Abstract: The present disclosure provides alternative nucleosides, nucleotides, and nucleic acids, and methods of using them.

Abstract: The current landscape for preparative chromatographic RNA purification uses reversed phase HPLC, but this technique presents many issues with process scale up and ion exchange for preparative purification has only been used for short RNAs. The invention provides preparative purification of RNA (e.g., mRNA) using ion (e.g., anion) exchange chromatography that allows for separation of longer RNAs up to 10,000 nucleotides in length via a scalable method. This method avoids problems with current techniques by using low pressure chromatography that is agreeable with existing equipment in cGMP commercial facilities, that uses aqueous-bases solutions as the mobile phase (rather than flammable of greater than 10 mg RNA/mL resin (e.g., using larger pore sorbents, >500 Angstroms, that display greater mRNA binding capacities), and that yields desired RNA salt forms for downstream formulation with no additional manipulation necessary (unlike ion pair reverse phase techniques).