Fig. 2: Ligand binding by the guanidine-II aptamer candidate associated with the elephant CA8 gene.
a Sequence and secondary structure for the African elephant representative 62 CA8, encompassing 62 nucleotides from the CA8 mRNA antisense plus two additional guanosine nucleotides (gg) added to the 5′ end to support production by in vitro transcription. The asterisk identifies the site of the 32P-radiolabel. b Representative image of the polyacrylamide gel electrophoresis (PAGE) separation of the products of in-line probing reactions using the elephant CA8 aptamer. See Supplementary Fig. 3 for two additional replicates. In-line probing reactions were conducted in the absence of ligand (‒), or in the presence of guanidine ranging from 1 µM to 10 mM. NR, T1, and ‒OH indicate precursor RNAs subjected to no reaction, partial digestion with RNase T1 (cleaves after G nucleotides), and partial digestion at elevated pH, respectively. Pre indicates the band corresponding to the precursor (full-length) 5′ 32P-labeled RNA. Some bands corresponding to RNase T1 digest are indicated with nucleotide numbers to assist in mapping sites of spontaneous cleavage. Nucleotides involved in forming base-paired stems P1 and P2 are indicated on the right. Bands undergoing substantial change in intensity are designated as sites 1, 2, and 3 (see vertical bars). c Plot of the fraction of RNA bound to ligand versus the logarithm of the molar concentration of guanidine. Fraction bound values are derived from the band intensity changes at sites 1, 2 and 3 as depicted in b. The error values are the standard error of the mean determined by goodness of fit to a sigmoidal curve. See Supplementary Fig. 3 for two additional replicates.
