3366 Nucleic Acids Research, 2011, Vol. 39, No. 8 G = RT ln(KD) and G = H – T S, with T being the reaction temperature (in K) and R being the gas constant (1.986 cal/K/mol). Measurements were repeated at least twice. NMR spectroscopy Prior to NMR experiments RNA solutions were heated to 95 C for 5 min, diluted 5-fold in ice cold water and snap-cooled on ice for 10 min. After folding, the RNA was concentrated to 500 ml using Vivaspin 2 centrifugal concentrators (Sartorius). Concentrated RNA was diluted 4-fold in NMR buffer (25 mM potassium phosphate buffer pH 6.2, 50 mM KCl) and again concentrated to 500 ml. Dilution and concentration steps were repeated seven times and RNA concentrated to a ﬁnal volume of 220 ml. The RNA concentration of the NMR samples was 150–250 mM for 1D-1H-experiments and titrations with neomycin and 0.6–1 mM when 2D-1H,1H-NOESY experiments were recorded. All spectra were recorded at 283 K in 90% H2O/10% D2O in NMR buffer on Bruker AVANCE 600-MHz or Bruker AVANCE 700-MHz spec- trometers equipped with cryogenic TXI-HCN probes and triple axis gradients. Water suppression in the 1D-1H-experiments was achieved using the jump-and- return water suppression scheme (19). In the 2D-1H,1H- NOESY- and the 2D-1H,15N-HSQC-experiments water suppression was achieved by using WATERGATE. For each 1D spectrum, 128–512 transients were accumulated. NOESY-spectra were recorded with 128 transients for each t1 increment and 400 complex points in the F1-dimension. Spectra were processed and analyzed using the Bruker Topspin 2.1 software. RESULTS A 3-nt element at the 50 part of the internal loop of N1 is important for regulation The secondary structure of the aptamer N1 consists of a 5-bp closing stem, an asymmetric internal loop and a terminal loop separated by two Watson–Crick G:C base pairs. The internal loop contains a 50-GUC-30/50-GUC-30 sequence (GUC motif) in the upper part and a 50-CUU-30 sequence 50 of the GUC motif (Figure 1B). Mutagenesis studies indicated that both loop regions are important for ligand binding (13). The ribosomal A-site also contains the GUC motif (Figure 1B, shaded in gray) but ﬂanked by three adenine residues, two at the 50 and one at the 30 site. The neomycin-binding aptamer, R23, was the most abundant sequence in the in vitro selected aptamer pool [12 out of 21 sequenced clones (14)] but shows no regula- tion [Figure 1B and D and Table 1; (13)]. It forms a stem loop structure with a neomycin-binding pocket consisting of three consecutive G:U wobble base pairs together with an adenine nucleotide reaching down from the highly structured terminal loop (20). We mutated the CUU sequence in the lower part of the internal loop of N1 towards the A-site creating the N1-A- site-hybrid N1(A) (Figure 1C). However, this mutation renders N1 inactive [Figure 1D and Table 1; (13)]. We then exchanged the terminal loop of N1 with the loop Aptamer N1 [N1-3/0-CUU] N1(R23) N1(Neo5) Neo5 R23 N1(A) N1-4/0-CCUU N1-3/0-CUC N1-3/0-CUA N1-3/0-CUG N1-2/0-CU N1-1/0-C N1-1/0-U N1-0/0 N1-2/1-CU/A N1-3/1-CUU/A Table 1. Activity of neomycin-binding aptamers Relative ﬂuorescence (%) 0 mM neomycina Relative ﬂuorescence (%) 100 mM neomycina Regulatory Factorb 35.5 54.5 56.1 12.4 66.4 65.6 44.6 44.2 39.6 15.7 26.1 22.2 17.8 2.3 35.3 33.5 9.0 35.0 28.1 7.2 59.2 65.4 11.7 12.7 15.4 9.9 7.1 11.9 9.7 2.3 33.2 29.2 3.9 1.6 2.0 1.7 1.1 1.0 3.8 3.5 2.6 1.6 3.7 1.9 1.8 1.0 1.1 1.1 agfp expression in the absence and presence of 100 mM neomycin. The ﬂuorescence emission of the vector pWHE601 expressing gfp without an aptamer in its 50UTR was set as 100%. Background level of a vector with no gfp expression was subtracted from all data. Values are mean of three independently grown cultures with SD <10%. Measurements were repeated at least twice. bEfﬁciency of regulation is given as the ratio of relative ﬂuorescence with and without neomycin. from R23 and Neo5, respectively, resulting in the hybrids N1(R23) and N1(Neo5) (Figure 1C). Neo5 is also from the in vitro selected pool like R23 and shows marginal regulation [Table 1; (13)]. Both hybrid constructs showed regulation albeit with a reduced dynamic range (Figure 1D and Table 1). This indicates that the CUU sequence of the internal loop is important for regulation whereas the terminal loop has only a modulating effect. This prompted us to investigate the importance of the CUU sequence by saturating mutagenesis. In a ﬁrst attempt we cloned four pools with three and two nucleo- tides, respectively, inserted 50 of the GUC motif in com- bination with one or no nucleotide 30 of the motif. The respective pools were named N1-X/Y with X indicating the number of nucleotides at the 50 and Y at the 30 site of the motif (N1-3/0, N1-2/0, N1-3/1 and N1-2/1, Figure 2A). Randomized aptamer sequences were inserted in the 50UTR of a constitutively expressed gfp reporter gene (6). We obtained complete coverage for the plasmid pools N1-3/0, N1-2/0 and N1-2/1 and 40% coverage for N1-3/1 (Supplementary Table S1). Yeast cells were transformed with these plasmid pools. A total of 192 individual colonies were grown for each pool in 96-well plates and gfp expression was determined in the absence and presence of 100 mM neomycin. Candidates with decreased ﬂuorescence in the presence of neomycin were sequenced and the regulation was veriﬁed. The activity of selected candidates is summarized in Figure 2, Table 1 and Supplementary Table S2. Interestingly, the wild-type sequence CUU was the most active sequence and no candidates with an increased dynamic range of regulation have been identiﬁed.