Activity of in vitro transcribed SNORD113 purified from different PAGE systems. As shown in panel A, SNORD113 was initially purified by denaturing PAGE (8% polyacrylamide, 8 M urea; see Materials and Methods) and then subjected to additional treatments (EDTA, CIP) and subsequent gel purification (B). SNORD113 adopted two primary conformers visible by UV shadow of the native gel, referred to as N-Top (top band), or N-Bot (bottom band); Den indicates RNA purified from a denaturing gel. Each band imaged was extracted from the gel and rerun on either a denaturing or native PAGE stained with Sybr Gold to visualize the RNA (C), or subjected to an autophosphorylation assay (D). Autophosphorylation activity was normalized to activation by ds106. Average activation values ± SD are shown below. n = 3 with at least two biological replicates. SNORD113, 500 nM; ds106, 10 nM; PKR, 100 nM.
