Both, the active aptamers N1 and N1(R23) bind neomycin in the low nanomolar range, but also, yet somewhat weaker, the inactive R23. In contrast to this N1(A) and N1-2/1-CU/A (both with a nucleotide 30 of the GUC motif) show very weak binding only (Figure 3B and Supplementary Figure S2). Mutating the terminal loop of N1 towards R23 in N1(R23) does not inﬂuence the binding strength but results in reduced regulation (Figure 1D and Supplementary Figure S2). These data show that a high binding afﬁnity is necessary but not suf- ﬁcient for regulation. All aptamers with high binding afﬁnity show a strongly favorable binding enthalpy H indicating a substantial energy gain from the formation of intra- and intermolecu- lar interactions (Figure 3C and Table 3). The H values correlate with the thermal stabilization, with N1 showing the highest value with 44 kcal/mol. The high enthalpic contribution is counteracted by a signiﬁcant and Table 3. Dissociation binding constants (KD) and thermodynamic parameters for neomycin binding to N1, R23 and N1(R23) at 37 C Aptamer N1-3/0-CUU R23 N1(R23) KD (nM) 9.2 ± 1.3 34.1 ± 2.1 9.5 ± 0.3 G (kcal/mol) H (kcal/mol) 11.4 ± 0.1 10.6 ± 0.0 11.4 ± 0.0 44.5 ± 0.1 33.6 ± 0.4 38.1 ± 0.3 –T S (kcal/mol) 33.1 ± 0.0 23.0 ± 0.4 26.7 ± 0.2 Parameters extracted from ITC measurements (Figure 3 and Supplementary Figure S2). Nucleic Acids Research, 2011, Vol. 39, No. 8 3369 unfavorable entropic contribution (Figure 3C and Table 3), indicating a loss of ﬂexibility for both RNA and ligand, and/or water hydration rearrangement upon binding. In line with this, N1 shows the highest –T S value and R23 the lowest. Taken together, genetic and biochemical data reveal that the presence of a sequence element at the 50 end of the internal loop is important for regulation. The length and sequence of the element show only a modulating effect. In contrast, additional nucleotides 30 of the GUC motif completely abolish regulation. A high binding afﬁnity as well as a considerable thermal stabilization is necessary for riboswitch activity indicating that major conformational changes occur upon neomycin binding. To characterize the degree and the nature of these struc- tural changes upon ligand binding in more detail we per- formed NMR spectroscopy. A largely unstructured ground state is important for regulation The recently solved structure of the aptamer N1 shows that its ground state is largely unstructured at tempera- tures >10 C (24). Thus, the free form shows only the 5 bp of the closing stem and two G:C base pairs of the upper helix. All nucleotides within the internal and the terminal loop are apparently not stabilized by hydrogen bonding. However, dramatic conformational changes occur upon neomycin binding (Figure 4). The upper helix is extended by three additional base pairs and stacks on the closing stem. The terminal loop becomes highly Figure 4. Conformation and neomycin binding of N1 and N1(R23). (A and B) Proposed secondary structure of the free form of N1 and N1(R23) based on the number of observable imino proton signals and NOE-patterns. The additional base pair in the terminal loop of N1(R23) is highlighted in red. (C and D) Comparison of the imino proton region of 1D-1H-spectra of free N1 and free N1(R23). Signal assignments are indicated. The assignment of the signal at 10.7 ppm to the imino proton of G13 (marked in red) is based on the comparison with spectra of the original R23 aptamer in its ligand-free state (20). (E and F) Imino proton region of the 1D-1H-spectrum of N1 and N1(R23) in the presence of one equivalent of neomycin. Compared to the spectrum of free N1 and N1(R23) shown in (D and E) novel signals and chemical shift changes are observed as expected due to the formation of stable 1:1 neomycin RNA-complexes in slow exchange on the NMR-time scale.