG AGA AGC AGG CCA TTA TCG CCG GCA TGG CGG CCG ACG CGC TGG GCT ACG TCT TGC TGG CGT TCG CGA CGC GAG GCT GGA TGG CCT TCC CCA TT3′. The complementary target strands of the above molecules were synthesized without the biotin label. For the production of a “Y-shape” dsDNA, the following target oligo was synthesized and applied to the surfaceimmobilized oligo21: oligo21“Y”, 5′-AAA CCT ATG AAG GGA GCT CTA-3′. For the detection of the SNP located in the TP53 cancer biomarker sequence the following oligonucleotides were also designed40 and synthesized by DNA Technology (Denmark): “long” hairpin (33 nts), 5′-GAG GTC ATG GTG GGG GCA GCG CCT CAC AAC CTC−biotin-3′ (Figure 3C); “short” hairpin (20 nts), 5′-GTT GTG CAG CGC CTC ACA AC− biotin-3′ (Figure 3F); “full” complementary (28 nts), 5′-GAG GTT GTG AGG CGC TGC CCC CAC CATG-3′; SNPcontaining mutant (28 nts), 5′-GAG GTT GTG AGG CAC TGC CCC CAC CATG-3′. The first two oligos were used as the surface-immobilized probes, whereas the last two were the TP53 target strands. The SNP is indicated in bold.

Figure 1. Schematic representation of the three different-length oligonucleotide probes in PBS (A) and Tris/Mg (C) buffer. The invaginations/hairpins in part A reflect possible conformations predicted by thermodynamic calculations (ref 81). The corresponding hybridization products upon interaction with each complementary target (red strand) in both buffers are also shown (B). The neutravidin protein layer is depicted (green) along with the biotinylated (first) probe strand (black).

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RESULTS Hybridization Studies of Straight DNAs in Different Buffer Solutions. In the first part of this work we have used the QCM-D technique to monitor the binding of ssDNA targets to their fully complementary probes under continuous flow and in the presence of two buffers. Three different oligonucleotides, i.e., oligo21, 75, and 110, were used as immobilized probes. Hybridization of the above oligos with their complementary strands is known to produce straight dsDNA products52 (Figure 1). DNA probes were biotinylated at the 5′-end in order to achieve oriented immobilization on a neutravidin-covered crystal surface. A neutravidin layer formation by physisorption on the gold electrode was verified by typically detecting ΔF ≈ −300 Hz and ΔD ≈ +1.3 × 10−6 (at the 35 MHz overtone), measurements in agreement with other works.53,55,56 In a typical experiment, the procedure comprises two steps; first, the immobilization of the biotinylated probe and then the addition of the complementary target (Figure 2). Buffer rinsing was always performed before and after each addition. In either step, frequency change represents mass accumulation,57 whereas the acoustic ratio (ΔD/ΔF) reflects on the conformation of the attached molecule. The acoustic ratio of the second step corresponds to the formation of the final duplex product. Addition of nonbiotinylated DNA to the neutravidin surface produced no signal change. All the above hybridizations were performed in the presence of monovalent (Na+) or divalent (Mg2+) cations, from the PBS and Tris/Mg buffers, respectively. On the basis of the fact that the acoustic ratio reflects the conformation of the formed product, we compared the acoustic ratio of in situ hybridized molecules with that of ex situ prehybridized or polymerase chain reaction (PCR)-produced dsDNA molecules. Experimental values for the acoustic ratio for 21, 75, and 110 base pairs were in agreement with recently published results53 and are summarized in Table 1.

Figure 2. Acoustic monitoring of two-step in situ hybridization; here, the first step (ΔD1, ΔF1) corresponds to the immobilization of 125 nM of oligo110 probe on the device surface followed by the binding of 50 nM of its complementary strand (ΔD2, ΔF2). Red and blue arrows indicate frequency and dissipation changes, respectively, during firstand second-strand addition. The initial neutravidin addition is omitted.

Hybridization Studies of Straight and Y-Shaped Molecules. The in situ hybridization of oligo21 with a target strand produced by altering the sequence of the fully complementary target 21 base strand was further investigated. In this new target (oligo21 “Y”) the final seven bases were changed to be identical to the bases of the first strand, therefore preventing the formation of hydrogen bonds in the upper part of the molecule and altering the structure of the final product from a straight (Figure 3A) into a “Y” form (Figure 3B). Measuring as previously the addition of the oligo21“Y”, in both buffers, we noticed that the new conformation was distinguishable from the full 21 bp structure through acoustic ratio comparisons. The corresponding acoustic ratios in Tris/Mg buffer are shown in Figure 3. Hybridization Studies Using DNA Hairpin Structures. Hairpin DNA structures (termed molecular beacons when a label is attached to them for optical or electrochemical detection) are commonly used to discriminate between intact and mutated sequences through hybridization.40,58 For QCM-D measurements we have used two hairpin sequences, a 33 base “long” DNA chain which contained two loops (Figure 3C) and 1856



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Table 1. Acoustic Ratiosa PBS buffer

Tris/Mg buffer

DNA length (bases)

first strand

second strand

first strand

second strand

preformed or PCR dsDNAb

21 75 110

166 ± 9 180 ± 19 213 ± 12

149 ± 11 225 ± 24 284 ± 17

77 ± 17 105 ± 18 123 ± 10

138 ± 16 210 ± 4 257 ± 6

138 ± 12 211 ± 33 272 ± 27

Acoustic ratio (ΔD/ΔF × 10−10/Hz) values measured for three different oligonucleotides and their respective duplexes (results from 3 to 13 experiments). bThe ratio of the preformed duplex is not affected by the type of the buffer solution; reported values are in agreement with Tsortos et al. (ref 53). a

Figure 4. Real-time monitoring of frequency and dissipation changes during addition of the “short” hairpin probe (0−25 min) and the TP53 targets (25−60 min). The red line corresponds to the addition of the full target, and the blue line corresponds to the mutant.

Figure 3. Suggested conformation of (A) a 21 bp long fully complementary dsDNA molecule and (B) the same molecule with 14 of the bases in a double-strand and 7 as single-strand extended noncomplementary overhangs (“Y” shape). Part C shows the 33 base “long” hairpin DNA probe, and parts D and E show the identical duplexes formed upon hybridization with either one of the 28 base TP53 targets; the five bases are in an extended protrusion form. (F) The 20 base “short” hairpin DNA probe and the produced duplexes with the 28 base TP53 “full” target (G) and mutant target (H). The (extended) overhangs in part G are 5 and 10 bases, and in part H they are 10 (extended) and 15 (contracted coil) bases long. The acoustic ratio (ΔD/ΔF × 10−10/Hz) is also shown for each structure measured in the Tris/Mg buffer. Mean values and standard deviations are from 3 to 10 measurements. The star * indicates the position of the mutation. All chain lengths are roughly drawn to scale.

formed.34−38 If two to five base pairs are able to form consecutively and create a stable nucleus, the addition of new base pairs leads to favorable negative contributions to the free energy and the duplex is able to rapidly complete the hybridization process and zipper the strands together.34,37 Although nucleation is considered as the rate-limiting step in solution-phase DNA hybridization of short strands, it was recently suggested16 that surface analytical methods such as QCM and SPR do not detect complete duplex hybridization but, instead, detect duplex nucleation or “initial target recognition”. In addition, voltammetry studies indicate that complete zippering of the duplex on the surface requires hours.16,40 Our data clearly suggest that this is not the case. We measured the acoustic ratio of three DNA molecules (21, 75, and 110 bp) produced by the addition of the second strand on the device surface during the two-step hybridization in two buffer solutions. We then compared these ratios to those obtained from the attachment under the same conditions of the same molecules, preformed by PCR or in vitro bulk mixing procedures, i.e., procedures that guarantee them to be doublestranded. The rational of the work is that if both experiments give the same acoustic ratio for the final products, then it can be arguably said that the DNA structures are the same. By the same token difference in the acoustic ratio for similar molecules is a strong indication of a different molecular conformation. Note that the criterion for distinguishing between identical and different acoustic ratios is set by the resolution capability of the system which, for these particular molecules, is ∼5−10%; thus, acoustic ratios differing by less than ∼10% are considered within experimental error identical, whereas differences larger that 10% indicate different ratios. Due to this threshold multiple measurements (≥3) are needed in order to get secure and meaningful data.

a 20 base “short” one containing only one loop (Figure 3F). Both probe sequences contained complementary regions to two target TP53 (28 base) gene-specific DNA sequences: one fully complementary and another with a hot-spot mutation of G to A (SNP at codon 175). This particular mutation is known to lead to cancer-triggering deactivation of tumor suppressor protein p53, responsible for cell cycle regulation.59 The two hairpin probes, in different experiments, were immobilized on a neutravidin layer, and each of the two TP53 targets were added in order to hybridize as in previous experiments. Measurements were made in Tris/Mg buffer, and the results are shown in Figure 4.

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DISCUSSION In order to develop solid-phase hybridization assays based on probing the structure of the DNA product, one should ensure that the binding of a target molecule to a surface-immobilized ssDNA probe results, indeed, in the final “correct” hybridized molecule, i.e., the fully hybridized structure produced spontaneously during solution-phase hybridization. It is generally accepted that nucleic acid hybridization is initiated by the formation of a nucleation complex in which stable intermolecular base pairing between complementary regions is 1857



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Figure 5. Acoustic ratio ΔD/ΔF dependency on the surface coverage. (A) Data are shown for the oligo75 probe in three different buffer systems: (red triangles) in Tris/1 M NaCl, (black triangles) in Tris/Mg, and (open circles) in PBS. Note that ∼270 Hz corresponds to ∼100% coverage. (B) Data for the in situ hybridization of the oligo75 at different surface coverages with 1 μM of its fully complementary second strand in two buffer systems: (black triangles) in Tris/Mg and (open circles) in PBS. All the actual (constant) values for the ratios are presented in Table 1.

hybridization values (“second strand” columns in Table 1). This finding is consistent with biophysical studies regarding DNA structure: even though multivalent cations are capable of softening and bending dsDNA66−68 and even collapsing it on the surface in the extreme cases,65,69 they do not have a big influence on the persistence length P.68,70,71 The stiff dsDNA with a Pds of about 50 nm (∼150 bp) is not greatly affected, whereas a ssDNA, having a Pss value around 1.0−2.4 nm72−74 can much more easily be bent and contract; even in high ionic strength solutions Pds is ∼10-fold higher than Pss. From microscopy and other studies it is well-established that the elongation per base in dsDNA is b ≈ 0.34 nm, whereas that for ssDNA varies anywhere from ∼0.275 to 0.461,72,76 or even ∼0.6 nm.74,77,78 A choice of values Pss = 2 nm and b = 0.4 then gives approximately five bases that can be thought of as a “stiff” segment on a ssDNA chain. Use of the above-mentioned choice for Pss and b makes the stretched-out 21 base ssDNA (in PBS) a little longer than the corresponding double-stranded molecule which justifies the (marginally) higher observed acoustic ratio (166 vs 149 × 10−10/Hz) shown in Table 1. Having established that the structure of the surfacehybridized molecules is identical to the fully hybridized products obtained in solution, the effect of surface coverage, i.e., the packing density and proximity of the probe strands to each other, was investigated. As mentioned in the introduction this parameter is of primary concern for the efficient design of DNA microarrays.10,11 We conducted experiments in three different buffers using many different loading concentrations of DNA on the sensor surface. Regarding the first step and the binding of the oligo75 probe, Figure 5A shows that the acoustic ratio of this molecule is independent of the coverage for the range between 10% and ∼95% (corresponding to probe concentrations between 50 nM and 2.5 μM). Figure 5B presents the results this time for the acoustic ratio of the same concentration (1 μM) of the fully complementary second strand when it binds to surfaces with different coverages of the oligo75 probe; again no dependency on probe surface coverage is observed. Similar conclusions were drawn in the case of preformed dsDNA molecules at the surface of both QCM-D and SAW devices.52,53 These findings are indeed in accord with our discrete molecule binding theory52 which predicts the acoustic ratio to be characteristic of the intrinsic viscosity [η] of a molecule, i.e., its conformation; as long as molecular shape is

In PBS solution the 21oligo (Figure 1A) is in an extended conformation60−62 having no self-complementarity in any long parts of its sequence. Contrary to this, the 75 and 110 base molecules are designed to have the potential of forming extended structures but with the possibility to contain small but significant hairpin parts in different locations of their sequences due to built-in self-complementary regions (Figure 1A). We attached the same oligos in the presence of Mg2+ which results in rather compact, more collapsed on the surface ssDNAs (Figure 1C) for all the examined structures.63−65 Note that the effect of magnesium on the structure of ssDNA could also be obtained by use of the Tris buffer solution containing 1 M NaCl (see also Figure 5A). Expected differences in the structure of the single-strand probes in the two buffers are indeed reflected in the measured acoustic ratios (Table 1); first we observe that acoustic ratios increase with the length of the oligonucleotide in both buffers as expected from the theory52 since larger, more protruding molecules are in general more dissipative. Second, for the same ssDNAs, all ratios in Tris/Mg are significantly lower than in PBS, reflecting the rather contracted more compact and less dissipative forms of the chains in Tris/Mg as opposed to the more extended and protruding ones in PBS. Following the addition of the complementary second strand in both buffers the corresponding acoustic ratio represents the transition of the ssDNA probe to the hybridized product (Figure 1B). The data in Table 1 strongly suggest that regardless of the structure of the ssDNA probe or method used to produce the double-stranded products, i.e., during the two-step hybridization procedure on the device surface or by simple loading of the sensor with prehybridized molecules, the resulting acoustic ratios are, within experimental error, the same and in full agreement with those predicted and measured in previous work for the 21, 75, and 110 bp extended straight duplexes using the discrete molecules binding approach.53 Focusing in Table 1, we notice the following: although the single-strand probes give different acoustic ratios when divalent cations (or very high concentration of monovalent ones) are present in the buffer solution instead of lower concentration of monovalent cations, the double-stranded products are not similarly affected. Preformed dsDNAs have identical acoustic ratios both in PBS- and in Mg2+-containing solutions (see last column in Table 1), and the same is true for the in situ 1858



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experiments of the two TP53 targets with the “short” hairpin DNA probe are presented. In both experiments the hairpin probe was first immobilized on the sensor surface, resulting in similar frequency changes (∼60 Hz), followed by addition of either the full or the mutated TP53 targets. In both cases, frequency changes were approximately 10 Hz, which proves that it is impossible to distinguish the SNP during targets addition through mass monitoring; dissipation changes were what made the difference. Moreover, the kinetics of these events were analyzed. One way to gain an appreciation of how fast all these processes occur is to look at the time scale of the acoustic experiments. It is apparent from Figures 2 and 4 that both of the measured signals, frequency and energy dissipation, equilibrate within a 5−10 min period after the addition of the second, complementary strand; this means that the acoustic ratio also stabilizes within this 10 min time period. Given the previous analysis this suggests that hybridization is complete within these few minutes at the surface of our sensor and this is true for all DNA systems presented here. Let us emphasize again that the term “hybridization” here means obtaining the “final” equilibrium shape of two strands coming together. We have offered ample experimental evidence that this shape is indeed not a “nucleation”, recognition, temporary structure, but the complete equilibrium structure. Finally, the following comments can be made in relation to Figure 3 and the conformation of molecules B, G, and H. From the acoustic ratio data, when one compares the values of the acoustic ratios corresponding to structures B and G these appear to be the same (169, 174 × 10−10/Hz) suggesting that when (both) overhang arms are 5, 7, and 10 bases long the ratio increases compared to A (138 × 10−10/Hz). This means that B and G are similar and more open structures than A. On the other hand, H gives a ratio (122 × 10−10/Hz) smaller than G (174 × 10−10/Hz) (and even than A); this requires that H is more compact than G. Given that G and H have identical structures for about half of the molecule configuration, it is an inescapable conclusion that the difference is due to the different structures of the overhangs, i.e., that the 15 base overhang in H is in a coiled up, rather contracted formation, and this is how it is drawn. Figure 1 drawings further exemplify this understanding of the situation. Our data here, like the results from another DNA structure, the Holliday junction,51 seem to be consistent in suggesting that strands with ∼8−10 (or fewer) bases are rather extended in shape and that more than 10 appear coiled up and contracted in the presence of magnesium ions. Such length-dependent transitions have also been suggested/observed by other researchers.64

not affected by the conditions and processes occurring at the surface then this ratio should remain constant and independent of surface coverage. The very significant practical implication of these observations is that one should not be greatly concerned with the parameter of surface coverage when probing molecular conformation by acoustic sensors in hybridization assays. In addition, since the acoustic detection principle is independent of the mass of the bound strand, the minimum amount of target DNA required for producing a detectable signal can be used and for multiple sample additions on the same surface (until surface saturation occurs). These observations together with the label-free nature of the acoustic assay point to simpler and more cost-effective hybridization assays. What happens if one needs to detect the binding of not fully complementary strands? With a system that relies only on mass detection (or, in general, the quantity) it becomes problematic. For example, if one tries to decide on the Y-form molecule depicted in Figure 3B it will be impossible to discriminate it from the fully matched duplex (Figure 3A); similar mass doubling would be detected. Our approach takes advantage of the extra information on conformation provided by the acoustic ratio; from the data given in Figure 3 it can be seen that molecules A and B, although identical in mass, can be distinguished based on their different shapes. The straight dsDNA produces a different acoustic ratio from the “Y-shaped” molecule (138 vs 169 × 10−10/Hz), and the question of complementarity can be answered. The above findings suggest that SNP detection should be feasible if a hybridization assay employed a carefully designed probe DNA. According to Figure 3, if one needs to detect a point mutation using the “long” hairpin DNA probe 3C (Figure 3), this is not possible; the resulting structures 3D and 3E (Figure 3) have identical mass and, based on the acoustic ratio (128 vs 133 × 10−10/Hz), the same shape. As follows from these data and thermodynamic calculations40 the mutation has a very little if any effect on the form of the produced duplex. On the other hand, hybridization of the “short” hairpin DNA probe 3F (Figure 3) to its fully matched and SNP-containing complementary target DNA results in structures 3G and 3H (Figure 3), respectively, producing completely different acoustic ratios (174 vs 122 × 10−10/Hz). Our results are fully consistent with those reported by Farjami et al. in their recent electrochemical study of TP53-specific hairpin molecular beacons,40 where it was demonstrated that the short DNA beacon probe used in the design of the “off−on” genosensor system is highly selective for SNP analysis and that the response of the developed genosensor primarily depends on the length of the produced hybrid. Besides the important finding that SNP detection is feasible these data also provide additional information on the “nucleation” question. The results of the “short” hairpin DNA experiments support the notion that the molecular event observed is not a mere recognition step but full hybridization. A simple recognition event would have a really small chance of being affected by a single mutation; the resulting structures (Figure 3, parts G and H) would be identical, and this would be reflected in the (same) acoustic ratio (unless the position of the mutation is right on the “nucleation” site, of course). Again, here the presence of SNP would be impossible to detect by simply monitoring the mass accumulation during the addition of the target oligonucleotide to the immobilized probe. The advantage of conformation probing versus mass detection is clearly shown in Figure 4 where two separate hybridization

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CONCLUSIONS Analysis of mass changes does not always provide the much needed answer on whether hybridization occurs in a detection scheme. Our results prove that conformation probing provides a simple and much more improved methodology also offering insights on the mechanisms involved in molecular recognition and binding. Furthermore, analyzing the acoustic ratio by using the “discrete molecule binding” approach offers advantages over the film-based models since the former can confirm the length of straight double-stranded DNA molecules and even allows prediction of the conformation of single-stranded DNA overhangs. This study suggests a paradigmatic change in solidphase DNA analysis during hybridization studies, exploiting label-free conformation-sensitive acoustic techniques. It also 1859



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paves the way for the development of highly sensitive acoustic detection platforms for aptamer/ligand and DNA/drug analysis, two areas primarily concerned with structural changes of DNA induced by the binding of small molecules, as well as protein−protein recognition. Toward this direction it is anticipated that the application of integrated SAW devices may be proven quite advantageous.79,80

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected].

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ACKNOWLEDGMENTS The authors thank ELKE−University of Crete, Grant No. KA 3123, EU Grant ProSA (MRTN-CT-2005-019475), the IMBBFO.R.T.H, and the Danish National Research Foundation for their support to the Center for DNA Nanotechnology (CDNA).

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Direct Detection of DNA Conformation in Hybridization Processes

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