High‐Density RNA Microarrays Synthesized In Situ by Photolithography,Angewandte Chemie International Edition

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High‐Density RNA Microarrays Synthesized In Situ by Photolithography
Angewandte Chemie International Edition ( IF 16.1 ) Pub Date : 2018-10-19 , DOI: 10.1002/anie.201806895
Jory Lietard ₁ , Dominik Ameur ₁ , Masad J Damha ₂ , Mark M Somoza ₁

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While high‐density DNA microarrays have been available for over three decades, the synthesis of equivalent RNA microarrays has proven intractable until now. Herein we describe the first in situ synthesis of mixed‐based, high‐density RNA microarrays using photolithography and light‐sensitive RNA phosphoramidites. With coupling efficiencies comparable to those of DNA monomers, RNA oligonucleotides at least 30 nucleotides long can now efficiently be prepared using modified phosphoramidite chemistry. A two‐step deprotection route unmasks the phosphodiester, the exocyclic amines and the 2′ hydroxyl. Hybridization and enzymatic assays validate the quality and the identity of the surface‐bound RNA. We show that high‐density is feasible by synthesizing a complex RNA permutation library with 262144 unique sequences. We also introduce DNA/RNA chimeric microarrays and explore their applications by mapping the sequence specificity of RNase HII.

中文翻译：

通过光刻法原位合成高密度 RNA 微阵列

虽然高密度 DNA 微阵列已经存在了三十多年，但迄今为止，等效 RNA 微阵列的合成已被证明是棘手的。在此，我们描述了首次使用光刻和光敏 RNA 亚磷酰胺原位合成混合型高密度 RNA 微阵列。偶联效率与 DNA 单体相当，现在可以使用改良的亚磷酰胺化学方法有效制备至少 30 个核苷酸长的 RNA 寡核苷酸。两步脱保护路线揭示了磷酸二酯、环外胺和 2' 羟基。杂交和酶测定验证了表面结合 RNA 的质量和身份。我们通过合成具有 262144 个独特序列的复杂 RNA 排列文库来证明高密度是可行的。我们还介绍了 DNA/RNA 嵌合微阵列，并通过绘制 RNase HII 的序列特异性来探索其应用。

更新日期：2018-10-19

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