Biostable aptamers with antagonistic properties to the neuropeptide nociceptin/orphanin FQ
Abstract
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 IC50 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 GTPγS 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.
Keywords
- Spiegelmer
- nociceptin
- neuropeptide
- biostable aptamer
- RNA, pain
- BSA, bovine serum albumin
- DEPC, diethylpyrocarbonate
- dNTP, deoxyribonucleotide triphosphate
- HBSS, Hank’s balanced salt solution
- NTP, ribonucleotide triphosphate
- PBS, phosphate buffered saline
- PCR, polymerase chain reaction
- PMSF, phenylmethylsulfonyl fluoride
- WGA, wheat germ agglutinin
Footnotes
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↵4 These authors contributed equally to this work.
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↵5 Present addresses: Amersham Buchler GmbH & Co. KG, Fraunhoferstrasse 7, 85737 Ismaning, Germany;
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↵6 Novartis Pharma AG, WKL-490.1.55, 4002 Basel, Switzerland;
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↵7 Philogen S.r.l., La Lizza 7, 53100 Siena, Italy;
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↵8 P.B., Am Marktplatz 8, 82152 Planegg, Germany;
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↵9 Degussa AG, CVPH-BT, Rodenbacher Chaussee 4, 63457 Hanau-Wolfgang, Germany;
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↵10 MicroDiscovery GmbH, Marienburger Strasse 1, 10405 Berlin, Germany.
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Article and publication are at http://www.rnajournal.org/cgi/doi/10.1261/rna.5186504.
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- Accepted November 7, 2003.
- Received September 12, 2003.
- Copyright 2004 by RNA Society