Thymine/adenine diblock-oligonucleotide monolayers and hybrid brushes on gold: a spectroscopic study
© Howell et al.; licensee Springer. 2013
Received: 14 December 2012
Accepted: 13 February 2013
Published: 27 February 2013
The establishment of spectroscopic analysis techniques for complex, surface-bound biological systems is an important step toward the further application of these powerful experimental tools to new questions in biology and medicine.
We use a combination of the complementary spectroscopic techniques of X-ray photoelectron spectroscopy, Infrared reflection-absorption spectroscopy, and near-edge x-ray absorption fine structure spectroscopy to monitor the composition and molecular orientation in adenine/thymine diblock oligonucleotide films and their hybridized brushes on gold.
We demonstrate that the surface-bound probe molecules, consisting of a binding adenine block, d(A), and a sensing thymine block, d(T), deviate from the ideal L-shape model due to the internal intra- and intermolecular hybridization. This effect becomes more pronounced with increasing length of the d(A) block. Nevertheless, these films were found to hybridize well with the complementary target d(A) strands, especially if they were treated in advance to reduce internal interaction between the molecules. In spite of the structural complexity of these films, the hybridization efficiency correlated well with the potential accessibility of the sensing d(T) blocks, defined by their lateral spacing.
These findings are a good demonstration of the strength of multi-technique spectroscopic analysis when applied to assemblies of biological molecules intrinsically prone to complex interactions.
Understanding the properties of and processes involving biological molecules on surfaces and interfaces is at the heart of many critical issues in biology and medicine [1–3]. The organization and hybridization of surface-bound DNA in particular is an important component in many established, new, and emerging medical and other biology-based technologies, including microarrays [3–5], DNA-decorated nanoparticles [6, 7], and DNA computing [8, 9]. In systems such as these, the tethering of the DNA strands to a solid surface is necessary for greater control over density, orientation, and hybridization processes. Nevertheless, precise measurement or reconstruction of these parameters and processes through conventional means such as microscopy and fluorescent labeling  or surface plasmon resonance (SPR) spectroscopy [11, 12] can be challenging due to the nanoscale nature of the DNA molecules involved as well as the non-specific character of these techniques.
Different types of spectroscopy have been shown to be useful for such nanoscale analysis, as they inherently give specific, label-free, and molecular-level information. Techniques such as X-ray photoelectron spectroscopy (XPS) [13, 14], near-edge X-ray absorption fine structure (NEXAFS) spectroscopy [15–18], time-of-flight secondary ion mass spectrometry (ToF-SIMS) [19–21], sum-frequency-generation (SFG) spectroscopy [22, 23], and infrared reflection-absorption spectroscopy (IRRAS) [24, 25] can be used to determine composition, content, molecular orientation and hybridization behavior of DNA assemblies. Yet despite the large amounts of potential information obtainable through use of these methods, the interpretation of spectra from biological samples, including surface-bound DNA, can be difficult, often resulting in the restriction of the use of these techniques to simplified or relatively straightforward systems. In order to expand the use of spectroscopic methods, comparative studies on biological molecules capable of acting in increasingly complex ways are needed.
Here, we use the abovementioned diblock-oligonucleotides to systematically increase the degree of complexity in DNA hybrid brushes. Using a combination of high-resolution XPS, IRRAS, and NEXAFS spectroscopy to gather complimentary information on molecular density, content, and orientation, we present a molecular-level picture of the effects of increasing complexity on these parameters. We find that as the DNA hybrid systems become capable of more complex interactions the predictability of orientation becomes increasingly difficult, even as the molecular content continues to change as expected. These results highlight the importance of using multiple spectroscopic techniques to gain a complete picture of parameters of and processes involving biological systems on the molecular scale, especially as the level of complexity increases.
Gold substrates were produced by using radio frequency magnetron sputtering to coat silicon wafers (Si-Mat Silicon Materials, Kaufering, Germany) with a chromium adhesion later, followed by a gold layer. The result was a 100 nm-thick polycrystalline gold film with a predominant (111) orientation. The coated wafers were cut into appropriate sizes, cleaned by exposure to UV/ozone (UV cleaner 42–220, Jelight) for 2.5 hrs, and rinsed thoroughly with HPLC-grade water immediately prior to film deposition.
High performance liquid chromatography-purified DNA sequences were purchased from Sigma–Genosys (Steinheim, Germany): 5’-(dA)5-(dT)15-3’, 5’-(dA)10-(dT)15-3’, 5’-(dA)15-(dT)15-3’, probe sequences, as well as a complimentary (dA)15 target. These will be referred to hereafter as A5T15, A10T15, A15T15, and A15, respectively (see Scheme 1). Block-DNA containing only adenine and thymine was chosen over block-DNA containing a random sequence of all four nucleobase types in order to facilitate interpretation of the spectra. The Tris–HCl, EDTA, and NaCl used in the deposition and hybridization buffers were obtained from Sigma-Aldrich (Seelze, Germany).
2.2 DNA Immobilization and hybridization
DNA brushes were formed by incubating gold wafers for 40 h at 37°C in 1 M CaCl2–TE buffer (10 mM Tris–HCl and 1 mM EDTA, pH=7.08) containing a 3 μM DNA solution. After incubation, unhybridized samples were rinsed for 1 min under flowing deionized water (resistivity >18.2 MΩ cm) to remove excess DNA and dried with N2.
Samples intended for hybridization were rinsed for either one full minute under deionized water or for 30 seconds under deionized water, followed by 30 seconds under flowing 0.1 M NaOH, followed again by 30 seconds under deionized water. The NaOH rinse was added in an effort to destabilize any partial inter-molecular hybrids and improve overall hybridization and orientation . After rinsing, these samples were placed in a 1M NaCl buffer containing 3 μM of the (dA)15 target sequence for 7 h at room temperature. Following incubation, samples were rinsed with 1 M NaCl buffer for 1 min, briefly dipped in deionized water to remove excess salts , and finally dried under flowing N2 according to previously described procedures . It should be noted that exposing surface-bound DNA hybrids to pure water, while necessary to prepare them for HRXPS and NEXAFS spectroscopy measurements, is known to affect their stability [11, 26]. However, a recent systematic study on the subject has shown that when the samples are rinsed with small volumes of water (~0.5 mL), such as those used here, breakup of such hybrids is minimal .
2.3 High Resolution X-ray Photoelectron Spectroscopy
HRXPS measurements were performed at the HE-SGM beamline of the synchrotron storage ring BESSY II in Berlin, Germany. The measurements were conducted under UHV conditions at a base pressure better than 1.5 × 10–9 mbar and at room temperature. The spectral acquisition time was selected such that no noticeable damage by the primary X-rays occurred during the measurements [27–30]. The acquisition of the spectra was carried out in normal emission geometry with an energy resolution of ~0.4 eV. The energy scale was referenced to the Au 4f7/2 peak at 83.95 eV .
The spectra were fitted with symmetric Voigt functions and either a Shirley or linear background. The fits were carried out self-consistently, i.e. the same peak parameters were used for identical spectral regions. The film thickness was determined on the basis of the intensity ratios of the C 1s and Au 4f emissions , assuming a standard exponential attenuation of the photoelectron signal and using the attenuation lengths reported by Lamont and Wilkes .
The thickness was then used to calculate the density of the surface-adsorbed DNA molecules in the A5T15/Au film according to the procedures developed by Petrovykh et al. [13, 14]. Due to minor carbon contamination in the A10T15/Au sample, the density values for this and the A15T15/Au films were calculated on the basis of the averaged intensity of the characteristic N 1s, P 2p, and O 1s signals and corrected for molecular composition. Density values of 1.40 × 1013, 1.02 × 1013, and 8.96 × 1012 molecules cm-2 were obtained using this method for the A5T15/Au, A10T15/Au, and A15T15/Au films, respectively. These values are comparable to those reported for similar films , although somewhat lower. This is most likely due to the fact that the C1s/Au4f intensity ratio was used to calculate thickness rather than the intensity of the Au 4f emission from sputter-cleaned gold [13, 14].
2.4 Infrared reflection-adsorption spectroscopy
IRRAS was performed on a Vertex 80 Infrared spectrometer (Bruker Optics, Ettlingen, Germany) using an A518 IRRAS sampling stage with an 80° fixed-angle gracing incidence. The signal was recorded by a D313 narrow-band MCT detector cooled by liquid N2. A gold substrate covered with a perdeuterated dodecanethiol self-assembled monolayer was used as a reference. Background measurements were performed with 1024 scans, and suppression of the water signal in samples was usually achieved between 1000 and 3000 scans. Baseline correction and data evaluation were performed using the Opus 6.5 IR software package (Bruker Optics).
2.5 Near-edge X-ray absorption fine structure spectroscopy
NEXAFS and HRXPS spectra were collected at the same beamline. The acquisition of the NEXAFS spectra was performed at the C, N, and O K-edges in the partial electron yield (PEY) mode with retarding voltages of −150, –300, and −350 V, respectively. Linear polarized synchrotron light with a polarization factor of ~0.91 was used. The energy resolution of the whole setup was estimated to be on the order of ~0.3 eV, slightly dependent on the photon energy. To monitor the orientational order in the films, the incidence angle of the light was varied from 90° (E-vector in the surface plane) to 20° (E- vector near the surface normal) in steps of 10-20° (the angles are defined with respect to the surface plane) . This approach is based on the dependence of the cross-section of the resonant photoexcitation process on the orientation of the electric field vector of the synchrotron light with respect to the molecular orbital of interest (so-called linear dichroism in X-ray absorption) . This effect results in a characteristic dependence of an adsorption resonance intensity on the incidence angle of X-rays, as long as there is some orientational order present in the probed system. The photon energy (PE) scale was referenced to the pronounced π* resonance of highly oriented pyrolytic graphite at 285.38 eV .
3 Results and discussion
3.1 Unhybridized DNA brushes
Beyond the chemical composition, information on average molecular orientation can be obtained from NEXAFS data by considering the angular dependence of the resonance intensity . A suitable way to monitor such dependence is to plot the spectra acquired at the normal and gracing incidence angles of the X-ray beam. The orientational order present in the probed system will then manifest itself as difference peaks at the positions of the characteristic absorption resonances. The sign and intensity of these peaks will then provide information on the exact molecular geometry and degree of the orientational order. To this end, the difference spectra of the A n T15 films and derived brushes of DNA hybrids were considered in detail.
Generally, the nucleobases are assumed to be oriented perpendicularly to the ssDNA backbone, even though they possess some flexibility in orientation due to rotation relative to the backbone and from irregular bends in the backbone. But even taking into account this flexibility, a very strong positive peak would arise in the difference NEXAFS spectra from a film in which the DNA strands are parallel to the surface (nucleobases would then be predominantly oriented perpendicular to the substrate), and a very strong negative peak would be seen when all DNA molecules are oriented perpendicular to the surface (the nucleobases would then be predominantly oriented parallel to the substrate) [15, 17, 38]. In the case of ideal L-shape diblock-oligonucleotides [24, 36], we would therefore expect to see a very strong positive peak from the parallel surface-binding adenine block and a very strong negative peak from the perpendicular sensing thymine block. In the case of deviations from the ideal parallel or perpendicular alignment, as presumably occurs in a real diblock-oligonucleotide system , we would expect to see less intense (but still clearly visible) positive adenine peaks and negative thymine peaks. In the ultimate case of partial or complete disordering of either the d(A) or d(T) blocks, no respective difference peaks would be perceptible.
The right-hand panel of Figure 3 shows the difference between the spectra of the A n T15 films collected at 90˚ and 20˚. The A5T15 film shows a strong positive adenine peak and a very weak negative thymine peak indicating, as expected, a predominantly parallel-to-the-substrate alignment of the adenine blocks of the DNA strands. Unexpectedly, however, this also indicates a near lack of any sort of orientational order in the thymine blocks. The most probable reason for this behavior is the interaction of the d(T) blocks with the surface-bound d(A) nucleotides , which can be explained by partial intramolecular and intermolecular hybridization. These hybrids could have formed either in solution prior to deposition, or after contact with the substrate. More sophisticated explanations are possible as well, including complex hybridization scenarios involving several molecules [26, 39, 40]. However, which of these configurations is dominant is not something that can be directly determined via NEXAFS spectroscopy but, presumably, requires its combination with theoretical calculation of most energetically favorable structural motifs.
The A10T15 film, in contrast, shows a predominantly perpendicular alignment of the thymine block as well as a predominantly parallel orientation of the adenine block. However, the intensities of the difference peaks are small, indicating that the extent of nucleobase alignment is small as well. As the A10T15 molecules have the potential to form twice the number of base pairs compared to the A5T15 molecules, it would be expected that any intermolecular hybrids formed in solution prior to deposition would be more stable. This may be one explanation for the results seen here, as a greater number of intermolecular hybrids would result in a smaller overall parallel ordering of the adenine nucleotides due to the presence of d(A) blocks with perpendicular orientation. In turn, the greater stability of these remaining inter- and intramolecular hybrids, compared to those of the A5T15 molecules, would result in a more stable perpendicular order in the thymine nucleotides . However, the still relatively low maximum number of base pairs overall would likely mean that these types of intermolecular hybrids are not very stable [41, 42] and thus unlikely to be the most common species in the A10T15/Au film. This would explain the continued dominance of the parallel orientation in the adenine nucleotides in this sample seen in the NEXAFS data. Note that the molecular orientations implied by the 90˚–20˚ difference spectra in Figure 3 for the A5T15/Au and A10T15/Au films are in full agreement with previous NEXAFS results .
The d(A) and d(T) blocks of the A15T15 molecules, in contrast to the A5T15 and A10T15 species, are fully complimentary in length, which should create the most stable inter- and intramolecular hybrids in solution. The most stable configuration of these molecules should be the full 30-base pair double helix formed from aligning two A15T15 strands. Upon exposure to the gold surface and subsequent rinsing and drying, the NEXAFS data reveal that both the adenine and thymine portions of these molecules are preferentially oriented perpendicular to the surface, suggesting that a large portion of the thymine blocks and at least a part of the adenine blocks are remaining upright. One molecular configuration which would be consistent with these data would be the attachment of the DNA molecules in complete hybrid form via only a portion of the d(A) block. Other arrangements could also exist, including an L-shaped configuration in which a second molecule is hybridized only with the d(T) portion, however the intensity of the adenine peak in the NEXAFS 90˚–20˚ data suggests that on average in this film more d(A) blocks are oriented upright than lying flat, in clear contrast to the A5T15/Au and A10T15/Au layers.
Summarizing, the HRXPS, IRRAS, and NEXAFS composition data give expected results for the unhybridized A n T15 films, confirming their quality and composition compared to previously tested films of this type . The NEXAFS results presented here support the theory of complex hybridization interactions affecting these layers, as the simplest molecular arrangements necessary to produce the observed spectra become more complex from the A5T15/Au, to A10T15/Au, to the A15T15/Au film.
3.2 DNA hybrid brushes
Hybridization was performed using two approaches: first, by rinsing the initial unhybridized films with only pure water, and second, by rinsing the films with water, followed by a 0.1 M solution of NaOH, then water a final time. The latter approach was intended to further destabilize any inter- or intra-molecular hybrids in the probe film with the aim of improving both the number and quality of the hybrids formed upon exposure to the complimentary A15 target .
Beyond this qualitative conclusion, it is possible to perform a numerical analysis of the intensity values in Figure 4 to get at least a coarse estimate of the degree of hybridization. Normalizing the intensities to the number of nitrogen atoms in adenine (5) and thymine (2) and assuming the same attenuation of the photoemission signal for the d(A) anchor of the A n T15 probe species (a factor of 2.5; see above), we obtained the following values for the extent of hybridization in the H2O-rinsed A n T15/Au:A15 brushes: 24%, 46%, and 87% for n = 5, 10, and 15, respectively. For the films rinsed with alternating H2O and NaOH, these values were calculated to be 39%, 57%, and ~100% for n = 5, 10, and 15, respectively. Note that these values can be considered as coarse estimates only, since the attenuation factor for the photoemission signal from the d(A) anchor of the A n T15 probe can change after the hybridization event. It is most likely that this factor would decrease as the inter- and intramolecular (probe) hybrids are disrupted and upright target-probe hybrids are formed, resulting in a partial uncovering of the d(A) anchors. Thus, the values of the hybridization efficiency calculated here are presumably the upper limits of the real values. Note also that these values represent coarse estimates only since they are based on a single sample for each particular system. However, qualitatively similar differences between the outcomes of the H2O and H2O-NaOH rinsing procedures were observed for all A n T15 samples, which strengthens the reliability of the conclusions.
Apart from this uncertainty, two tendencies are apparent. First, the H2O-NaOH rinsing procedure results in a greater number of hybrids than the H2O rinse alone. Second, the number of hybrids in the A n T15 films increases with increasing length of the d(A) block. This is expected, as longer d(A) blocks increase the potential for a larger separation between the sensing d(T) blocks and thus greater accessibility for the target strands.
In the case of the A15T15, we expect that in its as-deposited state this film has a rather high degree of intramolecular hybridization. However, any molecules that remain after the post-deposition 1 min rinsing with pure water would have to be either at least partially attached to the surface via the d(A) block or hybridized to a surface-bound molecule. Previous work examining the stability of surface-bound DNA hybrids has shown them to be quite unstable and readily broken apart and replaced by a fully complimentary, non-surface-bound target molecule , although it should be noted that those hybrids were not formed first in solution. In diblock-oligonucleotide system used here, the lowest energy state will be with the d(A) portion of the molecule fully adsorbed to the gold substrate and the d(T) portion fully hybridized. Thus, it is expected that the introduction of the A15 targets would break apart most of the inter or intra-molecular hybrids in the A15T15 layer.
Hybridization of the A10T15/Au film also causes a previously positive adenine-assigned difference peak (Figure 3) to become negative, indicating a change in the orientation of the majority of the adenine strands from parallel to perpendicular to the surface after hybridization. This behavior also agrees qualitatively with the idealized model of the A10T15/Au:A15 hybrids (Scheme 1), assuming that the ideal L-shape is significantly distorted. Also in this case, the spectral contribution of the perpendicularly-oriented A15 probes is clearly higher than that of the parallel A10 anchors. This is similar to what was observed for the A5T15/Au:A15 film, and can likely be explained by the same reasoning. However, in contrast to A5T15/Au:A15, there is no significant change in either the orientation or intensity of these peaks between the H2O-rinsed samples and those subjected to the H2O-NaOH rinsing procedure. As previously mentioned, this may be due to the relative instability of hybrids with few base pairs [26, 40]. The few intermolecular hybrids that were present in the film after rinsing but before hybridization may have been unstable enough to be replaced by a more energetically stable A10T15:A15 hybrid, regardless of whether the brush was exposed to NaOH or only H2O.
Both of the hybridized A15T15/Au samples show negative adenine- and thymine-assigned difference peaks, as was the case in the unhybridized layer. However, the dichroism becomes more pronounced after hybridization, which is especially obvious for the adenine-assigned difference peak. This increase in the dichroism can be associated with the contribution of the perpendicularly-oriented A15 targets, which have a particularly high spectral weight due to the high degree of hybridization (see above). The ideal L-shape model (Scheme 1) is likely also applicable in this case, at least qualitatively (under the assumption of distortion), given the high degree of hybridization. However, the comparably large spacing between the individual hybrids should diminish the orientational order, as usually occurs for loosely packed molecular films. This effect should be less pronounced in A10T15/Au:A15 and least pronounced in A5T15/Au:A15. Indeed, the latter film exhibits the highest extent of dichroism as compared to the two others, especially in the case of the film rinsed with alternating H2O and NaOH.
Returning to A15T15/Au:A15 and comparing the difference spectra for the cases of H2O and H2O-NaOH rinsing procedures, we can state that the addition of NaOH only slightly improves the orientational order in the hybrid films. Presumably, as mentioned above, the orientational order is limited by the low packing density and the improvement is solely related to the higher extent of hybridization in the case of the film treated with the H2O-NaOH rinsing procedure.
We have examined a series of adenine/thymine diblock-oligonucleotide films and their hybrid brushes using a combination of HRXPS, IRRAS, and NEXAFS spectroscopy to monitor sample composition as well as molecular orientation and ordering. As probe films, we used adenine-thymine diblock oligonucleotides composed of either 5, 10, or 15 adenine nucleotides [d(A)] connected to 15 thymine nucleotides [d(T)]. The quality and composition of these films was confirmed by the HRXPS, IRRAS, and NEXAFS composition data, while the binding function of the adenine block was confirmed by the HRXPS data. It was found that the orientational order in these films differed significantly from the ideal L-shape model, in which the binding d(A) block is oriented strictly parallel to the substrate while the d(T) portion of the molecule extends away and can be used to form hybrids with a complimentary target. In reality, the situation appeared to be more complex, a fact which was explained by inter- and intramolecular hybridization within the monomolecular assemblies. Deviations from the ideal model were observed in all probe films of the present study but became more pronounced with increasing length of the d(A) binding block and subsequently increased potential for stable inter- and intramolecular hybridization.
In spite of the complex molecular arrangements, all probe films performed well upon hybridization with the A15 target, exhibiting the expected correlation between the lateral density (i.e. accessibility) of the probe T15 strands and the extent of hybridization. Whereas this extent was relatively low for the most densely packed A5T15 films, it was found to increase the A10T15 samples, and was highest in the most loosely packed A15T15 monolayers. Furthermore, it was observed that the probe films rinsed with alternating H2O and NaOH exhibited higher hybridization efficiency than the films rinsed with H2O only. This confirms the ability of NaOH to reduce the number of surface-bound inter- and intramolecular hybrids, favoring more efficient hybridization.
The NEXAFS data for the hybrid A n T15/Au:A15 brushes do not contradict the ideal L-shape model but do not unequivocally support it. One can only say for sure that the T15:A15 hybrids have the expected predominantly upright orientation, with the highest orientational order present in the most densely packed A5T15/Au:A15 brush. It is, however, difficult if not impossible to monitor the orientation of the binding A n blocks since their contributions to the spectra are masked by those of the of the A15 targets. However, a parallel-to-the-substrate orientation of the binding blocks can be assumed to be most likely in view of the extensive disruption of the inter- and intramolecular hybrids in the probe film upon rinsing and subsequent hybridization.
These results are a good illustration of the strength of spectroscopic analysis in its specific application to biological systems, demonstrating that the use of different techniques to observe multiple facets of the system can often reveal unexpected effects. In some cases, such effects may prove to be key factors in understanding biological properties and processes on surfaces and interfaces at the molecular level.
We thank Prof. M. Grunze and Dr. P. Koelsch for support of this work, A. Nefedov and C. Wöll for the technical cooperation at BESSY II, the BESSY II staff for technical assistance during the experiments at the synchrotron, Dr. M. Bruns and V. Oberst for gold wafer fabrication, and S. Heissler for IR spectroscopy assistance. C.H. acknowledges support from a U.S. National Science Foundation Graduate Research Fellowship Award No. 0840595. This work has been supported by Deutsche Forschungsgemeinschaft (DFG) (Grants ZH 63/9-3 and ZH 63/14-1) and the Biointerfaces Programme.
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