A Structured Chitosan-Based Platform for Biomolecule Attachment to

Istituto di Tecnologie Biomediche, Consiglio Nazionale delle Ricerche, LITA, via F.lli Cervi, 93 20090 Segrate (MI) Italy,. Istituto di Biologia e Bio...
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Bioconjugate Chem. 2006, 17, 371−377

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A Structured Chitosan-Based Platform for Biomolecule Attachment to Solid Surfaces: Application to DNA Microarray Preparation Clarissa Consolandi,†,* Marco Severgnini,† Bianca Castiglioni,§ Roberta Bordoni,† Andrea Frosini,‡ Cristina Battaglia,‡ Luigi Rossi Bernardi,‡ and Gianluca De Bellis† Istituto di Tecnologie Biomediche, Consiglio Nazionale delle Ricerche, LITA, via F.lli Cervi, 93 20090 Segrate (MI) Italy, Istituto di Biologia e Biotecnologia Agraria, Consiglio Nazionale delle Ricerche, and Dipartimento di Scienze e Tecnologie Biomediche, Universita’ degli Studi di Milano, Milan, Italy. Received September 26, 2005; Revised Manuscript Received January 16, 2006

A structured chemical platform based on chitosan, an amine-rich polysaccharide, is presented as an alternative chemistry to functionalize solid support (in this case, glass slides) for grafting biomolecules. This approach has been adopted for generating arrays using amino-modified oligonucleotides with two different lengths (25-mer and 70-mer) for different purposes. Results using these chitosan-activated surfaces indicate high oligonucleotide loading capacity, good availability to hybridization against targets, and effectiveness in enzyme-mediated single nucleotide polymorphism (SNP) detection procedures by DNA polymerase and DNA ligase enzymes with low background. Universal arrays have been prepared and extensively used with excellent results in different applications. The chitosan-treated surfaces were also evaluated for their performance in a gene expression experiment.

INTRODUCTION Chemical binding of biomolecules to solid surfaces is a key step in a very large set of applications. Among many others, the generation of arrays of biomolecules is a relevant field where proper chemical platforms are needed. Microarrays consisting of oligonucleotides, polynucleotides, peptide nucleic acids, peptides, proteins, and other molecules are becoming a valuable tool in modern molecular biology (1), opening a way for largescale screening of mutations and studies of gene polymorphisms, gene expression analysis, and functional biology. The most relevant example of the application of arrays is their use for massive DNA analyses in parallel using oligonucleotides or polynucleotides as molecular probes. Oligonucleotide microarrays can be mass produced by photolithographic techniques in situ (2), but many users prefer homemade microarrays prepared by the spotting of modified oligonucleotides (3). This widely adopted option requires direct attachment of presynthesized oligonucleotides to an activated surface. This approach permits a very quick set up of new microarrays and their upgrading, incorporating new genetic information. A key step in this approach is the preparation of surfaces modified with appropriate functional groups allowing for the attachment of oligonucleotides to the solid support. Many different approaches have been proposed including those compatible with 5′-thiolated oligonucleotides (4), although in general amino-modified oligonucleotides are preferred. Gel pads have been proposed (5) as well as the use of branched linkers (6). Similarly, chemically preactivated microscope slides have been commercially proposed offering 3D reactive structures (7). * To whom correspondence should be addressed. Telephone: +39 0226422724/5. Telefax: +39 0226422770. E-mail: [email protected]. † Istituto di Tecnologie Biomediche, Consiglio Nazionale delle Ricerche. § Istituto di Biologia e Biotecnologia Agraria, Consiglio Nazionale delle Ricerche. ‡ Universita’ degli Studi di Milano.

We started exploring the potentials and pitfalls of a structured approach to the preparation of coated surfaces aimed especially at the microarray preparation. This approach requires proper functionalization of the glass surface with silanes chosen according to the functional moiety to be reacted, the scaffold creation using a polymer (either natural or synthetic), and finally the activation of the resulting surface using proper moieties. We presented our results using poly-L-lysine and acrylamide/ acrylic acid copolymer as scaffolds for oligonucleotide microarray preparation (8). These two polymers are well-known in the field and have been already used. Here we present our results using chitosan, an amine-rich polysaccharide derived by partial deacetylation of chitin (9). Its deprotonated amino groups can react with a variety of electrophiles. As a result, various chemistries can be exploited to cross-link chitosan and to graft substituents onto this polymer (10-14). We prepared three chemical supports using chitosan with three different molecular weights (120 kDa, 400 kDa, 650 kDa), each of these with or without KCl. In all these cases, the polymer is covalently bound to glass slides activated with 3-glycidoxypropyltrimethoxysilane (GOPS) as a scaffold for generating arrays. Polymer-coated slides have been activated with 1,4phenylene diisothiocyanate (PDITC) for generating arrays with presynthesized oligonucleotides bearing amino groups. Results obtained using this coupling platform show simple preparation of the surface, high oligonucleotide loading capacity, good binding efficiency, remarkably uniform performance over the entire surface, and no particular care required for their storage. We also demonstrate that the attachment of aminomodified oligonucleotides consistently yields arrays of readily accessible probes with different lengths (25-mer and 70-mer) to hybridization. Mutation detection by enzyme (ligase or polymerase)-mediated approaches has been successfully performed with low background. In combination with a universal array prepared using this platform, a PCR-ligation detection reaction approach was used for the single nucleotide polymorphism (SNP) detection in different fields with excellent specific-

10.1021/bc050285a CCC: $33.50 © 2006 American Chemical Society Published on Web 02/22/2006

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Table 1. Oligonucleotide Sequences Used in the Experiments as Probes and Templates sequencea

name 25-mer 70-mer 25-mer-compl 70-mer-compl

1450-1 ATTINO1 1450-1 ATTINO2 1450-1 ATTINO3 1336 PSEUDO Actinomycetes 16S RNA template a

5′-modification

a. Oligonucleotides Used in All Experiments for Hybridization Efficiency and Surface Loading 5′-AAAAAAAAAATACCGGCGGCAGCACCAGCGGTAAC-3′ NH2 5′-AAAAAAAAAGAGAAGGATTATGAGGAGGTTGGTG NH2 TGGATTCTGTTGAAGGAGAGGGTGAGGAAGAAGGAGAGGAATAC-3′ 5′-GTTACCGCTGGTGCTGCCGCCGGTA-3′ Cy5 5′-GTATTCCTCTCCTTCTTCCTCACCCTCTCCTTCA Cy5 ACAGAATCCACACCAACCTCCTCATAATCCTTCTCT-3′ b. Oligonucleotides Used as Probes and Template in the Minisequencing Experiment 5′-TTGTACACACCGCCCGTCACGT-3′ NH2 5′-TTGTACACACCGCCCGTCACGTC-3′ NH2 5′-TTGTACACACCGCCCGTCACG-3′ NH2 5′-CTAATCCCAIAAAACCGATCGT-3′ NH2 (5′-GGTGTTTCCGACTTTCCTGACGTGACGGGCGGTGTGTACAA-3′) none

3′-modification Cy3 Cy3 none none

none none none none none

Bases printed in bold and underlined are targeted by the ATTINO probes in the minisequencing reaction.

ity and high sensitivity. Finally, a large microarray for the detection of RNA expression levels has been prepared.

EXPERIMENTAL PROCEDURES All chemicals and solvents were purchased from SigmaAldrich (Milan, Italy) and, unless otherwise stated, used without further purification. Precleaned nonderivatized microscope slides (25 × 75 × 1 mm) were used (Sigma-Aldrich). Oligonucleotides were purchased from Thermo Hybaid (Ulm, Germany). They were HPLC purified and checked by MALDI-MS. Cleaning and Silanization of Microscope Slides. Glass slides were cleaned by soaking in 1 M NaOH for 2 h on a shaker followed by rinsing with distilled water, immersed in 1 N HCl solution overnight on a shaker, and then rinsed again in distilled water. Slides were immersed in 96% ethanol for 10 min and then washed three times with distilled water. Slides were immersed in acetone for 10 min, removed, and dried. Then they were treated with 1% (v/v) GOPS (3-glycidoxypropylsilane) in 95% ethanol for 1 h. Excess silane was removed by dipping the slides in 95% ethanol for 1 min. Finally they were dried at 150 °C for 20 min. Polymeric Coating Synthesis and Surface Activation. The polymer solution was prepared by dissolving 10 g of chitosan (120, 400, or 650 kDa) in a liter of HCl 50 mM. Each different polymer solution was prepared with or without KCl (0.5 M) in order to have six different coatings. The solutions were heated at 70 °C, stirred for 1 h, and then filtered. Treated slides were left in a water solution containing 10% PBS and 10% polymer (0.1% w/v) for 1 h on a shaker. The slides were washed five times with distilled water, centrifuged at 800 rpm for 3 min. and dried for 10 min at 45 °C. The slides were then activated by immersion into a 0.2% solution of 1,4-phenylene diisothiocyanate in 90% N,N-dimethylformamide and 10% pyridine. The activation reaction was carried out at room temperature for 2 h. After being washed with methanol and acetone (2 min each), the activated slides were dried and stored, until used, in a dark closed box under vacuum. Oligonucleotide Immobilization. Amino-modified oligonucleotides were diluted in printing buffer (150 mM sodium phosphate, pH 8.5) and spotted onto the treated slides. The spotting was performed using a contact system (MicroGrid II Compact, Biorobotics, UK). The covalent attachment to activated supports and the deactivation process were performed according to the following protocol: (a) The printed slides were placed in a saturated NaCl humidification chamber. (b) After overnight incubation at RT, the deactivation step was carried out by treatment with a solution consisting of 50 mM ethanolamine, 0.1 M Tris (pH 9), and 0.1% SDS at 50 °C for 15 min. (c) After being rinsed twice with

distilled water, the modified surfaces were immersed into a 4X SSC/0.1% SDS solution (prewarmed to 50 °C) for 15 min on a shaker. (d) Finally, printed surfaces were washed twice with distilled water and spun at 800 rpm for 3 min. We chose to use two different deposition schemes to test the loading and the binding capacity of our surfaces. (a) Coupling Efficiency (loading capacity). The loading capacity of the six chemically modified surfaces was tested by spotting at 10 different final concentrations (0.1-50 µM) of two oligonucleotides with two different lengths (25-mer and 70-mer) (Table 1a) bearing a 5′-amino modification and a 3′Cy3 fluorescent label. Each slide contains eight identical subarrays in a 2 × 4 conformation: the spots are arranged in a 16 × 13 matrix. Each concentration was replicated four times (15). (b) Binding Capacity. The activated surfaces were tested in hybridization experiments. These were performed using two 5′Cy5-labeled oligonucleotides with a sequence complementary to the 25-mer and 70-mer covalently attached amino-modified oligonucleotides (Table 1a), respectively. The two different oligonucleotides were spotted at a final concentration of 50 µM in a 150 mM sodium phosphate buffer (ph 8.5) onto the six different surfaces, using the attachment protocol described above. The deposition scheme has the usual structure of eight identical subarrays in a 2 × 4 conformation. Each oligonucleotide was replicated 64 times in each subarray. An amount of 65 µL of hybridization solution consisting of the Cy5-labeled oligonucleotides (0.1-1 µM), 5X SSC, 0.1% SDS, and 0.1 mg/mL denatured salmon sperm DNA was used to perform hybridization in a homemade multiple sample chamber (eight-well chamber), using Press-To-Seal silicone isolators (Schleicher & Schuell, Germany). The hybridization solution was heated in a boiling water bath for 2 min and then cooled in ice before the application onto the array. Hybridization experiments were carried out at 65 °C for 1.5 h. After incubation, slides underwent four washing steps at 65 °C by using 1X SSC/ 0.1% SDS for 15 min in a oven. Finally, hybridized oligo-arrays were spun at 800 rpm for 3 min. Ligation Detection Reaction and a Universal Array Format. We chose to use chitosan with medium molecular weight to prepare universal arrays. Solutions of 5′-aminomodified oligonucleotides with a 5′-polyA tail, called zip-codes, were transferred into a 384-well microtiter plate in a volume of 30 µL and spotted at a final concentration of 50 µM in 150 mM sodium phosphate buffer (pH 8.5) onto the surface using the attachment protocol described above. The oligonucleotides were applied onto the slides by contact printing using the MicroGrid II Compact arrayer. Each microscope slide contains eight identical subarrays, represented as a universal array whose layout (15) consisted of zip-codes

A Chitosan-Based Platform for Oligo-Arrays

printed in quadruplicate distributed within the printing area; two zip-codes, namely 63 and 66, were associated to a ligation control and a hybridization control, respectively (15); as a negative control (blank), printing buffer was used. Each universal array was a subarray consisting of 208 spots of 13 rows × 16 columns. As described in detail in Consolandi et al. (15), two discriminating probes (allele-specific probes designed to have their 3′-position placed just over the polymorphic site) labeled with two distinct fluorocromes (cyanine 3 and cyanine 5) and a common probe were selected for each SNP. Common probes were designed with a complementary zip-code sequence affixed to their 3′-end to unify the kinetics of the hybridization. Each zip-code sequence has a unique specific array location. The LDR mixture contained 81 oligonucleotides (27 common probes and 54 discriminating oligos), designed to identify 27 polymorphic sites in the HLA-A gene. As target, we used a heterozygous sample (A*0201/030101). The LDR mixture was cycled for 30 rounds of 94 °C for 30 s and 65 °C for 4 min in a thermal cycler (GeneAmp, PCR System 9700, Applied Biosystem, Foster City, CA) and then diluted to prepare the hybridization mixture. The mixture, after heating to 94 °C for 2 min and chilling on ice, was applied onto the subarray under a homemade multiple sample chamber (eight-well chamber), using PressTo-Seal silicone isolators (Schleicher & Schuell, Germany). Hybridization was carried out in the dark at 65 °C for 1.5 h, in a temperature-controlled system (Shack′n′Stack, Hybaid, England). After removal of the chamber, the slide was washed for 15 min in a prewarmed (65 °C) solution of 1X SSC/0.1% SDS with gentle shaking (Shack’n’Stack, Hybaid, England). Finally, the slide was spun at 800 rpm for 3 min. Solid-Phase Minisequencing Reaction Using a Synthetic Template. In the minisequencing technique (16), a primer extension reaction is performed, starting from a specifc primer that is designed to anneal directly adjacent to the mutation site, by the incorporation of a single fluorescent dideoxynucleotide (ddNTP) which is complementary to the variant base in the template. Each ddNTP is labeled with a different fluorophore with distinct spectral emission, thus allowing the identification of the incorporated base. The minisequencing reaction was performed onto an oligoarray, prepared by spotting of four oligonucleotides designed on the Actinomyces 16S ribosomal RNA sequence, according to Busti et al. (17). Three oligonucleotides (1450-1 ATTINO1, 1450-1 ATTINO2, 1450-1 ATTINO3) were designed in order to anneal directly adjacent to three consecutive bases while another oligonucleotide represents the negative control (1336 PSEUDO) (Table 1b). Each oligonucleotide was spotted 10 times in a row. We used a synthetic template as a sample at 1 µM final concentration and a labeled-ddNTPs mix (TamraddCTP, Cy5-ddUTP, R110-ddGTP, TexasRed-ddATP) at 0.1 µM final concentration. The reaction buffer consisted of 26 mM Tris-HCl pH 9.5, 6.5 mM MgCl2, and 0.2% Triton X-100 buffer with 0.25 U/µL Dynazime DNA polymerase (Finnzymes OY, Helsinki, Finland). The mixture, after heating to 94 °C for 2 min and chilling on ice, was applied onto the array under the homemade multiple sample chamber described above. The reaction was carried out in the dark at 55 °C for 1 h, in a temperature-controlled system (Shack’n’Stack, Hybaid, UK). After removal of the chamber, the slide was washed for 5 min in MilliQ-filtered water with gentle shaking (Shack’n’Stack, Hybaid, England). Finally, the slide was spun at 800 rpm for 3 min. Detection of RNA Expression Levels. Finally, the performance of the chitosan treated surface was evaluated in a gene expression experiment. A set of 5376 probes, comprising 4998 peach genes (Operon Technologies, Alameda, CA), 188 ran-

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domly generated controls, 30 human spike-in controls, 15 hybridization controls, and 145 Blanks negative controls, were deposited by a MicroGrid II Compact arrayer, equipped with 4 Apogent Discoveries Microspot 10K pins. Oligonucleotides were resuspended in 384-well plates in a sodium phosphate buffer, to reach a final concentration of 15 pmol/µL oligonucleotide in each well. All probes were 5′-amino-modified 70-mers. Each probe was spotted in duplicate. Slides were processed according to the protocol described in the Oligonucleotide Immobilization section. We evaluated the quality of the slides by using both a 9-mer complementary oligonucleotide and two aliquots of peach RNA, labeled with two different fluorochromes in a self-to-self-experiment. The 9-mer hybridization solution was prepared as follows: 7.5 pmol/mL 5′-Cy3-labeled 9-mer, 5X SSC, 0.1% SDS, and 0.1 mg/mL salmon sperm DNA. The hybridization was performed at 16 °C for 8 h in a GenTac HybStation (Genomic Solutions, Perkin-Elmer Life Sciences, Boston, MA); then the slides were washed with a 2X SSC, 0.1% SDS solution for 5 min at 16 °C. Target cDNAs, labeled with Cy3 and Cy5, were independently generated according to the SuperScript Indirect cDNA Labeling System (Invitrogen, Life Technologies, Paislay, UK). A 20 µg amount of RNA was used in each reaction. The resultant cDNA targets labeled with Cy3 and Cy5 were then purified using a PCR purification kit (Invitrogen, Life Technologies, Paislay, UK), as described by the manufacturer, and mixed together just before hybridization. The hybridization solution for the self-to self-experiment was the same as that used in the experiment with the 9-mer oligonucleotide. Hybridization was performed in the hybridization station for 8 h at 42 °C. The slides were washed with a preheated solution that contained 2X SSC, 0.1% SDS for 5 min at 42 °C and then with SSC 0.2% for 5 min. Signal Detection. Fluorescent signals were acquired at 5 µm resolution using a ScanArray 4000 laser scanning system (Perkin-Elmer Life Sciences, Boston, MA). The Green laser was used for CY3 dye ( λex 543 nm/λem 570 nm), Tamra dye ( λex 552 nm/λem 575 nm), the Red laser for CY5 dye (λex 633 nm/ λem 670 nm), the Yellow laser for Texas Red ( λex 593 nm/λem 612 nm), and finally the Blue laser for Rhodamine 110 (R110) ( λex 505 nm/λem 530 nm). Both the laser and photomultiplier tube (PMT) power were set in order to have no saturated signals in any case. Data Analysis. To quantitate the fluorescent intensity of spots, we used the QuantArray Quantitative Microarray Analysis software (Perkin-Elmer Life Sciences, Boston, MA). The quantitation method we chose was the “fixed circle” method. Spot intensities were calculated using the mean intensity option. Data analysis of each experiment was performed by the following calculations: local background was subtracted from the intensity of each spot; fluorescent intensities (IF) of the replicates spots were averaged and the coefficient of variation was calculated.

RESULTS AND DISCUSSION Since the advent of microarray technology, several chemistries have been proposed for oligonucleotide attachment to glass surfaces (16, 18). We have tested many of the simplest strategies to attach amino-modified oligonucleotides. Our experience indicates that such chemistries are not suited for oligonucleotide microarray preparation aimed at polymorphism or mutation detection. More complex strategies have been proposed, including those that incorporate polyfunctional linkers such as polyacrylamide gel pads (5), branched chains (3), and poly(acrylic acid-co-acrylamide) copolymers generated in situ (18). Such approaches overcome some difficulties arising from poor

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Figure 1. Array loading capacity. GOPS-chitosan-modified surfaces were spotted by using increasing concentrations from 0.1 to 50 µM of two oligonucleotides with two different lengths (25-mer and 70-mer) bearing a 5′-amino modification and a 3′-Cy3 fluorescent label. The graph shows the relationship between the oligonucleotide concentration and the surface-loading capacity measured as spot fluorescence intensity for the six modified surfaces (MC ) medium chitosan, MCK ) medium chitosan plus KCl, HC ) high chitosan, HCK ) high chitosan plus KCl, LC ) low chitosan, LCK ) Low chitosan plus KCl. Low corresponds to 120 kDa as molecular weight, medium corresponds to 400 kDa, and high corresponds to 650 kDa).

loading capacity of the (two-dimensional) glass surface, thanks to the three-dimensional structure of the swollen polymers. Following such an approach, we recently started experimenting with a three stage-process for preparing such surfaces involving (a) the functionalization of the glass surface, (b) attachment of a natural or synthetic polymer creating the scaffold, and (c) proper polymer scaffold activation against moieties to be targeted on the probes. Following our preliminary studies (8) using poly-L-lysine and acrylamide/acrylic acid copolymer, we selected chitosan, a polycation, to create a chemical platform suitable for biomolecules attachment to solid surfaces. Our approach involves the creation of a polymeric layer covalently bound to the surface and activated with phenyldiisothiocyanate (PDITC). This allows the attachment of aminebearing molecules. We analyzed the performance of these chemical platforms in order to characterize their behavior and ascertain their potential in oligonucleotide microarray preparation. All the sequences of the oligonucleotides used in the applications are reported in Table 1 (parts a and b). Efficiency and Specificity of Oligonucleotide Covalent Attachment: Array-Loading Capacity. The chitosan-modified surfaces were analyzed before oligonucleotide spotting by laser scanning at 543 nm (suitable for cyanine 3 dye excitation) and at 633 nm (suitable for cyanine dye excitation) to determine their overall fluorescent background, which was found in all cases to be very low at both wavelengths (data not shown). For DNA microarray fabrication, a pin contact arrayer was used. We tested a contact pin spotter (MicroGrid II Compact, Biorobotics, UK), demonstrating the mechanical stability of the polymeric coatings we propose (data not shown). We employed 5′-amino, 3′-Cy3-modified 25-mer and 70-mer oligonucleotides to investigate the influence of the six different coating solutions on the surface-loading capacity. To explore this issue, we analyzed our six derivatized surfaces by spotting the doubly modified oligonucleotides at different concentrations (ranging from 0.1 µM to 50 µM in 10 different concentrations) arranged in a 16 × 13 matrix. The corresponding signals were quantitated using QuantArray Quantitative Microarray Analysis

software. Sixty-four spots (two slides, eight subarray, four replicates) for each concentration were used to calculate mean and standard deviation. The results, shown in Figure 1, illustrate the relationship between the oligonucleotide concentration and the surfaceloading capacity measured as spot fluorescence intensity collected at 543 nm for the six modified surfaces. The loading capacity increases with increasing of the concentration of the spotted oligonucleotides in the both cases (25-70-mer). The 25-mer oligonucleotide shows a loading capacity higher than that of the 70-mer one. The surface prepared using the medium molecular weight chitosan with KCl shows the best spot fluorescence intensity (Figure 1), but the use of the same polymer without KCl gives a support with the best CV range (5% < CV < 17%), thus becoming the most reliable surface. The worst spot fluorescence intensity and the worst CV range are found for the surface prepared using the low molecular weight chitosan with KCl. Efficiency of Hybridization to Immobilized Oligonucleotides. A set of experiments was performed to evaluate the fluorescence signal produced in an oligo-oligo hybridization model experiment by determining the relationship between the loaded oligonucleotide concentration and the hybridization efficiency. This test is of great importance, as the major concern in oligonucleotide arrays preparation is the accessibility of the probe for hybridization, as well as the specificity obtained in the interaction. DNA microarrays described above were hybridized with two Cy5-labeled oligonucleotides complementary to the 25-mer and 70-mer spotted sequences using a range of oligonucleotide concentration from 0.1 to 1 µM. Figure 2a,b depicts the relationship between the loaded oligonucleotide concentration and the hybridization signal intensity for the both oligonucleotides. As shown in Figure 1, the loading capacity and availability to hybridization are good (Figure 2a,b). Above a probe concentration threshold, loading and availability remain nearly constant (tested at either 1 or 10 µM template concentration). This can be explained assuming that these chemistries yield

A Chitosan-Based Platform for Oligo-Arrays

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Figure 3. Scanner analysis of ligation detection reaction/universal array experiment. Fluorescent laser scanning of a heterozygous (A*0201/ 030101) sample analysis by using PCR/LDR/UA approach. This composite image is realized by overimposing Cy3 fluorescent signals (green palette) and Cy5 fluorescent signals (red palette). Yellow spots are the combination of Cy3 and Cy5 dyes.

Figure 2. Binding capacity. Printed slides with 10 different concentrations of covalently attached oligonucleotides were hybridized with five different concentrations (0.1-1 µM) of Cy5-labeled oligonucleotide complementary to the spotted ones. The graph depicts the relationship between the loaded oligonucleotide concentration and the hybridization signal intensity for the both oligonucleotides (a, b). (MC ) medium chitosan, MCK ) medium chitosan plus KCl, HC ) high chitosan, HCK ) high chitosan plus KCl, LC ) low chitosan, LCK ) low chitosan plus KCl. Low corresponds to 120 kDa as molecular weight, medium corresponds to 400 kDa, and high corresponds to 650 kDa).

surfaces with fairly well-spaced reactive groups that avoid excessive crowding of probes that could negatively affect the microarray performance. Applications. Efficiency of Ligation Detection Reaction Combined to a UniVersal Array. To explore the potential of the new surfaces, a ligation detection reaction (LDR) combined to a Universal Array (UA) platform has been optimized to analyze single nucleotide polymorphisms distributed within exons 2 and 3 of the HLA-A gene. The assay involves the amplification by PCR of the HLA-A genomic region (1900 bp), a multiplex LDR carried out to type twenty-seven HLA polymorphic sites followed by the capture of ligated products through hybridization onto a universal array. According to Consolandi et al. (15), a polymorphic site is typed by using two discriminating oligonucleotides labeled at their 5′-end with two fluorophores. Figure 3 shows the scanner analysis of the ligation detection reaction/universal array experiments obtained using a heterozygous sample (A*0201/030101). HLA genotyping by PCR/LDR/UA is in perfect agreement with typing obtained by direct sequencing. Our results clearly demonstrate that our treated surfaces are suitable to support hybridization reactions of complex targets, showing good specificity and efficiency. Good spot quality facilitates the subsequent evaluation of array data and has immediate effects on the reliability of the results. Enzymatic Reaction: Efficiency of Minisequencing Reaction onto the Surface. Our slides were also tested for their performance as supports for in-situ enzymatic reactions. In respect to the experiment described above, in which the enzymatic reaction was performed in liquid phase, we carried out the minisequenc-

ing reaction directly on the surface. The concept of the minisequencing assay is to anneal a detection primer to the nucleic acid sequence immediately 3′ of the nucleotide position to be analyzed and to extend this primer with a single labeled dideoxynucleotide using a DNA polymerase. To test this enzymatic procedure, we chose the Actinomyces 16S ribosomal RNA gene as reference sequence to design our synthetic template (see Experimental Procedures section) and probes (Table 1, part b). Three out of the four oligonucleotides (1450-1 ATTINO1, 1450-1 ATTINO2, 1450-1 ATTINO3) were designed in order to anneal directly adjacent to the targeted site. The 1336 PSEUDO oligonucleotide represent a negative control. The oligo-array contains the four oligonucleotides, acting as primers in the minisequencing reaction, each spotted 10 times in a row. During the reaction, the DNA polymerase carried out the incorporation of the fluorescent dideoxynucleotide (ddNTP), which is complementary to the targeted base in the template DNA. The extended probes, covalently bound to the solid support, can be detected thanks to the incorporation of fluorescent dyes. Figure 4 shows the fluorescent signals obtained using the four filters for the detection of the fluorescent signal due to the incorporation of the ddNTPs labeled with Tamra, Cy5, Rhodamine110, and Texas Red dyes. As expected, a fluorescent signal of the proper wavelength is detected identifying the targeted base on the template. No signal is detected from the negative control. This result suggests that our surfaces allow primer extension in-situ. Detection of RNA Expression LeVels. In the 9-mer hybridization, 99.1% of the 4998 “discovery” genes was present with at least one replicate per array. The randomly generated sequences and the spike-in controls were all present with at least one replicate per slide, whereas 98.5% of the hybridization controls performed as expected; at the same time 93.5% of the blank spots were not found by the quantitation software. In the self-to-self-hybridization, instead, we found that 3238 “discovery” probes (65%) were found as present by the quantitation software. The background subtracted intensities for the two channels were then drawn as a scatter plot against each other, to represent levels of up or down regulation. As the samples were aliquots of the same starting RNA, we expected no change or a very little number of transcripts to be differentially expressed. Given a threshold of 2-fold change as the limit of differential expression, only 131 out of the 3238 “discovery” genes were obtained. This means that about 96% of the transcripts were not recognized as differentially expressed. Pearson’s correlation coefficient between the two channels was

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Figure 4. Scanner analysis of the minisequencing experiment. An oligo-array, prepared by spotting of four oligonucleotides (Table 1b) designed on the Actynomices 16S rRNA sequence, is scanned by using the four filters for the detection of the fluorescent signal due to the incorporation of the ddNTPs labeled with Tamra, Cy5, Rhodamine110, and Texas Red dyes. The green frame includes the fluorescent image scanned by Tamra filter, showing a C extension for 1450-1 ATTINO1 as expected. The yellow frame includes the image scanned by Texas Red filter, showing an A extension for 1450-1 ATTINO2 as expected. The red frame includes the image scanned by using Cy5 filter, showing a T extension for 1450-1 ATTINO3 as expected. No extension is detected in the blue frame (Rhodamine 110 filter). The fluorescent signals are visible only for the three oligonucleotides specific for the 16S rRNA sequence. In the fourth case (1336 PSEUDO sequence not specific for the template used), no signal is detected.

and binding capacity. As demonstrated by the discrimination of SNPs, oligonucleotides attached to our surfaces reveal highly specific hybridization properties and the accessibility to polymerase for enzyme-mediated reactions. Also labeled cDNAs react with the oligonucleotide probes attached to the functionalized surfaces, indicating an excellent availability to hybridization.

ACKNOWLEDGMENT We gratefully acknowledge the financial support from CNR target project FIRB “Biochip”, RBNE01TZZ8_001, FIRB “MICRAM”, RBNE01ZB7A and FIRB 2003, RBLA03ER38_004.

LITERATURE CITED Figure 5. Scatter plot of intensities in the gene expression self-toself-experiment. Values for each channel are plotted one against each other in black dots. The solid line is the linear regression curve, whose equation is also reported on the plot. The dotted lines represent the 2-fold change thresholds for differential expression.

0.937, indicating a very high reproducibility of the sample preparation procedure, whereas the slope of the linear regression curve was found to be 0.957, indicating that the two fluorophores were well balanced and there was no need for normalization or correction of the data (Figure 5). We have reported here a robust chemical method for covalent immobilization of amino-modified oligonucleotides with two different lengths (25-mer and 70-mer) on glass surfaces based on polymeric coating. The DNA microarrays we prepared show good performance in terms of coupling specificity, and loading

(1) Ramsay, G. (1998) DNA chips: state-of-the art. Nature Biotechnol. 16, 40-44. (2) Lipshutz, R. J., Fodor, S. P. A., Gingeras, R. T., and Lockart, D. J. (1999) High-density synthetic oligonucleotide arrays. Nature Genet. 21, 20-24. (3) Beier, M. and Hoheisel, J. D. (1999) Versatile derivatisation of solid support media for covalent bonding on DNA-microchips. Nucleic Acids Res. 27, 1970-1977. (4) Rogers, Y. H., Jiang-Baucom, P., Huang, Z. J., Bogdanov, V., Anderson, S., and Boyce-Jacino, M. T. (1999) Immobilization of oligonucleotides onto a glass support via disulfide bonds: A method for preparation of DNA microarrays. Anal. Biochem. 266, 23-30. (5) Proudnikov, D., Timofeev, E., and Mirzabekov, A. (1998) Immobilization of DNA in polyacrylamide gel for the manufacture of DNA and DNA-oligonucleotide microchips. Anal. Biochem. 259, 34-41.

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A Chitosan-Based Platform for Oligo-Arrays (6) Benters, R., Niemeyer, C. M., Drutschmann, D., Blohm, D., and Wohrle, D. (2002) DNA microarrays with PAMAM dendritic linker systems. Nucleic Acids Res 15, 30(2), e10. (7) Ramakrishnan, R. et al. (2002) An assessment of Motorola CodeLink microarray performance for gene expression profiling applications. Nucleic Acids Res. 30(7), e30. (8) Consolandi, C., Castiglioni, B., Bordoni, R., Busti, E., Battaglia, C., Rossi Bernardi, L., and De Bellis, G. (2002) Two efficient polymeric platforms for oligonucleotide microarray preparation. Nucleosides, Nucleotides Nucleic Acids 21 (8, 9), 561-580. (9) Hudson, S. M. and Smith, C. (1998) In Biopolymers from Renewable Resources (Kaplan, D. L., Ed) pp 96-118, Springer, Berlin. (10) Hirano, S., Ohe, Y., and Ono, H. (1976) Selective N-acylation of chitosan. Carbohydr. Res. 47, 315-320. (11) Yalpani, M. and Hall, L. D. (1984) Some chemical and analytical aspects of polysaccharide modifications. III. Formation of branchedchain, soluble chitosan derivatives. Macromolecules 17, 272-281. (12) Gruber, J. V., Rutar, V., Bandekar, J., and Konish, P. N. (1995) Synthesis of N-[(3′-Hydroxy-2′,3′-dicarboxy)-ethyl]chitosan: A New, Water-Soluble Chitosan Derivative. Macromolecules 28, 8865-8867. (13) Xu, J., McCarthy, S. P., Gross, R. A., and Kaplan, D. L. (1996) Chitosan Film Acylation and Effects on Biodegradability. Macromolecules 29, 3436-3440.

(14) Kurita, K., Ikeda, H., Yoshida, Y., Shimojoh, M., and Harata, M. (2002) Chemoselective Protection of the Amino Groups of Chitosan by Controlled Phthaloylation: Facile Preparation of a Precursor Useful for Chemical Modifications. Biomacromolecules 3, 1-4. (15) Consolandi, C., Frosini, A., Pera, C., Ferrara, G. B., Bordoni, R., Castiglioni, B., Rizzi, E., Mezzelani, A., Rossi Bernardi, L., De Bellis, G., and Battaglia, C. (2004) Polymorphism analysis within the HLA-A locus by universal oligonucleotide array. Hum. Mut. 24 (5), 428-34. (16) Lindross, K., Liljiendahl, U., Raitio, M., and Syvanen, A. C. (2001) Minisequencing on oligonucleotide microarrays: comparison of immobilization chemistries. Nucleic Acids Res. 29, e69. (17) Busti, E., Bordoni, R., Castiglioni, B., Monciardini, P., Sosio, M., Donadio, S., Consolandi, C., Rossi Bernardi, L., Battaglia, C., and De Bellis, G. (2002) Bacterial discrimination by means of a universal array approach mediated by LDR (ligase detection reaction). BMC Microbiol. 2, 27. (18) Rehman, F. N., Audeh, M., Abrams, E. S., Hammond, P. W., Kennedy, M., and Boles, T. C. (1999) Immobilization of acrylamidemodified oligonucleotides by co-polymerization. Nucleic Acids Research 27 (2), 649-655. BC050285A