title: Two-dimensional combinatorial screening identifies specific aminoglycoside-RNA internal loop partners.
abstract: Herein is described the identification of RNA internal loops that bind to derivatives of neomycin B, neamine, tobramycin, and kanamycin A. RNA loop-ligand partners were identified by a two-dimensional combinatorial screening (2DCS) platform that probes RNA and chemical spaces simultaneously. In 2DCS, an aminoglycoside library immobilized onto an agarose microarray was probed for binding to a 3 x 3 nucleotide RNA internal loop library (81,920 interactions probed in duplicate in a single experiment). RNAs that bound aminoglycosides were harvested from the array via gel excision. RNA internal loop preferences for three aminoglycosides were identified from statistical analysis of selected structures. This provides consensus RNA internal loops that bind these structures and include: loops with potential GA pairs for the neomycin derivative, loops with potential GG pairs for the tobramycin derivative, and pyrimidine-rich loops for the kanamycin A derivative. Results with the neamine derivative show that it binds a variety of loops, including loops that contain potential GA pairs that also recognize the neomycin B derivative. All studied selected internal loops are specific for the aminoglycoside that they were selected to bind. Specificity was quantified for 16 selected internal loops by studying their binding to each of the arrayed aminoglycosides. Specificities ranged from 2- to 80-fold with an average specificity of 20-fold. These studies show that 2DCS is a unique platform to probe RNA and chemical space simultaneously to identify specific RNA motif-ligand interactions.
authors.affiliation: Department of Chemistry, University at Buffalo, The State University of New York, and the New York State Center of Excellence in Bioinformatics and Life Sciences, 657 Natural Sciences Complex, Buffalo, New York 14260, USA. mddisney@buffalo.edu
authors.forename: Matthew D
authors.initials: MD
authors.lastname: Disney
authors.forename: Lucas P
authors.initials: LP
authors.lastname: Labuda
authors.forename: Dustin J
authors.initials: DJ
authors.lastname: Paul
authors.forename: Shane G
authors.initials: SG
authors.lastname: Poplawski
authors.forename: Alexei
authors.initials: A
authors.lastname: Pushechnikov
authors.forename: Tuan
authors.initials: T
authors.lastname: Tran
authors.forename: Sai P
authors.initials: SP
authors.lastname: Velagapudi
authors.forename: Meilan
authors.initials: M
authors.lastname: Wu
authors.forename: Jessica L
authors.initials: JL
authors.lastname: Childs-Disney
mesh_terms: D000480:Alkynes / Q000737:chemistry; D000617:Aminoglycosides / Q000138:chemical synthesis / Q000737:chemistry*; D001386:Azides / Q000737:chemistry; D020029:Base Pairing; D002236:Carbohydrate Conformation; D020650:Combinatorial Chemistry Techniques; D005601:Framycetin / Q000737:chemistry; D007612:Kanamycin / Q000737:chemistry; D008024:Ligands; D009690:Nucleic Acid Conformation; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D009841:Oligonucleotides / Q000737:chemistry; D012313:RNA / Q000737:chemistry* / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D054852:Small Molecule Libraries; D014031:Tobramycin / Q000737:chemistry; D014158:Transcription, Genetic
chemical_list: D000480:Alkynes; D000617:Aminoglycosides; D001386:Azides; D008024:Ligands; D009841:Oligonucleotides; D054852:Small Molecule Libraries; D005601:Framycetin; D007612:Kanamycin; C488396:neamine; D012313:RNA; D014031:Tobramycin