Selection of RRE RNA binding peptides using a kanamycin antitermination assay.
The arginine-rich domains of several RNA-binding proteins have been shown to bind their cognate RNAs with high affinities and specificities as isolated peptides, adopting different conformations within different complexes. The sequence simplicity and structural diversity of the arginine-rich motif has made it a good framework for constructing combinatorial libraries and identifying novel RNA-binding peptides, including those targeted to the HIV Rev response element (RRE). Here we describe a modified transcription antitermination reporter assay engineered with kanamycin resistance that enables larger in vivo screens (approximately 10(9) sequences) than previously possible. We show that the assay detects only specific RNA-protein complexes, and that binders are enriched at least 300-fold per round of selection. We screened a large peptide library in which amino acids with charged, polar, and small side chains were randomly distributed within a polyarginine framework and identified a set of high affinity RRE-binding peptides. Most contain glutamine at one particular peptide position, and the best peptides display significantly higher antitermination activities than Rev or other previously described high-affinity RRE-binding peptides. The kanamycin antitermination (KAN) assay should be useful for screening relatively large libraries and thereby facilitate identification of novel RNA binders.
Peled-Zehavi
Hadas
H
Department of Biochemistry and Biophysics, University of California-San Francisco, 513 Parnassus Avenue, San Francisco, CA 94143-0448, USA.
Horiya
Satoru
S
Das
Chandreyee
C
Harada
Kazuo
K
Frankel
Alan D
AD
D001681:Biological Assay; D017930:Genes, Reporter; D015752:Genes, env; D015497:HIV-1 / Q000235:genetics* / Q000378:metabolism*; D007612:Kanamycin / Q000378:metabolism*; D019151:Peptide Library; D010455:Peptides / Q000378:metabolism; D012313:RNA / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000378:metabolism*
D019151:Peptide Library; D010455:Peptides; D016601:RNA-Binding Proteins; C015462:polyarginine; D007612:Kanamycin; D012313:RNA