3370 Nucleic Acids Research, 2011, Vol. 39, No. 8 Figure 5. Model of proposed secondary structure changes of N1, N1(R23) and R23 upon neomycin binding derived from NMR data. Unstructured elements are shaded in red, structured elements in blue. structured. The three nucleotides 50 of the GUC motif are bulged out and exposed to the solvent (Figure 5). They are not directly involved in ligand binding which corresponds to the genetic data. Since the insertion element does not interact with the ligand, its length and the identity of the nucleotides are variable. In contrast, the inactive R23 is largely pre-structured in its free state with pre-formed non-canonical base pairs and a pre-formed terminal loop [Figure 5; (20)]. The number of NMR signals is similar in the ground and in the ligand-bound form indicating that no new base pairs are formed. N1(R23) shows decreased regulation compared to N1 and we questioned if this is reﬂected in the structural pre-formation. The imino proton 1H-spectrum of N1(R23) is very similar to that of N1 in the region typical for imino groups in Watson–Crick base pairs (25) with the same number of signals (Figure 4). Between 10.0 and 12.0 ppm there is one additional signal with a narrow line width. Comparison with the spectrum of R23 (20) indicates that this signal most likely originates from the loop nucleotide G13 in the replaced terminal loop (Figure 4, marked in red). Thus, the free state of N1(R23) is unstructured in the vicinity of the internal bulge region similar to N1, whereas the terminal loop appears to be already structured in the absence of ligand (Figures 4 and 5). Neomycin binding leads to a change in the chemical shift and a reduction in the line width for the signal between 10.0 and 12.0 ppm as observed for the original R23 aptamer. The neomycin bound form of N1(R23) contains one additional signal in the Watson–Crick region of the spectrum and two additional signals between 10.0 and 12.0 ppm with chemical shifts typical for the imino protons of a U:U base pair (26). Thus, neomycin binding seems to stabilize N1(R23) by extension of the upper helix in a manner very similar to that observed for N1 (Figure 4). In addition, the terminal loop (50-GAGAA-30) of N1(R23) functionally replaces the terminal loop of N1 in interacting with the ligand as indicated by the reduced line width and shift change observed for the G13 imino proton signal. Taken together N1(R23) is a hybrid between N1 and R23 with respect to its regulatory abilities and structure which per- fectly ﬁts both the genetic and biochemical data. 30 nucleotide insertion leads to structure stabilization already in the absence of ligand Insertion of a nucleotide 30 of the GUC motif completely prevents regulation. We question if this can be explained by the structure of the aptamer in its ligand free state. Comparison of the imino proton 1H- and 2D-1H,1H-NOESY spectra of the free N1(A) with those of N1 reveals the presence of one additional signal with a chemical shift typical for a guanine in a Watson–Crick G:C base pair (Supplementary Figure S3). Another add- itional signal with a narrow line width is present in the region typical for U:U base pairs (26). NOEs link this uridine signal to two neighboring imino protons from guanines G9 and G20 in G:C base pairs (Supplementary Figure S3). Furthermore, there is a strong intra base pair NOE to one of the broad uridine imino signals in the same region. Thus, the NMR-data demonstrate that the upper helix is extended by a U:U base pair and an additional Watson–Crick G:C base pair in the free state of N1(A) compared to N1 in agreement with the higher melting temperature for free N1(A). Thus, the free forms of N1(A) and N1 differ signiﬁcantly in terms of structural stability. On the other hand, spectra of the neomycin bound N1(A) strongly resemble those of N1 (compare Supplementary Figure S3G and H). In particular, signals with a chemical shift very similar to those of U10, U13, U14 and U18 in N1 are observed in the 10.0–12.5-ppm region (27). This indicates that neomycin interacts with the terminal loop and the upper helix of N1(A) in a manner very similar to that of N1. For N1-2/1-CU/A, the imino proton spectrum contains at least one extra signal in the Watson–Crick region when compared to that of N1 (Supplementary Figure S3). Furthermore, for some of the signals in this region (e.g. U4, G5) a signiﬁcant reduction in line widths with respect to those observed in N1 is obvious which indicates a more stable structure. In addition, four observable signals with chemical shifts 10.3 ppm indicate the presence of transi- ently stable U:U base pairs and consequently a structure where the upper helix is already pre-formed. Thus, the