Amicon 3 000 mw cut off spin filters

Preparation phosphorylimidazolide oligonucleotides was adapted from a method used by Orgel and others^{31 (link)}. 3′ phosphorylated FANA or DNA was prepared by solid-phase chemical synthesis (see above) and re-suspended to 100 μM in 0.5 M imidazole (pH 6.0). 50 μl oligo/imidazole solution was added to 6.5 μmol solid 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC) (Pierce Biotechnology / Thermo, USA) and incubated at room temperature for 2 h. Oligos were desalted using Amicon 3,000 MW cut off spin filters (Merck Millipore, USA). Purification of (and analysis of reactions involving) all phosphorylimidazolide oligos were perfomed using Tris-free Urea-PAGE gels run using 10 mM NaOH, pH adjusted to 8.5 with Boric acid^{32 (link)}. FANA phorphorylimidazolides were analysed by mass spectrometry, phosphatase protection and Urea-PAGE mobility (Extended Data Fig. 10).