digestion was attached 30 to the aptamer sequences together with an optimized Kozak sequence for yeast (A AAATG). For in vitro transcription, aptamers where inserted into the plasmid pSP64 (Promega) digested with NheI and XbaI. Aptamers attached to a T7 promoter sequence were generated using complementary DNA oligonucleo- tides, designed in a way to comprise compatible 50 over- hangs. Aptamers were transcribed as hammerhead fusion to obtain deﬁned 30 ends. Stem 3 of the hammerhead ribozyme was mutagenized via PCR to base pair with the 30 ends of the aptamer sequences. All primer and vector sequences are available upon request. GFP ﬂuorescence measurement Saccharomyces cerevisiae strain RS453a (mat ade2-1 trp1-1 can1-100 leu2-3 his3-1 ura3-52) transformed with the respective constructs was grown at 28 C for 48 h in 5 ml of minimal medium [0.2% (w/v) yeast nitrogen base, 0.55% (w/v) ammonium sulfate, 2% (w/v) glucose, 12 mg/ ml adenine, MEM amino acids (Gibco BRL)] in the absence or presence of 100 mM neomycin trisulfate (Sigma). Cells were harvested by centrifugation and resus- pended in 2 ml phosphate-buffered saline (PBS). For each construct, three independently grown cultures were analyzed. Fluorescence measurements were carried out at 25 C on a Fluorolog FL3-22 (Horiba Jobin Yvon) with the excitation wavelength set to 482 nm and an emission wavelength of 510 nm. Optical density (OD600) was determined to ensure homogeneous cell growth. The vector pVT102-U (16) without gfp+ gene was analyzed in parallel as a blank and its value subtracted from all data. Measurements were repeated at least two times. In vivo screening Saccharomyces cerevisiae strain RS453a transformed with the respective plasmid pool was grown at 28 C in minimal medium. Colonies were transferred to 96-well plates con- taining 200 ml minimal medium and incubated for 24 h. An amount of 20 ml aliquots of each sample were transferred into fresh medium in new plates with and without 100 mM neomycin in a ﬁnal volume of 200 ml. Fluorescence meas- urements were performed 48 h after inoculation using an excitation wave length of 484 nm and an emission wave length of 512 nm with a SpectraFluor Plus ﬂuorescence reader (Tecan, Crailsheim). Optical density (OD600) was determined to ensure homogeneous cell growth. Individual candidates were streaked out to single colonies and plasmids were prepared according to Hoffman and Winston (17). After an Escherichia coli passage plasmids were sequenced, retransformed in yeast cells and the ﬂuorescence measurements repeated (see above). In vitro transcription The aptamers were transcribed in vitro from an EcoRI-linearized pSP64 plasmid using a T7 promoter. Transcription reactions contained 25 mM magnesium acetate, 200 mM Tris–HCl pH 8.0, 20 mM DTT, 2 mM spermidine, 0.1 mg/ml linearized plasmid, 4 mM of each Nucleic Acids Research, 2011, Vol. 39, No. 8 3365 NTP and 2.5 ml self-made T7 polymerase [prepared ac- cording to Davanloo et al. (18)]. After overnight incuba- tion at 37 C precipitated pyrophosphate was pelleted by centrifugation. Residual traces were resolved by adding 20% (v/v) 0.5 M EDTA pH 8.0 to the supernatant. After ethanol/aceton precipitation the RNA products were separated on a 15% denaturing polyacrylamid gel. Aptamer RNA was detected by UV shadowing and eluted from gel slices in 300 mM sodium acetate pH 6.5 overnight at 4 C. The supernatant was ﬁltered using a 0.45 mM ﬁlter (Sarstedt) and again ethanol/aceton precipitated. RNA was resuspended in H2O and stored at 20 C. An amount of 3 mM of each 15N-labeled NTP (Silantes) was used for preparation of 15N-labeled N1 RNA. UV-melting studies Prior to melting studies 50 mM RNA solutions were heated to 95 C for 5 min, diluted 35- to 40-fold in ice cold water and snap-cooled on ice for 10 min. After folding, buffer was added to a ﬁnal concentration of 20 mM Na-cacodylate pH 6.8 and 100 mM NaCl. The ﬁnal con- centration of RNA was 1 mM. For ligand-dependent melting studies 10 mM neomycin was added to the sample. Heating and cooling was performed on a UV spectrophotometer V-650 (Jasco) from 15–95 C with a rate of 0.5 C/min. Melting was followed by measuring the OD260 every minute. For each RNA three independent samples were analyzed. Buffer with or without neomycin was analyzed in parallel as a blank and its value was sub- tracted from all data. Tm values were determined by calculating the ﬁrst derivative. ITC measurements Prior to titration RNA solutions were heated to 95 C for 5 min and snap-cooled on ice for 10 min. After that, folding buffer was added to a ﬁnal concentration of 20 mM Na-cacodylate, 200 mM NaCl, 10 mM MgCl2, 1 mM spermidine, pH 6.8. The ﬁnal concentration was 4.5 mM for R23, N1(A) and N1-2/1-CU/A and 4 mM for N1 and N1(R23). Neomycin solution (38 mM) was prepared in the same buffer. ITC experiments were done on a VP-ITC microcalorimeter (MicroCal Inc. Northampton, MA) with the sample cell (1.44 ml) contain- ing RNA and the neomycin solution in the injector syringe. Following thermal equilibration at 37 C, an initial 120 s delay and two initial 1 ml injections, we did 28 serial injections of 10 ml at intervals of 180 s and at a stirring speed of 310 rpm. Raw data were recorded as power (mcal/s) over time (min). The heat associated with each titration peak was integrated and plotted against the respective molar ratio of neomycin and RNA and the re- sulting experimental binding isotherm was corrected for the effect of titrating neomycin into buffer alone. Thermodynamic parameters were extracted from a curve ﬁt to the corrected data using the one site binding model in the Origin software provided by MicroCal. These are the change in enthalpy H and the dissociation constant KD. As a consequence, the change in Gibbs free energy G and the change in entropy S can be calculated from