Fig. 2: Selective binding of Na⁺ by a natural RNA aptamer.

a, Sequence and secondary structure of the WT 66 kefB RNA construct used to assess ligand-binding characteristics of a DUF1646 motif RNA. The two lowercase letters designate guanosine nucleotides appended to the natural bacterial sequence from the kefB gene to facilitate efficient production by in vitro transcription. Mutant constructs M1 and M2 carry the boxed nucleotide changes at the sites indicated. Circles identify positions of notable strand scission from the in-line probing data depicted in b. b, PAGE separation of product bands resulting from in-line probing assay reactions reveal RNA shape changes induced by the addition of Na⁺. 5′ ³²P-labelled precursor (Pre) RNA was subjected to no reaction (NR), partial digestion with RNase T1 (T1) (cleaves after G nucleotides), incubation in alkali conditions (^–OH), or incubation in in-line probing reactions with buffer alone (including 2 mM MgCl₂ but lacking KCl) (−) or supplemented with various amounts of Na⁺ ranging from 10 μM to 100 mM. Bands corresponding to some products generated by RNase T1 digestion are identified by the guanosine nucleotide position located immediately 5′ of the cleavage site. Bands of interest were mapped according to their sites of spontaneous strand scission on the RNA construct depicted in a. c, Plot of the fraction of RNA bound to Na⁺ as estimated from the in-line probing data depicted in b. Band intensities at sites 1 and 2 were used to generate the plot. The line depicts an idealized binding curve for a 1-to-1 interaction and a K_D of 2.2 mM. d, PAGE analysis of in-line probing assays using 5′ ³²P-labelled 66 kefB RNA and conducted in the presence of various alkali metal cations or NH₄⁺ at 10 mM. e, PAGE analysis of in-line probing assays using 5′ ³²P-labelled WT 66 kefB RNA or the mutant M1 or M2 versions of this construct. In-line probing reactions were conducted in the absence (−) or presence (+) of 10 mM Na⁺.