Biostable aptamers with antagonistic properties to the neuropeptide nociceptin/orphanin FQ

The neuropeptide nociceptin/orphanin FQ (N/OFQ), the endogenous ligand of the opioid receptor-like 1 (ORL1) receptor, has been shown to play a prominent role in the regulation of several biological functions such as pain and stress. Here we describe the isolation and characterization of N/OFQ bindin...

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Bibliographic Details
Published in:RNA (Cambridge) 2004-03, Vol.10 (3), p.516-527
Main Authors: Faulhammer, Dirk, Eschgfäller, Bernd, Stark, Sandra, Burgstaller, Petra, Englberger, Werner, Erfurth, Jeannette, Kleinjung, Frank, Rupp, Johanna, Dan Vulcu, Sebastian, Schröder, Werner, Vonhoff, Stefan, Nawrath, Hermann, Gillen, Clemens, Klussmann, Sven
Format: Article
Language:English
Subjects:
Animals
Base Sequence
Humans
Ligands
Molecular Sequence Data
Nociceptin
Nociceptin Receptor
Nucleic Acid Conformation
Oocytes
Opioid Peptides - antagonists & inhibitors
Receptors, Opioid - metabolism
RNA - metabolism
Tritium - metabolism
Xenopus
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Summary:The neuropeptide nociceptin/orphanin FQ (N/OFQ), the endogenous ligand of the opioid receptor-like 1 (ORL1) receptor, has been shown to play a prominent role in the regulation of several biological functions such as pain and stress. Here we describe the isolation and characterization of N/OFQ binding biostable RNA aptamers (Spiegelmers) using a mirror-image in vitro selection approach. Spiegelmers are L-enantiomeric oligonucleotide ligands that display high affinity and specificity to their targets and high resistance to enzymatic degradation compared to D-oligonucleotides. A representative Spiegelmer from the selections performed was size-minimized to two distinct sequences capable of high affinity binding to N/OFQ. The Spiegelmers were shown to antagonize binding of N/OFQ to the ORL1 receptor in a binding-competition assay. The calculated IC(50) values for the Spiegelmers NOX 2149 and NOX 2137a/b were 110 nM and 330 nM, respectively. The competitive antagonistic properties of these Spiegelmers were further demonstrated by their effective and specific inhibition of G-protein activation in two additional models. The Spiegelmers antagonized the N/OFQ-induced GTPgammaS incorporation into cell membranes of a CHO-K1 cell line expressing the human ORL1 receptor. In oocytes from Xenopus laevis, NOX 2149 showed an antagonistic effect to the N/OFQ-ORL 1 receptor system that was functionally coupled with G-protein-regulated inwardly rectifying K(+) channels.
ISSN:1355-8382
1469-9001
DOI:10.1261/rna.5186504