Nucleic Acids Research Advance Access published online on September 2, 2009
Nucleic Acids Research, doi:10.1093/nar/gkp594
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Two-dimensional combinatorial screening and the RNA Privileged Space Predictor program efficiently identify aminoglycoside–RNA hairpin loop interactions
Department of Chemistry and Center of Excellence in Bioinformatics and Life Sciences, University at Buffalo, The State University of New York, 657 Natural Sciences Complex, Buffalo, NY 14260, USA
*To whom correspondence should be addressed. Tel: +1 716 645 4242; Fax: +1 716 645 6963; Email: mddisney{at}buffalo.edu
Received May 8, 2009. Revised June 27, 2009. Accepted June 29, 2009.
Herein, we report the identification of RNA hairpin loops that bind derivatives of kanamycin A, tobramycin, neamine, and neomycin B via two-dimensional combinatorial screening, a method that screens chemical and RNA spaces simultaneously. An arrayed aminoglycoside library was probed for binding to a 6-nucleotide RNA hairpin loop library (4096 members). Members of the loop library that bound each aminoglycoside were excised from the array, amplified and sequenced. Sequences were analyzed with our newly developed RNA Privileged Space Predictor (RNA-PSP) program, which analyzes selected sequences to identify statistically significant trends. RNA-PSP identified the following unique trends: 5'UNNNC3' loops for the kanamycin A derivative (where N is any nucleotide); 5'UNNC3' loops for the tobramycin derivative; 5'UNC3' loops for the neamine derivative; and 5'UNNG3' loops for the neomycin B derivative. The affinities and selectivities of a subset of the ligand–hairpin loop interactions were determined. The selected interactions have Kd values ranging from 10 nM to 605 nM. Selectivities ranged from 0.4 to >200-fold. Interestingly, the results from RNA-PSP are able to qualitatively predict specificity based on overlap between the RNA sequences selected for the ligands. These studies expand the information available on small molecule–RNA motif interactions, which could be useful to design ligands targeting RNA.