Interdisciplinary Nanoscience Center (iNANO) and Department of Chemistry, Aarhus University, 8000 Aarhus, Denmark.
Research Chemistry, Novo Nordisk A/S, Novo Nordisk Park, 2760 Måløv, Denmark.
Nucleic Acids Res. 2024 Jan 11;52(1):49-58. doi: 10.1093/nar/gkad1015.
Conjugates of therapeutic oligonucleotides (ONs) including peptide conjugates, provide a potential solution to the major challenge of specific tissue delivery faced by this class of drugs. Conjugations are often positioned terminal at the ONs, although internal placement of other chemical modifications are known to be of critical importance. The introduction of internal conjugation handles in chemically modified ONs require highly specialized and expensive nucleoside phosphoramidites. Here, we present a method for synthesizing a library of peptide-siRNA conjugates by conjugation at internal phosphorous positions via sulfonylphosphoramidate modifications incorporated into the sense strand. The sulfonylphosphoramidate modification offers benefits as it can be directly incorporated into chemically modified ONs by simply changing the oxidation step during synthesis, and furthermore holds the potential to create multifunctionalized therapeutic ONs. We have developed a workflow using a novel pH-controlled amine-to-amine linker that yields peptide-siRNA conjugates linked via amide bonds, and we have synthesized conjugates between GLP1 peptides and a HPRT1 siRNA as a model system. The in vitro activity of the conjugates was tested by GLP1R activity and knockdown of the HPRT1 gene. We found that conjugation near the 3'-end is more favorable than certain central internal positions and different internal conjugation strategies were compared.
治疗性寡核苷酸 (ONs) 的缀合物，包括肽缀合物，为这类药物面临的特定组织递药的主要挑战提供了潜在的解决方案。缀合物通常位于 ONs 的末端，尽管已知内部其他化学修饰的位置对于至关重要。在化学修饰的 ONs 中引入内部缀合位置需要高度专业化和昂贵的核苷亚磷酰胺。在这里，我们通过在有义链中掺入的磺酰基磷酰胺修饰物在内部磷原子位置进行缀合，提出了一种合成肽-siRNA 缀合物文库的方法。磺酰基磷酰胺修饰物具有直接通过在合成过程中改变氧化步骤就能被掺入化学修饰的 ONs 的优点，并且具有潜在的可能性来构建多功能治疗性 ONs。我们开发了一种使用新型 pH 控制的胺-胺连接子的工作流程，该连接子通过酰胺键连接肽-siRNA 缀合物，并且我们已将 GLP1 肽和 HPRT1 siRNA 作为模型系统合成了缀合物。通过 GLP1R 活性和 HPRT1 基因的敲低测试了缀合物的体外活性。我们发现 3'-末端附近的缀合比某些中央内部位置更有利，并且比较了不同的内部缀合策略。
