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

Abstract

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.

Strategies in the Preparation of DNA Oligonucleotide Arrays for Diagnostic Applications

“…imaging indicated sequence-specific hybridization with an average signal to background ratio greater than 6 [80]. These results compare well in terms of hybridization fidelity to those obtained earlier from an array of oligonucleotides prepared from 5'-(1"-methyl-6"-nitropiperonyl)oxycarbonylprotected deoxyribo-nucleoside phosphoramidites [2].…”

“…Photochemical deprotection rates of 5'-(3",5"'-dimethoxybenzoin)carbonyl-protected nucleosides on glass surfaces depended on irradiation wavelengths and solvent polarity. Photodeprotection occurred faster at 310 nm (t 1/2 = 5.5 to 13 sec) than at 365 nm (t 1/2 = 5.6 to 17 sec) in a nonpolar solvent or without solvent [80]. The 5'-(3",5"'-dimethoxybenzoin)carbonyl-protected deoxyribonucleoside phosphoramidites were used to prepare an array of the sequence 5'-AANTANCTAC where N is A, C, G, or T. Fig.…”

“…(23) shows the detailed preparation of these phosphoramidites [79,80]. The coupling efficiency of these phosphoramidites in the synthesis of homopolymers up to dodecamers was: 91-98% for poly-T; 82-95% for poly-C; 79-92% for poly-A and 74-84% for poly-G [80]. Photochemical deprotection rates of 5'-(3",5"'-dimethoxybenzoin)carbonyl-protected nucleosides on glass surfaces depended on irradiation wavelengths and solvent polarity.…”

Solid-phase synthesis of base-sensitive oligonucleotides

“…Advantage is the neutral cleavage, in which potential reagent contamination may be omitted. Applicability of the photolabile protections has also been demonstrated for the 5'-OH group (in photolithographic DNA synthesis), 74,75 for the 2'-OH group of ribonucleosides, 76 for the internucleotidic phosphates 77 and, finally, for the exocyclic amino groups of nucleobases, 35,78 when a base sensitive conjugate groups are incorporated. Deprotection is generally performed by irradiation at wavelengths > 300 nm to avoid damage of nucleic acids.…”

Topographical and Physicochemical Modification of Material Surface to Enable Patterning of Living Cells

“…This method has been most important in developing arrays of oligonucleotides for gene chip assays. 148,149 …”