An RNA molecule that specifically inhibits G-protein-coupled receptor kinase 2 in vitro

Abstract

G-protein-coupled receptors are desensitized by a two-step process. In a first step, G-protein-coupled receptor kinases (GRKs) phosphorylate agonist-activated receptors that subsequently bind to a second class of proteins, the arrestins. GRKs can be classified into three subfamilies, which have been implicated in various diseases. The physiological role(s) of GRKs have been difficult to study as selective inhibitors are not available. We have used SELEX (systematic evolution of ligands by exponential enrichment) to develop RNA aptamers that potently and selectively inhibit GRK2. This process has yielded an aptamer, C13, which bound to GRK2 with a high affinity and inhibited GRK2-catalyzed rhodopsin phosphorylation with an IC 50 of 4.1 nM. Phosphorylation of rhodopsin catalyzed by GRK5 was also inhibited, albeit with 20-fold lower potency (IC 50 of 79 nM). Furthermore, C13 reveals significant specificity, since almost no inhibitory activity was detectable testing it against a panel of 14 other kinases. The aptamer is two orders of magnitude more potent than the best GRK2 inhibitors described previously and shows high selectivity for the GRK family of protein kinases.

Development of Inflammatory Immune Response-Related Drugs Based on G Protein-Coupled Receptor Kinase 2

“…C13, as an RNA aptamer, can selectively inhibit GRK2 with high affinity. It can stabilize a unique inactive conformation of GRK2 through multiple interactions leading to the inhibition of GRK2 [68]. Both within and outside the kinase domain active site pocket, the terminal stem of the aptamer indirectly contributes to its selectivity by constraining a selected portion of the RNA.…”

Die chemische Biologie von Aptameren

“…B. molekulare Parasiten (DNA-Sequenzen mit hervorragenden Replikationseigenschaften -meist kürzer oder länger als die DNA-Bibliothek), akkumulieren. Die Verwendung automatisierter Arbeitsstationen setzt eine Immobilisierung des Zielmoleküls an magnetischen Partikeln [37,39] voraus. Entsprechende Verfahren sind so optimiert, dass keine Reinigung von Nucleinsäureintermediaten notwendig ist, was eine simultane Selektion von Aptameren gegen bis zu acht Zielmoleküle ermöglicht.…”

“…Für In-vitro-Selektionen wurden außer linearen Nucleinsäurebibliotheken (Abbildung 1 B) auch strukturell eingeschränkte Bibliotheken mit definierten Sekundärstrukturelementen eingesetzt. [39,40] Diese Bibliotheken haben den Vorteil, dass eine Verkürzung und Strukturbestimmung eines repräsentativen Aptamers unkompliziert ist. [41] Eine effiziente Kombination von chemischer Synthese mit Selelektionsprozessen wurde kürzlich von Bugaut et al demonstriert.…”

An Aptamer Targeting the Apical‐Loop Domain Modulates pri‐miRNA Processing

“…We performed chemical probing experiments of pri-mi18a in the presence and absence of aptamer 7 and with aptamer F as a control. [17] As shown in Figure 2 and Figure S5 in the Supporting Information, the presence of 7 results in the protection of the apical-loop domain modification by the chemical probes whereas F had no effect. The two studiesthe interaction analysis with the apical-loop domain mutants and the footprinting analysis-support the conclusion that aptamer 7 recognizes the apical-loop domain of pri-miR18a.…”