Hoffmann Stefanie, Hoos Johannes, Klussmann Sven, Vonhoff Stefan
NOXXON Pharma AG, Berlin, Germany.
Curr Protoc Nucleic Acid Chem. 2011 Sep;Chapter 4:Unit 4.46.1-30. doi: 10.1002/0471142700.nc0446s46.
This unit describes the solid-phase synthesis and downstream processing for RNA oligonucleotides with a length of up to 40 to 50 nucleotides on a 1- to 4-mmol scale with subsequent conjugation to PEG using the L-RNA spiegelmer NOX-E36 as an example. Following synthesis and two-step deprotection, the crude oligonucleotide is purified by preparative reversed-phase HPLC and desalted by tangential flow ultrafiltration. The resulting intermediate amino-modified oligonucleotide is reacted with NHS-ester-activated PEG, and the oligonucleotide-PEG conjugate is obtained after preparative AX-HPLC purification, followed by ultrafiltration and lyophilization. Critical process parameters are described, as well as time considerations and examples for analytical methods used as in-process and quality controls.
本单元以L-RNA镜像寡聚核苷酸NOX-E36为例，描述了1至4毫摩尔规模、长度达40至50个核苷酸的RNA寡核苷酸的固相合成及下游处理，以及随后与聚乙二醇（PEG）的偶联。合成及两步脱保护后，粗制寡核苷酸通过制备型反相高效液相色谱法纯化，并通过切向流超滤脱盐。所得的中间氨基修饰寡核苷酸与NHS-酯活化的PEG反应，经制备型AX-高效液相色谱法纯化、超滤和冻干后得到寡核苷酸-PEG偶联物。文中描述了关键工艺参数、时间考量以及用作过程控制和质量控制的分析方法示例。
