Precision Aptamers Against a Native GPCR through Ligand-Guided Selection.

Area of Science:

• Biochemistry
• Molecular Biology
• Drug Discovery

Background:

• G protein-coupled receptors (GPCRs) are crucial membrane proteins involved in numerous physiological processes.
• GPCRs represent a major class of drug targets, yet identifying ligands that bind them in their native state is challenging.
• The dynamic and heterogeneous conformational states of GPCRs complicate study in purified systems.

Purpose of the Study:

• To develop and validate an aptamer discovery platform for isolating ligands targeting GPCRs in their native cellular context.
• To identify conformationally sensitive aptamers that recognize GPCRs on the cell surface.
• To demonstrate the utility of ligand-guided selection for discovering functional aptamers against GPCRs.

Main Methods:

• Developed an expanded aptamer discovery platform utilizing ligand-guided selection (LIGS).
• Employed the β2-adrenergic receptor (β2AR) as a model system, using agonists and antagonists as competing ligands.
• Selected aptamers against cell-surface expressed β2AR, assessing binding affinity and functional engagement.

Main Results:

• Identified three high-specificity aptamers targeting the β2AR.
• Demonstrated selective binding of aptamers to cell-membrane bound β2AR, with higher apparent affinity than for purified receptors.
• Observed that selected aptamers induce rapid receptor internalization, indicating functional activity.

Conclusions:

• Ligand-guided selection is a generalizable strategy for discovering conformationally sensitive aptamers.
• This platform enables aptamer selection against GPCRs in their native membrane environments.
• The identified aptamers show specific binding and functional modulation of cell-surface GPCRs.