Orientation and Ordering in Sequence- and Length-Mismatched

Apr 26, 2012 - The effects of target sequence mismatches and target length mismatches on the orientation and ordering of surface-bound, poly(dT)·poly...
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Orientation and Ordering in Sequence- and Length-Mismatched Surface-Bound DNA Hybrids Caitlin Howell,†,‡ Yekkoni Lakshmanan Jeyachandran,‡ Patrick Koelsch,†,‡,§ and Michael Zharnikov*,‡ †

Institute of Toxicology and Genetics, Karlsruhe Institute of Technology, Hermann-von-Helmholtz-Platz 1, 76344 Eggenstein-Leopoldshafen, Germany ‡ Applied Physical Chemistry, University of Heidelberg, Im Neuenheimer Feld 253, 69120 Heidelberg, Germany ABSTRACT: The effects of target sequence mismatches and target length mismatches on the orientation and ordering of surface-bound, poly(dT)·poly(dA) hybrids on gold were investigated using a combination of high-resolution X-ray photoelectron spectroscopy and near-edge X-ray absorption fine structure spectroscopy. It was found that the overall orientation of the probe and target strands remained upright and relatively unchanged despite the presence of up to four sequence mismatches in the target strands, although the total number of hybrids decreased with higher numbers of mismatches. Upright probes were also observed in hybrids formed with targets of shorter length, with a greater number of hybrids present in these films as compared to those formed with longer targets. These results suggest that in DNA films of relatively high densities, such as those tested here, the presence of sequence or length mismatches in target strands does not result in a large disruption of orientation and ordering in surface-bound hybrids. This finding may be useful for those seeking to maintain consistent DNA strand orientation for precise nanotechnological applications or those using DNA microarrays for biosensing purposes.

1. INTRODUCTION The formation of DNA hybrids is an important component in many established, new, and emerging biotechnological systems, including DNA-decorated nanoparticles,1,2 DNA computing,3,4 and the directing of cell adhesion using DNA microarrays.5−7 In these, the tethering of the DNA strands to a solid surface is often crucial for the precise control of the hybridization process and, in many cases, the subsequent positioning of the attached moiety. Whereas the hybridization process can be monitored by different techniques such as surface plasmon resonance (SPR) or quartz crystal microbalance (QCM), the direct detection of orientation and order in surface-bound DNA hybrids can be challenging. Nevertheless, this is an important step toward controlling and manipulating their formation. The effects of target sequence and length mismatches on these parameters is a particularly interesting question as DNA target strands in reallife applications may not always be perfectly matched to their probes. To date, most studies on this topic have involved in situ kinetic measurements examining the hybridization rates of surface-bound DNA.8,9 These works have revealed that hybridization can be significantly affected by the presence of sequence mismatches as well as differences in length between the probe and target strands. In particular, Peterson et al.8 examined the effects on the presence of none, one, or two mismatches in a 25-base sequence on the hybridization kinetics of surface-bound DNA and found that the presence of a single or double internal sequence mismatch dramatically altered these kinetics, in contrast to predictions based on standard models of hybrid formation. Shamsi and Kraatz10 reported on the electrical impedance of surface-bound DNA hybridized with targets of different lengths and found a lower charge transfer in © 2012 American Chemical Society

hybrids with short targets hybridized near the top of the probe strand. These authors and others9,11 have hypothesized that differences in hybridization rates or electrical impedance may be due to probe−probe or probe−target formation of exotic structures and/or disruption in orientation and ordering in the surface-bound DNA strands; however, direct testing of such a hypothesis could not be performed. Previous work in our groups and others has established the use of near-edge X-ray absorption fine structure (NEXAFS) spectroscopy as well as high-resolution X-ray photoelectron spectroscopy (HRXPS) for the examination of films of singlestranded DNA12−15 and more recently on surface-bound DNA hybrids.16,17 These techniques can provide molecular-level, label-free information on the orientation and content of biorelevant assemblies, but their operation requires in vacuo examination of the substrates. This is particularly challenging for surface hybrids that are only stable in the presence of salts. Drying the samples leaves macrosized salt crystals on the surface which need to be removed to avoid scattering and background noise. Salt removal can be accomplished by dipping the samples in deionized water, a procedure that might disrupt the hybridized strands to some extent. However, relative trends within data sets can be used to compare efficiencies, ordering, and orientation within hybridized films.16,17 Here, we use HRXPS and NEXAFS spectroscopy in tandem to examine the effects of target sequence and length mismatches on the final ordering and orientation of surfacebound poly(dT)·poly(dA) hybrids (Scheme 1). HRXPS is used Received: March 12, 2012 Revised: April 23, 2012 Published: April 26, 2012 11133

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were chosen for these studies to make final analysis of the quantities and orientation of adenine and thymine possible, as both adenine and thymine give unique signatures using these techniques; a mixed sequence would have yielded indeterminate results. All final reconstituted DNA concentrations were confirmed via UV absorption measurements. For DNA film formation, sputter-coated gold wafers (100 nm Au/20 nm Cr/Si) cleaned via UV for 2.5 h were incubated for 40 h at 37 °C in 1 M CaCl2−TE buffer (10 μM Tris and 1 μM EDTA, pH 7.01) containing a 3 μM DNA solution, similar to previously described procedures.18 After incubation, samples were rinsed for 1 min with deionized water to remove excess DNA and ions. After rinsing, unhybridized samples were dried under flowing N2. Samples intended for hybridization were placed in a 3 μM solution of the target sequence for 7 h, an amount of time previously found to be sufficient for overcoming the most drastic differences in percent hybridization in targets containing mismatches.8 The chemical structure of a hybridized adenine− thymine pair is shown in Scheme 1. After hybridization, 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.19 It should be noted that treatments of these types of films to prepare them for HRXPS and NEXAFS spectroscopy measurements, in particular rinsing with pure water, are known to affect the stability of any hybrids present.17,19 However, a recent systematic study on the subject has shown that when the samples are rinsed with low volumes of water (∼0.5 mL), such as those used here, this effect is small.17 Samples were stored under an inert gas atmosphere in glass containers for transportation to the synchrotron for measurement, while the density of unhybridized witness DNA films was determined via standard XPS (K-alpha, Thermo Fischer Scientific) using the procedures developed by Petrovykh et al.18,20 The final density was found to be ∼5.4 × 1013 probes cm−2, a value close to the maximum reported in previous systematic studies of thiolated (dT)25 oligonucleotides deposited in 1 M CaCl2−TE buffer.18 Such high molecular densities were chosen to produce films as uniform and as free from random contamination as possible, parameters which are of critical importance in the use of such sensitive spectroscopic techniques as HRXPS and NEXAFS spectroscopy. Both HRXPS and NEXAFS spectroscopy experiments were performed at the D1011 beamline (bending magnet) at the MAX II storage ring of the MAX-lab synchrotron radiation facility in Lund, Sweden. The measurements were carried out at room temperature under ultra-high-vacuum (UHV) conditions at a base pressure better than 1.5 × 10−9 mbar. The spectrum acquisition time was selected in such a way that no noticeable damage by the primary X-rays occurred during the measurements.21−24 The HRXPS spectra were acquired in normal emission geometry at photon energies (PEs) ranging from 350 to 580 eV. The energy resolution was better than 100 meV, allowing a clear separation of individual spectral components. The energy width of the individual emissions was close to the intrinsic energy spread of the respective core-level photoemission process. The binding energy (BE) scale of every spectrum was individually calibrated using the Au 4f7/2 emission line of the underlying Au substrate at 83.95 eV,25 which is the value given by the International Organization of Standardization (ISO) standard.26 HRXPS spectra were fitted by symmetric

Scheme 1. Schematic of the Chemical Structure of a Single Nucleotide (dT) of a Thiolated Probe Hybridized with a Single Nucleotide (dA) of a Target in a Watson−Crick Pairing

to give information about the chemical composition of the surface, while NEXAFS is mostly used to determine ordering and orientation. Using these techniques, we show that DNA hybrids in densely packed films remain upright, despite the introduction of one, two, or four internal sequence mismatches in the target sequence. We also show that an unequally matched target−probe length does not lead to a significant disruption in the upright orientation of hybrids at sufficiently high initial probe densities. This work represents, to the best of our knowledge, the first direct, label-free measurements directly probing the orientation of surface-bound DNA hybrids formed with targets containing internal sequence mismatches and with targets of mismatched length.

2. EXPERIMENTAL SECTION Thymine poly(dT)25 probe sequences modified with C6 thiol linkers on the 5′ ends (Table 1) were purchased from SigmaTable 1. DNA Sequences Used in These Experiments probe and targets probe: (dT)25-S target: (dA)25, perfect match target: (dA)25M1, one mismatch target: (dA)25M2, two mismatches target: (dA)25M4, four mismatches target: (dA)15 target: (dA)6

description thiol-C6-5′TTTTTTTTTTTTTTTTTTTTTTTTT-3′ 5′-AAAAAAAAAAAAAAAAAAAAAAAAA-3′ 3′-AAAAAAAAAAAAAAAACAAAAAAAA-5′ 3′-AAAAAAAACAAAAAAACAAAAAAAA-5′ 3′-AAAACAAACAAAAAAACAAACAAAA-5′ 3′-AAAAAAAAAAAAAAA-5′ 3′-AAAAAA-5′

Aldrich and used as received after reconstitution in HPLCgrade H2O (pH 6.8). The thiol anchor groups were originally protected with a short-chain alkanethiol, but were deprotected by dithiothreitol shortly before delivery. The adenine target sequences were also purchased from Sigma-Aldrich. For the experiments involving sequence mismatches, target sequences with one, two, or four internal cytosine mismatches were used, while for the experiment involving target length mismatches, targets of approximately half and approximately one-fifth of the probe length were used (Table 1). Homo-oligonucleotides 11134

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Voigt functions and a Shirley-type background. The fits were performed self-consistently, with identical fit parameters used for the same spectral regions. The acquisition of the NEXAFS spectra was carried out at both the carbon and nitrogen K-edges in the partial electron yield mode with retarding voltages of −150 and −300 V, respectively. Linear polarized synchrotron light with a polarization factor of ∼95% was used. The energy resolution was better than 100 meV. The incidence angle of the light was varied from 90° (E vector parallel to the surface plane) to 20° (E vector nearly normal to the surface) in steps of 10−20° to monitor the orientational order of the target molecules within the films. This approach is based on the linear dichroism in Xray absorption, i.e., the strong dependence of the cross-section of the resonant photoexcitation process on the orientation of the electric field vector of the linearly polarized light with respect to the molecular orbital of interest.27 The raw NEXAFS spectra were normalized to the incident photon flux by division through the spectrum of a clean, freshly sputtered gold sample. Furthermore, the spectra were reduced to the standard form by subtraction of a linear pre-edge background and normalization to the unity edge jump (as determined by a nearly horizontal plateau 40−50 eV above the respective absorption edges). The energy scale was referenced to the most intense π* resonance of highly oriented pyrolytic graphite (HOPG) at 285.38 eV.28

Figure 1. N 1s HRXPS spectra of an unhybridized (dT)25-S/Au probe film (bottom spectrum) and the films containing hybrids formed with either perfectly matched (dA)25 targets or targets containing one, two, or four internal sequence mismatches. The spectra of the hybrid samples are broken down into their constituent T and A components. The values given to the left of each spectrum represent the final percentage of A relative to T. Blue and red color codes are used for T and A, respectively.

3. RESULTS AND DISCUSSION 3.1. Sequence Mismatch Effects. HRXPS measurements were performed to determine the quality of the initial unhybridized films as well as the adenine content of the hybrid films. The C 1s, O 1s, P 2p, and S 2p spectra were similar to those previously reported from well-formed, high-density thiolated ssDNA layers.16,18,20 The N 1s spectra for the pristine (dT)25-S film and those containing hybrids formed with either perfectly matched (dA)25 targets or those containing one, two, or four internal cytosine mismatches (M) are shown in Figure 1. The spectrum from the unhybridized film consists of a single N 1s peak centered at ∼400.8 eV, which is characteristic of thymine not in direct contact with gold.12 In the samples with hybrids, two additional peaks at 399.3 and 401.1 eV appear in a 1:2 ratio, consistent with the N 1s spectrum of adenine.14,29,30 To determine the ratio of adenine to thymine strands (Figure 1), the joint N 1s spectra were decomposed into their respective adenine and thymine components and the intensity of the thymine component was then multiplied by 5/2 to take into account the two nitrogen atoms present in the thymine bases relative to the five nitrogen atoms in the adenine bases (a length correction was not necessary since the probe and target strands had the same length). It should be noted that, in the case of the targets containing mismatches, there should also be small contributions from the cysteine nucleobases at approximately 399.0 and 400.6 eV, with the intensity ratio opposite that of adenine.30 Although these contributions are expected to be minimal due to the low numbers of C vs A, they would result in a slight contribution to both the thymine and adenine portions of the spectra and thus result in an apparent percentage of adenine nucleotides slightly different from the actual value. Similar amounts of adenine were found between the samples containing hybrids with perfectly matched (dA)25 and singlemismatch (dA)25M1 target strands. Castelino and colleagues31 conducted experiments with fluorescent probes in which they examined the effect of the presence of one or three mismatches

on the hybridization density at varying salt concentrations and found that at high concentrations it was only possible to distinguish multiple mismatches. This is in good agreement with the results presented here, as our samples were prepared at a high salt concentration. Samples with targets containing either two or four internal mismatches show less adenine, in agreement with previous studies.8,31 Peterson et al., in their work examining the hybridization kinetics of sequences with one or two mismatches using SPR, demonstrated a clear difference in equilibrium hybridization efficiency in the strands with mismatches at low densities (1.5 × 1012 probes cm−2), but an eventual near convergence of the equilibrium hybridization efficiency at higher densities (3.0 × 1012 probes cm−2) after 7−10 h.8 In this work, samples with one or two mismatches still show differences in the percentage of hybrids present more closely agreeing with the results of Peterson and colleagues after 4−5 h. This may be due to the higher density of the samples used here (5.4 × 1013 probes cm−2), which has been shown to slow the hybridization process,9,32 combined with a hybridization time of only 7 h. The amount of adenine present in these samples is fairly substantial. At first glance, this may be somewhat surprising given that the molecular density of the probe films is close to maximum reported values for thiolated (dT)25 films in 1 M CaCl2−TE buffer.18 Previous work examining the effect of surface density on hybridization efficiency has shown that at higher densities this process is severely slowed due to steric inhibition,9,32 and most work examining hybridization tends to use lower density samples to minimize this effect. However, higher density samples were chosen for these experiments as they are well-defined and less prone to random contamination that may otherwise stick to the gold surface. Furthermore, work by Lee et al.13 demonstrated that, in complex media such as serum, high-density single-stranded DNA layers actually hybridize more effectively than low-density layers. The authors proposed that this may be due to the greater overall availability 11135

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Figure 2. N K-edge NEXAFS spectra of a (dT)25-S/Au probe film before hybridization (bottom spectra) and the samples containing hybrids formed with either a perfectly matched (dA)25 target or a (dA)25 target containing one, two, or four mismatches (M) (upper spectra). Left: Spectra acquired at an X-ray incidence angle of 55°. Right: Difference spectra (90° − 20°). The positions of the most pronounced, π*-like absorption resonances of T and A are marked by vertical thin solid lines. Horizontal dashed lines correspond to zero for the individual difference spectra. Blue and red color codes are used for T and A, respectively.

of probe strands in the high-density films, as the nonspecific adsorption of proteins onto the surface was more likely to block most available binding sites. Note that the relatively high densities reported in this work have not often been investigated in the literature. Lee and coworkers13 reported a 45% hybridization efficiency after 20 min in a target solution for 20-mer probe layers with a density of 6 × 1012 molecules cm−2. At a concentration of 2 or 3 × 1013 molecules cm−2, this value dropped to 15−20%. Peterson et al.8 found around 50% hybridization efficiency for 25-mer probe films with 3 × 1012 molecules cm−2 after about 8 h in a target solution. Since the unhybridized film examined here was found to contain ∼5.4 × 1013 molecules cm−2, the calculated 45% hybridization efficiency of the samples with the perfectly matched and single-mismatched targets is therefore higher than what we would expect. However, this may be partly due to the fact that these values were estimated by HRXPS N 1s measurements, as opposed to the SPR8,13 or fluorescence33 techniques. Adenine target strands are introduced onto the surface of the DNA film and therefore are more likely to hybridize with the upper parts of their probe strands than diffuse down into the lower parts of the film.8 XPS by its nature probes the upper portion of a sample more than the buried portions, which may result in a slight overestimate of the adenine targets in this work. Nevertheless, the use of HRXPS was essential in these experiments to measure the samples under conditions similar to those used in the NEXAFS experiments (UHV) and to make direct comparisons. Furthermore, the self-consistent nature of these calculations makes comparison among the hybridized samples accurate, even if the absolute amount of adenine may be somewhat different from that reported by other methods. Figure 2 shows the N K-edge NEXAFS spectra of pristine and hybridized (dT)25-S/Au films acquired at an X-ray incidence angle of 55° (the so-called “magic angle”) and the difference between the spectra acquired at X-ray incidence angles of 90° (normal incidence) and 20° (grazing incidence).

Whereas 55° spectra give insight into the chemical identity of the molecules on the surface, the difference spectra additionally provide information about the orientation and orientational order of the molecules.16,27 In the 55° plot (Figure 2, left) the spectrum of the unhybridized (dT)25-S/Au film shows the characteristic π*-like resonances of thymine in the pre-edge region: a primary peak at 401.1 eV, corresponding to the N 1s → LUMO transition, and a smaller secondary peak at 402.0 eV, corresponding to the N 1s → LUMO + 1 transition.34 The films containing hybrids additionally show π*-like resonances unique to adenine at 399.4 and 401.3 eV, corresponding to the N 1s → LUMO transition and the N 1s → LUMO + 2 transition for this nucleobase, respectively.34 The latter low-intensity resonance is not clearly distinguishable from the primary thymine feature at 401.1 eV due to overlap. The former resonance is therefore best suited to monitor the efficiency of hybridization, and as expected, the hybridized samples show a decrease in intensity of this resonance relative to the thymine-specific feature at two and four mismatches. This is in agreement with the decreasing amount of adenine in these films as calculated via HRXPS (Figure 1). Note that, beyond the above qualitative agreement, the relative intensity decrease for the multiple-mismatch cases as compared to the perfect-match situation correlates quantitatively with the percentage values derived from the HRXPS data. The 90° − 20° difference spectra (Figure 2, right) may exhibit either positive or negative peaks at the positions of the characteristic π*-like absorption resonances of adenine and thymine as long as there is a predominant molecular orientation. A positive peak occurs when the planes of the nucleobases are oriented upright with respect to the substrate, while a negative peak indicates a parallel-to-the-substrate orientation of the nucleobases, corresponding to an upright orientation of the DNA strands.35 The lack of a peak suggests a lack of order. The distinct negative peak visible in the spectrum of the unhybridized (dT)25-S/Au film is indicative of an upright 11136

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orientation of DNA strands with respect to the substrate, as is expected for a film of this type and density.12,16,17,35,36 For the samples containing hybrids, this negative character of the thymine peak persists, indicating that overall there are no significant changes in the upright ordering of the thymine probe strands in the hybrid samples formed with any of the targets. The spectra in this plot also show distinct negative adenine-specific peaks in each of the hybrid samples. The negative nature of these peaks indicates that the adenine strands in all of these samples are also oriented upright (predominantly). By comparing the intensity of the characteristic π* resonance in the 55° spectrum to its complementary peak in the 90° − 20° curves, a more quantitative idea of the persistence or change in the degree of orientational order among the samples can be obtained: a relatively large 55° resonance intensity to a low 90° − 20° peak intensity indicates low overall order and vise versa. Given that the intensity of the 55° peak is similar to that of its corresponding 90° − 20° peak in the case of each sample, it can be concluded that there is no significant difference in the average orientation of the target strands among the films tested in our study. Such an observation is in agreement with the findings of Castelino et al.31 that at high salt concentrations the hybridization densities of single-mismatch targets were not distinguishable from those of perfect-match targets. The results shown in Figure 2 further show that, in the samples tested here, up to four mismatches do not result in a difference in orientation of the final hybrid. This is most likely due to the high density of these films, which is known to be a major factor in controlling surface-bound DNA probe orientation.13 Since the molecular density of the DNA probe strands is high enough to force an upright molecular orientation, it may, at first, seem unexpected that it would be possible for the adenine target strands to penetrate between the probe molecules enough for a significant number of hybrids to be formed. The explanation may lie in the preparation procedures used for these films. As mentioned above, the unhybridized samples were rinsed under flowing water for 1 min prior to the measurement. This would have resulted in a near-complete removal of any residual ions in the film and thus the removal of any screening charges, minimizing electrostatic repulsion between the neighboring probe molecules and forcing the upright conformation. However, for hybrid formation these films were placed in a relatively highly concentrated (1 M) NaCl buffer, which would have again provided screening charges and allowed the target molecules to approach the probes. Thus, it would have been possible to obtain the reasonable number of hybrids seen in the HRXPS and NEXAFS spectra. The fact that HRXPS was used to calculate these values may also be playing a role, as discussed above. 3.2. Sequence Length Effects. In addition to sequence mismatches, previous studies have also suggested that mismatches in length between the probe and target strands can also affect ordering and orientation in surface-bound hybrids.10,16,37 To test this, we examined hybrids formed between (dT)25-S probe molecules and (dA)25, (dA)15, or (dA)6 targets. The N 1s HRXPS spectra from the samples are presented in Figure 3, along with the estimated percentage of A relative to T. As these films were fabricated under the same conditions as the layers used in the previous experiments involving sequence mismatches, the N 1s spectra of the unhybridized film and the

Figure 3. N 1s HRXPS spectra of a (dT)25-S/Au probe film (bottom spectrum) and films with hybrids formed using (dA)n targets of varying length (upper spectra). Where applicable, the spectra are broken down into their constituent T and A components. The values given to the left of each spectrum represent the final percentage of A relative to T. Blue and red color codes are used for T and A, respectively.

film formed with perfectly matched targets are identical to those presented in Figure 1. They are shown here once again for comparison. The estimated percentage of A relative to T in (dT)25-S/Au:(dA)25 is 45%, which coarsely correspond to one target strand for every two probe strands. The percentages of A relative to T in (dT)25-S/Au:(dA)15 and (dT)25-S/Au:(dA)6 are 55% and 24%, respectively, which, after correction for the length of the target stands, indicates roughly one target strand for every probe strand in both cases (92% for (dT)25-S/Au: (dA)15 and 100% for (dT)25-S/Au:(dA)6). The difference in total percentage of A relative to T in the (dA)25 targets versus the shorter (dA)15 and (dA)6 targets may be partially explained by steric effects, as shorter strands are expected to diffuse more easily into the probe film than longer strands. However, one may also expect that if this were the only contributing factor, there would be a significantly higher percentage of targets present in the film hybridized with the (dA)6 moieties, as the size of these species should make them even more diffusible than (dA)15. It is also imaginable that the shorter targets would be more likely to hybridize more than one per probe strand, further increasing the adenine percentage, which also may result in more stable hybrids due to an increase in base stacking. However, it is known that hybrids with fewer hydrogen bonds, such as the (dT)25-S/Au: (dA)6 assemblies here, will be less stable than those with more hydrogen bonds, such as (dT)25-S/Au:(dA)15 hybrids, and more likely to dissociate.9,16 In the process of preparing these samples for ex situ characterization via HRXPS and NEXAFS, the samples containing hybrids were rinsed under flowing 1 M NaCl buffer and then briefly dipped into water to remove excess salt. Although this process by no means disrupts all the hybrids, it is more likely to affect a greater number of hybrids in the (dT)25-S/Au:(dA)6 sample than in the (dT)25-S/Au:(dA)15 film, which may explain why there are not even more hybrids present in the former. However, the fact that there are also not fewer hybrids present in the (dT)25-S/Au:(dA)6 film may be an indication that more of these targets are buried within the film, where they are less likely to be disrupted. 11137

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Figure 4. N K-edge NEXAFS spectra of a (dT)25-S/Au probe film before hybridization (bottom spectra) and the films containing hybrids formed with adenine targets of varying length (upper spectra). Left: Spectra acquired at an X-ray incidence angle of 55°. Right: Difference spectra (90° − 20°). The positions of the most pronounced, π*-like absorption resonances of T and A are marked by vertical thin solid lines. Horizontal dashed lines correspond to zero for the individual difference spectra. Blue and red color codes are used for T and A, respectively.

difference to an increased flexibility of the unhybridized lower portion of the probe strand in the hybrids in the former case. In the (dT)25-S/Au:(dA)15 and (dT)25-S/Au:(dA)6 samples examined here, the high density of the initial unhybridized film would make it more likely that the target strand would bind to the upper part of the probe, and thus, it may be expected that an orientational change such as that observed by Shamsi and Kraatz would occur in this film. However, those researchers used samples with densities in the range of (1.1− 2.4) × 1012 molecules cm−2. The higher densities of the (dT)25S/Au:(dA)15 and (dT)25-S/Au:(dA)6 samples examined here (∼5.4 × 1013 thymine probe molecules cm−2) would leave significantly less room for the resulting hybrid to bend over and would explain the lack of drastic change observed here. Previous work in our laboratories16 examining orientation and ordering of hybrids in a lower density (dT)25-S/Au:(dA)15 film (∼2.0 x1013 probes cm−2) also showed a striking lack of order in both the adenine target and thymine probe strands, suggesting that density is indeed a major factor in controlling the orientation of surface hybrids with shorter targets. A graphic description of some potential hybrid orientations in each of the samples is shown in Scheme 2. 3.3. Drying Effects. It is also worth noting that, as part of these experiments, the effect of drying the sample with flowing N2 before placing it in the hybridization solution was also investigated. No large difference was found in the final overall adenine content or hybrid orientation between the samples that were dried prior to hybridization and those that were not (Figure 5). 3.4. Sequence Mismatch Effects in Lower Density Films. As previously stated, Peterson et al.8 found significant differences in SPR hybridization vs time curves for perfectly matched and mismatch-containing targets, suggesting the involvement of unknown orientational or structural changes in the hybridization process. Levicky and Horgan11 assumed that one mechanism involved in this might be the formation of target bridges facilitated by the mismatches, in which one target strand binds to two (or more) probe strands, creating a cross-

Figure 4 shows the N K-edge NEXAFS spectra for these samples. As was the case for the N 1s HRXPS data, the unhybridized sample and the sample with hybrids formed from the perfectly matched (dA)25 targets are identical to those shown in Figure 2 and are presented again here for ease of comparison. The films containing hybrids show characteristic absorption resonances of both thymine and adenine (see the previous section). The intensity of the adenine-specific π* resonance in the 55° plot (Figure 4, left) is similar in films with (dA)25 and (dA)15 targets and decreases noticeably in the film with (dA)6 targets. This is in agreement with the percentages of adenine in these films calculated via HRXPS (45%, 55%, and 24% for (dA)25, (dA)15, and (dA)6, respectively). In the 90° − 20° difference spectra of the probe and hybridized films (Figure 4, right), the peak associated with the resonances of thymine at 401.1 and 402 eV is negative for all films, indicating an upright orientation of the thymine probe molecules that was also observed in the sequence mismatch experiments. As previously stated, this is most likely due to the effects of film density, as the molecules are tightly packed, an arrangement which favors an upright orientation.13,36 The films containing hybrids all show a clear negative adenine-specific peak at 399.4 eV in the difference spectra (Figure 4, right), indicating that the target molecules in these films are also oriented upright. A more qualitative comparison of the adenine or thymine peak intensities between the 55° and 90° − 20° plots, as discussed above, again showed no significant change in the orientational order between the hybrids formed with the (dA)25 or (dA)15 complements and those formed with the (dA)6 targets. Recently, Shamsi and Kraatz10 used electrical impedance to examine ordering in surface-bound 51-mer probe strands hybridized with targets of different lengths. They found that when a 25-mer target strand was hybridized to the upper part of the probe, the resulting hybrid bent over toward the surface. This did not occur when the target was hybridized to the center or lower portion of the probe strand. They attributed this 11138

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similar films.12 However, it is known that surface interactions can significantly disrupt the stability of hybrids15 and that a lower surface density of molecules leaves more room for potential contaminants to infiltrate the film. It may then be the case that the lack of systematic response in these lower density films was due to a less well defined and non-uniform system.

Scheme 2. Schematic Representation of Hypothesized Ordering and Orientation of a (dT)25-S/Au Film Alone (Blue) and Hybridized with (dA)n Targets of Varying Length (Red)a

4. CONCLUSIONS In summary, we have investigated orientation and ordering in surface-bound DNA hybrids on gold made from thiolated thymine homo-oligonucleotide probes and adenine homooligonucleotide targets containing internal sequence mismatches, as well as those with adenine targets of different lengths. To accomplish this, we used the complementary techniques of HRXPS, which gives insight into the content of the samples, and NEXAFS spectroscopy, which also provides information about the orientation of the molecules in the film. Both hybridized and unhybridized samples were found to show signatures of predominantly upright molecular orientation, which persisted even in the hybrids formed with internal mismatches. This was attributed to the density of the film, which was high enough to restrict significant rearrangement of the molecules. However, it was also observed that films formed with targets containing an increasing number of mismatches showed a decreasing total number of hybrids, in agreement with the current understanding of the effects of sequence mismatches on the stability of such structures. Hybrids formed with target sequences approximately half and one-fifth the length of the probe strand also showed a general upright orientation, also most likely due to the initial density of the molecules in the film. The total amount of adenine from the targets was also found to be lower in the films containing hybrids formed with the shortest target, as expected due to the lower stability of hybrids with fewer hydrogen-bonded base pairs. At the same time, the relative number of the hybridized target strands increased with decreasing chain length, corresponding to the higher permeability of the shorter molecules. Taken together, these results suggest that target sequence and length mismatches do not have noticeable effects on the overall order and molecular orientation of surface-bound hybrids in films of sufficiently high densities (∼5.4 × 1013 probes cm−2 in this case), as such high densities cause steric

a Molecules in the unhybridized film are relatively well ordered, as are the hybrids in the films after hybridization with different targets.

hybrid. Presumably, the formation of such exotic structures is more prevalent in lower density films, where more free space is available and where more hybridization occurs.32,38,39 To more closely examine how the presence of sequence mismatches would affect hybrids formed from such lower density films, (dT)25-S/Au monolayers were fabricated using the procedures described in the Experimental Section using an initial incubation time of 20 rather than 40 h. This resulted in probe films with a density of ∼2.8 × 1013 molecules cm−2, which is approximately 2 times less than that of the layers used for the previously described experiments. However, it was found upon analysis with HRXPS and NEXAFS that neither the molecular orientation nor the adenine target content changed in a systematic way in these samples. The NEXAFS difference spectrum of the initial unhybridized (dT)25-S/Au sample revealed a relatively low degree of order and suggested an association of some of the nucleobases with the gold substrate, a phenomenon which has been observed via NEXAFS by other researchers examining

Figure 5. N 1s HRXPS spectra (left panel) and N K-edge NEXAFS difference (90° − 20°) spectra (right panel) of (a) a (dT)25-S/Au probe film that was dried prior to hybridization with its (dA)25 target and (b) a (dT)25-S/Au probe film that was kept wet prior to hybridization with its (dA)25 target. The decomposed HRXPS spectra reveal similar adenine contents in both films, and the NEXAFS difference spectra exhibit similar orientations of the final hybrids under both conditions. Blue and red color codes are used for T and A, respectively. 11139

dx.doi.org/10.1021/jp302381s | J. Phys. Chem. C 2012, 116, 11133−11140

The Journal of Physical Chemistry C

Article

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and electrostatic repulsion of the tightly packed molecules. This finding may be advantageous in systems where strict control over the orientation of DNA strands and their attached moieties is necessary.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +49-6221-54-4921. Fax: +49-6221-54-6199. Present Address §

National ESCA and Surface Analysis Center for Biomedical Problems (NESAC/BIO), Department of Bioengineering, University of Washington, Box 351750, Seattle, WA 98195, United States. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS

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REFERENCES

We thank Prof. M. Grunze for support of this work, the Maxlab staff and Dr. A. Preobrajenski in particular for technical assistance during the experiments at the synchrotron, Dr. M. Bruns for assistance with standard XPS density determination, and Dr. D. Petrovykh for helpful discussions. C.H. acknowledges support from a U.S. National Science Foundation Graduate Research Fellowship. This work has been supported by Deutsche Forschungsgemeinschaft (DFG) (Grant ZH 63/93) and the Biointerfaces Programme.

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