extra adenine residue opposite the bulge region leads to a signiﬁcant structural stabilization of the free form of the RNA in comparison to N1 (Supplementary Figure S3C and F). In contrast, neomycin binding results in a spectrum that closely resembles N1 with the distinctive signals for U10, U13, U14 and U18 present (Supplementary Figure S3I). Taken together, NMR data show that aptamers with a nucleotide 30 of the GUC motif are already pre-formed in the absence of neomycin. In addition, N1 shows contacts of the uppermost G:C base pair of the closing stem to neomycin which enforces coaxial stacking of the upper helix on the closing stem (24). This contact is prevented by insertion of a 3 nucleotide, thereby reducing ligand contacts, probably resulting in the low binding afﬁnity. DISCUSSION The initial starting point of our study was the observation that the four neomycin-binding RNA aptamers N1, R23, N1(R23) and N1(A) showed different levels of regulation when employed as riboswitches. Despite obvious similarities with respect to size and secondary structure, only two of them are able to control gene expression. The aim of this study was to investigate differences between active and inactive aptamers and to unravel the determin- ants for regulation. Our study shows that differences in the conformation between the ground and ligand bound state is the most important factor in conferring regulation. A sequence element of nucleotides 50 of the GUC motif keeps the RNA in an open conformation. Ligand binding induces extensive structural changes. The upper helix is extended by three additional base pairs and stacks directly on top of the closing stem, forming a solid continuous 10-bp helix. These conformational changes are accompanied by a dramatic thermal stabilization, also reﬂected in an ex- tremely high favorable enthalpy for ligand binding. Inactive aptamers, in contrast, are highly pre-formed. R23 is largely pre-structured in its ground state with pre-formed non-canonical base pairs and a pre-formed terminal loop (20). This is reﬂected by a lower degree of thermal stabilization and a reduced H value. N1(R23) as an intermediate has the terminal loop already pre-structured whereas the internal loop originating from N1 is open and gets stabilized upon ligand binding (Figure 5). The difference in the ground states is also sup- ported by the observation of virtually identical binding afﬁnities for N1 and N1(R23), which, importantly, are composed of different constituting energy terms. This is an example of enthalpy-entropy compensation, which in- dicates that the same ﬁnal state is reached, however, on different routes (28,29). As little as one nucleotide insertion 30 between the GUC motif and the closing stem results in a complete loss of regulation, independent of the sequence. Structural data show that this nucleotide allows the pre-formation of the GUC motif and interferes with the stacking of the upper helix on the closing stem which abrogates the stabilizing Nucleic Acids Research, 2011, Vol. 39, No. 8 3371 effect of ligand binding. In addition, it dramatically reduces the binding afﬁnity for neomycin. The asymmetry of the internal loop is clearly the im- portant feature for regulation with the nucleotides 50 to the GUC motif destabilizing the ground state. The one to four nucleotide small element, which is not implicated in ligand binding, is the functional element rendering the aptamer N1 active. In addition this element allows the tuning of the riboswitch efﬁciency. Small changes in size and sequence allow the adjustment of the regulatory window of the riboswitch towards different applications. A ﬂexible unbound state is also present in natural riboswitches. They ensure high selectivity and afﬁnity by interacting with nearly all functional groups provided by their target (30), with the ligand nearly completely en- veloped inside the RNA-binding pocket. This suggests that there has to be enough ﬂexibility in the unbound state for the ligand to access the binding pocket. The binding pocket of the purine riboswitches, e.g. is comprised of a three-way junction which is locally dis- ordered in the free state and collapses around the ligand upon binding (31,32). This ﬂexibility in the unbound state is similar for the regulatory aptamer N1, which binds neomycin via conformational selection (24,33). Riboswitches found in nature are mostly comprised of an aptamer domain and an expression platform, con- nected by a switching sequence which acts as a communi- cation module. Dependent on the binding status, the switching sequence interacts either with the aptamer domain or the expression platform, thereby favoring one of two mutually exclusive structures and subsequent modulation of gene expression (31). The N1 riboswitch combines aptamer and expression platform in the same domain. Here the ‘switching element’ (the CUU bulge) does not communicate between two otherwise independ- ent domains, but prevents extensive pre-formation of the binding pocket, thereby distinguishing the unbound and bound conformations. This difference in thermal stability between the ‘on-’ and ‘off’-state of an RNA regulator is reminiscent of thermoswitches in prokaryotes. Thermoswitches function in a similar way, just by opening and closing of base pairing interactions with the SD sequence (34). It will be interesting to analyze if thermal stabilization is a common feature determining riboswitch activity of regulating aptamers. If so, this information is invaluable (i) in predicting the applicability of newly selected aptamers, (ii) in further improving the performance of existing engineered riboswitches or (iii) in creating new riboswitches by rendering regulatory inactive aptamers active. A detailed knowledge of the necessary molecular determinants for riboswitch activity will be of immense value to provide more versatile RNA switches for synthet- ic biology. SUPPLEMENTARY DATA Supplementary Data are available at NAR Online.