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

P. Kumar 0 Jyoti Choithani 0 K. C. Gupta 0 0 Nucleic Acids Research Laboratory, Institute for Genomics and Integrative Biology , Mall Road, Delhi University Campus , Delhi 110 007, India 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 mercaptoalkylfunctionalized oligonucleotides. Immobilization of oligonucleotides on a glass surface has been realized via two routes. In the first one (A), 5aminoalkyl- 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 fluoresceinlabeled complementary oligonucleotides. Furthermore, the proposed method was compared with the existing methods with respect to immobilization efficiency of oligonucleotides. - Last decade has witnessed the emergence of microarray technology, a very powerful and promising tool for gene discovery (1), genome analysis (2), DNA sequencing by hybridization (3), medical diagnostics for genetic diseases (4) and the detection of single nucleotide polymorphisms (5). This also allows undertaking studies relating to nucleic acidligand interaction (6) and DNA computing (7). The surface-bound oligonucleotides (microarrays) offer several advantages over the conventional gel-based format. The success of microarrays not only depends on the chemistry used for the immobilization of oligonucleotides but also depends on the good accessibility and functionality of the surface-bound oligonucleotides, density of attachment, thermal stability of the array under experimental conditions and reproducibility of attachment chemistry. Basically, two approaches are used for fabrication of DNA/ oligonucleotide arrays. In one approach, oligonucleotide probes are directly synthesized on the surface at a pre-selected positions (in situ synthesis) (813) following conventional and photolithographic techniques such as the Affimetrix on-chip synthesis. This methodology is by far the most efficient method for the construction of high-density oligonucleotide arrays, however, it has practical limitations in terms of flexibility and affordability. The second method, called the deposition method, where pre-fabricated nucleic acids are covalently (1220) or non-covalently (21) immobilized on solid surfaces (organic or inorganic), offers an excellent flexibility. This latter approach has thus become the most widely used method for creating low- to medium-density DNA microarrays. Two important factors which influence the quality of microarrays fabricated by the latter method, are (i) the nature of the solid surface used, and (ii) the chemistry employed for fixing of oligonucleotides on solid surfaces. Though a large number of chemical methods have been reported for immobilization of oligonucleotides on pre-functionalized surfaces, there are only two reagents (3-mercaptopropyltriethoxysilane and 3-glycidyloxypropyltrimethoxysilane) available that can directly be used for fixing oligonucleotides on virgin glass surface. 3-Mercaptopropyltriethoxysilane results in immobilization of oligonucleotides via disulfide linkage (22), which is a labile linkage and 3-glycidyloxypropyltrimethoxysilane requires longer reaction time (8 h) (23,24). Therefore, a glass-specific reagent, which could fix oligonucleotides on a virgin glass surface rapidly and via a stable linkage, is still elusive. A number of solid surfaces such as polypropylene, polyethylene, nylon, poly(methyl methacrylate) (PMMA), glass, silicon, etc., have been proposed for this purpose. Of these, glass and PMMA appear attractive because these supports can easily be derivatized generating reactive functional groups such as aminoalkyl, mercaptoalkyl, carboxyl, aldehyde, etc. on the surface. Glass, in particular, being an inexpensive material having low intrinsic fluorescence and a relatively homogeneous chemical surface, offers an added advantage in the sense that laser scanners can be used for visualization of fluorescent spots on the surface. In this communication, we describe a method for construction of oligonucleotide arrays using a novel heterobifunctional reagent, [N-(2-trifluoroethanesulfonatoethyl)-N-(methyl)-triethoxysilylpropyl-3-amine] (NTMTA) (25), on an unmodified (virgin) glass surface. The method is suitable for the construction of oligonucleotide microarrays on a glass surface without employing any additional coupling reagent, thus making the strategy cost-effective, efficient and rapid. The NTMTA reagent has been explored in two ways; in the first one, the NTMTA reagent was reacted with a 5-aminoalkyl- or mercaptoalkyl-functionalized oligonucleotide through its trifluoroethanesulfonate (tresyl) ester function to generate a oligonucleotide conjugate, which subsequently reacts with the unmodified glass surface to obtain a surface-immobilized oligonucleotide. Whereas, in the second route, the NTMTA reagent was allowed to react first with the unmodified glass surface through its triethoxysilyl function to generate reactive tresyl functions on the surface, which in the subsequent step react with 5-aminoalkyl or mercaptoalkyl groups bearing oligonucleotides to generate glass surface-bound oligonucleotides. The constructed microarrays were successfully used in detection of nucleotide mismatches by hybridization with fluorescein-labeled complementary oligonucleotides. Furthermore, the proposed method was compared with some of the existing glass-specific methods. MATERIALS AND METHODS Reagents and chemicals employed in the present investigation were purified prior to their use. 3-Glycidyloxypropyltrimethoxysilane, 3-mercaptopropyltriethoxysilane, 2,2,2-trifluoroethanesulfonyl chloride (tresyl chloride), 3-chloropropyltriethoxysilane, N-methyl-2-aminoethanol and N,Ndiisopropylethylamine were procured from Aldrich Chemical Co., St Louis, MO. Fluorescein-DMTrdT-phosphoramidite (dTF (...truncated)