Saraya Jagandeep S, O'Flaherty Derek K
Department of Chemistry, University of Guelph, Guelph, Canada.
Curr Protoc. 2025 Aug;5(8):e70174. doi: 10.1002/cpz1.70174.
Chemical modifications of oligonucleotides are widely used to improve their functional properties. Amino modifiers provide versatile chemical handles for post-synthetic (bio)conjugation, nucleic acid immobilization on solid supports, and studies of nonenzymatic genome replication related to the origins of life. This protocol describes an economical disulfide-containing solid-support linker that enables on-column synthesis of nucleic acids bearing 3'-amino or 3'-phosphate modifications. The orthogonal nature of this linker allows for an on-column protecting group strategy, facilitating the synthesis of DNA and RNA containing 3'-amino-2',3'-dideoxyribosides from commercially available unprotected mononucleosides. This protocol further extends the on-column construction of oligomers containing 3'-amino-2',3'-dideoxyribosides to those containing a 2'-amino-2'-deoxynucleoside at the 3'-end, using an orthogonal protecting group strategy. An improved on-column deprotection procedure for DNA and RNA is also presented, eliminating the precipitation step typically utilized in conventional RNA workflows, resulting in an enhanced strand recovery for certain sequences. Finally, the disulfide-containing solid support is applied to the preparation of amino acid-oligonucleotide conjugates, wherein the identity of the final product was contingent upon the specific deprotection conditions employed. All oligonucleotide conjugates and derivatives were purified using standard methods [(e.g., strong anion exchange high-performance liquid chromatography (SAX-HPLC)] and characterized by mass spectrometry (MS). © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Synthesis of 4,4'-dimethoxytrityl (DMTr) disulfide succinimide and solid-support functionalization reaction Basic Protocol 2: Solid-phase synthesis of DNA containing a 3'-amino-2',3'-dideoxyguanosine using 3'-Disulfide CPG Alternate Protocol 1: Synthesis of DNA containing terminal 2'-amino-2'-deoxyadenosine Alternate Protocol 2: Alternate RNA (terminated with 3'-amino modifiers or phosphate) workflow using D-TentaGel Basic Protocol 3: Amino acid-oligonucleotide conjugate synthesis using Lys and D-TentaGel.
寡核苷酸的化学修饰被广泛用于改善其功能特性。氨基修饰剂为合成后（生物）共轭、核酸在固体支持物上的固定以及与生命起源相关的非酶促基因组复制研究提供了通用的化学处理方法。本方案描述了一种经济的含二硫键的固体支持物连接体，它能够在柱上合成带有3'-氨基或3'-磷酸修饰的核酸。这种连接体的正交性质允许采用柱上保护基策略，便于从市售的未保护单核苷合成含有3'-氨基-2',3'-二脱氧核糖核苷的DNA和RNA。本方案进一步将含有3'-氨基-2',3'-二脱氧核糖核苷的寡聚物的柱上构建扩展到那些在3'-末端含有2'-氨基-2'-脱氧核苷的寡聚物，采用正交保护基策略。还提出了一种改进的DNA和RNA柱上脱保护程序，消除了传统RNA工作流程中通常使用的沉淀步骤，从而提高了某些序列的链回收率。最后，将含二硫键的固体支持物应用于氨基酸 - 寡核苷酸共轭物的制备，其中最终产物的特性取决于所采用的特定脱保护条件。所有寡核苷酸共轭物和衍生物均使用标准方法[例如强阴离子交换高效液相色谱（SAX - HPLC）]进行纯化，并通过质谱（MS）进行表征。© 2025作者。由Wiley Periodicals LLC出版的《当前方案》。基本方案1：4,4'-二甲氧基三苯甲基（DMTr）二硫代琥珀酰亚胺的合成及固体支持物功能化反应 基本方案2：使用3'-二硫键控制孔玻璃（CPG）固相合成含有3'-氨基-2',3'-二脱氧鸟苷的DNA 替代方案1：合成含有末端2'-氨基-2'-脱氧腺苷的DNA 替代方案2：使用D - TentaGel的替代RNA（以3'-氨基修饰剂或磷酸终止）工作流程 基本方案3：使用赖氨酸和D - TentaGel合成氨基酸 - 寡核苷酸共轭物
