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 .
Neomycin b
Manufactured by Merck Group
Sourced in United States
Neomycin-B is a broad-spectrum antibiotic that is commonly used in laboratory settings. It is effective against a variety of gram-negative and gram-positive bacteria. Neomycin-B functions as an inhibitor of bacterial protein synthesis, ultimately leading to bacterial cell death.
Automatically generated - may contain errors
Lab products found in correlation
Kanamycin a, by Merck Group (3 mentions)
Rna oligonucleotides, by Integrated DNA Technologies (3 mentions)
Ribostamycin, by Merck Group (2 mentions)
Tobramycin, by Merck Group (2 mentions)
Paromomycin, by Merck Group (2 mentions)
Netilmicin, by Merck Group (2 mentions)
Sisomicin, by Merck Group (2 mentions)
Amikacin, by Merck Group (2 mentions)
Cacodylic acid, by Avantor (2 mentions)
Kanamycin b, by Merck Group (2 mentions)
Geneticin, by Merck Group (2 mentions)
Sodium cacodylate, by Merck Group (2 mentions)
Geneticin g418, by Merck Group (1 mentions)
Mitoxantrone, by Santa Cruz Biotechnology (1 mentions)
Clomiphene, by Merck Group (1 mentions)
Ciprofloxacin, by Merck Group (1 mentions)
5 protocols using neomycin b
1
Aminoglycosides Procurement and Expression
2
2AP-Labeled RNA Aptamer Synthesis
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Neomycin-B was obtained as its sulfate
salt from Sigma-Aldrich. All RNA oligonucleotides were from Integrated
DNA Technologies Inc. (IDT, Coralville, IA) and were maintained at
−20 °C in deionized distilled water (ddH2O)
until use. Sigma-Aldrich was the source of sodium cacodylate, and
cacodylic acid was obtained from Amresco. All other chemicals were
from Fisher Scientific. The buffer contained 200 mM NH4CI, 80 mM KCl, 80 mM Na-cacodylate, and 5 mM MgCl2 at
pH 7.4. The sequence of the 2AP-labeled RNA aptamer 2AP15NEO1A
is given by GGACUGGGCGAXAAGUUUAUCC where
X is 2-aminopurine.
salt from Sigma-Aldrich. All RNA oligonucleotides were from Integrated
DNA Technologies Inc. (IDT, Coralville, IA) and were maintained at
−20 °C in deionized distilled water (ddH2O)
until use. Sigma-Aldrich was the source of sodium cacodylate, and
cacodylic acid was obtained from Amresco. All other chemicals were
from Fisher Scientific. The buffer contained 200 mM NH4CI, 80 mM KCl, 80 mM Na-cacodylate, and 5 mM MgCl2 at
pH 7.4. The sequence of the 2AP-labeled RNA aptamer 2AP15NEO1A
is given by GGACUGGGCGAXAAGUUUAUCC where
X is 2-aminopurine.
3
Binding Affinity of Aminoglycoside Antibiotics
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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 .
4
Aminoglycoside-RNA Aptamer Binding Assay
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Neomycin-B, paromomycin, ribostamycin, tobramycin, kanamycin-A, kanamycin-B, sisomicin, geneticin, netilmicin, and amikacin (Figs. 1 A, 3 B) were obtained as their sulfate salts from Sigma-Aldrich. All RNA oligonucleotides were from Integrated DNA Technologies Inc., and were maintained at −20°C in deionized distilled water (ddH2O) until use. Sodium cacodylate was from Sigma-Aldrich and cacodylic acid was from Amresco. All other chemicals were from Fisher Scientific. Buffers were as follows: A (13.5 mM NaCl, 150 mM KCl, 20 mM HEPES, 0.22 mM Na2HPO4, 0.44 mM KH2PO4, 120 μM MgCl2, 120 nM CaCl2, 100 μM MgSO4 at pH 7.3), N (10 mM Na2HPO4 at pH 7.3), and F (5 mM MgCl2, 200 mM NH4CI, 80 mM KCl, 80 mM Na-cacodylate at pH 7.4). All buffers were prepared with deionized distilled water and their pH's were measured at 25°C. Sequences of the RNA aptamers used in this study are shown in Supplemental Table S1. The buffer components used to determine the effect of [I] on aminoglycoside binding are listed in Supplemental Table S2.
5
Screening FDA-approved drugs for antiviral activity
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The primary fluorescence anisotropy screen used a collection of 1,120 FDAapproved drugs dissolved in DMSO at a concentration of 5 mM, acquired from Prestwick Chemical (Illkirch France). According to the manufacturer, the compounds were selected for their high chemical and pharmacological diversity and accessible information on bioavailability and safety in humans. For subsequent experiments, neomycin B, clomiphene and ciprofloxacin were obtained from Sigma-Aldrich (St. Louis USA), neamine from Toronto Research (Toronto Canada), and cyproheptadine and mitoxantrone from Santa Cruz Biotechnology (Dallas USA). Additional amounts of other compounds were obtained from the Prestwick stocks. The antiretroviral drugs zidovudine and nelfinavir used as positive controls in the anti-HIV activity assay were obtained through the AIDS Reagent Program (NIAID, NIH USA).
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