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
NOXXON Pharma AG, 10589 Berlin, Germany.
RNA. 2004 Mar;10(3):516-27. doi: 10.1261/rna.5186504.
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.
神经肽痛敏肽/孤啡肽FQ（N/OFQ）是阿片样物质受体1（ORL1）的内源性配体，已被证明在调节疼痛和应激等多种生物学功能中发挥重要作用。在此，我们描述了使用镜像体外筛选方法分离和鉴定N/OFQ结合生物稳定RNA适配体（镜像寡核苷酸）的过程。镜像寡核苷酸是L-对映体寡核苷酸配体，与D-寡核苷酸相比，它们对其靶标显示出高亲和力和特异性，并且对酶促降解具有高抗性。从所进行的筛选中选出的一个代表性镜像寡核苷酸被最小化至两个能够与N/OFQ高亲和力结合的不同序列。在结合竞争试验中，这些镜像寡核苷酸被证明可拮抗N/OFQ与ORL1受体的结合。镜像寡核苷酸NOX 2149和NOX 2137a/b的计算IC50值分别为110 nM和330 nM。在另外两个模型中，这些镜像寡核苷酸对G蛋白激活的有效且特异性抑制进一步证明了它们的竞争性拮抗特性。这些镜像寡核苷酸拮抗了N/OFQ诱导的GTPγS掺入表达人ORL1受体的CHO-K1细胞系的细胞膜中。在非洲爪蟾的卵母细胞中，NOX 2149对与G蛋白调节的内向整流钾通道功能偶联的N/OFQ-ORL 1受体系统表现出拮抗作用。
