Kanamycin a

Aminoglycosides, neomycin-B, kanamycin-A and Geneticin (G418), were purchased as their sulfate salts from Sigma-Aldrich (St. Louis, MO). Bacterial and yeast expression vectors and their expected expressed RNA products are listed in Tables S1–S3.

Growth inhibition was measured as previously described [15 (link),23 (link)]. Briefly, overnight cultures of MJF376, MJF451, MJF455, and MJF612 strains carrying constructs were diluted at 1:50 (v:v) in CphM and grown, until an OD₆₀₀ of 0.2 was reached. Expression was then induced by the addition of 1 mM isopropyl-b-D-thiogalactopyranoside (IPTG) for 30 min, 10 mM stocks of compound 262 solubilized in sterile dimethyl sulfoxide (DMSO) (Sigma-Aldrich, St. Louis, MO, USA) were diluted two times to achieve its final concentration in pre-warmed CphM with a final DMSO concentration of 2%, and 100 µL were added to wells of a pre-warmed, sterile 96-well flat bottom plate (Greiner bio-one, Monroe, NC, USA). Cultures were then diluted at 1:200 (v:v) in pre-warmed CphM, 100 µg/mL ampicillin, and 2 mM IPTG with diluted experimental antibiotics and were indicated, at 2× their concentration or mock (DMSO only). One hundred microliters of culture mixture with kanamycin A at a final concentrations of 0.2 µM (Sigma-Aldrich, St. Louis, MO, USA) were added to 96-well plates for a total of 200 µL, sealed with a sterile breathable film (Axygen, Union City, CA, USA), wrapped in aluminum foil and placed in a 37 °C shaker and rotated at 110 Cycles per minute for 16–17 h. OD₆₂₀ was then taken with a Multiskan Ascent 354 (Thermo Fisher Scientific, Waltham, MA, USA) plate reader.

All RNA oligonucleotides used in this study were purchased from Integrated DNA Technologies Inc. (IDT, Coralville, IA, USA) and stored at −20 °C in ddH₂O (deionized-distilled water) until tested in the ITC or in 2AP fluorescence experiments. Sequences of the oligonucleotides used in this study are shown in Table S1. Neomycin-B, ribostamycin, paromomycin, tobramycin, kanamycin-A, kanamycin-B, sisomicin, geneticin, netilmicin, and amikacin were obtained as their sulfate salts from Sigma-Aldrich (St. Louis, MO, USA). Cacodylic acid was purchased from Amresco (Solon, OH) and sodium cacodylate was from Sigma-Aldrich (St. Louis, MO, USA). All other chemicals were reagent grade and obtained from Fisher Scientific (Pittsburgh, PA, USA). The constituents of the low ionic strength buffer (A) and high ionic strength buffer (F) are listed in Table S1.