Interaction of RNA with phage display selected peptides analyzed by capillary electrophoresis mobility shift assay.
A sensitive capillary electrophoresis mobility shift assay (CEMSA) to analyze RNA/peptide interactions has been developed. Capillary electrophoresis (CE) has been adapted for investigating the interaction between variously methylated 17-nt analogs of the yeast tRNAPhe anticodon stem and loop domain (ASL(Phe)) and 15-amino-acid peptides selected from a random phage display library (RPL). A peptide-concentration-dependent formation of RNA/peptide complex was clearly visible during CEMSA. In the presence of peptide, the UV-monitored CE peak for ASLPhe with three of the five naturally occurring modifications (2'-O-methylcytidine (Cm32), 2'-O-methylguanine (Gm34) and 5-methylcytidine (m5C40) shifted from 18.16 to 20.90 min. The mobility shift was observed only for methylated RNA. The negative effects of diffusion, electroosmotic flow and adhesion of molecules to the capillary internal wall were suppressed by using a buffer containing a sieving polymer and a polyacrylamide-coated capillary. Under these conditions, well-shaped peaks and resolution of RNA free and bound to peptide were achieved. Peptide tF2, the most populated ligand in the RPL, specifically bound triply methylated ASLPhe in a methylated nucleoside-dependent manner. CE was found to be an efficient and sensitive method for the qualitative analysis of RNA-peptide interaction and should be generally applicable to the study of RNA-peptide (protein) interactions.
Mucha
Piotr
P
Department of Chemistry, University of Gdansk, Poland. fly@chemik.chem.univ.gda.pl
Szyk
Agnieszka
A
Rekowski
Piotr
P
Guenther
Richard
R
Agris
Paul F
PF
D000595:Amino Acid Sequence; D000926:Anticodon / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D019075:Electrophoresis, Capillary / Q000379:methods*; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D019151:Peptide Library; D010455:Peptides / Q000737:chemistry / Q000378:metabolism*; D012360:RNA, Transfer, Phe / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D000926:Anticodon; D019151:Peptide Library; D010455:Peptides; D012360:RNA, Transfer, Phe