Efficiency of Digital Photolithographic Synthesis of Large, High-Quality DNA Libraries and Microarrays using a Guanine O⁶ Dephosphitylation Strategy

Large-scale de novo nucleic acid synthesis is a powerful tool enabling researchers to better understand and engineer biological systems. Fields ranging from genomics to nucleic acid therapeutics to synthetic biology make use of high-throughput experimental approaches requiring access to large pools or libraries of DNA, RNA, synthetic nucleic acid analogs, non-nucleosidic building blocks, or combinations of these. Large oligonucleotide libraries are synthesized as microarrays and used in situ for surface-based assays or cleaved for off-array applications. Here, using a digital maskless photolithographic approach, we address an important source of error in DNA microarray synthesis, oligonucleotide fragmentation arising from the O⁶-phosphitylation of guanine during the potentially hundreds of coupling cycles required for complex library synthesis. Introducing a very short debranching step using standard capping reagents suppresses depurination-based fragmentation and greatly enhances synthetic yield.