Enzymes are the major players for many biological processes.Fundamental studies of the enzymatic activity at the single-molecule level provides important information that is otherwise inaccessible at the ensemble level.Yet,these single-molecule experiments are technically di±cult and generally require complicated experimental design.Here,we develop a Holliday junction(HJ)-based platform to study the activity of restriction endonucleases at the single-molecule level using single-molecule FRET(sm-FRET).We show that the intrinsic dynamics of HJ can be used as the reporter for both the enzyme-binding and the substrate-release events.Thanks to the multiple-arms structure of HJ,the fluorophore-labeled arms can be different from the surface anchoring arm and the substrate arm.Therefore,it is possible to independently change the substrate arm to study different enzymes with similar functions.Such a design is extremely useful for the systematic study of enzymes from the same family or enzymes bearing different pathologic mutations.Moreover,this method can be easily extended to study other types of DNA-binding enzymes without too much modi fication of the design.We anticipate it can find broad applications in single-molecule enzymology.
Xin WangJingyuan NieYi LiHai PanPeng ZhengMeng QinYi CaoWei Wang
A new method of light-powered dissipative supra-molecular polymerization is established,in which supramolecular polymerization is implemented in the far-from-equilibrium state.A bifunctional mono-mer containing two viologen moieties was designed.Upon inputting energy by light,the sys-tem was driven far from equilibrium,and the mono-mers were photoreduced and activated to form supramolecular polymers driven by 2∶1 host–guest complexation of the viologen cation radical and cucurbit[8]uril.As the system returned to equilibri-um,the supramolecular polymers depolymerized spontaneously by air oxidation.This method works in both linear and in cross-linked supramolecular polymerization.The strategy of light-powered dis-sipative supramolecular polymerization is anticipat-ed to have potential in the fabrication of functional supramolecular materials,especially in creating novel“living”materials.
