Elucidation of the RNA target of linezolid by using a linezolid-neomycin B heteroconjugate and genomic SELEX.
A covalently modified heteroconjugate between linezolid and neomycin B leads to an enhanced and more specific binding affinity to hairpin RNA targets in comparison to neomycin B itself. This heteroconjugate was used as a lure to select linezolid-specific hairpin RNA from an Escherichia coli genome RNA. The selected RNA obtained after eight cycles not only has typical stem-loop structures but also includes known sequences of the linezolid binding site. The results of RNA footprinting show that the binding site of the heteroconjugate encompasses both stem and loop regions, suggesting that the possible binding site for linezolid is in the terminal loop. In addition, findings from application of a surface plasmon resonance assay clearly demonstrate that linezolid binds to selected hairpin RNA in a highly specific manner with a low millimolar affinity. The results suggest that heteroconjugates might represent a generally useful approach in studies aimed at uncovering loop-specific RNA binding ligands that would be otherwise difficult to identify owing to their weak affinities.
Kim
Hyun Jin
HJ
Department of Chemistry and Education, Seoul National University, Seoul 151-742, Republic of Korea.
Kwon
Miyun
M
Yu
Jaehoon
J
D000081:Acetamides / Q000737:chemistry* / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D001710:Biotin / Q000737:chemistry; D016503:Drug Delivery Systems; D004926:Escherichia coli / Q000235:genetics; D005601:Framycetin / Q000737:chemistry* / Q000378:metabolism; D000069349:Linezolid; D008969:Molecular Sequence Data; D015394:Molecular Structure; D023303:Oxazolidinones / Q000737:chemistry* / Q000378:metabolism; D011500:Protein Synthesis Inhibitors / Q000737:chemistry; D015347:RNA Probes / Q000737:chemistry* / Q000235:genetics; D052156:SELEX Aptamer Technique
D000081:Acetamides; D023303:Oxazolidinones; D011500:Protein Synthesis Inhibitors; D015347:RNA Probes; D005601:Framycetin; D001710:Biotin; D000069349:Linezolid