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<p>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 O<sup>6</sup>-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.</p>

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

  • 期刊论文

  • 出版模式：

    开放获取

  • 发表日期：

    2025年10月31日

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  • Santra Santhosh,
  • Sharon Istvánffy,
  • Omer Sabary,
  • Eitan Yaakobi,
  • Maya Giridhar,
  • Jürgen Behr,
  • Mark M. Somoza

摘要

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.