A Novel Approach for Single Molecule Observation of DNA Loops Formed by ToxR-RNA Polymerase Complex, LacI, and DmpR

Abstract

DNA looping gets involved in DNA transcription, replication and recombination. These DNA loops, that are formed by interaction between proteins, protein complexes, and DNA molecules, prominently regulate gene expression. DNA looping has been extensively studied; however, there are a limited number of tools to observe DNA looping phenomena. Current approaches for observation of DNA looping are mutation of protein binding sites succeeded by transcriptional regulation analysis, DNase footprinting, EMSA (electrophoretic mobility shift analysis), electron microscopy, TPM (tethered particle motion), and AFM (atomic force microscopy). We developed a novel approach to directly visualize the DNA looping phenomena. We utilized a fluorescent microscope for visualization of DNA loops, formed by ToxR-RNA polymerase complex, LacI, and DmpR; furthermore, we quantitatively analyzed the DNA looping efficiency by counting DNA molecules at single molecule level. We used a flowcell system to synthesize substrate DNA molecules containing ToxR-RNA polymerase complex binding sites, LacO sites, and DmpR binding sites. As a result, we observed bright dots at the DNA looping position on the DNA backbones. The dots result from comparatively higher density of DNA at DNA looping sites compared with DNA backbones. This approach has advantages over other approaches in simplicity and sensitivity; therefore, the approach will be used for detection of DNA looping. Moreover, this approach will also be applied in studies of new therapeutics for genetic disease caused by gene expression errors and incorrect DNA looping. Key Word: DNA looping, fluorescent microscopy, flowcell, ToxR, RNA polymerase, LacI, DmpR