In vitro selection of RNA ligands to substance P.
RNA ligands to the tachykinin substance P have been selected from a large pool of random sequence RNA molecules. Substance P is an undecapeptide that plays a variety of roles as a neurotransmitter and neuromodulator in the central and peripheral nervous system of mammals. A systematic evolution of ligands by exponential enrichment (SELEX) procedure was used to isolate RNAs that bind substance P immobilized on a solid support. RNAs that also bind substance P in solution were identified, and the tightest binder was subjected to mutagenesis in a second SELEX procedure to evolve ligands with a higher affinity for the peptide. A comparative analysis of 36 ligands isolated from the second SELEX experiment revealed two main sequence classes with highly conserved secondary structures within each class. Dissociation constants for the interaction of these ligands with substance P in solution were determined by equilibrium dialysis. The amino acid residues involved in the interaction with the highest affinity ligand (190 nM Kd) were mapped by determining which of a set of overlapping fragments of substance P can compete with the intact peptide for binding. A binding competition experiment also demonstrated the ability of the same ligand to discriminate between substance P and the reverse orientation of the same amino acid sequence. The results from this study demonstrate that SELEX can yield high affinity RNA ligands to small nonconstrained peptides.
Nieuwlandt
D
D
Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder 80309, USA.
Wecker
M
M
Gold
L
L
D000595:Amino Acid Sequence; D001483:Base Sequence; D001667:Binding, Competitive; D004277:DNA, Single-Stranded / Q000737:chemistry; D008024:Ligands; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009843:Oligoribonucleotides; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D012313:RNA / Q000737:chemistry* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry; D015183:Restriction Mapping; D012685:Sepharose; D012689:Sequence Homology, Nucleic Acid; D013373:Substance P / Q000737:chemistry / Q000378:metabolism*; D013698:Templates, Genetic
D004277:DNA, Single-Stranded; D008024:Ligands; D009843:Oligoribonucleotides; D010446:Peptide Fragments; D011994:Recombinant Proteins; D013373:Substance P; D012313:RNA; D012685:Sepharose