In Vitro Selection of RNA Ligands to Substance P

Abstract

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.

RNA aptamers selectively modulate protein recruitment to the cytoplasmic domain of β-secretase BACE1 in vitro

“…These affinities match with those obtained for other nonconstrained, non-nucleic acid binding peptides for which aptamers have been selected (Nieuwlandt et al 1995;Weiss et al 1997;Blind et al 1999;Proske et al 2002b). To evolve these tight-binding aptamers we used affinity maturation of the originally selected binders.…”

“…Indeed, the membraneproximal epitope contains three arginine residues and fewer acidic amino acids, whereas the C-terminal half does not contain any basic amino acids but a cluster of acidic ones. Tight-binding aptamers have been observed before to preferentially evolve to epitopes that contain positively charged amino acid residues, presumably utilizing electrostatic interactions (Nieuwlandt et al 1995;Kimoto et al 1998;Blind et al 1999). In addition, our anti-B1-CT aptamers add to a series of other aptamers that target proteins implicated in neurodegenerative diseases, in particular aptamers directed against various forms of the prion protein (Weiss et al 1997;Proske et al 2002a;Rhie et al 2003;Sayer et al 2004).…”

A DNA aptamer population specifically detects Leishmania infantum H2A antigen

“…It has been shown that aptamers form stable and specific complexes with a range of different targets, including small molecules such as amino acids and highly complex proteins and whole viruses. [3][4][5][6][7][8][9][10] These specialized molecules are analogs to antibodies in specificity and affinity, but have the potential advantage of being reproduced by chemical synthesis and be more easily labeled with fluorescent or other reporters during their synthesis. Consequently, they are considered potential therapeutic agents and also thought to rival antibodies in immunoassay-like analyses.…”

Clinical applications of nucleic acid aptamers in cancer

“…The term 'aptamer' means 'to fit' (aptus) in Latin (6), which indicates two important properties of aptamers: i) their ability to fold into complex tertiary structures and recognize their targets with high affinity (low nM to high pM equilibrium dissociation constants); and ii) their specificity, somewhat analogous to antigen-antibody interactions. Using this technique, a number of aptamers that specifically recognize targets, such as metal ions (11)(12)(13), organic dyes and amino acids (14)(15)(16)(17), antibiotics (18,19) and peptides (20,21), as well as proteins of various sizes and functions (6,22,23), whole cells (24)(25)(26)(27), whole organisms (28), viruses (29) and bacteria (30), have been obtained (31)(32)(33). The SELEX process is based on the ability of these small oligonucleotides to fold into unique 3D structures that interact with a target with high specificity and affinity through such interactions as van der Waals surface contacts, hydrogen bonding and base stacking.…”