Proofing of Photolithographic DNA Synthesis with 3‘,5‘-Dimethoxybenzoinyloxycarbonyl-Protected Deoxynucleoside Phosphoramidites

We have evaluated in a microchip format the photochemical solid-phase phosphoramidite DNA synthesis method we previously developed. A set of nucleoside building blocks with “easy-off” base protecting groups was prepared bearing photolabile 5‘-O-dimethoxybenzoincarbonate (DMBOC) groups. Photolysis rates and cycle yields for these DMBOC-protected nucleotides covalently attached to planar, derivatized glass surfaces were determined by fluorescence imaging-based methods earlier developed by McGall et al. and described in detail elsewhere. Data were obtained for both 280/310 and 365/400 nm irradiation in a range of solvents. Deprotection of the DMBOC occurs fastest in a nonpolar medium or without solvent. The coupling efficiency of these amidites in the synthesis of homopolymers was determined to be in the range 80−97%, with purines generally showing lower efficiency than pyrimidines. These DMBOC-protected monomers were used to prepare a 4 × 4 array of 16 decanucleotides of the sequence 5‘-AAXTAXCTAC−chip, where X = A, C, G, or T. The array was hybridized with a target deoxyeicosanucleotide of the sequence fluorescein-5‘-CTGAACGGTAGCATCTTGAC. Surface fluorescence imaging demonstrated sequence-specific hybridization to this probe.