Edge-State Programmed Carbonized Nanogels Enable Precise Hot-Start PCR by Multivalent Enzyme Inhibition

Abstract

Hot-start polymerase chain reaction (PCR) improves DNA amplification specificity by inhibiting polymerase activity before denaturation. We present carbonized polysaccharide nanogels (CNGs) as protein-free, thermoresponsive inhibitors for precise hot-start control. Mild pyrolysis of sodium alginate yields alginate-derived CNGs (Alg-CNGs) with graphitic carbon domains and oxygen-rich edge chemistries, including phenolic and lactone groups. Alg-CNGs inhibit Taq DNA polymerase over 1000-fold compared to native alginate at ambient temperatures, with activity restored above approximate to 75 degrees C. Isothermal titration calorimetry, limited proteolysis-mass spectrometry, molecular docking, and dynamics simulations reveal multivalent interactions with polymerase subdomains that restrict open-closed transitions for catalysis, which weaken upon heating, consistent with enthalpy-driven binding and entropy-enabled release. Chemical modifications, such as phenol oxidation and lactone ring opening, reduce inhibition and identify phenolic and lactone edge groups as key contact determinants. Alg-CNGs outperform antibody-based alternatives, with a binding affinity one order of magnitude higher than reported hot-start antibodies, ensuring high-fidelity and single-target amplification. Unlike antibodies or aptamers, Alg-CNGs serve as universal hot-start inhibitors, improving specificity across various commercial Taq DNA polymerases and showing strong potential to replace protein- and aptamer-based hot-start PCR systems in nucleic acid diagnostics.