Specificity of Phage Display Selected Peptides for Modified Anticodon Stem and Loop Domains of tRNA |
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Authors: | Matthewos Eshete Marie T. Marchbank Susan L. Deutscher Brian Sproat Grazyna Leszczynska Andrzej Malkiewicz Paul F. Agris |
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Affiliation: | (1) Department of Molecular and Structural Biochemistry, North Carolina State University, 128 Polk Hall, Campus Box 7622, Raleigh, NC 27695-7622, USA;(2) Present address: Chemistry Department, Mississippi Valley State University, Itta Bena, MS 38941, USA;(3) Department of Biochemistry, University of Missouri, Columbia, MO 65212, USA;(4) RNA-TEC NV, Provisorium 2, Minderbroedersstraat 17-19, 3000 Leuven, Belgium;(5) Institute of Organic Chemistry, Technical University, Łodz, 90-924, Poland |
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Abstract: | Protein recognition of RNA has been studied using Peptide Phage Display Libraries, but in the absence of RNA modifications. Peptides from two libraries, selected for binding the modified anticodon stem and loop (ASL) of human tRNALys3 having 2-thiouridine (s2U34) and pseudouridine (Ψ39), bound the modified human ASLLys3(s2U34;Ψ39) preferentially and had significant homology with RNA binding proteins. Selected peptides were narrowed to a manageable number using a less sensitive, but inexpensive assay before conducting intensive characterization. The affinity and specificity of the best binding peptide (with an N-terminal fluorescein) were characterized by fluorescence spectrophotometry. The peptide exhibited the highest binding affinity for ASLLys3(s2U34;Ψ39), followed by the hypermodified ASLLys3 (mcm5s2U34;ms2t6A37) and the unmodified ASLLys3, but bound poorly to singly modified ASLLys3 constructs (Ψ39, ms2t6A37, s2U34), ASLLys1,2 (t6A37) and Escherichia coli ASLGlu (s2U34). Thus, RNA modifications are potentially important recognition elements for proteins and can be targets for selective recognition by peptides. |
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Keywords: | Anticodon domain protein-RNA interaction pseudouridine 2-thiouridine |
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