Maximizing Mismatch Discrimination by Surface-Tethered Locked

Dec 26, 2012 - Ulla Karhunen , Eeva Malmi , Ernesto Brunet , Juan Carlos Rodríguez-Ubis , Tero Soukka. Sensors and Actuators B: Chemical 2015 211, 29...
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Maximizing Mismatch Discrimination by Surface-Tethered Locked Nucleic Acid Probes via Ionic Tuning Sourav Mishra, Srabani Ghosh, and Rupa Mukhopadhyay* Department of Biological Chemistry, Indian Association for the Cultivation of Science, Jadavpur, Kolkata-700032, India S Supporting Information *

ABSTRACT: Several investigations on DNA-based nucleic acid sensors performed in the past few years point toward the requirement of an alternative nucleic acid that can detect target DNA strands more efficiently, i.e., with higher sensitivity and selectivity, and can be more robust compared to the DNA sensor probes. Locked nucleic acid (LNA), a conformationally restricted DNA analogue, is potentially a better alternative than DNA, since it is nuclease-resistant, it can form a more stable duplex with DNA in a sequence-specific manner, and it interacts less with substrate surface due to presence of a rigid backbone. In this work, we probed solid-phase dehybridization of ssDNA targets from densely packed fully modified ssLNA probes immobilized onto a gold(111) surface by fluorescence-based measurement of the “on-surface” melting temperatures. We find that mismatch discrimination can be clearly improved by applying the surface-tethered LNA probes, in comparison to the corresponding DNA probes. We show that concentration as well as type of cation (monovalent and polyvalent) can significantly influence thermal stability of the surface-confined LNA−DNA duplexes, the nature of concentration dependence contradicting the solution phase behavior. Since the ionic setting influenced the fully matched duplexes more strongly than the singly mismatched duplexes, the mismatch discrimination ability of the surface-confined LNA probes could be controlled by ionic modulations. To our knowledge, this is the first report on ionic regulation of melting behavior of surface-confined LNA−DNA duplexes.

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of changes in various parameters like oligonucleotide length,14 salt concentration,14 type of cations,14 and type of buffers18 have been monitored to understand their role in stability of the surface-confined DNA−DNA duplexes as well as to find out the optimum situation to maximize single base mismatch discrimination.14 Though DNA-based biosensors have found wide applications in the microscale9 as well as nanoscale nucleic acid sensing experiments,19−22 its reduced bioactivity due to potential DNA−surface interactions through relatively exposed nucleobases and degradability by the nuclease compel one to search for alternatives so that these drawbacks can be overcome. Recently, it has been shown that locked nucleic acid (LNA) probes can potentially be a better alternative than the DNA probes for single base mismatch discrimination in target DNA sequences onto a gold(111) surface.23 In the present study, we explored an approach for solid-state single base mismatch discrimination in target DNA sequences using the LNA sensor probes, based on measurement of the “onsurface” melting temperature (Tm) values of the LNA−DNA and the DNA−DNA duplexes. This was done by means of fluorescence detection of the Cy3 labeled target DNA probes that remained on the surface at the end of each heat-induced

dentification of single base mismatch in DNA sequences is of great importance to understand genetic variations present among individuals; to know how the individuals develop response to external agents like drugs, pathogens, chemicals etc.; and to recognize an individual’s propensity toward development of a specific disease. There are around 10 million single nucleotide polymorphisms (SNPs) that have been detected in the human genome.1 Generally identification of these SNPs relies upon detection of the differential response between hybridization of the fully matched sequences and singly mismatched sequences or between dehybridization of the fully matched duplexes and singly mismatched duplexes. The SNPs are usually detected/analyzed by real-time polymerase chain reaction (PCR),2−5 microarrays,6,7 and nanobiosensor technologies.8,9 A significant number of approaches that are based on microarray and biosensor technologies may require detection of solid-phase hybridization, in which surfaceimmobilized capture probes bind to target molecules from solution. In the past two decades, these technologies have made considerable advancements and have been applied in important areas like gene expression profiling, genotyping, and biological detection.10−12 Though the underlying facts behind nucleic acid hybridization in bulk solution are fairly well-understood, the understanding of the nucleic acid hybridization onto solid surfaces is much less developed.10,13 Recently, there have been some reports on DNA-biosensors that are based on solid-phase DNA−DNA hybridization.8,10,14−18 In these reports, the effects © 2012 American Chemical Society

Received: October 3, 2012 Accepted: December 26, 2012 Published: December 26, 2012 1615

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extent. Very recently, Springer et al.14 addressed these facts, i.e., the effect of cations (both the type and the concentration) on the duplex stability, mismatch discrimination for solid-phase DNA−DNA hybridization to an extent but the effect of these parameters on solid phase LNA-DNA hybridization, with an aim to maximize single base mismatch discrimination, is yet to be addressed. Herein, for the first time, we have investigated the effects of varying concentrations of monovalent and polyvalent cations, namely Na+, Mg2+, spermidine (3+), and spermine (4+) on solid-phase hybridization of fully matched and singly mismatched DNA targets to densely packed immobilized LNA probes onto a gold(111) surface. We found that in case of both LNA and DNA probes, single base mismatch discrimination is better performed on the surface and that LNA excels DNA in this respect. Importantly, the type as well as concentration of the cations is found to influence the mismatch discrimination ability of the LNA probes.

dehybridization step. The sensitivity of this approach was estimated from the differences between the melting temperatures of the respective duplexes and maximized by controlling the ionic parameters like salt concentration and type of cation. LNA is a conformationally restricted molecule since it contains a modified ribose moiety in which the 2′-oxygen and the 4′-carbon are linked by a methylene bridge, in effect, locking the sugar in a RNA mimicking sugar conformation (Ntype) (Figure 1).24,25 LNA can bind with complementary

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MATERIALS AND METHODS Preparation of LNA Sensor Probe Solutions. The solutions of thiolated and fully modified LNA sequences (Table 1) (HPLC purified, procured from Exiqon, Denmark) were Table 1. Nucleic Acid Sequences Used in the Present Study

Figure 1. Chemical structures of (A) deoxyribonucleic acid (DNA) and (B) locked nucleic acid (LNA).

DNA/RNA sequences in a sequence-specific manner obeying the Watson−Crick base pairing rule with higher affinity compared to DNA, which is reflected in the higher values of the solution melting temperatures of LNA−DNA/LNA−RNA duplexes compared to those of DNA−DNA/DNA−RNA duplexes.24,25 LNA is nuclease-resistant;26,27 its higher structural rigidity may prevent interactions with the solid substrates,28 and it can have multiple water bridges that provide it with extra stability compared to DNA or RNA.29 Though it was found that LNA can enhance single base mismatch discrimination in both bulk solutions30 as well as on the surface,23 the basic framework of hybridization “in solution phase” and “on surface” can differ significantly with respect to various aspects as following. Since the probe density and local concentration of the negatively charged oligonucleotides could be considerably higher on the surface compared to that in bulk solution, both steric effects as well as electrostatic effects could have an important role in solid-phase hybridization.14 In general, cations compensate the negative charge of the oligonucleotide backbone and therefore stabilize the oligonucleotide duplexes.14 So, it is expected that the effect of cations will be more pronounced in solid-phase hybridization compared to hybridization processes in bulk solution because of high surface density of negatively charged probes onto the solid surface. It has been reported that hybridization efficiency could be profoundly influenced by the total concentration of sodium ion irrespective of the type of buffer used.18 The majority of the investigations so far dealing with the effect of cations on solid-phase nucleic acid hybridization have focused more on probe density, while the effect of different types and concentrations of the cations, present in the hybridization buffer, on duplex stability has been investigated to a little

DNA/LNA

sequence

DNA-1 DNA-2 DNA-3 LNA-1 LNA-2 LNA-3 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′ 5′-HS-C6-CTA-TGT-CAG-CAC-3′ 5′-HS-C6-CTA-TGT-AAG-CAC-3′ 5′-HS-C6-CGA-TCT-GCT-AAC-3′ 5′-Cy3-GTG-CTG-ACA-TAG-3′ 5′-Cy3-CGA-TCT-GCT-AAC-3′ 5′- GTG-CTG-ACA-TAG-3′

prepared in phosphate buffered saline (20 mM sodium phosphate, 100 mM sodium chloride, pH 7.0) or PBS. The LNA concentrations were determined by UV−visible spectroscopy, considering the ε260 (L/(mol × cm)) values for LNA-1, LNA-2, and LNA-3 as 112 700, 118 100, and 111 100, respectively (all the ε260 values presented here or later were obtained from the manufacturer-provided data sheets). LNA-1 and LNA-2 were the sensor probes, LNA-1 for a complete match situation and LNA-2 for a single base mismatch situation. LNA-3 was the fully mismatched sensor probe used for control experiments. Preparation of DNA Sensor Probe Solutions. The solutions of thiolated DNA sequences (Table 1) (HPLC purified, procured from alpha DNA, Canada) were prepared in the PBS medium. The DNA concentrations were determined by UV−visible spectroscopy, considering the ε260 (L/(mol × cm)) values for DNA-1, DNA-2, and DNA-3 as 123 020, 131 350, and 123 020, respectively. DNA-1 and DNA-2 were the sensor probes, DNA-1 for complete match situation and DNA2 for a single base mismatch situation. DNA-3 was the fully mismatched sensor probe used for control experiments. Preparation of Cy3 Labeled Target DNA Solutions. The solutions of Cy3 labeled DNA sequences (Table 1) (HPLC purified, procured from IDT, Canada) were prepared in the sodium phosphate buffer (20 mM Na2HPO4/20 mM NaH2PO4, pH 7.0) containing a selected amount of 50, 100, 1616

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labeled target molecules were removed from the surface, as confirmed by fluorescence imaging (see Figures S1 and S2 in the Supporting Information). Heating was performed in several steps till the complete removal of the Cy3-labeled targets from the surface was ensured. Exactly the same protocol was applied to monitor the thermal denaturation behavior of the surfaceconfined DNA−DNA duplexes by taking thiolated DNA oligonucleotides as sensor molecules. The exposure time was kept fixed for all the fluorescence imaging experiments. The fluorescence intensity corresponding to every respective image was determined by Image-pro MC6.1 software (Media Cybernetics, Bethesda, MD). Then, the decrease in fluorescence intensity with an increase in temperature was plotted. To calculate the melting temperature from the experimental data, a sigmoidal fit was accomplished employing the Boltzman function using the data evaluation software Origin8 (OriginLab Cooperation, Northampton, MA). 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 = time constant where the constraint is dx! = 0. The melting temperatures were calculated from the inflection point of the fit function as described earlier.31 The standard error of melting temperature measurement was ±0.2 °C. Determination of Tm in Solution. The oligonucleotide solutions were prepared in 20 mM sodium phosphate buffer (pH 7.0) containing a selected amount of 50, 100, 500 mM NaCl. Hybridization was performed by mixing equal volumes of equimolar solutions of target DNA and the relevant LNA/DNA sensor probe sequences at room temperature and allowing the resulting solution to stand for 30 min. Then, the absorbance at 260 nm was measured with Peltier control Perkin-Elmer DTP1 UV−vis spectrophotometer. The heating cycles were performed considering the temperature range of 25−96 °C at a heating 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 = 0.5.32 To check the reversibility of melting transitions, cooling curves were collected for fully matched and singly mismatched LNA− DNA duplexes for the salt concentration of 100 mM at an annealing rate of 1 °C/min.

500, 1000, 2000, 3000 mM NaCl or 1.5, 5, 10, 15, 20 mM MgCl2, or 5, 10, 15 mM spermidine, or 5, 10 mM spermine. The DNA concentrations were determined by UV−visible spectroscopy, considering the ε260 (L/(mol × cm)) value for Cy3 DNA-1 and Cy3 DNAnc as 124 400 and 123 200, respectively. Cy3 DNA-1 was the labeled target probe, being fully matched to LNA-1 and DNA-1, and bearing a single base mismatch with respect to both LNA-2 and DNA-2. Cy3 DNAnc was the fully mismatched target probe used for control experiments. Preparation of Unlabeled Target DNA Solution. The solution of unlabeled target DNA sequence T-DNA-1 (Table 1) (HPLC purified, procured from alpha DNA, Canada) was prepared in the PBS medium and its concentration was determined by UV−visible spectroscopy considering the ε260 (L/(mol × cm)) value as 133 300. T-DNA-1 was fully matched to LNA-1 and DNA-1 and singly mismatched to LNA-2 and DNA-2. Preparation of Gold(111) Surface. Gold on mica (Phasis, Switzerland) substrate (gold layer thickness, 200 nm) was flame annealed until a reddish glow appeared. This procedure was repeated 7−8 times. After a short period (1−2 s) of cooling in air, the substrate was subjected to further modification steps. Preparation of DNA/LNA Sensor Probe Modified Gold(111) Surface. In all the cases, immobilization of the nucleic acid sensor probes on gold(111) surface was performed in the PBS medium at room temperature (24 ± 1 °C). Hybridization was carried out in 20 mM sodium phosphate buffer (20 mM Na2HPO4, 20 mM NaH2PO4, pH 7.0) containing a selected amount of 50, 100, 500, 1000, 2000, 3000 mM NaCl, or 1.5, 5.0, 10, 15, 20 mM MgCl2, or 5, 10, 15 mM spermidine, or 5, 10 mM spermine at room temperature. Fluorescence Intensity Measurement and Determination of Melting Temperature (Tm) on Surface. Freshly annealed gold(111) substrate was immersed in thiolated LNA solution of 0.5 μM concentration and incubated for 4 h at room temperature. After incubation, the substrate was first washed with 1 mL (2 × 500 μL) of PBS solution followed by 2 mL (4 × 500 μL) of filtered autoclaved Milli-Q water to remove the nonspecifically adsorbed molecules and then dried under gentle nitrogen jet. Then, the LNA-modified gold pieces were subjected to hybridization by incubating into 1 μM Cy3 DNA-1 solution, having different hybridization environment, i.e., hybridization buffer containing a selected amount of 50, 100, 500, 1000, 2000, 3000 mM NaCl, or 1.5, 5, 10, 15, 20 mM MgCl2, or 5, 10, 15 mM spermidine, or 5, 10 mM spermine as desired, for 1 h at room temperature. Next, the gold pieces were taken out from the hybridization solution, washed thoroughly with 1 mL (2 × 500 μL) of corresponding hybridization buffer followed by 2 mL (4 × 500 μL) of filtered autoclaved Milli-Q water to remove the nonspecifically bound target molecules and dried under a mild nitrogen jet. The fluorescence images were obtained at room temperature in a dark condition with an Olympus BX61 fluorescence microscope by excitation (λexc) at ∼550 nm and emission (λem) at ∼570 nm. Then, to monitor the thermal denaturation behavior of the surface-confined LNA−DNA duplexes, the gold piece was placed into 600 μL of 20 mM sodium phosphate buffer (20 mM sodium phosphate, 100 mM sodium chloride, pH 7.0) and heated to a desired temperature for 15 min. Then, the gold piece was taken out, washed and fluorescence images were recorded again in order to find out the amount of Cy3-labeled target molecules left on the surface. This process was continued until all the Cy3-

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RESULTS AND DISCUSSION In this study, the single base mismatch discrimination capability of fully modified thiol-LNA probes immobilized onto gold(111) surface has been maximized by ionic tuning, i.e., by varying salt concentration and the type of cations present in hybridization buffer. The applied condition for formation of the self-assembled LNA sensor films on a gold(111) surface was optimized before for achieving high coverage and a molecular orientation away from the surface.23 The sensor strand modified gold samples were always prepared by the immersion method so that the nucleic acid molecules could be kept in well-solvated conditions during the preparative stage. Effectiveness of the immersion method over the other sample preparation methods, e.g., droplet contact method and droplet deposition method, was exemplified earlier.33 Gold(111) substrate was selected since this surface is widely used in biosensor applications,19,20,34,35 especially where immobilization of the sensor molecules via gold−thiol linkage formation36 1617

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particular temperature for each type of duplex, quite obviously because the duplexes were increasingly dehybridized with an increase in temperature. The fluorescence intensity values were plotted against temperature in each case, and from the denaturation profiles the Tm values were determined (Figure 2). The “on-surface” Tm values differed significantly from the

is relevant. Since cleanliness of the substrate surface is an absolute necessity for effective anchoring of the thiolated nucleic acid probes on the gold surface,36 the gold pieces were always freshly annealed prior to nucleic acid modification. Three 12-mer thiolated ssLNA sequences, namely, LNA-1, LNA-2, and LNA-3, were employed as the sensor probes (Table 1). The 12-mer 5′-Cy3 labeled ssDNA sequence (Cy3 DNA-1), which was fully matched to LNA-1 and singly mismatched to LNA-2, was employed as the target strand (Table 1). The Cy3 DNAnc was the fully mismatched target probe used for control experiments (Table 1). Three DNA sequences DNA-1, DNA-2, and DNA-3 having the same base sequence as LNA-1, LNA-2, and LNA-3, respectively, were applied as the DNA sensor probes (Table 1). The sensor probes were kept small in size, and the mismatch in both LNA and DNA sensor probes was centrally placed in order to maximize mismatch discrimination capacity of the probes. In all the sensor probe molecules, a hexyl spacer [−(CH2)6−] was introduced at the 5′-end, which helps to keep the nucleic acid part away from the gold surface so that nonspecific adsorption via nucleobases could be avoided to a considerable extent. The LNA sequence taken was fully LNA-modified since Owczarzy et al. reported very recently that additional LNA moieties adjacent to the initial modification seem to enhance stacking and H-bonding interactions,37 which is expected to result in high affinity to the complementary target DNA strands. Moreover, full modification could ensure the maximum level of nuclease resistance of the sequence. Both the sensor and the target sequences were kept short in length (12 mer) since it has been shown that duplex stabilization is achieved best with short oligonucleotide sequences.14 LNA purine (adenine) was placed at the mismatch site in order to achieve the maximum mismatch discrimination as per an earlier finding that LNA purines offer great potential to recognize mismatches than LNA pyrimidines and DNA purines.38 Fluorescence microscopy was employed to determine the melting temperatures of the surface-confined LNA−DNA and DNA−DNA duplexes, formed in different ionic environments, to identify the optimum situation where the single base mismatch discrimination ability of the LNA film could be maximized. The solution phase melting temperatures of the sensor−target duplexes, same as those used for on-surface measurements, were determined by UV−vis absorption spectroscopy. Tm Measurement of the Surface-Confined LNA−DNA and DNA−DNA Duplexes. To investigate the melting behavior of the surface-bound LNA−DNA and DNA−DNA duplexes, effective anchoring of the thiolated LNA and DNA sensor probes onto the gold(111) surface was ensured first. The modified gold surfaces were then exposed to the labeled target DNA probes, and the fluorescence images were captured. To determine the Tm values, the gold pieces were heated to desired temperatures by starting from the lower temperatures and 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). Since a thorough wash was given after each step of heating that should ensure effective removal of the dehybridized Cy3 DNA-1 strands from the surface, the measure of the fluorescence intensity obtained after each heating step should be directly proportional to the remaining portion of the duplexes on the surface. With an increase in temperature, fluorescence intensity corresponding to the labeled target molecules on the surface was found to reduce and it became almost negligible after reaching a

Figure 2. Graphs show the melting behavior of the (A) DNA-1−Cy3 DNA-1 duplex (fully matched), (B) DNA-2−Cy3 DNA-1 duplex (singly mismatched) on a gold(111) surface, (C) LNA-1−Cy3 DNA-1 duplex (fully matched), and (D) LNA-2−Cy3 DNA-1 duplex (singly mismatched) on a gold(111) surface.

corresponding melting temperatures obtained in bulk solution, indicating largely different energetics playing on the surface compared to that in solution. Importantly, single base mismatch discrimination was found to be enhanced on the surface in the case of both LNA and DNA sensor probes, LNA being more efficient than DNA in this respect as in solution (Table 2). Table 2. Differences in Melting Temperatures between Fully Matched and Singly Mismatched Situations for Solution Phase Measurements and On-Surface Measurements sensor probe

solution (°C)

surface (°C)

DNA LNA

9.9 21.5

18.3 23.6

In the present scenario, as the melting reaction proceeds to achieve the equilibrium between the solution and the probe concentrations on the gold surface, the removal of the solution after each heating step could cause denaturation of some more duplexes even if at the same temperature. So 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.39 and Wartell et al.,40 it is likely that the reported Tm values do not largely deviate from the equilibrium Tm values. Also, when a comparative view is taken, i.e., the Tm values and/ or the mismatch discrimination ability of the surface-confined LNA probes for different ionic conditions are compared, the 1618

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reduction in fluorescence intensity with increase in temperature was solely due to duplex dissociation or it was influenced by the surface probe desorption, Cy3-tagged thiolated LNA was immobilized onto gold substrate and heated to different temperatures by following the same procedure as was for all the other experiments and fluorescence images were captured. It was observed that the produced film was more or less homogeneous having the probe density of about (1−10) × 1015 molecules/cm2 (from our ongoing experiments) whereas in the case of DNA the probe density reported on the gold surface was about (1−2) × 1013 molecules/cm2.41 We also found that no significant amount of thiolated immobilized probe was desorbed from the gold surface upon heating up to 80 °C, as the captured fluorescence images at different temperatures were almost identical, having very similar fluorescence intensity values (see Figure S4 in the Supporting Information). Earlier, in several studies,42,43,17,8 the thiol-DNA SAM on the gold surface was heated at 70 °C or beyond 70 °C for inducing a dehybridization event. Peterson et al. showed that only the target DNA strands were removed from the gold surface when the surface modified with the duplex film was heated at 70/80 °C.17 No thiol-DNA was lost from the surface and, moreover, the films did not loose their specificity upon heating at that temperature.43,17 Therefore, in our study too, the reduction of fluorescence emission upon heating is expected not to have any significant contribution from detachment of the probes from the surface, and it is more likely that the fluorescence reduction is primarily due to dehybridization of the surface-bound duplexes. As far as fluorescence quenching close to the gold(111) surface is concerned, the quenching effects should largely remain the same for all the experiments, since similar preparative conditions were maintained all through. If a comparative view is taken (i.e., if the Tm values at different ionic conditions or the mismatch discrimination at different ionic conditions are compared), such effects are expected to be generally canceled out. Tm Measurement of the LNA−DNA and DNA−DNA Duplexes in Solution. The Tm profiles of all the hybridized nucleic acid samples (LNA−DNA and DNA−DNA duplexes, in both fully matched and singly mismatched combinations) were obtained by measuring absorbance over a temperature range of 25−96 °C. All the melting temperature profiles were monophasic and the melting curves were sigmoidal in nature, which indicate cooperative melting behavior of the nucleic acid duplexes in solution (see Figures S5 and S6 in the Supporting Information). Role of Salt Concentration in “On-Surface” Mismatch Discrimination by LNA Probes. In order to assess the possibilities of enhancement of the mismatch discrimination ability of surface-tethered LNA sensor probes by ionic adjustments, we varied the concentration of NaCl in hybridization buffer within the range 50−1000 mM. With an increase in salt concentration, the Tm of the fully matched duplexes increased significantly, whereas the singly mismatched duplexes were least affected, in the case of both LNA and DNA probes (Table 3). The interstrand repulsion between the negatively charged strands of the fully matched duplexes could be increasingly compensated by the positive counterions as the salt concentration was increased. This resulted in a melting temperature rise. The interstrand repulsion is expected to be less severe in the case of the singly mismatched duplexes due to a relatively insignificant extent of hybridization and therefore lesser proximity of the two strands. This rendered the Tm values

factor of inaccuracy in the Tm values should be largely canceled out. In order to analyze the denaturation profiles of the surfaceconfined LNA−DNA and DNA−DNA duplexes, the plots for melted fractions ( f) of the single stranded LNA/DNA against temperature (T) were obtained by fitting the melting profile in a two-state transition model that assumes that only two species (fully associated and fully dissociated) are present (Figure 3).

Figure 3. Typical melted fraction (f) vs temperature curves for the fully matched DNA−DNA (dotted line) and LNA−DNA (solid line) duplexes on the gold(111) surface. In both the cases, immobilization of sensor strands and subsequent hybridizations were carried out in sodium phosphate buffer (20 mM sodium phosphate, 100 mM sodium chloride, pH 7.0).

From the plots, it was observed that melting transition of the LNA−DNA and the DNA−DNA duplexes were quite similar and took place within a broad temperature range of about 12 °C, which is likely to be due to a lack of conformational homogeneity of the surface-tethered LNA−DNA and DNA− DNA duplexes. The robustness of the nucleic acid layers upon storage after first use was checked by assessing the hybridization efficiency for a second or third time hybridization, which was carried out on the same day and after a week’s storage using the same chip. The efficiency of both the DNA-modified chip and the LNAmodified chip reduced, though the LNA-modified chip retained a higher degree of hybridization efficiency even after a week’s storage (Table S1 in the Supporting Information). The experiments for assessing the level of hybridization efficiency retained, after storage, were carried out for the physiologically relevant salt concentration of 100 mM only. For control experiments, the thiolated LNA-3 and DNA-3 sensor strands were immobilized onto the gold(111) surface keeping all the sample preparation conditions the same as in the case of the Tm measurement experiments (see the Supporting Information). The modified gold substrates were then treated with the fully mismatched Cy3 DNAnc strands. No fluorescence signal could be detected, indicating that no significant level of nonspecific adsorption of the target strands took place in either of the two cases (see Figure S3a,b in the Supporting Information). Furthermore, in order to test whether heating could alter the fluorescence capacity of the fluorophorelabeled oligonucleotides and thereby interfere with the Tm measurements, the Cy3 DNA-1 solution was heated to 70 °C, then cooled down to room temperature, and applied onto a thiolated LNA-1 modified gold(111) surface. The fluorescence image and the intensity were found to be similar to the sample prepared using unheated Cy3 DNA-1 solution (see Figure S3c,d in the Supporting Information). In order to check the quality of immobilization as well as to confirm whether the 1619

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was reported that mismatch discrimination ability of LNAmodified probes was similar, whether the buffer contained 69 mM Na+ or 1 M Na+.30 In the case of fully LNA-modified sequences, however, we found that though mismatch discrimination ability of LNA probes remained almost the same for 50 and 100 mM Na+, it could be increased when the Na+ concentration was raised to 500 mM (Table S3 in the Supporting Information). On the gold(111) surface, the mismatch discrimination ability of the surface-tethered LNA probes was found to be more susceptible to ionic modulation and excelled solution behavior in this respect for the salt concentrations ≥100 mM onward (see Table 3 and Table S3 in the Supporting Information). We further observed that in the case of the fully matched LNA−DNA duplexes, the effect of change in the salt concentration on Tm values was rather pronounced within the salt concentration range 50−100 mM, whereas for higher salt concentration values up to 1000 mM, the Tm increased with a uniform rate (Table 3). In case of the fully matched DNA−DNA duplexes, however, the rate of increase in Tm values was more or less consistent throughout the entire range of salt concentration (50−1000 mM) applied (Table 3). This means that mismatch discrimination by LNA probes could be relatively drastically enhanced within the salt concentration range 50−100 mM compared to the higher salt concentrations, while for DNA probes, mismatch discrimination ability was more or less uniformly affected throughout the entire range of salt concentration applied (Table 3). The mismatch discrimination ability of LNA probes could be maximized for the salt concentration of 1 M (Table 3). However, with a further increase in NaCl concentration to 2 and 3 M, the Tm values of the fully matched LNA−DNA duplexes reduced, the singly mismatched duplexes remained unaffected, and therefore the mismatch discrimination ability of the LNA probes reduced (Figure 4B). As both LNA and DNA are negatively charged species, an electrostatic penalty needs to be paid as the repulsive interactions increase when the ssLNA and the ssDNA strands come in close proximity prior to duplex formation. This penalty can be reduced by increasing the amount of salt and/or by adjusting the type of cation in hybridization buffer. It has been recently reported by Fuchs et al. that the electrostatic interactions can clearly influence DNA−DNA duplex stability for salt concentrations up to 620 mM of NaCl in the case of 16-

Table 3. Melting Temperatures of Nucleic Acid Duplexes on Gold(111) Surface for Different Sodium Chloride Concentrations in 20 mM Sodium Phosphate Buffer (pH 7.0)a NaCl concn (mM) nucleic acid duplexes DNA-1−Cy3 DNA-1 DNA-2−Cy3 DNA-1 ΔTm (DNA) LNA-1−Cy3 DNA-1 LNA-2−Cy3 DNA-1 ΔTm (LNA)

50 44.7 29.3 15.4 41.2 28.4 12.8

°C °C °C °C °C °C

100 47.8 29.5 18.3 52.7 29.1 23.6

°C °C °C °C °C °C

500 53.9 30.0 23.9 57.4 29.2 28.2

°C °C °C °C °C °C

1000 57.8 30.2 27.6 61.1 29.4 31.7

°C °C °C °C °C °C

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

of the singly mismatched duplexes almost independent of salt concentration, and with increase in sodium concentration from 50 to 1000 mM, we observed a Tm rise of only about 1 °C. This observation is consistent with that made earlier in a study on detection of DNA sequences using DNA sensor probes,14 where it was shown that at high sodium ion concentrations (>100 mM), destabilization of the mismatched duplexes was slightly lower in comparison to the lower sodium ion concentrations. Effectively, such a disparity between the ways the Tm values of the fully matched and the singly mismatched duplexes depended on salt concentration could result in enhancement of the single base mismatch discrimination ability of both LNA and DNA probes as the salt concentration was increased (Table 3). Plot of Tm vs log[Na+] was found to be linear (Figure 4A) within the salt concentration range 50−1000 mM for both LNA and DNA. Positive slope of the plots indicates counterion association during duplex formation. The slopes were more positive for the fully matched duplexes compared to the singly mismatched duplexes indicating that counterion association along with duplex stabilization was significant for the fully matched duplexes, whereas for singly mismatched duplexes such an effect was almost negligible. Because of this reason, maximization of the single base mismatch discrimination ability of the LNA sensor layer could be achieved by varying salt concentration in the hybridization buffer. In solution phase studies on LNA−DNA duplex formation, using partially LNA-modified sequences, it

Figure 4. Variation of melting temperature (Tm) of the LNA−DNA duplexes with (A) log [Na+] and (B) with [NaCl] on the gold(111) surface compared to corresponding DNA−DNA duplexes. In part A, the empty symbols correspond to DNA−DNA duplexes and the filled symbols correspond to LNA−DNA duplexes, while in both of the cases the circles symbolize fully matched and the squares symbolize a single base mismatch situation. In part B, the solid circles symbolize fully matched LNA−DNA duplexes, and the solid squares symbolize singly mismatched LNA−DNA duplexes. 1620

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Table 4. Melting Temperatures of LNA−DNA Duplexes on Gold(111) Surface for Different Magnesium Chloride, Spermidine, and Spermine Concentrations in 20 mM Sodium Phosphate Buffer (pH 7.0)a MgCl2 concn (mM)

a

spermidine concn (mM)

spermine concn (mM)

nucleic acid duplexes

10

15

20

5

10

15

5

10

LNA-1−Cy3 DNA-1 LNA-2−Cy3 DNA-1 ΔTm

48.5 °C 29.9 °C 18.6 °C

62.7 °C 30.1 °C 32.6 °C

62.9 °C 30.6 °C 32.3 °C

53.9 °C 34.5 °C 19.4 °C

63.2 °C 33.6 °C 29.6 °C

54.5 °C 33.5 °C 21.0 °C

56.8 °C 34.1 °C 22.7 °C

63.8 °C 34.3 °C 29.5 °C

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

discrimination ability of the LNA sensor film could be maximized at a 15−20 mM Mg2+ concentration. In comparison, the effect of Na+ was most pronounced for the concentrations 50−100 mM. Maximization of mismatch discrimination could therefore be better achieved by applying Mg2+. In the solution phase study, it was reported that the interactions of Na+ and Mg2+ ions with probe−target duplexes are not significantly mismatch-specific.30 However, on the surface, we observed that the singly mismatched duplexes remained almost unaffected compared to the fully matched duplexes whatever were the salt concentration and the type of cation. This indirectly implies that on the surface the interactions of Na+ and Mg2+ with the probe−target duplex is mismatch-sensitive and that might arise from the spatial restrictions due to higher probe density on the surface. In the presence of spermidine and spermine in the hybridization buffer, we found almost the same results as we obtained for Mg2+ except for the fact that the results were obtained with lower concentrations of the polyamines compared to Mg2+. However, in the case of DNA, it was shown by Hou et al. that polyamines, especially spermine, can stabilize the mismatched duplex to such an extent that it could be as stable as the perfectly matched duplex.50 So it is quite obvious that the mode of interaction of spermine with the surface-tethered LNA−DNA duplexes differ in some aspects compared to the DNA−DNA duplexes. That might arise from the lesser backbone flexibility of the LNA molecules as well as spatial restrictions on the surface. It was observed that there is minimal hysteresis present between the solution heating and the cooling curves (Figure S6 in the Supporting Information), which indicates the largely reversible character of the melting transitions. Given this condition of reversibility to have met in our experiments, it is likely that the observed differences in melting temperature between the fully matched and the mismatched duplexes and between LNAs and DNAs are primarily determined by the factors related to base stacking and H-bonding. Enhanced base stacking and H-bonding in the case of the fully matched duplexes compared to the singly mismatched duplexes (where the mismatch is centrally placed) should primarily control the differences in melting temperature between fully matched and singly mismatched duplexes. In order to account for the differences between LNAs and DNAs, it has been observed earlier in solution phase experiments that single base mismatch discrimination can be performed better using the LNA probes, compared to the DNA probes.24,2 This superior discrimination capability of LNA has been attributed to the fact that LNA modification enhances base stacking of fully matched base pairs and decreases stabilizing stacking interactions of mismatched base pairs.30 This trend that is observed in the solution phase experiments has been reflected in the case of the immobilized LNA strands onto the gold(111) surface as shown by Mishra et al.23 The trend could be reproduced on the surface probably because of the structural rigidity of the LNA backbone that

mer thiol-DNA strands self-assembled on the gold(111) surface. 44 In the present study, we varied the NaCl concentration in the hybridization buffer from 50 mM to 3 M. It was observed that the Tm of the duplexes increased for salt concentrations up to 1 M. This fact can be explained by the shielding effect of the sodium ion that can minimize the electrostatic repulsion between the negatively charged LNA and DNA backbones, thereby stabilizing the duplexes. Beyond 1 M however, the Tm was found to reduce. It has been reported earlier that at high salt concentration (NaCl > 1 M) where the electrostatic contributions saturate, the presence of backbone charges can no longer influence the interactions.45 However, since there can be water structure rearrangement at the water− solute interface as a result of high salt concentration,46,47 salt can still influence the stability of the macromolecular structure indirectly at high salt concentrations. In the present case, the decrease in melting temperature for salt concentrations beyond 1 M could most likely be a result of destabilization of the duplexes due to water structure rearrangement. Role of Cationic Charge in “On-Surface” Mismatch Discrimination by LNA Probes. In order to investigate how the charge of cation could affect the melting behavior of the LNA−DNA duplexes, we incorporated magnesium ion (2+), spermidine (3+), and spermine (4+) in the hybridization buffer at varied concentrations. It was observed that with an increase in the magnesium ion (Mg2+) concentration, the Tm values of the fully matched LNA−DNA duplexes increased noticeably, whereas for the singly mismatched duplexes, the increase in Tm was negligible (Table 4), effectively resulting in enhanced mismatch discrimination with increasing Mg2+ concentration. Similar trends, i.e., clear increase in Tm of the fully matched duplexes and almost no change in Tm of the singly mismatched duplexes, were observed in the case of Na+ treatment too (Table 3). Within the concentration range of 10−15 mM, the effect of Mg2+ on Tm of the fully matched LNA−DNA duplexes was most pronounced. Below 10 mM, the increase in Tm with increasing salt concentration was much less prominent (data not shown). Above 15 mM, the effect of salt concentration seemed to become saturated. It is evident from the Tm values that a significantly lower concentration of the divalent magnesiun ion could be applied compared to the concentration of monovalent sodium ion for achieving a similar level of the stability of the LNA−DNA duplexes (Tables 3 and 4). It has been shown earlier that the Mg2+ distribution around an isolated DNA duplex is more compact than the Na + distribution.48 Therefore, the total sum of positive charges is higher for magnesium under the restricted spatial conditions, leading to a stronger stabilization effect of Mg2+ over Na+.14 In the case of LNA, a similar situation might be prevailing since it has been shown that the mode of binding of Mg2+ to nucleic acids depends essentially on the backbone negative charge density.49 As LNA residues do not introduce any significant charges, this result is quite expected. The single base mismatch 1621

dx.doi.org/10.1021/ac3028382 | Anal. Chem. 2013, 85, 1615−1623

Analytical Chemistry

Article

nonspecific interactions with gold surface and be oriented more upright with respect to the surface.23 It is likely that this orientational advantage of the ssLNA strands could positively influence its mismatch discrimination capacity compared to the ssDNA strands.

could result in reduced nonspecific interactions with the surface28 helping the LNA strand to adopt a more upright orientation and therefore making it least susceptible to the surface effects. The upright orientation also makes the LNA strand more accessible to the target DNA strand, ensuring greater fidelity in sequence recognition and therefore superior single base mismatch discrimination. Piao et al. reported that LNA purines offer great potential to recognize mismatches than LNA pyrimidines and DNA purines.38 In addition to that, Owczarzy et al. reported that a centrally placed A.G mismatch, in the case of LNA, has the largest discriminatory boost.37 In our case, since there is a centrally placed A.G mismatch (Table 1), enhanced mismatch discrimination in the case of the LNA probes, compared to the DNA probes, is expected for thermodynamic reasons. Melting of a nucleic acid duplex in solution means total unzipping of the duplex, where the individual strands can freely move away from each other. The same pathway is not expected to be followed within a film at a solid−liquid interface since each duplex is closely surrounded by a number of other duplexes within a film structure. The local concentration of the negatively charged oligonucleotides and therefore the probe density could be considerably higher on the surface compared to that in bulk solution since the probes can be chemically anchored on the surface. This allows both the steric effects as well as the electrostatic effects to play an important role in solid-phase hybridization. Interpretation of experimental data on hybridization/dehybridization events occurring in a film at the solid−liquid interface can pose a considerable amount of challenges and is often not straightforward. A number of sources of deviation from ideal behavior need to be considered. For example, immobilization may not be best achieved for all the sensor probes due to the presence of surface defects; second, the sensor probes may not all be oriented suitably for target-binding due to inhomogeneities in the film structure and/or nonspecific interactions with the surface, e.g., gold− nitrogen (nucleobase) interactions; third, the probe density may be too high that would inhibit effective target entry into the sensor film prior to hybridization. As a consequence of a complex interplay of such deviations from a “solution-like” situation, the “on-surface” Tm values are unlikely to follow the same pattern of change as in the case of solution measurements. The thermal melting experiments on surface-confined nucleic acid duplexes can therefore provide only an approximate idea about the thermodynamic stability of the nucleic acid duplexes. Despite the possible sources of ambiguities as described, two clear conclusions could be made in the present study. First, single base mismatch discrimination could be better performed by LNA probes compared to DNA probes on the gold(111) surface. Second, the single base mismatch discrimination ability of the LNA probes could be enhanced by increasing the monovalent salt (NaCl) concentration to 1 M. Mismatch discrimination could also be improved by using polyvalent cations like magnesium ion (2+), spermidine (3+), and spermine (4+), the maximum by a magnesium ion considering comparable concentrations in each case (Table 4). Importantly, we found that the same extent of duplex stabilization could be achieved by applying nearly 2 orders of magnitude lower concentrations of the polyvalent cations compared to the concentration of monovalent sodium ion. Unlike DNA, LNA has the modified ribose moiety which reduces the conformational flexibility and increases local organization of the phosphate backbone,51 and the ssLNA strands could avoid

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CONCLUSIONS In conclusion, we report for the first time how the single base mismatch discrimination ability of surface-anchored LNA probes can be maximized by ionic adjustments. While mismatch discrimination by both DNA and LNA probes was found to be improved on the surface, in comparison to the solution phase, LNA probes excelled DNA probes both in solution and on the surface. Both the type and the concentration of cations were found to play a decisive role in solid phase LNA−DNA hybridization. As the effect of salt concentrations on singly mismatched duplexes was almost negligible compared to the fully matched duplexes, single base mismatch discrimination ability of the LNA sensor layer could be enhanced with an increase in salt concentration. When we compare these results to the behavior of DNA sensor probes, it is revealed that ionic tuning results in more prominent effects on LNA-based single base mismatch discrimination compared to the DNA-based discrimination. This means that LNA probes are more susceptible to changes in the ionic environment and can therefore respond to smaller changes in ionic conditions.

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ASSOCIATED CONTENT

S Supporting Information *

Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +91 33 2473 4971 ext. 1506. Fax: +91 33 2473 2805. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We acknowledge financial support (Grant No. BT/PR-11765/ MED/32/107/2009) from DBT, Government of India, and research fellowships of S.M. and S.G. from CSIR, Government of India.

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REFERENCES

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