The Efficiency of Light-Directed Synthesis of DNA Arrays on Glass Substrates

Abstract

New methods based on photolithography and surface fluorescence were used to determine photodeprotection rates and stepwise yields for light-directed oligonucleotide synthesis using photolabile 5‘-(((α-methyl-2-nitropiperonyl)oxy)carbonyl)(MeNPOC)-2‘-deoxynucleoside phosphoramidites on planar glass substrates. Under near-UV illumination (primarily 365 nm) from a mercury light source, the rate of photoremoval of the MeNPOC protecting group was found to be independent of both the nucleotide and length of the growing oligomer (t1/2 = 12 s at 27.5 mW/cm2). A moderate dependence on solvent polarity was observed, with photolysis proceeding most rapidly in the presence of nonpolar solvents or in the absence of solvent (e.g., t1/2 = 10−13 s at 27.5 mW/cm2). In solution, the photolysis rate was linearly dependent on light intensity over the range 5−50 mW/cm2. Average stepwise yields for the synthesis of dodecamer oligonucleotides were in the range of 92−94%, using monomers based on N6-(phenoxyacetyl)-2‘-deoxyadenosine, N2-isobutyryl-2‘-deoxyguanosine, N4-isobutyryl-2‘-deoxycytidine, and thymidine. By comparison, an efficiency of 98%/step was obtained using a conventional 5‘-dimethoxytrityl monomer with acid deprotection on the same support. The lower yields associated with the photochemical process appears to be due to incomplete recovery of free 5‘-hydroxyl groups after photolysis on the support, although high yields of 5‘-OH nucleosides (≥96%) are consistently observed when 5‘-MeNPOC monomers are photolyzed in solution.