Abstract
L-Oligonucleotide aptamers (Spiegelmers) consist of non-natural L-configured nucleotides and are of particular therapeutic interest due to their high resistance to plasma nucleases. The anaphylatoxin C5a, a potent inflammatory mediator generated during complement activation that has been implicated with organ damage, can be efficiently targeted by Spiegelmers. Here, we present the first crystallographic structures of an active Spiegelmer, NOX-D20, bound to its physiological targets, mouse C5a and C5a-desArg. The structures reveal a complex 3D architecture for the L-aptamer that wraps around C5a, including an intramolecular G-quadruplex stabilized by a central Ca2+ ion. Functional validation of the observed L-aptamer:C5a binding mode through mutational studies also rationalizes the specificity of NOX-D20 for mouse and human C5a against macaque and rat C5a. Finally, our structural model provides the molecular basis for the Spiegelmer affinity improvement through positional L-ribonucleotide to L-deoxyribonucleotide exchanges and for its inhibition of the C5a:C5aR interaction.
Highlights
L-Oligonucleotide aptamers (Spiegelmers) consist of non-natural L-configured nucleotides and are of particular therapeutic interest due to their high resistance to plasma nucleases
Spiegelmers are generated by the SELEX process (Systematic Evolution of Ligands by EXponential enrichment)[21] from oligonucleotide libraries in the natural D-configuration but with the addition of two chiral inversion steps
Through postSELEX optimization, the initial C5a-binding, 44 nucleotides-long, L-RNA Spiegelmer NOX-D19 was modified by six positional riboto-deoxyribonucleotide exchanges, leading to improved affinity and allowing a further truncation by four nucleotides, yielding the 40 nucleotide-long Spiegelmer NOX-D20
Summary
L-Oligonucleotide aptamers (Spiegelmers) consist of non-natural L-configured nucleotides and are of particular therapeutic interest due to their high resistance to plasma nucleases. The NOX-D20:C5a binding mode observed in the structure is further validated by mutational studies using surface plasmon resonance (SPR) and allows to explain NOX-D20 specificity for human and mouse C5a species as opposed to macaque and rat C5a22, as well as the increased affinity obtained following ribo-to-deoxyribonucleotide exchange. These data provide a molecular basis for the inhibitory properties of NOX-D20 towards the C5a:C5aR interaction
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