Evidence of 5′ heterogeneity revealed from affinity purification of an SLI RNA transcribed from an SLI-ARiBo precursor. (A) Schematic representation of SLI(2)-ARiBo fusion RNA. (Gray arrowhead) Points to the VS ribozyme cleavage site in the internal loop between Stems Ia and Ib. (B) Small-scale affinity batch purification of SLI(2) analyzed on a 20% denaturing polyacrylamide gel stained with SYBR Gold. The SLI(2) RNA was transcribed as an ARiBo-fusion RNA [SLI(2)-ARiBo] and purified by affinity. Aliquots from each purification step were loaded on the gel [(LE) load eluate; (W1–3) washes; (E1–3) elutions; and (NaCl) matrix regeneration with 2.5 M NaCl] in the amounts shown, where 1× correspond to ∼50 ng of SLI(2)-ARiBo precursor present in the transcription reaction or the equivalent of 8.23 ng of SLI(2) to be purified. In addition, standard quantities of SLI(2)-ARiBo from the transcription reaction, gel-purified control RNA (29 nt), and SLI(2) cleaved in the transcription reaction were loaded as controls. Bands corresponding to the SLI(2)-ARiBo (176 nt), the ARiBo tag (147 nt), and SLI(2) (29 nt) RNAs are indicated on the right side of the gel.
