AFM IMAGING AND SINGLE-MOLECULE FORCE SPECTROSCOPY STUDIES ON MACROMOLECULAR INTERACTIONS AT SINGLE-MOLECULE LEVEL

Abstract

The investigations on macromolecular interactions at the single-molecule level using atomic force microscopy(AFM) imaging and AFM-based single-molecule force spectroscopy(SMFS),with an emphasis on the combination of these two methods,have been discussed on the base of the author's recent work in related fields.First,AFM imaging(including static air phase and in situ liquid phase AFM imaging) is used to detect the effect of protein-binding on the topology of double stranded DNA(dsDNA) based on the characteristic(spaghetti-like) AFM image of dsDNA.Then,SMFS study on the nature of force-induced conformation transition of dsDNA is demonstrated.The force-fingerprint of dsDNA can then be used to study protein-DNA interactions revealing the effects of protein-binding on the dynamic conformation of dsDNA.In addition,the application of AFM-based SMFS in the investigation of RNA-protein coat interactions in an intact plant virus,tobacco mosaic virus(TMV),has been demonstrated.To realize this,a TMV particle is immobilized perpendicularly on a gold-coated substrate.The genetic RNA can then be pulled step-by-step out of the TMV particle through its 5′ opening.This study extends the force spectroscopy technique to the investigation of nucleic acid-protein interactions in more complicated biological systems(i.e.,in the virus particle).Finally,the attempt in the quantitative study of polymer interactions in a polyethylene oxide(PEO) single crystal is successfully accomplished by pulling a single PEO chain via its thiol-labelled end out of its single crystal by a good in situ combination of AFM imaging and SMFS.The generality of the established method in the investigation of crystallization of other polymers is discussed.This quantitative information on inter-and intramolecular interactions of macromolecules is useful in revealing the mechanism of many important biological processes(such as DNA replication) as well as in the design of high-performance polymer materials.