title: Conformationally selective RNA aptamers allosterically modulate the β2-adrenoceptor.
abstract: G-protein-coupled receptor (GPCR) ligands function by stabilizing multiple, functionally distinct receptor conformations. This property underlies the ability of 'biased agonists' to activate specific subsets of a given receptor's signaling profile. However, stabilizing distinct active GPCR conformations to enable structural characterization of mechanisms underlying GPCR activation remains difficult. These challenges have accentuated the need for receptor tools that allosterically stabilize and regulate receptor function through unique, previously unappreciated mechanisms. Here, using a highly diverse RNA library combined with advanced selection strategies involving state-of-the-art next-generation sequencing and bioinformatics analyses, we identify RNA aptamers that bind a prototypical GPCR, the β2-adrenoceptor (β2AR). Using biochemical, pharmacological, and biophysical approaches, we demonstrate that these aptamers bind with nanomolar affinity at defined surfaces of the receptor, allosterically stabilizing active, inactive, and ligand-specific receptor conformations. The discovery of RNA aptamers as allosteric GPCR modulators significantly expands the diversity of ligands available to study the structural and functional regulation of GPCRs.
authors.affiliation: Department of Medicine, Duke University Medical Center, Durham, North Carolina, USA.
authors.forename: Alem W
authors.initials: AW
authors.lastname: Kahsai
authors.affiliation: Department of Medicine, Duke University Medical Center, Durham, North Carolina, USA.
authors.forename: James W
authors.initials: JW
authors.lastname: Wisler
authors.affiliation: Department of Medicine, Duke University Medical Center, Durham, North Carolina, USA.|Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts, USA.
authors.forename: Jungmin
authors.initials: J
authors.lastname: Lee
authors.affiliation: Department of Medicine, Duke University Medical Center, Durham, North Carolina, USA.
authors.forename: Seungkirl
authors.initials: S
authors.lastname: Ahn
authors.affiliation: Department of Medicine, Duke University Medical Center, Durham, North Carolina, USA.|Department of Biochemistry, Duke University Medical Center, Durham, North Carolina, USA.
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Cahill Iii
authors.affiliation: Duke Human Vaccine Institute, Duke University Medical Center, Durham, North Carolina, USA.
authors.forename: S Moses
authors.initials: SM
authors.lastname: Dennison
authors.affiliation: Department of Medicine, Duke University Medical Center, Durham, North Carolina, USA.
authors.forename: Dean P
authors.initials: DP
authors.lastname: Staus
authors.affiliation: Department of Medicine, Duke University Medical Center, Durham, North Carolina, USA.
authors.forename: Alex R B
authors.initials: AR
authors.lastname: Thomsen
authors.affiliation: Duke Human Vaccine Institute, Duke University Medical Center, Durham, North Carolina, USA.
authors.forename: Kara M
authors.initials: KM
authors.lastname: Anasti
authors.affiliation: Department of Medicine, Duke University Medical Center, Durham, North Carolina, USA.
authors.forename: Biswaranjan
authors.initials: B
authors.lastname: Pani
authors.affiliation: Department of Medicine, Duke University Medical Center, Durham, North Carolina, USA.
authors.forename: Laura M
authors.initials: LM
authors.lastname: Wingler
authors.affiliation: The University of North Carolina School of Medicine, Chapel Hill, North Carolina, USA.
authors.forename: Hemant
authors.initials: H
authors.lastname: Desai
authors.affiliation: Department of Surgery, Duke University Medical Center, Durham, North Carolina, USA.|Duke Translational Research Institute, Duke University Medical Center, Durham, North Carolina, USA.|Moores Cancer Center, University of California, San Diego, La Jolla, California, USA.
authors.forename: Kristin M
authors.initials: KM
authors.lastname: Bompiani
authors.affiliation: Department of Pharmacology, University of North Carolina School of Medicine, Chapel Hill, North Carolina, USA.
authors.forename: Ryan T
authors.initials: RT
authors.lastname: Strachan
authors.affiliation: Genome Sequencing and Analysis Core Resource, Duke University, Durham, North Carolina, USA.
authors.forename: Xiaoxia
authors.initials: X
authors.lastname: Qin
authors.affiliation: Duke Human Vaccine Institute, Duke University Medical Center, Durham, North Carolina, USA.
authors.forename: S Munir
authors.initials: SM
authors.lastname: Alam
authors.affiliation: Department of Surgery, Duke University Medical Center, Durham, North Carolina, USA.|Duke Translational Research Institute, Duke University Medical Center, Durham, North Carolina, USA.
authors.forename: Bruce A
authors.initials: BA
authors.lastname: Sullenger
authors.affiliation: Department of Medicine, Duke University Medical Center, Durham, North Carolina, USA.|Department of Biochemistry, Duke University Medical Center, Durham, North Carolina, USA.|Howard Hughes Medical Institute, Duke University Medical Center, Durham, North Carolina, USA.
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Lefkowitz
mesh_terms: D000494:Allosteric Regulation / Q000187:drug effects; D052157:Aptamers, Nucleotide / Q000737:chemistry / Q000378:metabolism*; D048588:Benzoxazines / Q000737:chemistry / Q000494:pharmacology; D006801:Humans; D008958:Models, Molecular; D011487:Protein Conformation; D018343:Receptors, Adrenergic, beta-2 / Q000737:chemistry / Q000378:metabolism*
chemical_list: D052157:Aptamers, Nucleotide; C584569:BI167107; D048588:Benzoxazines; D018343:Receptors, Adrenergic, beta-2