Generation of RNA aptamers to the G-protein-coupled receptor for neurotensin, NTS-1.
G-protein-coupled receptors (GPCRs) are integral membrane proteins involved in signal transduction and constitute major drug targets for disease therapy. Aptamers, which are globular RNA or DNA molecules evolved to specifically bind a target, could represent a valuable tool with which to probe the role of such receptors in normal tissue and disease pathology and for cocrystallization with receptors for structure determination by X-ray crystallography. Using the bacterially expressed rat neurotensin receptor NTS-1 as an example, we describe a strategy for the generation of GPCR-specific RNA aptamers. Seven rounds of a "subtractive," paramagnetic bead-based selection protocol were used to enrich for neurotensin receptor-specific aptamers, while circumventing the evolution of aptamers reactive to minor protein contaminants. Representatives of each aptamer family were analyzed in Escherichia coli membrane nitrocellulose filter binding assays. Eight aptamers demonstrated specificity for the neurotensin receptor. One aptamer, P19, was characterized in detail and shown to bind to both the rat receptor and the human receptor with nanomolar affinity. P19 was also shown to interact with rat neurotensin receptor expressed in CHO cells, in both membrane preparations and intact cells. P19 represents the first example of a GPCR-specific RNA aptamer.
Daniels
Dion A
DA
Gene Expression and Protein Biochemistry, Medicines Research Centre, Stevenage, SG1 2NY, United Kingdom.
Sohal
Awinder K
AK
Rees
Stephen
S
Grisshammer
Reinhard
R
D000818:Animals; D019204:GTP-Binding Proteins / Q000378:metabolism; D009496:Neurotensin / Q000378:metabolism; D012313:RNA / Q000378:metabolism*; D051381:Rats; D018028:Receptors, Neurotensin / Q000302:isolation & purification / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017423:Sequence Analysis, RNA; D013696:Temperature
D018028:Receptors, Neurotensin; D011993:Recombinant Fusion Proteins; C114036:neurotensin type 1 receptor; D009496:Neurotensin; D012313:RNA; D019204:GTP-Binding Proteins