Brush‐on‐Brush Architecture Enables Durable and Reversible Aptamer Therapeutics With Minimal PEG Associated Immunogenicity

We present a "Brush-on-Brush" (BOB) nanocarrier as a next-generation alternative to PEGylation for nucleic acid aptamer therapeutics. This architecture, comprising aptamers and dense brush-shaped oligo(ethylene glycol) (b-OEG) side chains co-grafted onto a peptide backbone, was validated using the thrombin-binding HD1 aptamer. BOB-HD1 exhibited exceptional nuclease resistance, retaining >40% integrity after 2 h of S1 nuclease exposure (versus complete degradation of free aptamer within 3 min), while fully preserving G-quadruplex folding, thrombin-binding activity, and antidote-mediated reversibility. In mice, BOB-HD1 achieved a 7.5-fold increase in systemic exposure and sustained anticoagulation for 4 h, compared to <20 min for unmodified or linearly PEGylated aptamer. Critically, unlike linear PEG conjugates that induced anti-PEG antibodies and accelerated blood clearance upon repeated dosing, BOB-HD1 induced no detectable anti PEG IgM or IgG responses under the tested conditions and maintained full efficacy upon repeated administration. This dense brush architecture thus addresses key stability, clearance, and PEG associated immunogenicity barriers that have impeded aptamer clinical translation, offering a versatile and controllable platform for durable aptamer therapeutics while minimizing detectable anti PEG antibody responses under the tested conditions.