In vitro selection of a novel catalytic RNA: characterization of a sulfur alkylation reaction and interaction with a small peptide.
An in vitro RNA selection for catalytic activity was used to co-select for binding activity to a small peptide. 5'-phosphorothioate-modified RNA (GMPS-RNA) sequences were selected from a randomized pool of oligoribonucleotides for their ability to accelerate a halide substitution reaction with N-bromoacetyl-bradykinin (BrBK). One RNA selected shows a 2,420-fold increase in rate of reaction with BrBK relative to the starting pool. This reaction is specifically inhibited by free bradykinin (Ki 230 microM), indicating that interactions with bradykinin contribute to the rate enhancement. Inhibition of the reaction by the peptide requires both the amino- and carboxy-terminal arginine residues of the peptide for optimal inhibition activity. Reaction with N-bromoacetamide is not enhanced, indicating that the intrinsic reactivity of the 5' phosphorothioate is not increased in the selected RNA. Through 3'-end boundary analysis, much of the catalytic activity of the selected GMPS-RNA is shown to reside in a hairpin structure in the selected region of the molecule. This hairpin structure is also implicated in the recognition of the peptide substrate.
Wecker
M
M
Department of Molecular, Cellular, and Developmental Biology, University of Colorado at Boulder 80304, USA. wecker@beagle.colorado.edu
Smith
D
D
Gold
L
L
D000447:Aldehydes; D000478:Alkylation; D001120:Arginine / Q000502:physiology; D001483:Base Sequence; D001920:Bradykinin / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D002074:Butanones; D019020:Directed Molecular Evolution / Q000379:methods*; D006157:Guanosine Monophosphate / Q000031:analogs & derivatives* / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009843:Oligoribonucleotides; D011485:Protein Binding; D012313:RNA / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D016337:RNA, Catalytic / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D013379:Substrate Specificity; D013455:Sulfur / Q000378:metabolism; D013873:Thionucleotides
D000447:Aldehydes; D002074:Butanones; D008024:Ligands; C101934:N-bromoacetylbradykinin; D009843:Oligoribonucleotides; D016337:RNA, Catalytic; D016601:RNA-Binding Proteins; D013873:Thionucleotides; C101935:guanosine-5'-monophosphorothioate-RNA; D012313:RNA; D013455:Sulfur; C066524:guanosine 5'-monophosphorothioate; D006157:Guanosine Monophosphate; D001120:Arginine; C005135:kethoxal; D001920:Bradykinin