Michael Jahnz

A New Embedded Process for Compartmentalized Cell‐Free Protein Expression and On‐line Detection in Microfluidic Devices

During the last century, an enormous number of protein functions were identified, many of which can be generally described as catalytic. Ongoing research currently focuses on two aspects, analysis of the native proteome to reveal new cellular functions, and adaptation of proteins with known functions to technical processes, with the additional aspect of how these functions could be modified or improved. To this end, directed evolution of proteins has in many cases proved to be a successful strategy for designing new biocatalysts for chemical, pharmaceutical or even household use. It relies on the sensitive detection of mutants with new or improved properties, as well as their efficient singling out and amplification. In this work, a combined approach based on ultrasensitive spectroscopy, microfluidic chips and artificial cells is provided, in order to contribute a new analytical tool for both proteome discovery and evolutionary biotechnology. What is the artificial cell concept? Artificial cells reduce the features of a complex microbiological organism down to the two basic properties that are needed for defined protein analysis: compartmentalization and in vitro protein expression. A promising approach for compartmentalization in miniature environments is the formation of a water-in-oil emulsion, in which micrometer-sized water droplets embedded in a hydrophobic layer serve as artificial biocontainers. All essential compounds for transcription and translation, including the protein-encoding gene as well as a substrate to probe for catalysis, can be included within the same compartment, thus providing an essential requirement for in vitro evolution by linking genotype and phenotype. However, with classical methods for the generation of emulsions in batch (e.g. magnetic stir bar), individual droplets cannot be accurately addressed, and automated droplet handling, analysis and sorting is limited. For in vitro protein evolution, tools for automated highthroughput formation, analysis and isolation of individual biocontainers are highly desirable. 10] Therefore downscaling and integration of all functional steps on a single microchip represents an exciting advance. In microfluidic channel networks, the formation of water-in-oil emulsions at high rates and with monodisperse droplets becomes feasible, while small sample quantities can be controllably introduced, while automated droplet handling, sensitive detection of products and potential isolation of individual droplets can be performed. In this work, we demonstrate the efficient implementation of microstructured devices to generate water-in-oil emulsions, and at the same time perform in vitro expression of proteins inside the water droplets (Figure 1). By using the red-shifted

Light-induced flickering of DsRed provides evidence for distinct and interconvertible fluorescent states.

Green fluorescent protein (GFP) from jellyfish Aequorea victoria, the powerful genetically encoded tag presently available in a variety of mutants featuring blue to yellow emission, has found a red-emitting counterpart. The recently cloned red fluorescent protein DsRed, isolated from Discosoma corals (), with its emission maximum at 583 nm, appears to be the long awaited tool for multi-color applications in fluorescence-based biological research. Studying the emission dynamics of DsRed by fluorescence correlation spectroscopy (FCS), it can be verified that this protein exhibits strong light-dependent flickering similar to what is observed in several yellow-shifted mutants of GFP. FCS data recorded at different intensities and excitation wavelengths suggest that DsRed appears under equilibrated conditions in at minimum three interconvertible states, apparently fluorescent with different excitation and emission properties. Light absorption induces transitions and/or cycling between these states on time scales of several tens to several hundreds of microseconds, dependent on excitation intensity. With increasing intensity, the emission maximum of the static fluorescence continuously shifts to the red, implying that at least one state emitting at longer wavelength is preferably populated at higher light levels. In close resemblance to GFP, this light-induced dynamic behavior implies that the chromophore is subject to conformational rearrangements upon population of the excited state.

Triple-Color Coincidence Analysis: One Step Further in Following Higher Order Molecular Complex Formation

Confocal fluorescence spectroscopy is a versatile method for studying dynamics and interactions of biomolecules in their native environment with minimal interference with the observed system. Analyzing coincident fluctuations induced by single molecule movement in spectrally distinct detection channels, dual-color fluorescence cross-correlation, and coincidence analysis have proven most powerful for probing the formation or cleavage of molecular bonds in real time. The similarity of the optical setup with those used for laser scanning microscopy, as well as the non-invasiveness of the methods, make them easily adaptive for intracellular measurements, to observe the association and dissociation of biomolecules in situ. However, in contrast to standard fluorescence microscopy, where multiple fluorophores can be spectrally resolved, single molecule detection has so far been limited to dual-color detection systems due to the harsh requirements on detection sensitivity. In this study, we show that under certain experimental conditions, employing simultaneous two-photon excitation of three distinct dye species, their successful discrimination indeed becomes possible even on a single molecule level. This enables the direct observation of higher order molecular complex formation in the confocal volume. The theoretical concept of triple-color coincidence analysis is outlined in detail, along with an experimental demonstration of its principles utilizing a simple nucleic acid reaction system.

Accessing Molecular Dynamics in Cells by Fluorescence Correlation Spectroscopy.

Abstract Fluorescence correlation spectroscopy (FCS) analyzes spontaneous fluctuations in the fluorescence emission of small molecular ensembles, thus providing information about a multitude of parameters, such as concentrations, molecular mobility and dynamics of fluorescently labeled molecules. Performed within diffractionlimited confocal volume elements, FCS provides an attractive alternative to photobleaching recovery methods for determining intracellular mobility parameters of very low quantities of fluorophores. Due to its high sensitivity sufficient for single molecule detection, the method is subject to certain artifact hazards that must be carefully controlled, such as photobleaching and intramolecular dynamics, which introduce fluorescence flickering. Furthermore, if molecular mobility is to be probed, nonspecific interactions of the labeling dye with cellular structures can introduce systematic errors. In cytosolic measurements, lipophilic dyes, such as certain rhodamines that bind to intracellular membranes, should be avoided. To study free diffusion, genetically encoded fluorescent labels such as green fluorescent protein (GFP) or DsRed are preferable since they are less likely to nonspecifically interact with cellular substructures.

Two-Photon Fluorescence Coincidence Analysis: Rapid Measurements of Enzyme Kinetics

Dual-color fluorescence cross-correlation analysis is a powerful tool for probing interactions of different fluorescently labeled molecules in aqueous solution. The concept is the selective observation of coordinated spontaneous fluctuations in two separate detection channels that unambiguously reflect the existence of physical or chemical linkages among the different fluorescent species. It has previously been shown that the evaluation of cross-correlation amplitudes, i.e., coincidence factors, is sufficient to extract essential information about the kinetics of formation or cleavage of chemical or physical bonds. Confocal fluorescence coincidence analysis (CFCA) (Winkler et al., Proc. Natl. Acad. Sci. U.S.A. 96:1375-1378, 1999) emphasizes short analysis times and simplified data evaluation and is thus particularly useful for screening applications or measurements on live cells where small illumination doses need to be applied. The recent use of two-photon fluorescence excitation has simplified dual- or multicolor measurements by enabling the simultaneous excitation of largely different dye molecules by a single infra-red laser line (Heinze et al., Proc. Natl. Acad. Sci. U.S.A. 97:10377-10382, 2000). It is demonstrated here that a combination of CFCA with two-photon excitation allows for minimization of analysis times for multicomponent systems down to some hundreds of milliseconds, while preserving all known advantages of two-photon excitation. By introducing crucial measurement parameters, experimental limits for the reduction of sampling times are discussed for the special case of distinguishing positive from negative samples in an endonucleolytic cleavage assay.

C-terminal fluorescence labeling of proteins for interaction studies on the single-molecule level.

Homogeneously labeled protein samples are generated by expressed protein ligation for studying protein interactions. Miniscule amounts of samples are used in single-molecule-sensitive measurements and fluorescence resonance energy transfer experiments.

An ultrasensitive site-specific DNA recombination assay based on dual-color fluorescence cross-correlation spectroscopy

Site-specific exchange of genetic information is mediated by DNA recombinases, such as FLP or Cre, and has become a valuable tool in modern molecular biology. The so far low number of suitable recombinating enzymes has driven current research activities towards alteration of catalytic properties, such as thermostability or recognition sequences. However, identification and analysis of new mutants requires sensitive in vitro activity assays, which traditionally are based on gel electrophoresis. Here, we describe the development of a new sensitive DNA recombination assay based on dual-color fluorescence cross-correlation spectroscopy (DC-FCCS), which works in homogenous solution and does not require any separation step such as electrophoresis. The assay was validated with unlabeled FLP recombinase and different fluorescently labeled DNA substrates containing the FLP recognition target (FRT). This strategy fulfills all requirements for possible application in high throughput screening and engineering of new site-specific DNA recombinases starting from the FLP-FRT system, and is easily adjustable to other systems like Cre/loxP.

A novel homogenous assay for topoisomerase II action and inhibition.

Topoisomerase II is the only enzyme able to cleave and religate double‐stranded DNA; this makes it essential for many vital functions during normal cell growth. Increased expression of topoisomerase II is a common occurrence in neoplasia, and different topoisomerase II inhibitors have indeed been proven to be powerful anticancer drugs. For this reason, the topoisomerase II catalytic cycle has attracted strong interest, but only a few techniques contributing to studies in this field have emerged. All of the currently used conventional methods to elucidate the action and inhibition of topoisomerase II require separation steps and are therefore unsatisfactory in terms of sensitivity, speed, and throughput. Here, for the first time, we present an assay that works in homogenous solution. The assay is based on dual‐color fluorescence cross‐correlation spectroscopy (DC‐FCCS) and allows monitoring of topoisomerase II action and, especially, detection and discrimination of different topoisomerase II inhibitor classes. The effectiveness of our new assay was confirmed by measuring the effects of a catalytic inhibitor (novobiocin) and a topoisomerase poison (m‐AMSA) with bacteriophage T4 topoisomerase as a model system, thus showing the strategy to be easy, fast, and extremely sensitive. Further development of the DC‐FCCS‐based assay and subsequent application in high‐throughput drug screening of new anticancer drugs is proposed and discussed.