Combined optical trapping and nanometer-precision localization for the single-molecule study of DNA-binding proteins

Abstract

The development of an increasing variety of single-molecule techniques has provided remarkable insights on several biological processes. Recently, tremendous improvements have been achieved in the precision of localization of single fluorescent molecules, allowing localization and tracking of biomolecules at the nm level. In the present work, we describe a single-molecule assay, based on the combination of two different single-molecule techniques in the same experimental setup: nanometer-precision Fluorescence Imaging and optical Trapping (FIAT). A microfluidic chamber allows fast exchange of the sample buffer between two different buffer compositions. The main advantage of the FIAT assay is the possibility of detecting the position of a single fluorescently labeled biomolecule and characterize its dynamics of interaction with the substrate, while precisely controlling the mechanical properties of the substrate itself. These features make FIAT well suitable for the study of several biological systems, including DNA-binding proteins and molecular motors. Here, we present preliminary results obtained with two proteins: RNA polymerase (RNAp) and the lactose repressor (LacI): two crucial proteins involved in prokaryotic gene expression and its regulation. RNAp, in a stalled ternary complex, was labeled with a quantum dot and localized on the T7 promoter. The DNA molecule containing the promoter was suspended between two optical traps and the position of RNAp was measured with a precision of ∼ 4 nm. For the study of LacI, the protein is labeled with a quantum dot through a genetically-encoded biotin tag at the C-terminal (after the tetramerization domain) and a DNA construct containing two primary operators (O1) is suspended between the two traps. The positions at which binding of LacI takes place are measured. These methods will be extended to the study of dynamics of RNAp and LacI in different mechanical conditions.