Construction of oligonucleotide arrays on a glass surface using a heterobifunctional reagent, N-(2-trifluoroethanesulfonatoethyl)-N-(methyl)-triethoxysilylpropyl-3-amine (NTMTA)

Abstract

A rapid method for construction of oligonucleotide arrays on a glass surface, using a novel heterobifunctional reagent, N-(2-trifluoroethanesulfonatoethyl)-N-(methyl)-triethoxysilylpropyl-3-amine (NTMTA), has been described. The heterobifunctional reagent, NTMTA, carries two different thermoreactive groups. The triethoxysilyl group on one end is specific towards silanol functions on the virgin glass surface, while the trifluoroethanesulfonyl (tresyl) group on the other end of the reagent reacts specifically with aminoalkyl- or mercaptoalkyl- functionalized oligonucleotides. Immobilization of oligonucleotides on a glass surface has been realized via two routes. In the first one (A), 5'- aminoalkyl- or mercaptoalkyl-functionalized oligonucleotides were allowed to react with NTMTA to form a oligonucleotide-triethoxysilyl conjugate which, in a subsequent reaction with unmodified (virgin) glass microslide, results in surface-bound oligonucleotides. In the second route (B), the NTMTA reagent reacts first with a glass microslide whereby it generates trifluoroethanesulfonate ester functions on it, which in a subsequent step react with 5'-aminoalkyl or mercaptoalkyl oligonucleotides to generate support-bound oligonucleotides. Subsequently, the oligonucleotide arrays prepared by both routes were analyzed by hybridization experiments with complementary oligonucleotides. The constructed microarrays were successfully used in single and multiple nucleotide mismatch detection by hybridizing these with fluorescein-labeled complementary oligonucleotides. Further more, the proposed method was compared with the existing methods with respect to immobilization efficiency of oligonucleotides.

Scheme 1. Preparation of NTMTA reagent and immobilization of oligonucleotides.

Scheme 1. Preparation of NTMTA reagent and immobilization of oligonucleotides.

Scheme 2. Comparison of proposed method with the standard methods.

Scheme 2. Comparison of proposed method with the standard methods.

Scheme 2. Comparison of proposed method with the standard methods.

Figure 1

Time kinetics to determine the optimal time required to immobilize oligonucleotides on unmodified glass surface via route A (a) and route B (b). Squares, 5′-mercaptoalkylated ligand; diamonds, 5′-aminoalkylated ligand.

Figure 1

Time kinetics to determine the optimal time required to immobilize oligonucleotides on unmodified glass surface via route A (a) and route B (b). Squares, 5′-mercaptoalkylated ligand; diamonds, 5′-aminoalkylated ligand.

Figure 2

Threshold concentration of oligonucleotide sequence d(T^FTT TTT TTT TTT TTT TTT TT)-OPO₃-(CH₂)₆SH required for visualizing fluorescence under a laser scanner. Lane I, concentration of spots (2.5, 5, 7.5, 10, 15 µM); lane II, immobilization efficiency.

Figure 3

(a) Immobilization of oligonucleotides via route A. H₂N-(CH₂)₅-OPO₃-d(CAG AGG TTC TTT GAG TCC TT) (entry 1, Table 1) and HS-(CH₂)₆-OPO₃-d(CAG AGG TTC TTT GAG TCC TT) (entry 2, Table 1) visualized after hybridization with FAM-d(AAG GAC TCA AAG AAC CTC TG) (entry 7, Table 1). (b) Immobilization of oligomers via route B. H₂N-(CH₂)₅-OPO₃-d(CTC CTG AGG AGA AGG TCT GC) (entry 5, Table 1) and HS-(CH₂)₆-OPO₃-d(CTC CTG AGG AGA AGG TCT GC) (entry 3, Table 1) visualized after hybridization with FAM-d(GCA GAC CTT CTC CTC AGG AG) (entry 8, Table 1).

Figure 4

A correlation sketch between concentration of immobilized probe and fluorescence intensity. Fluorescent oligonucleotide was spotted in 0.025–1.0 µM concentrations. The spotted microslide was scanned under a laser scanner.

Figure 5

Detection of nucleotide mismatches and specificity of immobilization via hybridization with fluorescein-labeled complementary oligonucleotides. Lane 1, H₂N-(CH₂)₅-OPO₃-d(CTC CTG AGG AGA AGG TCT GC) (entry 5, Table 1); lane 2, H₂N-(CH₂)₅-OPO₃-d(CTC CTG AGG CGA AGG TCT GC) (entry 6, Table 1); lane 3, H₂N-(CH₂)₅-OPO₃-d(CTC CTG CGG AGA ACG TCT GC) (entry 10, Table 1); lane 4, H₂N-(CH₂)₅-OPO₃-d(CTC CTG CGG CGA ACG TCT GC) (entry 11, Table 1); lane 5, H₂N-(CH₂)₅-OPO₃-d(TTT TTT TTT TTT TTT TTT TT) (entry 12, Table 1). Hybridization was carried out using FAM-d(GCA GAC CTT CTC CTC AGG AG) (entry 8, Table 1) followed by laser scanning.

Figure 6

(a) Morphology of spots obtained after hybridization with labeled complementary oligonucleotide. I, NTMTA method; II, epoxide method; III, disulfide method. (b) Comparison of immobilization efficiency. I, NTMTA method; II, epoxide method; III, disulfide method.

Figure 7

Thermal stability of immobilized oligonucleotides (microarray). Comparison on the basis of fluorescence signals obtained after hybridization of heat-treated and untreated arrays of oligonucleotides. I, untreated microslide; II, heat-treated microslide.