Oligonucleotide Microarrays: Immobilization of Phosphorylated

Aug 30, 2006 - signal-to-noise ratio (∼98) and resulted in high hybridization efficiency ... of phosphorylated oligonucleotides on epoxylated glass ...
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Bioconjugate Chem. 2006, 17, 1184−1189

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Oligonucleotide Microarrays: Immobilization of Phosphorylated Oligonucleotides on Epoxylated Surface S. Mahajan, P. Kumar, and K. C. Gupta* Nucleic Acids Research Laboratory, Institute of Genomics and Integrative Biology, Mall Road, Delhi University Campus, Delhi-110 007, India. Received April 24, 2006; Revised Manuscript Received July 19, 2006

A facile and efficient method for direct immobilization of phosphorylated oligonucleotides on an epoxy-activated glass surface is described. The new immobilization strategy has been analyzed for its performance in DNA microarray under both microwave and thermal conditions. It reflects high immobilization efficiency (∼23%), and signal-to-noise ratio (∼98) and resulted in high hybridization efficiency (∼36%) in comparison to those obtained with standard methods, viz., NTMTA (∼9.76%) and epoxide-amine (∼9.82%). The probes immobilized through the new strategy were found to be heat-stable, since the performance of microarray decreased by only ∼7% after subjecting it to 20 PCR-like heat cycles, suggesting that the chemistry could be used in integrated PCR/microarray devices. The immobilization of probes following the proposed chemistry resulted in spots of superior quality in terms of spot morphology, spot homogeneity, and signal reproducibility. The constructed microarrays have been successfully used for the discrimination of nucleotide mismatches. In conclusion, these features make the new immobilization strategy ideal for facile, efficient, and cost-effective manufacturing of DNA microarrays.

INTRODUCTION DNA microarrays have rapidly evolved to become one of the essential tools to study expression or mutation of thousands of genes simultaneously (1-4). DNA microarrays are mostly based on hybridization of targets with double-stranded PCR amplicons or single-stranded oligonucleotides (probes) immobilized on a polymer surface (biochip), followed by detection via an imaging or a scanning process. These biochips are replacing labor-intensive and reagent-consuming conventional gel-based methods. Basically, oligonucleotide microarrays can be fabricated in two different ways, viz., (i) by in situ synthesis following conventional and photolithographic techniques (58) and (ii) by deposition method, i.e., immobilization of presynthesized oligonucleotides on selected polymer surfaces (9-16). The deposition method, where prefabricated oligonucleotides are immobilized either covalently or noncovalently onto various solid surfaces (organic or inorganic), offers flexibility insofar as a variety of ligands can be fixed after purification (17-24). In this approach, reactive groups are generated on the chip surface, which can form covalent bonds with the reactive functionalities available in presynthesized oligonucleotide sequence in the presence of an activator or condensing agent (25-28). The methodology allows the attachment of probes in a regioselective manner with a good surface coverage as well as retention of the full activity of probes. In the present report, we describe a novel approach to synthesize oligonucleotide arrays that employs immobilization of 3′-phosphorylated oligonucleotides onto epoxy-functionalized glass microslides (Scheme 1) under microwave irradiation, where the reaction completes in just 10 min with remarkably high immobilization efficiency (∼23% at 0.2 µM probe concentration). The uniqueness of the proposed strategy lies in opening of the epoxy ring anchored on the polymer support by the phosphate group of the 3′-phosphate oligomers, which can be synthesized simply by employing a commercially available * Tel. +91 11 27662491; Fax +91 11 27667471; Email: kcgupta@ igib.res.in.

aminoalkylated polymer support (universal support) instead of conventional nucleoside-loaded polymer supports and hence eliminates the use of engineered polymer supports. Even the larger fragments of DNA as well as PCR amplicons can be immobilized after incorporating phosphate groups by enzymatic phosphorylation.

EXPERIMENTAL PROCEDURES Glass Silanization. The glass slides (75 × 22 mm2) were first cleaned according to published protocol (29). The silanization was accomplished using GOPTS (2%, v/v) in toluene (75 mL) for 4-5 h at 50 °C (30). After washing and drying, the slides were stored under an inert atmosphere. Oligonucleotide Synthesis and Purification. Oligonucleotides were synthesized at 0.2 µmol scale on a Pharmacia Gene Assembler Plus (see Supporting Information) using the standard phosphoramidite approach following the manufacturer’s protocol (Gene Assembler Plus Manual, Uppsala, Sweden, 1988) (31). Determination of Optimal pH Required for Immobilization of Phosphorylated Oligomers on Epoxy-Activated Glass Surface. In order to study the effect of pH on immobilization of phosphorylated oligonucleotides on epoxylated glass slide, the attachment of the probe was carried out at different pHs (6-12). Briefly, a labeled oligomer, viz., TET-d (TCT TGT ACG TCC GTC AAC GT)-OPO32- (3 µM) dissolved in reaction buffer (N-methylimidazole (0.1 M) containing 10% dimethylsulfoxide (DMSO) (v/v)) of different pHs 6, 8, 10, and 12, was spotted by a pipetteman (0.5 µL) on a functionalized glass microslide and incubated in a humid chamber for 12 h, followed by capping of the residual epoxy functionalities with a capping buffer (0.1 M Tris containing 50 mM ethanolamine, pH 9.0) for 15 min at 50 °C. The slide was then subjected to washings with 1× SSC buffer (4 × 50 mL), pH 7.0, for 20 min and Milli Q water (2 × 50 mL), followed by drying under vacuum. Finally, the slide was scanned under a laser scanner. Immobilization Kinetics. The result obtained from the model experiment was used to study the immobilization kinetics of phosphorylated oligonucleotide onto the epoxy-functionalized glass surface under microwaves (see Supporting Information).

10.1021/bc0601065 CCC: $33.50 © 2006 American Chemical Society Published on Web 08/30/2006

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Scheme 1. Strategy for the Immobilization of Phosphorylated Oligonucleotides on Epoxy-Functionalized Glass Surface.

In order to determine the optimal time required for immobilization of 3′-phosphorylated oligonucleotide, TET-d (TCT TGT ACG TCC GTC AAC GT)-OPO32- was diluted in reaction buffer (pH 10) to concentrations of 5, 10, and 20 µM and spotted onto epoxy-functionalized glass microslides in duplicates and subjected to microwave (MW) irradiation (with no exposure longer than 10 s in a humid chamber) in a MW oven operating at 800 W. Slides were withdrawn at regular time intervals (2, 4, 6, 8, 10, and 12 min) and subjected to capping and washings as described above. After drying under vacuum, the spots were visualized under a laser scanner and quantified. Spotting and DNA Immobilization. In order to look into the effect of oligonucleotide concentration to achieve highest density of oligonucleotide attachment on the surface, the kinetics of the probe immobilization at various concentrations under microwave irradiation was investigated. The reactions were carried out by spotting a labeled probe, TET-d (TCT TGT ACG TCC GTC AAC GT)-OPO32-, in a dilution series ranging from 0.2 to 40 µM concentrations for 10 min (60 × 10 s) under microwaves (as mentioned above). After treatment with capping buffer and usual washings, the slide was visualized under a laser scanner and quantified. PCR Heat Cycling. To evaluate the thermal stability of the linkage formed by probe immobilization, a microarray was prepared by immobilizing labeled 3′-phosphorylated oligonucleotide TET-d (TCT TGT ACG TCC GTC AAC GT)-OPO32(5 µM) under microwave irradiation for 10 min. The constructed microarray was subjected to PCR-like conditions for 0, 5, 10, 15, and 20 cycles in 2× SSC buffer (pH 7). PCR parameters were as follows: 94 °C for 30 s (denaturing step), 54 °C for 30 s (annealing step), and 72 °C for 30 s (extension step). After thermocycling, the slide was washed, dried, and visualized under a laser scanner. Similarly, the pH stability of the projected chemistry was evaluated by subjecting the constructed microarray to washings (4 × 15 min) with buffers of different pHs, ranging from 7 to 9. Specificity of Immobilization Chemistry. In order to investigate the specificity of the system, four oligonucleotides, viz., d (ACG GTA ACA GGA AAA AGA AG)-OPO32-, d (ACG GTA ACA GGA AAA AGC AG)-OPO32-, d (ACG GTA ACA CGA AAA AGC AG)-OPO32-, and d (TTT TTT TTT TTT TTT TTT TT)-OPO32- (5 µM) having zero, one, and two mismatches and noncomplementary probe, were spotted on an epoxylated glass slide (under microwave irradiation) followed by capping and usual washings. Subsequently, the spots on the microslide were hybridized with a complementary labeled TET-d (CTT CTT TTT CCT GTT ACC GT) and kept at 45 °C for 1 h and then at room temperature for 12 h. After washings with hybridization buffer, 2× SSC buffer (pH 7) (5 × 5 min), the microslide was subjected to laser scanning followed by quantification.

For each experimental condition tested on the microarrays, the experiment was repeated 2-3 times. The immobilization and hybridization data presented are the average of these repetitions, and the error bars represent the percentage error ((2-4%) observed on this average. In order to determine the immobilization and hybridization efficiencies, a calibration curve was constructed using a labeled oligomer (see Supporting Information).

RESULTS AND DISCUSSION Oligonucleotide probes bearing a 3′-phosphate group bind covalently to the epoxy-functionalized glass slide under the influence of microwaves (MW), a nonconventional energy source, which has significantly reduced the chemical reaction time from hours to minutes with increased yields (immobilization efficiency) and improved reproducibility (32-34). Though a large number of articles describe rate enhancement of reactions by microwaves, in contrast to thermal/photochemical reactions, the use of microwaves to construct oligonucleotide arrays has largely remained unexplored. We have constructed oligonucleotide arrays following the proposed chemistry and evaluated them for parameters like thermal stability, specificity, sensitivity for detection of targets, and reproducibility of attachment chemistry. The proposed strategy has been compared with the standard methods, viz., NTMTA and epoxide-amine, to highlight its advantages over them. With the aim to determine the optimal time required to complete the reaction between 3′-phosphorylated oligomers and the epoxy-functionalized glass surface, a model experiment was designed for which a compound, 2-(4,4-dimethoxytrityloxy)ethyl phosphate {DMTrO(CH2)2OPO32-}, was synthesized and reacted with the epoxy-functionalized control pore glass (CPG 500 Å) under microwaves and thermal condition (see Supporting Information). The results with the model compound indicate that the reaction between phosphate and epoxy group essentially completes in 10 min under microwaves, while it takes ∼4 h at 35 °C under thermal conditions. The optimal pH required for effective immobilization of phosphorylated oligonucleotides on the epoxy-activated glass surface was determined by spotting a labeled probe (at 3.0 µM) at different pHs. The results show that maximum immobilization was achieved at pH 10.0 (Figures 1A,B); therefore, immobilization of modified probes was carried out at pH 10.0 for the rest of the experiments. Similarly, optimal time required for the immobilization process under microwaves was evaluated by constructing a microarray as mentioned in the Experimental Procedures. The results confirmed that attachment density reaches a maximum between 8 and 10 min under microwaves (Figure 2A). The immobilization of probes reaches saturation level above 20 µM as shown by the fluorescence intensity of

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Figure 1. (A) A graph representing influence of pH on immobilization reaction of phosphorylated probe with epoxy-activated surface. (B) Fluorescence map obtained by immobilization of 5′-labeled, 3′phosphorylated probe, TET-d (TCT TGT ACG TCC GTC AAC GT)OPO32-, at different pHs (6-12). Slide was scanned at 75% of laser power and 70% of photomultiplier gain.

the spots (Figure 2B). The pictorial image showing the detection limit of 0.2 µM achieved with a 20-mer oligonucleotide is shown in Figure 3. The extent of involvement of internucleotidic phosphodiester groups and amines of heterocyclic bases to the immobilization reaction was determined by taking 5′-labeled nonphosphorylated oligomer and 4,5,6,7-tetrachlorofluorescein (TET dye) as controls of the experiment. The 5′-labeled-3′-phosphorylated, 5′labeled nonphosphorylated oligonucleotides, and 4,5,6,7tetrachlorofluorescein were spotted at 0.5 µM concentration on the same slide and exposed to microwaves for 10 min followed by usual washings (Figure 4A). Fluorescence intensity of spots of the nonphosphorylated oligonucleotide was found to be ∼12% of the probe with the 3′-phosphate modification, whereas no signal was observed at the place where only TET dye was spotted, implying selective binding of the terminal phosphate to the epoxy-activated surface (Figure 4B). The low but noticeable amount of immobilization of unmodified oligonucleotide could have arisen from weaker binding of phosphodiester groups of the probe to the surface. This was significantly reduced by treating the slides with capping buffer. The thermal stability of the constructed microarray was evaluated by subjecting it to 0, 5, 10, 15, and 20 cycles of PCRlike conditions. The fluorescent signal of the heat-treated microarrays was found to decrease by only ∼3% after 10 cycles and ∼7% after 20 cycles. The fluorescent signals of heat-treated and untreated microarrays (Figure 5A) show that the oligonucleotides immobilized on an epoxy-glass slide following the proposed strategy are sufficiently stable to be used in biological research. In an analogous way, the vulnerability of the constructed microarray toward different pHs was evaluated. The quantification results revealed that fluorescence intensity decreases by ∼4.5% at pH 8, whereas it became slightly unstable with

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Figure 2. Graphs depicting (A) time kinetics to determine the optimal time required for immobilization of phosphorylated oligonucleotide on epoxy-glass slide under microwaves; O, 5 µM; 0, 10 µM; 4, 20 µM; (B) Immobilization kinetics using different oligonucleotide concentrations under microwaves (10 min).

∼15.3% of reduction at pH 9.0 as compared to the intensity obtained at pH 7 (Figure 5B). The dynamics of microarray hybridization was demonstrated by performing the kinetics of the hybridization reaction for different target oligonucleotide concentrations. The results depict that, in the initial stage of increasing target concentration, the hybridization signal increases and then reaches a steady state between 40 and 80 µM (Figure 6A). The kinetics of hybridization efficiency (defined as the percent of covalently attached oligonucleotides that participated in duplex formation) for different probe concentration were realized by plotting the hybridization efficiency for different amounts of immobilized probes. The hybridization efficiency reaches a maximum (∼36%) for an optimal immobilized probe concentration of 5 µM; above this concentration, the efficiency did not increase even if the concentration of immobilized probes was increased (Figure 6B). This could be explained on the basis that overloading the surface with probes might cause a crowding effect that could lower the accessibility of the surface-bound probes. In addition, the efficiency of hybridization depends on many factors like the number of accessible epoxy groups to the 3′-phosphorylated oligonucleotides on the surface, the attachment efficiency of oligonucleotides to the epoxy-functionalized surface, and the efficiency in the reaction of hybrid formation between immobilized and target oligonucleotide concentration. However, by using a more sensitive label in the target oligonucleotide, viz., Cy 3, immobilized probes can be recognized even at low concentration (1 nM) of the target (see Supporting Information). In order to demonstrate the specificity of the system, four oligonucleotides were immobilized onto an epoxy-functionalized glass microslide, each at 5 µM concentration. After treatment with capping buffer, the microslide was subjected to a hybrid-

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Figure 3. Fluorescence map of epoxy-glass slide spotted with TET-d (TCT TGT ACG TCC GTC AAC GT)-OPO32- in a concentration dependent manner in duplicates. Slide was scanned at 75% of laser power and 70% of photomultiplier gain.

Figure 4. (A) Fluorescence map of epoxy-glass slide spotted with TET-d (TCT TGT ACG TCC GTC AAC GT) (lane 3), 4,5,6,7tetrachlorofluorescein (TET) (lane 2), and TET-d (TCT TGT ACG TCC GTC AAC GT)-OPO32- (lane 1), each one spotted at 0.5 µM; (B) Comparison of fluorescence intensity obtained by spotting 3′-phosphorylated A and unmodified oligomer B on epoxy-glass slide. Slide was scanned at 75% of laser power and 70% of photomultiplier gain.

Figure 5. (A) Thermal stability of oligonucleotide probe attached to epoxy-glass slide and comparison of fluorescence signal obtained after 0, 5, 10, 15, and 20 cycles of PCR-like conditions. (B) pH stability of the constructed microarray.

ization step with a labeled oligomer (complementary to zero mismatch sequence). Fluorescence was only detected at spots exposed to the appropriate complement. Figure 7A clearly shows the base mismatches; the perfectly matched sequence gave the maximum intensity (lane 1), while spots having one (lane 2) and two (lane 3) mismatches showed fluorescence intensity in decreasing order. No measurable nonspecific hybridization signal from the noncomplementary control (lane 4) was detected, demonstrating that nonspecific hybridization did not occur. The signal-to-noise ratio was, on an average, >98 as calculated from the signals obtained from complementary hybridized spots and noncomplementary hybridized spots. The quantitative result of the scanned image is depicted in Figure 7B. The applicability of the projected strategy to recognize the target even at low concentration of spotted probe solution (0.2-1 µM) was determined by performing the hybridization experiment (see Supporting Information).

The proposed strategy for oligonucleotide microarray preparation was compared with the two existing standard methods, viz., NTMTA (20) and epoxide-amine (21). For comparison purposes, an equal concentration of the appropriate oligonucleotide (each at 5 µM) was used. However, different reaction times, as reported therein, were employed in all three methods. After immobilization, the slides were subjected to usual washings and drying. The spots were then scanned under a laser scanner for quantification, and the immobilization efficiencies for each method were calculated using the calibration curve (see Supporting Information). Similarly, to confirm the superiority of the proposed strategy, hybridization experiments were also performed at the same spotting concentration (5 µM), and the results obtained were quantified. The immobilization and hybridization efficiencies of the proposed method (∼10% and 36%) were found to be significantly higher than the method employing NTMTA reagent (∼4.5% and 9.76%) and epoxide-

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Figure 6. (A) Measurement of steady state hybridization by exposing immobilized probe (5 µM) to different target concentrations (20, 40, 60, and 80 µM). (B) Hybridization efficiency obtained by hybridization of phosphorylated oligonucleotide immobilized on to epoxy-glass slide, varying the amount of covalently immobilized probes (1, 3, 5, and 10 µM), with a complementary labeled target.

Mahajan et al.

Figure 8. Comparison of immobilization (A) and hybridization efficiencies (B) of proposed strategy (I) with standard ones, viz., (II) ) NTMTA and (III) ) epoxide-amine.

Figure 9. A histogram plotted between the number of washing cycles and fluorescence intensity obtained after hybridization: a, proposed strategy; b, NTMTA; and c, epoxide-amine.

amine immobilization chemistry (∼4.62% and 9.82%), as shown in Figures 8A,B. The effect of time of washing on fluorescence intensity for the above-discussed three approaches was also evaluated. Briefly, the microslides obtained after performing hybridization assay for the three methods, viz., the proposed, NTMTA, and epoxide-amine, were subjected to 5, 10, and 20 cycles of washing with 2× SSC buffer, pH 7 (each cycle of 5 min duration). The quantification results (Figure 9) reveal that the fluorescence intensities decrease by ∼1.28% and 1.75% (proposed method), ∼1.22% and 1.62% (NTMTA), and ∼0.81% and 2.42% (epoxide-amine method) after 10 and 20 cycles, respectively.

CONCLUSIONS Figure 7. (A) Fluorescence map showing the detection of nucleotide mismatches and specificity of immobilization via hybridization with TET-d (CTT CTT TTT CCT GTT ACC GT) (40 µM): lane 1, d(ACG GTA ACA GGA AAA AGA AG)-OPO32-; lane 2, d(ACG GTA ACA GGA AAA AGC AG)-OPO32-; lane 3, d(ACG GTA ACA CGA AAA AGC AG)-OPO32-; lane 4, d(TTT TTT TTT TTT TTT TTT TT)OPO32-, each spotted at 5 µM. Slide was scanned at 70% of laser power and 65% of photomultiplier gain. (B) Comparison of fluorescence intensity observed with perfect, single, and double mismatch in probe after hybridization with target (complementary to zero mismatch).

We have demonstrated a new, efficient, and fast method for fabrication of oligonucleotide arrays under microwaves. The technique relies on modification of oligonucleotide at the 3′end with a free phosphate group, an easy modification that can be performed by using commercially available aminoalkylated support, followed by reaction with epoxy-activated surface. The utility of the method is demonstrated by probing the chemical and thermal stability as well as the reproducibility of the constructed microarray. The highlighting feature of the proposed strategy is that DNA sequences as well as PCR amplicons can

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also be immobilized on an epoxylated glass surface after enzymatic phosphorylation. We hope that the projected attachment chemistry involved in this effort will enable a nonchemist, the actual user, to construct oligonucleotide microarrays for routine applications.

ACKNOWLEDGMENT Financial support for this work was provided by CSIR Task Force Project, NNIOSB. S.M. thanks the Council of Scientific and Industrial Research (CSIR), New Delhi, India, for the award of a Senior Research Fellowship. Supporting Information Available: Additional experimental data. This material is available free of charge via the Internet at http://pubs.acs.org.

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