Directing in Vitro Selection towards G-quadruplex-forming Aptamers to Inhibit HMGB1 Pathological Activity

Area of Science:

• Biochemistry
• Molecular Biology
• Drug Discovery

Background:

• High-Mobility Group Box1 (HMGB1) is implicated in inflammatory and autoimmune diseases, and cancer.
• Novel inhibitors are needed to target HMGB1's pathological activities.

Purpose of the Study:

• To discover and characterize novel anti-HMGB1 aptamers.
• To evaluate the therapeutic potential of these aptamers.

Main Methods:

• In vitro selection of guanine-rich oligonucleotides to form G-quadruplex (G4) structures.
• Spectroscopic, electrophoretic, and chromatographic analysis of aptamer folding and stability.
• Chemiluminescent binding assays to assess HMGB1 recognition and selectivity.
• Cell-based assays to evaluate anti-HMGB1 activity and toxicity.

Main Results:

• A set of G-quadruplex aptamers targeting HMGB1 were identified.
• Aptamers, particularly those forming dimeric parallel G4 structures, showed preferential binding to HMGB1.
• The lead aptamer, L12, demonstrated potent anti-HMGB1 activity (IC50 ~28 nM) with good binding affinity.
• L12 exhibited high thermal and enzymatic stability, low toxicity in healthy cells up to 5 μM, and inhibited HMGB1-induced cell migration.

Conclusions:

• Novel G-quadruplex aptamers, including L12, effectively inhibit HMGB1.
• L12 is a promising lead candidate for further in vivo studies due to its stability, potency, and safety profile.