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 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.
机构:Univ Leeds, N England Struct Biol Ctr, Leeds LS2 9JT, W Yorkshire, England
Convery, MA
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Rowsell, S
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机构:Univ Leeds, N England Struct Biol Ctr, Leeds LS2 9JT, W Yorkshire, England
Rowsell, S
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Stonehouse, NJ
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机构:Univ Leeds, N England Struct Biol Ctr, Leeds LS2 9JT, W Yorkshire, England
Stonehouse, NJ
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Ellington, AD
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机构:Univ Leeds, N England Struct Biol Ctr, Leeds LS2 9JT, W Yorkshire, England
Ellington, AD
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Hirao, I
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机构:Univ Leeds, N England Struct Biol Ctr, Leeds LS2 9JT, W Yorkshire, England
Hirao, I
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Murray, JB
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机构:Univ Leeds, N England Struct Biol Ctr, Leeds LS2 9JT, W Yorkshire, England
Murray, JB
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Peabody, DS
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机构:Univ Leeds, N England Struct Biol Ctr, Leeds LS2 9JT, W Yorkshire, England
Peabody, DS
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Phillips, SEV
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机构:Univ Leeds, N England Struct Biol Ctr, Leeds LS2 9JT, W Yorkshire, England
Phillips, SEV
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Stockley, PG
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Univ Leeds, N England Struct Biol Ctr, Leeds LS2 9JT, W Yorkshire, EnglandUniv Leeds, N England Struct Biol Ctr, Leeds LS2 9JT, W Yorkshire, England
机构:Univ Leeds, N England Struct Biol Ctr, Leeds LS2 9JT, W Yorkshire, England
Convery, MA
;
Rowsell, S
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机构:Univ Leeds, N England Struct Biol Ctr, Leeds LS2 9JT, W Yorkshire, England
Rowsell, S
;
Stonehouse, NJ
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机构:Univ Leeds, N England Struct Biol Ctr, Leeds LS2 9JT, W Yorkshire, England
Stonehouse, NJ
;
Ellington, AD
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机构:Univ Leeds, N England Struct Biol Ctr, Leeds LS2 9JT, W Yorkshire, England
Ellington, AD
;
Hirao, I
论文数: 0引用数: 0
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机构:Univ Leeds, N England Struct Biol Ctr, Leeds LS2 9JT, W Yorkshire, England
Hirao, I
;
Murray, JB
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机构:Univ Leeds, N England Struct Biol Ctr, Leeds LS2 9JT, W Yorkshire, England
Murray, JB
;
Peabody, DS
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机构:Univ Leeds, N England Struct Biol Ctr, Leeds LS2 9JT, W Yorkshire, England
Peabody, DS
;
Phillips, SEV
论文数: 0引用数: 0
h-index: 0
机构:Univ Leeds, N England Struct Biol Ctr, Leeds LS2 9JT, W Yorkshire, England
Phillips, SEV
;
Stockley, PG
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Univ Leeds, N England Struct Biol Ctr, Leeds LS2 9JT, W Yorkshire, EnglandUniv Leeds, N England Struct Biol Ctr, Leeds LS2 9JT, W Yorkshire, England