DARPins (an acronym for designed ankyrin repeat proteins) are genetically engineered antibody mimetic proteins typically exhibiting highly specific and high-affinity target protein binding. They are derived from natural ankyrin repeat proteins, one of the most common classes of binding proteins in nature, which are responsible for diverse functions such as cell signaling, regulation and structural integrity of the cell. DARPins consist of at least three repeat motifs or modules, of which the most N- and the most C-terminal modules are referred to as "caps", since they shield the hydrophobic core of the protein. The number of internal modules is indicated as number (e.g. N1C, N2C, N3C, ...) while the caps are indicated with "N" or "C", respectively. The molecular mass of e.g. 14 or 18 kDa (kilodaltons) for four- (N2C) or five- (N3C) repeat DARPins is rather small for a biologic (ca 10% of the size of an IgG).

DARPins constitute a new class of potent, specific and versatile small-protein therapeutics, and are used as investigational tools in various research, diagnostic and therapeutic applications. Molecular Partners AG, a clinical-stage biopharmaceutical company with several DARPin molecules in clinical and preclinical development, is currently pursuing development of therapeutic DARPins both through their own development and in parallel with partners. Athebio AG builds on further improving the DARPin scaffold for a partnering model approach.

In addition, DARPins can be used as crystallization chaperones for soluble and membrane proteins, including G protein-coupled receptors (GPCRs), either as binding partners or as rigid fusions to the target protein, a concept now being extended to structure determination by cryoEM.

The DARPin platform was discovered and developed in the laboratory of Andreas Plückthun at the University of Zurich, Switzerland while studying engineering and libraries of recombinant antibodies. DARPins are derived from naturally occurring ankyrin proteins, a protein class that mediates high-affinity protein-protein interactions in nature.

DARPin libraries were designed via sequence alignments of several thousand natural ankyrin repeat motifs (of about 33 amino acids each) combined with structure-based design and recombinant DNA methods. These proteins consist of repetitive structural units that form a stable protein domain with a large potential target interaction surface. Typically, DARPins comprise four or five repeats, of which the first (N-capping repeat) and last (C-capping repeat) serve to shield the hydrophobic protein core from the aqueous environment. DARPins correspond to the average size of natural ankyrin repeat protein domains. Proteins with fewer than three repeats (i.e., the capping repeats and one internal repeat) do not form a stable enough tertiary structure. The molecular mass of a DARPin depends on the total number of repeats, as shown in the following chart:

Libraries of DARPins with randomized potential target interaction residues, with diversities of over 10¹² variants, have been generated at the DNA level. From these libraries, biochemists can select DARPins to bind the target of choice with picomolar affinity and specificity can be selected using ribosome display or phage display using signal sequences allowing cotranslational secretion. DARPins can be designed to act as receptor agonists, antagonists, inverse agonists, enzyme inhibitors, or simple target protein binders.

DARPins are expressed in the cytoplasm of Escherichia coli at high levels (over 10 g/L in fermentation, 1 g/L in shake flask) in soluble form. The proteins exhibit high thermal and thermodynamic stability (denaturation midpoint: usually equilibrium unfolding: ∆G > 9.5 kcal/mol) increasing with increasing repeat number. DARPins are stable in human blood serum and can be engineered so as not to contain T-cell epitopes.

Due to the high specificity, stability, potency and affinity, as well as their flexible architecture, DARPins have a rigid body-binding mode. Multi-specific or multivalent constructs made by genetic fusion suggest that fused DARPins have similar binding properties as single-domain DARPins. The absence of cysteines in the scaffold enables engineering of site-specific cysteines, allowing site-directed coupling of chemicals to the molecule. Non-natural amino acids can be introduced for the same purpose.