Structural basis for the targeting of complement anaphylatoxin C5a using a mixed L-RNA/L-DNA aptamer

Area of Science:

• Biochemistry
• Structural Biology
• Medicinal Chemistry

Background:

• L-Oligonucleotide aptamers (Spiegelmers) are non-natural nucleic acids with high resistance to plasma nucleases, making them promising therapeutic agents.
• The anaphylatoxin C5a is a key inflammatory mediator in complement activation, linked to organ damage, and is a target for Spiegelmer-based therapies.

Purpose of the Study:

• To determine the crystallographic structures of an active Spiegelmer, NOX-D20, bound to its physiological targets, mouse C5a and C5a-desArg.
• To elucidate the molecular basis of Spiegelmer binding to C5a and understand the structural determinants of specificity and affinity.
• To provide insights into the inhibition mechanism of the C5a:C5aR interaction by Spiegelmers.

Main Methods:

• X-ray crystallography to determine the 3D structures of Spiegelmer-C5a complexes.
• Site-directed mutagenesis and functional assays to validate the binding mode and specificity.
• Biophysical techniques to assess binding affinity and inhibition of receptor interaction.

Main Results:

• The first crystallographic structures of an active Spiegelmer (NOX-D20) bound to mouse C5a and C5a-desArg were determined.
• The structures reveal a complex 3D architecture where the Spiegelmer wraps around C5a, featuring an intramolecular G-quadruplex stabilized by a Ca(2+) ion.
• Functional studies confirmed the binding mode and rationalized the specificity of NOX-D20 for mouse and human C5a over macaque and rat C5a.
• The structural model explains affinity improvements via nucleotide exchanges and inhibition of the C5a:C5aR interaction.

Conclusions:

• The determined structures provide a detailed molecular understanding of Spiegelmer-C5a interactions.
• This structural insight facilitates the rational design of improved Spiegelmer-based therapeutics targeting C5a.
• The findings support the therapeutic potential of Spiegelmers in inflammatory conditions mediated by the complement system.