Restriction endonuclease selective inhibition by β-cyclodextrin sulfate

Abstract Restriction endonucleases (REs) are sequence-specific DNA-cleaving enzymes that are widely used as tools in molecular biology. Although REs is not typically considered therapeutic targets, their inhibition provides a useful framework for understanding how DNA-binding enzymes can be differentially modulated. In this present study, we investigated the inhibitory activity of β-cyclodextrin sulfate (β-CDsul), a highly anionic macrocycle previously reported to act as a non-selective inhibitor of restriction enzymes. Using NdeI-linearised pBR322 as a defined substrate, we quantified the inhibition of EcoRI, HindIII, and VspI under optimised digestion conditions. β-CDsul inhibited EcoRI with substantially greater potency than HindIII or VspI, exhibiting a 13- to 27-fold difference in IC50 values. In contrast, EDTA inhibited all three enzymes with comparable potency, consistent with non-selective divalent metal ion chelation. Selective inhibition of EcoRI by β-CDsul was maintained in a dual-enzyme system containing both EcoRI and VspI, supporting an enzyme-dependent mode of action. These findings demonstrate that β-cyclodextrin sulfate acts as a selective inhibitor of EcoRI and highlight the potential of polyanionic macrocycles as biochemical probes for differential endonuclease activity.