Extended Data Figure 2. Annealing of labeled oligonucleotides to the 5′ domain RNA.

³²P-labeled oligonucleotides were annealed to the 3′ extension of h3 of 5′dom-h3 (h3P5, DNA) (a) or to the extended loop of h16 of 5′dom-h3h16 (h16P2-2, RNA) (b). Annealing reactions were performed in HK buffer and 6 mM 2-mercaptoethanol at 25 ºC. Binding data were fit to the quadratic form of a two-state binding isotherm. Apparent dissociation constants were ≤0.4 nM and 2.6 ± 0.2 nM for h3 and h16 oligonucleotides, respectively. Equilibrium constants are the average and S.D. of two or more independent trials. The lengths of the labeled oligonucleotides were varied to optimize affinity with the extended 5′ domain RNA, while avoiding perturbations to S4 binding (see Extended Data Fig. 1).