Facile detection of specific RNA-polypeptide interactions by MALDI-TOF mass spectrometry.
A simple method for the detection of specific RNA-polypeptide interactions using MALDI-TOF mass spectroscopy is described. Instead of direct observation of the RNA-polypeptide complex, we attempted the indirect observation of the binding event by focusing on the disappearance of the free polypeptide signal upon interaction with RNA. As a result, specific binding of the Rev-response element (RRE) RNA of the HIV with two RRE-binding peptide aptamers, DLA and RLA peptides, as well as the bacteriophage lambda boxB RNA with the lambda N peptide was observed. We also show that specific RNA-binding peptides can be identified from a mixture of peptides with varying RNA-binding affinity, showing that the method could be applied to high-throughput screening from simple peptide libraries. The method described in this study provides a quick and simple method for detecting specific RNA-polypeptide interactions that avoids difficulties associated with direct observation of RNA and RNA-polypeptide complexes, which may find various applications in the analysis of RNA-polypeptide interactions and in the identification of novel RNA-binding polypeptides.
Sugaya
Maki
M
Department of Materials and Life Science, Faculty of Science and Technology, Seikei University, Musashino, Tokyo 180-8633, Japan.
Saito
Ryota
R
Matsumura
Yuriko
Y
Harada
Kazuo
K
Katoh
Akira
A
D000595:Amino Acid Sequence; D052158:Aptamers, Peptide / Q000138:chemical synthesis / Q000737:chemistry* / Q000494:pharmacology; D010582:Bacteriophage lambda / Q000737:chemistry / Q000187:drug effects; D001665:Binding Sites; D001667:Binding, Competitive; D020650:Combinatorial Chemistry Techniques; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019151:Peptide Library; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry* / Q000494:pharmacology; D012367:RNA, Viral / Q000737:chemistry* / Q000187:drug effects; D020218:Response Elements / Q000187:drug effects; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D013329:Structure-Activity Relationship; D054334:Viral Regulatory and Accessory Proteins / Q000737:chemistry / Q000187:drug effects; D054321:rev Gene Products, Human Immunodeficiency Virus / Q000235:genetics*
D052158:Aptamers, Peptide; C059417:N protein, Bacteriophage lambda; D019151:Peptide Library; D010455:Peptides; D012367:RNA, Viral; D054334:Viral Regulatory and Accessory Proteins; D054321:rev Gene Products, Human Immunodeficiency Virus