New Fluorescence Technique For Ultra-Sensitive Enzyme Characterization Developed - Screening Catalytic Activity At The Single-Molecule Level

March 12, 1998

New fluorescence technique for ultra-sensitive enzyme characterization developed - Screening catalytic activity at the single-molecule levelEnzymes are characterized by and valued for their ability to catalyze a wide range of chemical reactions. Consequently, precise analytical techniques for monitoring enzyme functions are indispensable tools for the understanding of biological phenomena at the molecular level, and for the development of new pharmaceuticals. A research team at the Max Planck Institute for Biophysical Chemistry in Göttingen, Germany, headed by the Nobel laureate Manfred Eigen, published two articles in PNAS (Proceedings of theNational Academy of Sciences) introducing dual-color fluorescence cross-correlation spectroscopy as a new method for an ultra-sensitive characterization of enzyme activity (PNAS 95:1416-1420, abstract: and demonstrating its potential for high throughput screening (PNAS 95:1421-1426, abstract:

In recent years, fluorescence correlation spectroscopy (FCS) has become an attractive analytical tool for the investigation of biomolecular processes. Due to the interplay of modern confocal optics, new dyes as efficient fluorescent probes, sensitive photodetectors, and fast data processing, FCS allows the observation of the dynamics of single molecules in real time while they pass an open volume element of about one femtoliter, i.e. only the size of a common bacterial cell. This method was invented and worked out by groups at the Cornell University, Ithaca, at the Karolinska Institute, Stockholm, and at the Max Planck Institute, Göttingen; nowadays it has found its way into several laboratories and companies all over the world as a tool for basic research as well as for industrial applications such as drug screening.

Dual-color fluorescence cross-correlation spectroscopy (dual-color FCS) was proposed by Rudolf Rigler, Stockholm and Manfred Eigen, Göttingen, in the early nineties, and has recently been implemented by Eigen’s group in Göttingen. In contrast to conventional single-color FCS, the new method uses two perfectly superimposed laser foci, and detects correlated fluctuations that arise from single molecules carrying two spectrally distinguishable fluorescent labels. It allows precise and highly specific detection of molecules on a large unspecific fluorescent background; moreover, the method is fully compatible with biological environments. Two important applications were addressed by the authors: Real-time measurements of enzyme kinetics were successfully performed, and the suitability of dual-color FCS for high throughput screening has been demonstrated.

Kinetics of biomolecular interactions like nucleic acid hybridization or protein aggregation were previously investigated by Eigen and coworkers applying dual-color FCS. Now, it has been extended to real-time analyses of enzyme kinetics. As a first application, the kinetic parameters of the cleavage reaction of a double-helix DNA catalyzed by a specific endonucleolytic enzyme were determined. Furthermore, catalytic activity down to enzyme concentrations of one picomole per liter was detected with high reproducibility. This is at least one order of magnitude more sensitive as compared to other homogeneous endonucleolytic assays. As the authors pointed out, this technique can, in principle, be used for any reaction in which a (covalent or non-covalent) linkage between two different fluorophores is either broken or formed. This includes other hydrolytic enzymes like proteases, esterases or glycolyases, as well as ligating enzymes.

High-throughput screening with dual-color FCS was demonstrated in the second article; this combination is termed RAPID FCS (Rapid Assay Processing by Integration of Dual-color Fluorescence Cross-correlation Spectroscopy). While conventional FCS identifies molecules by their diffusion properties, requiring a considerable amount of analysis time, dual-color FCS simply counts doubly labeled molecules; therefore, analysis times for simple yes-or-no decisions are much shorter and data evaluation is faster. As reported by the authors, analysis times of one second per sample and less were achieved with endonucleolytic assays; sample volumes could even be reduced to submicroliters without decreasing the signal strength. RAPID FCS can probe 104 to 105 samples per day, and possibly more. Therefore, this technology is an ideal tool for ultra-high throughput screening when combined with nano-technology; and it will gain access to progressive selection strategies in evolutionary biotechnology, in which rare and specific binding or catalytic properties have to be screened in large numbers of samples.


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