DNA Polymerization in Optical Tweezers

Abstract

Molecular motors associated with nucleic acids ensure replication, maintenance and expression of genetic information, which is crucial for life. Biochemical and biological studies have generated considerable knowledge about these molecular motors. However, these studies assess only sum reactions/effects due to ensemble averaging. Single molecule (SM) measurements overcome this shortcoming. Such measurements allow learning about individual motor functions, such as processivity, fidelity, step size, template dependence and generated forces. Transient intermediate states and rarely occurring events can also be observed.We are developing an assay based on bacteriophage phi29 polymerase to study DNA/RNA polymerases in action using high resolution optical tweezers (OT) combined with fluorescence imaging (OT-SMF). We use an optically levitated ‘dumbbell’ assay: the nucleic acid (NA) construct features biotinylated and digixigeninated ends that tether two different kinds of microspheres (coated with streptavidin and anti-digoxigenin); the protein of interest is attached directly to the NA tether. Since in our double OT instrument one trap is stable whereas the other mobile, we can manipulate the tethers, detect changes in tether length and stiffness, apply different forces and simultaneously observe the fluorescently stained template/protein. We present preliminary results on DNA replication by phi29 polymerase.