FRep: A Fluorescent Protein-Based Bioprobe for in Vivo Detection of Protein–DNA Interactions

Abstract

We describe a bacterial reporter system, FRep, for rapid and facile detection of protein-DNA recognition. The bioprobe reporter comprises genes of two fluorescent proteins (FPs) separated by a potential DNA target. If a coexpressed transcription factor binds the DNA target, transcription of the second FP is impeded, resulting in loss of FRET partner. Using ratiometric FRET, we show that evaluation of protein-DNA recognition can be reliably made on bZIP and bHLHZ transcription factors and their DNA targets. FRep displays similar thresholds of detection regarding protein-DNA binding affinities, as compared to well-established electrophoretic and yeast assays, although we observed variations in the intensity of fluorescence signals and detection thresholds that may depend on differences between DNA-binding protein production levels and/or stability in the cell, or the expressed bioprobe linker between the two FPs. FRep can potentially be applied to high-throughput searches of both protein and DNA libraries; in a mock library screen, binding and nonbinding complexes can even be distinguished by visual inspection of colonies on plates. FRep presents notable advantages over existing technologies when applied to assessing protein-DNA interactions in vivo, and this approach has the potential for applications in assaying protein-protein interactions and screening molecules that influence specific macromolecular interactions.

Single Cell Optical Imaging and Spectroscopy

“…In general, applications using “traditional” FRET pairs for single cell analyses are excluded from this discussion. Some interesting applications of FRET for cellular analyses include measurements of the interaction of cell membrane or intracellular components; − ion, metabolite, or small molecule quantification; − nucleic acids, nucleic acid binding proteins, and the mechanisms of their action and degradation; − cell forces; pathogen discrimination and host/pathogen interactions; , drug discovery; and protein localization, activity, synthesis, and folding. − …”

Chemical Analysis of Single Cells

“…Motivated by the inherent difficulties of investigations of protein–DNA interactions in vitro, Sharavan et al have presented an in vivo FRET-based bioprobe called FREP . In vivo FRET assays, however, suffer from a relativity high instance of false positives and false negatives, in part because the acceptor–donor distance is critical.…”

“…Motivated by the inherent difficulties of investigations of protein−DNA interactions in vitro, Sharavan et al have presented an in vivo FRET-based bioprobe called FREP. 18 In vivo FRET assays, however, suffer from a relativity high instance of false positives and false negatives, in part because the acceptor−donor distance is critical. The unique solution based on works by Fields and Song 19 and Waldo et al, 20 described in this article, utilizes an E. coli cell containing expression vectors for a transcription factor and the reporter, which can be designed for optimal fluoresce transfer.…”

Choosing a suitable method for the identification of replication origins in microbial genomes

“…Owing to the pivotal role played by DNA-associating proteins in various cellular processes, many in vitro, in vivo, in silico , and biophysical techniques have been developed to study DNA–protein interactions. In vitro technique includes southwestern assay, yeast one-hybrid assay (Y1H), phage display and proximity ligation assay (PLA); scanning probe microscope (SPM) is a novel in vivo method on the interaction of protein–DNA; biophysical technique includes many methods, such as fluorescence-based techniques [time-resolved fluorescence depolarization, double labeled native gel electrophoresis and fluorescence-based imaging, fluorescence resonance energy transfer (FRET) techniques ( Clegg, 1995 )], capillary electrophoresis with laser-induced fluorescence (CE-LIF) ( Riddick and Brumley, 2008 ), also some fluorescence-based protein or nucleic acids bioprobe like FRep ( Shahravan et al, 2011 ), quantum dots (QDs) ( Michalet et al, 2005 ), SPR, nuclear magnetic resonance, circular dichroism (CD), atomic force microscopy (AFM), and microcalorimetry ( Dey et al, 2012 ).…”