Isolation of short RNAs with homogeneous 3'-ends using quaternary-amine anion exchange chromatography

Biol Methods Protoc. 2024 May 17;9(1):bpae033. doi: 10.1093/biomethods/bpae033. eCollection 2024.


Visualizing RNA-protein interactions through structural approaches requires the use of RNA molecules purified to homogeneity. We describe here a simple and effective method, free of acrylamide contamination and without using UV radiation, to separate in vitro synthesized, heterogeneous RNA transcripts (up to ∼15 nucleotides) at single-nucleotide resolution by quaternary-amine anion exchange chromatography. The quality of short RNAs isolated through this method is validated by gel electrophoresis, mass spectrometry, and crystallization with a protein-binding partner.

Keywords: capping; chromatography; in vitro transcription; short RNA; structural biology.