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 ≈75°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.