Facilitating Mismatch Discrimination by Surface-Affixed PNA Probes

Department of Biological Chemistry, Indian Association for the Cultivation of Science, Jadavpur, Kolkata-700 032, India. Langmuir , 2013, 29 (10), pp ...
1 downloads 6 Views 2MB Size
Download PDF
Article pubs.acs.org/Langmuir

Facilitating Mismatch Discrimination by Surface-Affixed PNA Probes via Ionic Regulation Srabani Ghosh, Sourav Mishra, Trambaki Banerjee, and Rupa Mukhopadhyay* Department of Biological Chemistry, Indian Association for the Cultivation of Science, Jadavpur, Kolkata-700 032, India S Supporting Information *

ABSTRACT: There has been a search for alternative nucleic acids that can be more effectively used in nucleic acid detection technologies compared to the DNA probes. Peptide nucleic acid (PNA), which contains a non-ionic peptidic backbone, offers such possibilities since it is nuclease-resistant, it binds to DNA with high affinity, and it can be readily self-assembled onto solid substrates, e.g., gold(111), with a molecular backbone orientation away from the substrate. Although application of PNA as a sensor probe has been exemplified, so far there is little or no account of the ionic modulation of single base mismatch discrimination capacity of surfacetethered PNA probes. Herein, we report “on-surface” melting temperatures of PNA-DNA duplexes formed on gold(111) surface, as obtained from fluorescence measurements. We show that surface-tethered PNA forms a stabler duplex than DNA, and is more effective in single base mismatch discrimination than DNA. Importantly, although PNA backbone is non-ionic, variation in the ionic components in hybridization buffer, i.e., varying concentration of monovalent sodium ion, and the nature of anion and the cation, exhibits clear effects on the mismatch discrimination capacity of PNA probes. In general, with decreasing cation concentration, PNA-DNA duplexes are stabilized and mismatch discrimination capacity of the PNA probes is enhanced. The stabilizing/destabilizing effects of anions are found to follow the Hofmeister series, emphasizing the importance of hydrophobic interaction between nucleobases for stability of the PNA-DNA duplexes. Interestingly, the nature of ionic dependence of “on-surface” mismatch detection ability of PNA probes differs significantly from the “solution” behavior of these probes.

■

INTRODUCTION Peptide nucleic acid (PNA) is a DNA analogue, in which the negatively charged phosphodiester units of DNA backbone are replaced by 2-aminoethyl-glycine linkages (Figure 1).1 As a result, in the absence of the strand−strand electrostatic repulsions, unlike in the case of DNA, formation of close association of ssPNA strands becomes relatively straightforward, and compact self-assembled PNA films can be readily generated on solid substrates like gold(111) surface by a simple immersion method.2 In such films, the immobilized ssPNA strands can be oriented away from the surface, as elicited from reflection absorption infrared spectroscopy (RAIRS) experiments,2 and nonspecific interactions with the underlying gold substrate can be largely avoided, creating an ideal situation for the target nucleic acid strands to access the immobilized sensor PNA probes. On the contrary, the DNA films comprising the negatively charged ssDNA strands have been found to be mostly disordered/poorly ordered,3 where nonspecific DNA− surface interactions could occur through the relatively exposed nucleobases, resulting in reduced bioactivity of the film.4 Further advantages of using PNA are that PNA probes can bind to DNA oligomers in a sequence-specific manner with higher affinity compared to the DNA probes obeying Watson− Crick hydrogen bonding rule5−8 and that PNA is not © 2013 American Chemical Society

susceptible to hydrolytic (enzymatic) cleavage. For these benefits, PNA appears to be an attractive candidate as a sensor probe in solid-state DNA detection technologies. So far, reports on solid-state DNA-DNA hybridization have been made.4,9−13 A number of reports on solid-state DNA detection by PNA have also been made.14−22 Important information, such as how the single base mismatch discrimination ability of surfaceconfined PNA probes can be controlled by ionic variations, is however lacking. In solution-phase studies, it is a standard observation that the sequence specificity of a sensor PNA probe toward the target oligonucleotide sequences is reflected in the melting temperature (Tm) values of the respective sensor-target duplexes formed.23−25 For example, the thermal stabilities of the PNADNA duplexes are considerably lowered by the presence of mismatches in the target DNA oligomers.23,24,26,27 Assuming that the sequence specificity would be reflected in the Tm values in the case of solid-state PNA-DNA hybridization too, we investigated the sequence specificity of 12-mer ssPNA sensor probes toward the DNA target oligomer, in fully matched and Received: July 27, 2012 Revised: February 8, 2013 Published: February 17, 2013 3370

dx.doi.org/10.1021/la400125x | Langmuir 2013, 29, 3370−3379

Langmuir

Article

completely non-complementary sequence used in control experiments. The exact concentrations of the PNA solutions were determined by UV−visible spectrophotometry, using absorbance value at 260 nm [(ε260 (L/(mol × cm)) for PNA-1, PNA-2, PNA-3, and Cy3-PNA 1 taken as 116700, 123800, 116700, and 122200, respectively (all the ε260 values presented here or later were obtained from the manufacturer-provided data sheets)]. Preparation of DNA Sensor Probe Solutions. The 12-mer ssDNA sensor probes DNA 1, DNA 2, and DNA 3 (Table 1), all having a −(CH2)6SH group at 5′ position (Alpha DNA, Canada), were dissolved in sodium phosphate buffer (20 mM sodium phosphate, 100 mM sodium chloride, pH 7.00). DNA 1 and DNA 2 were the pairs of oligomers having a single base difference between each other, while DNA 3 was the completely non-complementary sequence that served for control experiments. The exact concentrations of the DNA solutions were determined by UV−visible spectrophotometry, using absorbance values at 260 nm [(ε260 (L/(mol × cm)) for DNA-1, DNA-2, and DNA-3 taken as 123020, 131350, and 123020, respectively]. Preparation of Cy3 Labeled DNA Target Probe Solutions. The Cy3-labeled DNA target probe samples Cy3-DNA 1 and Cy3DNAnc (IDT, Canada) (Table 1) were taken in sodium phosphate buffer (20 mM sodium phosphate, x mM sodium chloride/sodium sulfate/sodium nitrate/tetra methyl ammonium chloride as per experimental design, pH 7.00, where x could be 2/50/100/500/ 1000 mM). The exact concentrations of the DNA solutions were determined by UV−visible spectrophotometry, using absorbance value at 260 nm [(ε260 (L/(mol × cm)) for Cy3-DNA-1 and Cy3-DNAnc taken as 124400 and 116000, respectively]. Preparation of Label-Free DNA Target Probe Solution. The DNA sequence T-DNA 1 (Table 1) (Alpha DNA, Canada) was used as the unlabeled target DNA probe. It was dissolved in sodium phosphate buffer (20 mM sodium phosphate, x mM sodium chloride, pH 7.00, where x could be 2/50/100/500/1000 mM). The exact concentrations of the DNA solutions were determined by UV−visible spectrophotometry, using absorbance value at 260 nm [(ε260 (L/(mol × cm)) taken as 133300]. Preparation of Gold(111) Surface. Gold on mica (Phasis, Switzerland) substrate was flame-annealed following a previously reported procedure2 and always immediately before the nucleic acid modification step, since cleanliness of the substrate surface is an essential requirement for effective anchoring of nucleic acid sequences on gold surface via gold−thiol bond formation.28 Melting Experiments in Solution. For obtaining melting temperatures of PNA-DNA and DNA-DNA duplexes, in fully matched and singly mismatched combinations, equal volumes of equimolar solutions of the target DNA and the PNA/DNA sensor probes were mixed and kept at room temperature (24 ± 1 °C) for 30 min. All the hybridization reactions were carried out in sodium phosphate buffer (20 mM sodium phosphate, x mM sodium chloride, pH 7.00, where x could be 2/50/100/500/1000 mM). All the melting experiments were performed using a Peltier control Perkin-Elmer DTP1 UV−vis spectrophotometer. The melting temperatures of the duplexes were determined by measuring the absorbance values at 260 nm over the temperature range 25 to 90 °C at a heating rate of 1 °C/min. In order to check the reversibility of melting transitions, cooling curves were collected for fully matched and singly mismatched PNA-DNA duplexes for the salt concentration of 100 mM at an annealing rate of 1 °C/min. In order to calculate the melting temperatures, the fraction of melted base pairs, θ, was calculated from the standard formula, θ = (A − AL)/(AU − AL), where A, AL, and AU are sample absorbance, absorbance of the lower baseline, and absorbance of the upper baseline, respectively. Tm is defined as the temperature where θ = 0.5.29 To calculate the melting temperature from the experimental data, a sigmoidal fit was carried out employing Boltzman function using the data evaluation software Origin 8 (OriginLab Cooperation, Northampton, MA, USA). The equation used for fitting was y = A2 + (A1 − A2)/(1 + exp((x − x0)/dx)), where A1 = initial y value, A2 = final y value, and x0 = center, i.e., the value of x at (A1 + A2)/2, dx =

Figure 1. Chemical structures of deoxyribonucleic acid (DNA) and peptide nucleic acid (PNA).

singly mismatched combinations, in comparison to the 12-mer ssDNA sensor probes, on gold(111) surface, by fluorescencebased measurement of the respective “on-surface” Tm values. We varied the ionic environment of the surface-confined duplexes by varying salt concentration and the nature of salt in the hybridization buffer. The purpose was to understand whether and how the ionic factors could control the mismatch discrimination ability of the surface-confined PNA probes. While our observations revealed an increase in the thermal stability of both PNA-DNA and DNA-DNA duplexes on gold(111) surface compared to solution, we also found that the single base mismatch discrimination was better achieved by PNA probes compared to DNA probes. Importantly, such discrimination could be fine-tuned by controlling the ionic settingsthe nature of “on-surface” ionic control differing noticeably from “solution” behavior of the PNA probes.

■

MATERIALS AND METHODS

Preparation of PNA Sensor Probe Solutions. The 12-mer ssPNA sensor probes PNA 1, PNA 2, PNA 3, and Cy3-PNA 1 (Table 1), all having a −(CH2)6SH group at N-ter position (Panagene, Korea), were dissolved in filtered autoclaved Milli-Q water (resistivity 18.2 MΩcm)/sodium phosphate buffer (20 mM sodium phosphate, x mM sodium chloride, pH 7.00, where x could be 2/50/100/500/1000 mM). PNA 1 and PNA 2 were the pairs of oligomers having a single base difference between each other, while the PNA 3 was the

Table 1. Nucleic Acid Sequences Applied in the Present Study DNA/PNA

sequence

DNA 1 DNA 2 DNA 3 PNA 1 PNA 2 PNA 3 Cy3-PNA 1 Cy3-DNA 1 Cy3-DNAnc T-DNA 1

5′-HS-C6-CTA-TGT-CAG-CAC-3′ 5′-HS-C6-CTA-TGT-AAG-CAC-3′ 5′-HS-C6-CGA-TCT-GCT-AAC-3′ N-ter-HS-C6-CTA-TGT-CAG-CAC-CONH2-C-ter N-ter-HS-C6-CTA-TGT-AAG-CAC-CONH2-C-ter N-ter-HS-C6-CGA-TCT-GCT-AAC-CONH2-C-ter N-ter-HS-C6-CTA-TGT-CAG-CAC-Lys(Cy3) 5′-Cy3-GTG-CTG-ACA-TAG-3′ 5′-Cy3-CGA-TCT-GCT-AAC-3′ 5′-GTG-CTG-ACA-TAG-3′ 3371

dx.doi.org/10.1021/la400125x | Langmuir 2013, 29, 3370−3379

Langmuir

Article

DNA duplexes were first formed on gold(111) surface in sodium phosphate buffer (20 mM sodium phosphate, 100 mM sodium chloride, pH 7.00) by usual procedure and then dehybridized by heating the respective sample in sodium phosphate buffer of the same composition as for hybridization. Rehybridization was performed after keeping the gold pieces for a few hours to few days. The fluorescence images of all the rehybridized samples were obtained at room temperature. The reusability of the sensor probe modified gold surfaces was assessed for the physiologically relevant salt concentration of 100 mM only. Fluorescence Data Acquisition and Analysis. The fluorescence images were obtained with an Olympus BX61 fluorescence microscope. All the images were recorded considering λexc = ∼550 nm and λem = ∼570 nm. The exposure time was kept fixed for all the experiments. All the fluorescence experiments were done in dark condition. The fluorescence images were taken from twenty different areas of two different samples and then averaged out. The fluorescence intensity was measured by the Image-pro MC6.1 software (Media Cybernetics, Bethesda, MD), which is provided with the Olympus IX61 fluorescence microscope. Then, the reduction in fluorescence intensity with increase in temperature was plotted to obtain the melting profile of the surface-confined duplexes and the melting temperature was determined. Sample Preparation for AFM Experiments. Freshly flameannealed gold on mica substrate was immersed in 150 μL of PNA/ DNA solution of 0.5 μM and incubated at room temperature (24 ± 1 °C) for 4 h. After incubation was complete, the substrate was washed with 2 mL (4 × 500 μL) filtered autoclaved Milli-Q water, dried in ambient condition, and imaged by AFM. AFM Data Acquisition and Analysis. Images were recorded in ambient condition at room temperature (24 ± 1 °C) using PicoLE AFM equipment of Agilent Corp. (USA). Imaging was carried out in the intermittent contact mode to minimize sample damage and using a 10 μm scanner. The cantilevers (μmasch, Estonia) having back side coated with Al, and frequencies within 208−232 kHz and force constant values 3.5−12.5 N/m, were used for the imaging experiments. The probe cleaning procedure, scan parameters, scan conditions, and image analysis procedure were applied as reported previously.2 The AFM images were taken at least from five to six different areas of each sample to check for reproducibility of the features observed. Control Experiments. In order to test the effect of heat on the fluorescence capacity of the labeled DNA probes, 20 μL of 1.0 μM Cy3-DNA 1 was heated at 70 °C for 15 min and then cooled down to room temperature and deposited on the modified gold surface. This sample was incubated in the humidity chamber for 1 h, washed with 2 mL (500 μL × 4) sodium phosphate buffer (20 mM sodium phosphate, 100 mM sodium chloride, pH 7.00), and dried under soft nitrogen, and the fluorescence images were captured. In order to test whether nonspecific adsorption of the DNA target probes occurs on the sensor probe modified gold(111) surfaces, the DNA 3 modified gold surface was incubated with 20 μL of 1.0 μM Cy3-DNAnc (fully mismatched sequence) in the humidity chamber for 1 h. The sample was then washed with 2 mL (500 μL × 4) phosphate buffer (20 mM sodium phosphate, 100 mM sodium chloride, pH 7.00), dried under soft nitrogen, and fluorescence images were obtained. For checking whether the thiol-PNA probes were desorbed from the gold surface upon heating, the labeled thiol-PNA probe modified gold(111) substrates were heated at 50 and 70 °C, and fluorescence images of the surface so obtained were taken. These images and the respective fluorescence intensities were compared with the image (and the fluorescence intensity) obtained at room temperature. For calculating the probe density onto the gold(111) surface, the labeled thiol-PNA probe was immobilized onto the gold(111) surface, and then, the modified substrate was immersed in 12 mM 2mercaptoethanol for 20 h for removal of the PNA probes from surface. The gold piece was removed and the fluorescence intensity of the solution was measured using a Perkin-Elmer PTP Fluorescence Peltier system.

time constant where the constraint is dx! = 0. The melting temperatures were calculated from the inflection point of the fit function as reported earlier.30 The standard error of melting temperature measurement was ±0.2 °C. Melting Experiments on Gold(111) Surface. To investigate the melting behavior of the PNA-DNA and DNA-DNA duplexes on gold(111) surface, the thiolated sensor PNA oligomers PNA 1/PNA 2/PNA 3 and the thiolated sensor DNA oligomers DNA 1/DNA 2/ DNA 3 were first immobilized onto gold(111) surface by the immersion method. For this, freshly annealed gold on mica pieces were immersed in thiolated PNA/DNA sensor probe solutions of 0.5 μM concentration and kept at room temperature (24 ± 1 °C) for 4 h. Then, the gold pieces were washed with 2 mL (500 μL × 4) of sodium phosphate buffer (20 mM sodium phosphate, 100 mM sodium chloride, pH 7.00) followed by deposition of a 20 μL droplet of the Cy3-labeled DNA target probe solution on the modified gold surface, and incubation in a humidity chamber for 1 h at room temperature. The gold pieces were then washed with 4 mL (500 μL × 8) of the respective sodium phosphate buffer, i.e., 20 mM sodium phosphate, x mM sodium chloride/sodium sulfate/sodium nitrate/tetramethyl ammonium chloride as per requirement, pH 7.00, where x could be 2/50/100/500/1000 mM, so that the washing buffer could be kept the same as the hybridization buffer, i.e., the buffer in which the DNA target probes were suspended. The gold pieces were dried with soft nitrogen jet and the fluorescence images were captured. For melting of the duplexes, the samples were placed in 600 μL of sodium phosphate buffer of the same composition as that of the hybridization buffer and heated at desired temperatures for 15 min. Heating of a sample was performed in steps that could be as small as 0.7 °C (near the anticipated melting temperature value) or as high as 5.0 °C (away from the melting temperature value). The samples were then taken out of the sample container for washing at a constant temperature (in our case, it is room temperature), so that an “isothermal wash” could be given, and the “nonequilibrium thermal dissociation” of the duplexes could be avoided. Washing was performed with 2 mL (500 μL × 4) of sodium phosphate buffer (same composition as that of the hybridization buffer) at room temperature using an accupipette, followed by drying the samples with soft nitrogen jet and the fluorescence images were captured. It was expected that the labeled target DNA strands would be separated from the surface-anchored sensor PNA probes primarily during the heating (i.e., melting) step. Since, in the present case, the melting reaction proceeds to achieve the equilibrium between solution and gold surface probe concentrations, further removal of target DNA strands from the PNA film, during washing, cannot be ruled out. The absolute Tm values that we report are therefore unlikely to be an accurate representation of thermal stability of the duplexes, and the accurate Tm values of the surface-confined duplexes would be somewhat higher than the Tm values reported herein. However, since for oligonucleotides the difference between the nonequilibrium Tm and equilibrium Tm could be small, as reasoned by Anshelevich et al.31 and Wartell et al.,32 it is likely that the reported Tm values would not deviate from the equilibrium Tm values to any significant extent. Also, the extent of target strand removal during washing step should nearly be the same in each instance, since the same wash procedure is applied in each case, making the differences in the Tm values for different ionic conditions depending primarily on the heat-induced denaturation of duplexes, and therefore the thermal stability of the duplexes. Importantly, when a comparative view (i.e., the relative values of Tm for different salt concentrations, or for different types of anions and cations) is considered, any such effect of wash should largely be canceled out. The melting temperatures were calculated from the experimental data employing Boltzman function using the data evaluation software Origin 8 (Origin Lab Cooperation, Northampton, MA, USA) in the same manner as in the case of solution measurements (see previous section). For assessing the ability of the PNA/DNA sensor probe modified gold(111) surfaces to retain the hybridization efficiency after storage for one day to one week after the first use, the PNA-DNA or DNA3372

dx.doi.org/10.1021/la400125x | Langmuir 2013, 29, 3370−3379

Langmuir

Article

Figure 2. Melting behavior of the duplexes (A) DNA 1−Cy3-DNA 1 (fully matched), (B) DNA 2−Cy3-DNA 1 (singly mismatched), (C) PNA 1− Cy3-DNA 1 (fully matched), (D) PNA 2−Cy3-DNA 1 (singly mismatched) on gold(111) surface.

■

RESULTS AND DISCUSSION In this study, the mismatch discrimination ability of surfaceanchored PNA probes (and the DNA probes, which were applied for drawing a comparison with the PNA probe performance) has been assessed in varied ionic conditions, by fluorescence-based measurement of the melting temperatures of the PNA-DNA duplexes. The ionic differences were introduced by varying sodium chloride concentration, and the nature of anionic/cationic components of the salt in hybridization buffer. Gold(111), which is widely used in biosensor applications,33,34 has been the substrate of choice, since the sensor molecules can be effectively anchored onto gold surface via gold−thiol bond formation.28 Thiolated 12-mer oligonucleotides and thiolated 12-mer ssPNA oligomers having a thiol group along with a hexyl spacer unit [−(CH2)6SH] at the 5′ position (for oligonucleotides) and the N-terminal (for PNA oligomers), respectively, were employed as the sensor probes. The hexyl spacer [−(CH2)6−] is one of the standard spacers, which is widely used for keeping the nucleic acid part away from the gold surface so that nonspecific adsorption via nucleobases can be avoided and the sequence can remain exposed for target binding in a biosensor experiment. The target DNA oligomers were formulated considering hybridization to the sensor probes in antiparallel fashion, since it was shown earlier that the duplexes formed in antiparallel orientation are more stable than the duplexes formed in parallel orientation.35 The thiolated PNA and DNA oligomers were immobilized onto gold(111) surface by incubating the gold pieces in 0.5 μM

nucleic acid solutions for 4 h in fully immersed condition. The applied condition for formation of the self-assembled PNA film on gold(111) surface was optimized before to attain high coverage of the two-dimensionally ordered molecular arrangement and an upright molecular orientation.2 The PNA and DNA films were characterized by AFM, a high-resolution imaging method for studying surface features. The T m measurements in solution phase were carried out by UV−vis spectrophotometry. For Tm measurements on gold(111) surface, the fluorescence intensity of the fluorophore-labeled (5′-Cy3 modified) target oligonucleotides was monitored by fluorescence imaging. “On-Surface” Melting Behavior of PNA-DNA Duplexes on Gold(111) Surface. The effective immobilization of the thiolated PNA probes (PNA 1, PNA 2, PNA 3) and the thiolated DNA probes (DNA 1, DNA 2, DNA 3) onto gold(111) surface was first ensured as per standardized procedures.2 The modified gold surfaces were then exposed to the labeled target DNA probes (Cy3-DNA 1, Cy3-DNAnc), and the fluorescence images were captured (the representative images for PNA-DNA duplexes are shown in Figures S1 and S2 in Supporting Information). To determine the Tm values, the gold pieces were heated to desired temperatures. Since the samples were thoroughly washed after each heating step, which should ensure total removal of the dehybridized Cy3-DNA 1 strands from the surface, the fluorescence intensity obtained after each heating step should be directly proportional to the remaining portion of the duplexes on the surface. With increase in temperature, the fluorescence intensity was found to be reduced as the duplexes were increasingly dehybridized, and finally, the fluorescence was non-detectable after reaching a 3373

dx.doi.org/10.1021/la400125x | Langmuir 2013, 29, 3370−3379

Langmuir

Article

solution difficult. The energy requirement to overcome this hindrance in target strand’s exit from the film could play a role in elevation of Tm values of the surface-confined duplexes. The other factors that may give rise to the altered “on-surface” Tm values could be a different duplex environment at the solid− liquid interface, compared to that in bulk solution, due to different dielectric constant of water near a solid surface37 and the hydrophilic nature of gold surface.38,39 For control experiments, the thiolated PNA 3 and DNA 3 sensor strands were immobilized on gold(111) surface keeping the sample preparation condition the same as in the case of immobilization of PNA 1/PNA 2 or DNA 1/DNA 2 oligomers. The modified gold substrates were then treated with the fully mismatched Cy3-DNAnc strands. No fluorescence signal could be detected, meaning that nonspecific attachment of the DNA target probes onto the PNA/DNA sensor probe modified gold(111) surface was negligible, in either of the two cases (Figures S3a, S3b in Supporting Information). In order to test whether heating of the samples could alter the fluorescence capacity of the fluorophore-labeled DNA target probes and thereby interfere with the Tm measurements, the Cy3-DNA 1 solution was heated and applied onto the PNA 1 modified gold(111) surface. The fluorescence image (Figure S3c in Supporting Information) and the intensities were found to be similar to those of the PNA 1−Cy3-DNA 1 sample, which was prepared by using unheated Cy3-DNA 1 solution. No significant loss of fluorescence intensity could be detected upon heating a labeled thiolated PNA probe modified gold(111) surface (see Figure S4 in Supporting Information) indicating that the PNA probes were not desorbed from the surface due to heating. The effectiveness of the PNA/DNA sensor films retained upon storage, after first use, was checked by assessing the hybridization efficiency for a second/third time detection that was carried out on the same day and after a week’s storage using the same chip. The efficiency was generally reduced compared to the original hybridization efficiency (Table S1 in Supporting Information). The PNA films appeared to be more robust than the DNA films, since the reduction in hybridization efficiency was less in the case of the PNA probes (Table S1 in Supporting Information). Apparently, the more sturdy nature of PNA film could be attributed to the nuclease-resistant PNA backbone and the more ordered compact structure of the PNA film compared to the DNA film (see Figure 3). Effects of Salt Concentration Variation on the Melting Behavior of PNA-DNA Duplexes on Gold(111) Surface.

particular temperature, which was different for different PNADNA/DNA-DNA duplexes. The fluorescence intensity values were plotted against temperature in each case, and from the denaturation profiles, the Tm values were determined (Figure 2). It is revealed from the Tm values that an increase in Tm took place in the case of the fully matched PNA-DNA/DNA-DNA duplexes, while the Tm of the singly mismatched PNA-DNA/ DNA-DNA duplexes remained almost the same on gold(111) surface, compared to the solution Tm values (Table 2). A Table 2. Melting Temperatures of the Respective Duplexes Formed in Solution (20 mM sodium phosphate, 100 mM sodium chloride, pH 7.00) and on Gold(111) Surfacea DNA/PNA

Tm/°C (in solution)

Tm/°C (on surface)

DNAfully matched DNAsingly mismatched ΔTm (DNA) PNAfully matched PNAsingly mismatched ΔTm (PNA)

38.6 28.7 9.9 52.2 39.1 13.1

47.8 29.5 18.3 61.8 39.0 22.8

a Differences in melting temperature between fully matched and singly mismatched situations are shown as ΔTm.

theoretical investigation reported by Schmitt et al.36 reveals that, in the case of DNA, hybridization between the fully matched sequences could be enhanced on surface. This was primarily attributed to a greater sensor probe density (i.e., no. of probes within a unit volume on the surface) achievable on surface due to anchoring of the probes on surface (here, gold(111)) compared to that achievable in solution phase, where the probes are free to diffuse, considering the same unit volume and probe concentration as applicable in the case of surface modification. The difference between the PNA probe density on surface, which is estimated to be ∼3.8 × 1013 strands/cm2 or 2.8 × 1021 strands/cm3 (considering a volume as relevant for single molecular layer thickness of 7.59 nm, which is the length of a PNA strand in stretched configuration, and where the PNA strand is assumed to be in perfectly upright condition) and that in solution, which is ∼9.03 × 1014 strands/ cm3, could result in a difference between the number of PNADNA duplexes formed on surface and in solution and, therefore, between the corresponding Tm values of the fully matched duplexes. In the case of the singly mismatched duplexes, since hybridization is largely inhibited anyway due to lack of complementarity, especially since the mismatch is located at the central region of the sequence, the probe density factor became less influential, and led to almost similar “on surface” and “solution” Tm values of the singly mismatched duplexes. In effect, the single base mismatch discrimination could be better performed on gold(111) surface than in solution phase, as reflected in the respective Tm values (Table 2). The considerable increase in Tm values for the surfaceanchored fully matched PNA-DNA and DNA-DNA duplexes, compared to their respective solution Tm values, could partly also arise due to spatial confinement of the duplexes within a film. Removal of the target strands from a film, upon dehybridization, could not be a single step event, especially if duplex density on the surface was too high, and therefore each duplex was sterically jammed by the surrounding duplexes that could make travel of the dehybridized target strands to the bulk

Figure 3. AFM topographs of the (a) PNA and (b) DNA modified gold(111) surface prepared using 0.5 μM PNA/DNA concentrations, at room temperature and for 4 h incubation time. Scale bar for (a) and (b) 150 nm and Z range for (a) 0−1.03 nm, (b) 0−1.04 nm. 3374

dx.doi.org/10.1021/la400125x | Langmuir 2013, 29, 3370−3379

Langmuir

Article

Table 3. Melting Temperatures of Nucleic Acid Duplexes on Gold(111) Surface for Different Sodium Chloride Concentrations in 20 mM Sodium Phosphate Buffer (pH 7.00)a salt concentration (mM)

a

nucleic acid duplexes

2

DNA 1−Cy3-DNA 1 DNA 2−Cy3-DNA 1 ΔTm (DNA) PNA 1−Cy3-DNA 1 PNA 2−Cy3-DNA 1 ΔTm (PNA)

31.1 28.7 2.4 67.8 39.6 28.2

50 °C °C °C °C °C °C

44.7 29.3 15.4 62.2 39.4 22.8

100

°C °C °C °C °C °C

47.8 29.5 18.3 61.8 39.0 22.8

°C °C °C °C °C °C

500 53.9 30.0 23.9 56.7 38.2 18.5

°C °C °C °C °C °C

1000 57.8 30.2 27.6 54.5 37.9 16.6

°C °C °C °C °C °C

Differences in melting temperature between fully matched and singly mismatched situations are shown as ΔTm.

Figure 4. (A) Plot of melting temperature [Tm] vs salt concentration [Na+]. (B) Tm vs log[Na+] plots. In both (A) and (B), blue lines correspond to DNA-DNA duplexes and maroon lines correspond to PNA-DNA duplexes. The circles symbolize fully matched and triangles symbolize singly mismatched situation.

respectively, the single base mismatch could be better discriminated by DNA as the salt concentration was increased or by PNA as the salt concentration was decreased (Table 3). A large increase in discrimination was observed at around 50−100 mM concentration in the case of DNA-DNA duplexes (Figure 4A). Beyond these concentrations, the increase in discrimination was more or less of a uniform rate. A notable increase in the discrimination was observed at around 2 mM concentration in the case of PNA-DNA duplexes, while at higher concentrations, the rate of decrease was more or less even (Figure 4A). The rate of increase of the single base mismatch discrimination as a result of increasing salt concentration, as observed in the case of DNA-DNA duplexes, was found to be considerably more drastic compared to the rate of increase of the single base mismatch discrimination with decreasing salt concentration, as observed in the case of PNA-DNA duplex formation (Figure 4A). This observation clearly indicates that variation in Na+ concentration could influence DNA-DNA duplex stability more drastically than the PNA-DNA duplex stability, which is probably to be expected, since DNA is a negatively charged species while PNA is non-ionic in nature as has been explained in detail before by Tomac et al.40 The plots of Tm vs log[Na+] followed a linear relationship in the case of both PNA-DNA and DNA-DNA duplexes (Figure 4B) (see Table S2 in Supporting Information for analysis of the fitted lines). The positive slope of the plot observed for the fully matched DNA-DNA duplexes indicates counterion association during duplex formation.40 The negative slope of the plot observed for the fully matched PNA-DNA duplexes can be explained by the counterion release upon helix formation.40 At the low sodium ion concentration (2−400 mM), counterion

In this part of the work, we studied the effects of variation in sodium ion (Na+) concentration (2−1000 mM) on melting temperatures of the surface-confined PNA-DNA duplexes and compared the results with response of the surface-confined DNA-DNA duplexes under similar variations. The Tm values for the fully matched DNA-DNA duplexes were found to increase with increasing Na+ concentration, whereas increasing the salt concentration resulted in a continuous decrease in the Tm values of the fully matched PNA-DNA duplexes (Table 3). Interestingly, these trends are in agreement with those observed in solution-phase measurements, which were reported earlier by Tomac et al.40 For single base mismatch situation, a similar trend, i.e., the DNA-DNA duplexes were more stabilized but the PNA-DNA duplexes were destabilized with increasing Na+ concentration, was observed (Table 3). However, the changes in Tm values for singly mismatched duplexes were insignificant compared to the changes observed in the case of the fully matched duplexes, which is clearly portrayed in the Tm vs Na+ concentration plots (Figure 4A). In order to check whether the Tm value of the PNA-DNA duplex could be maximized by not adding any salt in the buffer medium, we measured the Tm values for both the fully matched and the singly mismatched duplexes in “zero” salt condition. It was found that the Tm value of the fully matched PNA-DNA duplex was increased to 68.4 °C, and the Tm value of the singly mismatched duplex remained unchanged, compared to the value obtained for 2 mM salt concentration. Since the changes in Tm value for singly mismatched duplexes were insignificant, whereas the Tm value for fully matched duplexes increased noticeably with increase or decrease in salt concentration, in the case of DNA and PNA, 3375

dx.doi.org/10.1021/la400125x | Langmuir 2013, 29, 3370−3379

Langmuir

Article

polyamide backbone.14,43,44 In fact, in a study on molecular dynamics simulations of single-stranded PNA, DNA, and RNA in aqueous solvent, it has been revealed that fluctuations in the end-to-end distance of the oligomers are the least in the case of PNA strand, followed by DNA strand, and the highest in the case of RNA strand, making PNA globally less flexible than DNA and RNA.43 Experimentally, it has been shown by singlewavelength anomalous diffraction experiments that the double helix flexibility of the PNA strand is rather restricted despite the absence of cyclic moieties.44 The fact that the melting transition in the case of PNA-DNA duplexes was sharper than the transition for DNA-DNA duplexes means that the free energy change for the PNA-DNA duplex dehybridization is more temperature dependent. A sharper transition also indicates that the affinity constant would be more strongly temperature dependent. Consequently, at the physiologically relevant temperature, which is 37 °C, the PNA-DNA duplexes are expected to be more stable than the DNA-DNA duplexes.42 Effects of Salt Concentration Variation on “Solution” Melting Behavior of PNA-DNA Duplexes. The measurements carried out at the physiologically relevant salt concentration of 100 mM revealed monophasic profiles, which is characteristic of melting of duplexes (see Figures S5 and S6 in Supporting Information). It was also observed that heating and cooling curves were reversible, indicating that the transitions were at equilibrium (see Figure S6 in Supporting Information). The PNA-DNA duplexes were found to be considerably more stable than the corresponding DNA-DNA duplexes (Table 2), which is consistent with the previous findings.40 Single base mismatch discrimination was also found to be better performed by the PNA sensor probes than the DNA sensor probes, as shown earlier.40 The Tm values of the PNA-DNA duplexes (fully matched as well as singly mismatched) obtained for other salt concentrations, i.e., 20/50/500/1000 mM, in sodium phosphate buffer (20 mM sodium phosphate, x mM sodium chloride, pH 7.00), clearly indicate a distinctly different conduct (Table 4) compared to the “on-surface” behavior (Table 3). While the variation in Tm values of the singly mismatched PNA-DNA duplexes with changes in salt concentration (2 to 1000 mM) was found to be quite small (approximately 1.5 °C) in the case of “on-surface” measurement, the variation in Tm for a similar change in salt concentration was found to be approximately 5.0 °C in the case of solution measurement. Interestingly, the variation in Tm of the fully matched PNA-DNA duplexes was found to be more (approximately 13.0 °C) in the case of “onsurface” measurement compared to the variation observed (approximately 9.0 °C) in the case of solution measurement. Effectively, mismatch discrimination by the PNA probes could appreciably be better performed on surface than in solution for all the salt concentrations applied (see Tables 3 and 4).

release/uptake between the single-stranded and duplex state was greater, but at the moderate or high salt concentration (>400 mM), a significantly small amount of counterion release/ uptake occurred upon duplex formation (Figure 4A). This is in agreement with the “salting out” effect, where the water molecules that are involved in interaction with the peptidic linkages at low salt concentrations get increasingly more involved in interacting with the salt ions as the salt concentration increases, thereby assisting in increasing PNAPNA association. This enhanced interaction between the PNA strands of different PNA-DNA duplexes could reduce the base stacking interactions within a PNA-DNA duplex leading to destabilization of the duplex and therefore decrease the Tm value as the salt concentration was increased. The melted fractions ( f) of the single PNA/DNA strands against temperature (T) were obtained by fitting the melting profile in two-state transition model that assumes that only two species (fully associated and fully dissociated) are present (Figure 5). This model is often accurate for short

Figure 5. Typical melted fraction ( f) vs temperature curves, where hybridization buffer is 20 mM sodium phosphate, 100 mM sodium chloride, pH 7.00. Filled circles correspond to DNA-DNA duplexes and empty circles correspond to PNA-DNA duplexes.

oligonuceotides, i.e., less than 14mer oligonucleotides.41,42 From the plots, it is clearly observed that the melting transition in the case of PNA-DNA duplexes was more abrupt, since it took place within a much narrower range of temperature of about 6.5 °C, compared to the transition in the case of DNADNA duplexes that took place within the range of about 12 °C. The narrower melting transition observed in the case of PNA-DNA duplexes could be due to a greater extent of conformational homogeneity present in the PNA-DNA duplex population compared to the DNA-DNA duplexes. The relatively restricted conformational flexibility of PNA-DNA duplexes compared to that of the DNA-DNA duplexes could be related to the fact that the PNA strands are less flexible than DNA strands due to the presence of planar amide groups in its

Table 4. Melting Temperatures of PNA-DNA Duplexes in Solution for Different Sodium Chloride Concentrations in 20 mM Sodium Phosphate Buffer (pH 7.0)a NaCl conc. (mM)

a

nucleic acid duplexes

2

50

100

500

1000

PNA 1−T-DNA 1 PNA 2−T-DNA 1 ΔTm

55.6 °C 40.4 °C 15.2 °C

54.3 °C 40.2 °C 14.1 °C

52.2 °C 39.1 °C 13.1 °C

47.4 °C 36.1 °C 11.3 °C

46.2 °C 35.5 °C 10.7 °C

Differences in melting temperature between fully matched and singly mismatched situations are shown as ΔTm. 3376

dx.doi.org/10.1021/la400125x | Langmuir 2013, 29, 3370−3379

Langmuir

Article

Table 5. Melting Temperatures of PNA-DNA Duplexes on Gold(111) Surface Varying the Anion (salt concentration 100 mM) and Cation in 20 mM Sodium Phosphate Buffer (pH 7.00) salt monovalent anion

divalent anion

monovalent cation

nucleic acid duplexes

NaOCOCH3

NaCl

NaNO3

Na2SO4

NMe4Cl (0 mM)

NMe4Cl (2 mM)

NMe4Cl (1000 mM)

PNA 1−Cy3-DNA 1 PNA 2−Cy3-DNA 1 ΔTm

62.9 °C 39.3 °C 23.6 °C

61.8 °C 39.0 °C 22.8 °C

54.7 °C 39.6 °C 15.1 °C

55.3 °C 35.1 °C 20.2 °C

68.4 °C 39.6 °C 28.8 °C

68.4 °C 39.4 °C 29.0 °C

65.9 °C 38.9 °C 27.0 °C

Effects of Variation in Nature of Salt Components on the Melting Behavior of PNA-DNA Duplexes on Gold(111) Surface. Since the nature of the ions is known to affect the thermal stability of specific conformations of proteins/nucleic acids, we measured the Tm values of PNADNA duplexes for different types of salt components, which are monovalent anions CH3COO−, Cl−, NO3−, and divalent anion SO42−, for the fixed countercation, i.e., Na+, and monovalent cation NMe4+, having Cl− as the counteranion. An increasingly destabilizing effect was observed in the case of the fully matched duplexes as the anion was changed from CH3COO− to Cl− and NO3− (Table 5), in agreement with the solution phase behavior as reported earlier by Tomac et al.40 No distinct differences could however be observed in the case of the singly mismatched duplexes (Table 5). Interestingly, the divalent anion SO42−, which is expected to exert the least destabilizing effect due to its terminal position in the Hofmeister series (cation series: Mg2+ > Li+ > Na+ > K+ > NH4+; anion series: SO4 2− > HPO42− > CH3COO− > Cl− > Br− > NO3− > I− > ClO4− > SCN−)40,45,46 induced almost similar destabilizing effects as NO3− (Table 5). The destabilizing effect of SO42− on the singly mismatched duplex was rather prominent, in comparison to the effects of the monovalent anions (Table 5). The cation NMe4+ was selected on the basis of the very high degree of nondenaturing (or stabilizing) effect it can exert on the nucleic acid structures in the solution phase.45 In our study, NMe4+ was found to stabilize the fully matched PNA-DNA duplexes better than Na+, at the minimum concentration, i.e., 2 mM, and more prominently at the maximum concentration, i.e., 1000 mM (Tables 3 and 5). The singly mismatched duplexes were found to be much less affected and the Tm values were almost the same within the total concentration range of 2− 1000 mM for both Na+ and NMe4+ (Tables 3 and 5). The stabilizing influence of NMe4+ was also reflected in the observations that the Tm value was reduced by only 2.5 °C as the salt concentration was increased from 2 mM to 1000 mM (Table 5), while in the case of Na+, the reduction in Tm for a similar increase in salt concentration was 13.3 °C (Table 3). The Hofmeister series, which is considered to be a predictive guide for the stabilizing versus destabilizing effects of particular anions, is likely to be better followed at high salt concentrations (beyond 1000 mM), where the electrostatic contributions saturate,47 since this series is based on lyotropic propensities of a material which are most pronounced when there is a water structure rearrangement at the water−solute interface as a result of high salt concentration.45,46 In the case of PNA-DNA duplex formation in solution phase, it has been observed that the Hofmeister series of anions is clearly reflected in the Tm values, whereas the cation series has little or no influence on the Tm values.40 The present observation that the duplexes responded to anion variations at a moderate salt concentration of 100 mM could be due to the fact that, in the present study, we employed a close-packed PNA assembly formed at the

gold−water interface, where the water content and the water arrangement around the duplexes would be different than that in the solution phase. This argument would be especially relevant considering the fact that the hydrophobic effects that are exerted by PNA due to the presence of a non-ionic peptidic backbone in its structure will be more prominent when the PNA strands are closely spaced, as in the case of a close-packed film.

■

CONCLUSION In conclusion, mismatch discrimination ability of surfaceanchored PNA probes could be successfully enhanced via ionic adjustments, i.e., by varying salt concentration and the type of counterion. While the nature of ionic dependence of “onsurface” behavior of PNA probes deviates significantly from the “solution” behavior of these probes, e.g., in the case of the singly mismatched duplexes, considerable similarities were also observed, e.g., in the case of the fully mismatched duplexes. We not only provide an effective means for improving performance of the PNA probes, but also discuss a strategy for measurement of “on-surface” melting temperature values of PNA-DNA duplexesthe latter contribution requiring further refinement though to obtain accurate melting temperature values. Considering the need for developing more sensitive, targetspecific, and robust high-throughput array technologies, PNAbased nucleic acid detection assays, as presented in this report, could offer practical inputs in achieving better control on the on-surface DNA detection capabilities.

■

ASSOCIATED CONTENT

S Supporting Information *

Fluorescence images of PNA-Cy3-DNA duplexes on gold(111) surface at different heating stages, results of control experiments, solution melting curves, and information on rehybridization efficiency of the PNA film and the DNA film.This material is available free of charge via the Internet at http:// pubs.acs.org.

■

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS We gratefully acknowledge the financial support (Grant No. BT/PR-11765/MED/32/107/2009) from Department of Biotechnology, Govt. of India, and (Grant No. SR/FTP/CS66/2006) from SERC Fast Track scheme of Department of Science and Technology, Govt. of India; and the research fellowships of S.G., S.M., and T.B. from the Council of Scientific and Industrial Research, Govt. of India. 3377

dx.doi.org/10.1021/la400125x | Langmuir 2013, 29, 3370−3379

Langmuir

■

Article

(21) Park, H.; Germini, S.; Sforza, S.; Corradini, R.; Marchelli, R.; Knoll, W. Kinetic and affinity analyses of hybridization reactions between peptide nucleic acid probes and DNA targets using surface Plasmon field-enhanced fluorescence spectroscopy. Biointerphases 2006, 1, 113−122. (22) Park, H.; Germini, A.; Sforza, S.; Corradini, R.; Marchelli, R.; Knoll, W. Effect of ionic strength on PNA-DNA hybridization on surfaces and in solution. Biointerphases 2007, 2, 80−88. (23) Jensen, K. K.; Orum, H.; Nielsen, P. E.; Norden, B. Kinetics for hybridization of peptide nucleic acids (PNA) with DNA and RNA studied with the BIAcore technique. Biochemistry 1997, 36, 5072− 5077. (24) Ratilainen, T.; Holmen, A.; Tuite, E.; Haaima, G.; Christensen, L.; Nielsen, P. E.; Norden, B. Hybridization of Peptide Nucleic Acid. Biochemistry 1998, 37, 12331−12342. (25) Ratilainen, T.; Holmen, A.; Tuite, E.; Nielsen, P. E.; Norden, B. Thermodynamics of sequence-specific binding of PNA to DNA. Biochemistry 2000, 39, 7781−7791. (26) Wittung, P.; Nielsen, P. E.; Buchardt, O.; Egholm, M.; Norden, B. DNA-like double helix formed by peptide nucleic acid. Nature 1994, 368, 561−563. (27) Ratilainen, T.; Holmen, A.; Tuite, E.; Nielsen, P. E.; Norden, B. Thermodynamics of sequence-specific binding of PNA to DNA. Biochemistry 2000, 39, 7781−7791. (28) Nuzzo, R. G.; Allara, D. L. Adsorption of bifunctional organic disulfides on gold surfaces. J. Am. Chem. Soc. 1983, 105, 4481−4483. (29) Owczarzy, R. Melting temperatures of nucleic acids: Discrepancies in analysis. Biophys. Chem. 2005, 117, 207−215. (30) Proll, F.; Mohrle, B.; Kumpf, M.; Gauglitz, G. Label-free characterisation of oligonucleotide hybridisation using reflectometric interference spectroscopy. Anal. Bioanal. Chem. 2005, 382, 1889− 1894. (31) Anshelevich, V. V.; Vologodskii, A. V.; Lukashin, A. V.; FrankKamenetskii, M. D. Slow relaxational processes in the melting of linear biopolymers: a theory and its application to nucleic acids. Biopolymers 1984, 23, 39−58. (32) Wartell, R. M.; Benight, A. S. Thermal denaturation of DNA molecules: A comparison of theory with experiment. Phys. Rep. 1985, 126, 67−107. (33) Wang, J. Survey and summary: From DNA biosensors to gene chips. Nucleic Acids Res. 2000, 28, 3011−3016. (34) Drummond, T. G.; Hill, M. G.; Barton, J. K. Electrochemical DNA sensors. Nat. Biotechnol. 2003, 21, 1192−1199. (35) Eriksson, M.; Nielsen, P. E. Solution structure of a peptide nucleic acid−DNA duplex. Nat. Struct. Biol. 1996, 3, 410−413. (36) Schmitt, T. J.; Knotts, T. A. Thermodynamics of DNA hybridization on surfaces. J. Chem. Phys. 2011, 134, 205105−205109. (37) Soares, D. M.; Tenan, M. A.; Gomide, A. B.; Gomes, W. E. Physical properties of water near a gold surface: a nanorheological analysis. ChemPhysChem 2010, 11, 905−911. (38) Smith, T. The hydrophilic nature of a clean gold surface. J. Colloid Interface Sci. 1980, 75, 51−55. (39) Zheng, J.; Chin, W. C.; Khijniak, E.; Khijniak, E., Jr.; Pollack, G. H. Surfaces and interfacial water: Evidence that hydrophilic surfaces have long-range impact. Adv. Colloid Interface Sci. 2006, 127, 19−27. (40) Tomac, S.; Sarkar, M.; Ratilainen, T.; Wittung, P.; Nielsen, P. E.; Norden, B.; Graslund, A. Ionic effects on the stability and conformation of peptide nucleic acid complexes. J. Am. Chem. Soc. 1996, 118, 5544−5552. (41) Breslauer, K. J.; Sturtevant, J. M.; Tinoco, I. Calorimetric and spectroscopic investigation of the helix-to-coil transition of a ribooligonucleotide: rA7U7. J. Mol. Biol. 1975, 99, 549−565. (42) Mergny, J. L.; Lacroix, L. Analysis of thermal melting curves. Oligonucleotides 2003, 13, 515−537. (43) Sen, S.; Nilsson, L. MD simulations of homomorphous PNA, DNA, and RNA single strands: characterization and comparison of conformations and dynamics. J. Am. Chem. Soc. 2001, 123, 7414− 7422.

REFERENCES

(1) Nielsen, P. E.; Egholm, M.; Berg, R. H.; Buchardt, O. Sequence selective recognition of DNA by strand displacement with a thymine substituted polyamide. Science 1991, 254, 1497−1500. (2) Ghosh, S.; Mukhopadhyay, R. An atomic force microscopy investigation on self-assembled peptide nucleic acid structures on gold(111) surface. J. Colloid Interface Sci. 2011, 360, 52−60. (3) Wang, H.; Tang, Z.; Li, Z.; Wang, E. Self-assembled monolayer of ssDNA on Au(111) substrate. Surf. Sci. 2011, 480, L389−L394. (4) Herne, T. M.; Tarlov, M. J. Characterization of DNA probes immobilized on gold surfaces. J. Am. Chem. Soc. 1997, 119, 8916− 8920. (5) Egholm, M.; Buchardt, O.; Christensen, L.; Behrens, C.; Freier, S. M.; Driver, D. A.; Berg, R. H.; Kim, S. K.; Norden, B.; Nielsen, P. E. PNA hybridizes to complementary oligonucleotides obeying the Watson-Crick hydrogen-bonding rules. Nature 1993, 365, 566−568. (6) Wittung, P.; Nielsen, P. E.; Buchardt, O.; Egholm, M.; Norden, B. DNA-like double helix formed by peptide nucleic acid. Nature 1994, 368, 561−563. (7) Leijon, M.; Gräslund, A.; Nielsen, P. E.; Buchardt, O.; Norden, B.; Kristensen, S. M.; Eriksson, M. Structural characterization of PNADNA duplexes by NMR. Evidence for DNA in a B-like conformation. Biochemistry 1994, 33, 9820−9825. (8) Carlsson, C.; Jonsson, M.; Norden, B.; Dulay, M. T.; Zare, R. N.; Noolandi, J.; Nielsen, P. E.; Tsui, L. C.; Zielenski, J. Screening for genetic mutations. Nature 1996, 380, 207. (9) Levicky, R.; Herne, T. M.; Tarlov, M. J.; Satija, S. K. Using selfassembly to control the structure of DNA monolayers on gold: a neutron reflectivity study. J. Am. Chem. Soc. 1998, 120, 9787−9792. (10) Peterson, A. W.; Heaton, R. J.; Georgiadis, R. Kinetic control of hybridization in surface immobilized DNA monolayer films. J. Am. Chem. Soc. 2000, 122, 7837−7838. (11) Peterson, A. W.; Heaton, R. J.; Georgiadis, R. The effect of surface probe density on DNA hybridization. Nucleic Acids Res. 2001, 29, 5163−5168. (12) Harris, N. C.; Kiang, C. H. Defects can increase the melting temperature of DNA−nanoparticle assemblies. J. Phys. Chem. B 2006, 110, 16393−16396. (13) Nasef, H.; Ozalp, V. C.; Beni, V.; O’Sullivan, C. K. Melting temperature of surface tethered DNA. Anal. Biochem. 2006, 406, 34− 40. (14) Briones, C.; Marti, E. M.; Navarrob, G. C.; Parroa, V.; Romanb, E.; Gago, J . A. M. Ordered self-assembled monolayers of peptide nucleic acids with DNA recognition capability. Phys. Rev. Lett. 2004, 93, 2081031−2081034. (15) Marti, E. M.; Briones, C.; Pradier, C. M.; Gago, J. A. M. A DNA biosensor based on peptide nucleic acids on gold surfaces. Biosens. Bioelectron. 2007, 22, 1926−1932. (16) Calabretta, A.; Wasserberg, D.; Trumpie, G. A. P.; Subramaniam, V.; Amerongen, A. V.; Corradini, R.; Tedeschi, R.; Sforza, S.; Reinhoudt, D. N.; Marchelli, R.; Huskens, J.; Jonkheijm, P. Patterning of peptide nucleic acids using reactive microcontact printing. Langmuir 2011, 27, 1536−1542. (17) Wittung, P.; Rodahl, M.; Kasemo, B.; Nielsen, P. E.; Norden, B. Detection of point mutations in DNA by PNA-based quartz-crystal biosensor. Colloids Surf., A: Physicochem. Eng. Aspects 2000, 174, 269− 273. (18) Kambhampati, W.; Nielsen, P. E.; Knoll, W. Investigating the kinetics of DNA−DNA and PNA−DNA interactions using surface plasmon resonance-enhanced fluorescence spectroscopy. Biosens. Bioelectron. 2001, 16, 1109−1118. (19) Liu, J.; Tian, S.; Nielsen, P. E.; Knoll, W. In situ hybridization of PNA/DNA studied label-free by electrochemical impedance spectroscopy. Chem. Commun. 2005, 2969−2971. (20) Xu, F.; Pellino, A. M.; Knoll, W. Electrostatic repulsion and steric hindrance effects of surface probe density on deoxyribonucleic acid (DNA)/peptide nucleic acid (PNA) hybridization. Thin Solid Films 2008, 516, 8634−8639. 3378

dx.doi.org/10.1021/la400125x | Langmuir 2013, 29, 3370−3379

Langmuir

Article

(44) Menchise, V.; Simone, G. D.; Tedeschi, T.; Corradini, R.; Sforza, S.; Marchelli, R.; Capasso, D.; Saviano, M.; Pedone, C. Insights into peptide nucleic acid (PNA) structural features: the crystal structure of a D-lysine-based chiral PNA−DNA duplex. Proc. Natl Acad. Sci. U.S.A. 2003, 100, 12021−12026. (45) Hamaguchi, K.; Geiduschek, E. P. The effect of electrolytes on the stability of the deoxyribonucleate helix. J. Am. Chem. Soc. 1962, 84, 1329−1338. (46) Collins, K. D.; Washabaugh, M. D. The Hofmeister effect and the behaviour of water at interfaces. Q. Rev. Biophys. 1985, 18, 323− 422. (47) Arakawa, T.; Timasheff, S. N. Mechanism of protein salting in and salting out by divalent cation salts: balance between hydration and salt binding. Biochemistry 1984, 23, 5912−5923.

3379

dx.doi.org/10.1021/la400125x | Langmuir 2013, 29, 3370−3379