Rapid assay processing by integration of dual-color fluorescence cross-correlation spectroscopy: High throughput screening for enzyme activity

Abstract

Dual-color f luorescence cross-correlation spectroscopy (dual-color FCS) has previously been shown to be a suitable tool not only for binding but also for catalytic rate studies. In this work, its application as a rapid method for high-throughput screening (HTS) and evolutionary biotechnology is described. This application is called RAPID FCS (rapid assay processing by integration of dual-color FCS) and does not depend on the characterization of diffusion parameters that is the prerequisite for conventional f luorescence correlation spectroscopy. Dual-color FCS parameters were optimized to achieve the shortest analysis times. A simulated HTS with homogeneous assays for different restriction endonucleases (EcoRI, BamHI, SspI, and HindIII) achieved precise yes-or-no decisions within analysis times of about 1 s per sample. RAPID FCS combines these short analysis times with the development of fast and f lexible assays resulting in sensitive, homogeneous f luorescence-based assays, where a chemical linkage between different f luorophores is either cleaved or formed, or where differently labeled molecules interact by noncovalent binding. In principle, assay volumes can be reduced to submicroliters without decreasing the signal strength, making RAPID FCS an ideal tool for ultra-HTS when combined with nanotechnology. RAPID FCS can accurately probe 10 4 to 10 5 samples per day, and possibly more. In addition, this method has the potential to be an efficient tool for selection strategies in evolutionary biotechnology, where rare and specific binding or catalytic properties have to be screened in large numbers of samples.

Simultaneous two-photon excitation of distinct labels for dual-color fluorescence crosscorrelation analysis

“…Because no pinholes are required, a combination with two-photon imaging, by using the same experimental setup and scanning the excitation beam, seems particularly attractive. Further, the simplicity of the described measurement concept as well as the quality of the obtained data suggest a use of this technique in high-throughput screening where short analysis times per sample are required, such as crosscorrelation setups like RAPID FCS (15) or coincidence analysis such as Confocal Fluorescence Coincidence Analysis (16). Particularly interesting is the idea of extending this concept to multicolor two-photon excitation for simultaneous detection and analysis of more than two fluorescent species.…”

“…The results demonstrate that the performance, which can be obtained in terms of detection sensitivity and specificity, is indeed comparable or even superior to that achievable in one-photon setups with two excitation lines. This result is particularly promising for intracellular applications (23), analytic purposes (14), fast analytic purposes (15), or even high-throughput screening approaches (16) of dual-color crosscorrelation, where several known additional advantages of two-photon excitation, such as reduced background and probe depletion, come into play (17,23).…”

“…Dual-color crosscorrelation analysis (12,13), a conceptual modification of FCS using two spectrally separable fluorescent labels, yields a considerable improvement in signal specificity for heterogeneous systems where molecular interactions of different species are to be observed. The simplicity of the measurement process has recently even encouraged biotechnological applications leading to online kinetics of enzymatic reactions (14) and fast enzyme screening applications (15,16). In principle, any molecular association and dissociation, as well as the formation or cleavage of chemical bonds, can be studied simply by simultaneous detection of two spectrally separated fluorophores, following the amplitude of the crosscorrelation function in real time during the reaction.…”

Global analysis of fluorescence fluctuation data

“…In the last few years an increasing number of applications of dual-colour FCCS have been reported. These vary from enzyme kinetics Koltermann et al 1998, Rarbach et al 2001, nucleotide hybridisation (Schwille et al 1997;Rigler et al 1999b) and protein-DNA interactions (Rippe 2000) to the application of two-photon excitation (Heinze et al 2000). Dual-colour FCCS applications in live cells have been reported recently (Bacia et al 2002;Hink et al 2003).…”

Chemical complementation: A reaction-independent genetic assay for enzyme catalysis

“…Recently, attention has turned to direct selections for catalytic activity. While strategies ranging from in vitro fluorescence assays to physically linking the enzyme to its substrate have all recently been reported (10)(11)(12)(13)(14)(15), a general solution to this problem is yet to emerge.…”