3368 Nucleic Acids Research, 2011, Vol. 39, No. 8 Figure 3. Melting point analyses and determination of the equilibrium dissociation constants (KD) by ITC. (A) Melting curves in the absence (black curves) and presence (red curves) of 10 mM neomycin for 1 mM N1. Melting curves were recorded in triplicates. (B) Upper panel: power required to maintain the temperature (37 C) of the RNA solution (4 mM) recorded over the time of multiple injections (10 ml) of ligand (38 mM neomycin) until saturation was reached (baseline-corrected). Lower panels: integrated heats of interaction per mole of injectant plotted against the molar ratio of ligand over RNA and ﬁtted to a single binding site model. (C) Gibbs free energies ( G), enthalpic ( H) and entropic (–T S) contributions of N1, R23 and N1(R23). We have chosen 11 aptamers for melting analyses with varying degrees of regulation including six active [N1, N1-3/0-CUC, N1-2/0-CU, N1-1/0-C, N1-1/0-U, N1(R23)] and ﬁve inactive aptamers [R23, N1(A), N1-2/ 1-CU/A, N1-3/1-CUU/A and N1-0/0]. Melting curves were measured in the absence and presence of 10 mM neomycin and are displayed in Figure 3A and Supplementary Figure S1. All proﬁles showed monophasic melting. The only exception was N1-2/0-CU with a second melting event at higher temperatures in the absence of neomycin. All tested aptamers show a signiﬁcant increase in thermal stability upon neomycin binding (Figure 3A, Supplementary Figure S1 and Table 2). We observe a cor- relation between regulation and thermal stabilization. Regulating aptamers are stabilized >20 C compared to 6.3–15.6 C for the inactive variants. Exceptions are the aptamers N1-1/0-C and N1-1/0-U which are only stabilized by 16.1 C. This smaller degree of stabilization seems to result from a more stable ground state compared to the other active aptamers N1, N1-3/0-CUC and N1(R23). Consequently N1-1/0-C and N1-1/0-U markedly interfere with gene expression already in the absence of neomycin (Figure 2D) shifting the regulatory window. We propose that this minor stabilization is sufﬁ- cient to confer regulation due to the already impaired gfp expression. R23 in contrast which is stabilized to a similar Table 2. Determination of Tm values of different neomycin-binding aptamers Aptamer Tm in Ca Tm in Ca 0 mM neomycin 10 mM neomycin Tm Regulatory Factorb N1-3/0-CUU 50.5 ± 0.2 R23 57.5 ± 0.5 N1(R23) 51.3 ± 0.5 N1(A) 54.6 ± 0.5 N1-3/0-CUC 50.5 ± 0.2 N1-2/0-CU n.d. N1-1/0-C 61.0 ± 0.5 N1-1/0-U 61.6 ± 0.5 N1-0/0 74.2 ± 0.4 N1-2/1-CU/A 60.3 ± 0.3 N1-3/1-CUU/A 56.0 ± 0.5 71.8 ± 0.5 73.1 ± 0.6 71.5 ± 0.4 68.3 ± 0.8 72.5 ± 0.3 74.0 ± 0.4 77.1 ± 0.2 77.6 ± 0.7 80.5 ± 0.5 71.4 ± 0.5 69.3 ± 0.8 21.3 15.6 20.2 13.7 22.0 n.d. 16.1 16.1 6.3 11.1 13.3 3.9 1.1 1.6 1.0 3.5 3.7 1.9 1.8 1.0 1.1 1.1 n.d.: not determined. The aptamer N1-2/0-CU shows biphasic melting behavior in the absence of neomycin. aMelting analysis was performed using 1 mM aptamer RNA in 20 mM Na-cacodylate pH 6.8 and 100 mM NaCl. Melting curves were recorded in triplicates. Buffer with or without neomycin was analyzed in parallel as a blank and substracted from all data. bEfﬁciency of regulation is given as the ratio of relative ﬂuorescence with and without neomycin. degree (only 0.5 C less) is not active, because of the less impaired basal gene expression (Figure 1D). We determined the binding constants for two active N1, N1(R23) and three inactive aptamers [R23, N1(A), N1-2/ 1-CU/A] using isothermal titration calorimetry (Table 3).