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RNA Aptamers and Spiegelmers: Synthesis, Purification, and Post‐Synthetic PEG Conjugation

2011; Wiley; Volume: 46; Issue: 1 Linguagem: Inglês

10.1002/0471142700.nc0446s46

1934-9289

Stefanie Hoffmann, Johannes Hoos, Sven Klußmann, Stefan Vonhoff,

RNA and protein synthesis mechanisms

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.