A low cost, low concentration single molecule method for real time measurement of enzyme kinetics has been developed by Korean scientists. This could reduce the cost of research and development for enzymatic gene therapy and aid drug discovery. 

Single molecule methods are used extensively to study the dynamics and structures of various biological molecules. They usually require small sample sizes and yield fast and low-cost activity studies for biomolecules, as part of basic kinetic tests or as part of applications such as analytical assay units, therapeutics and molecular logic gates. Gel electrophoresis is often used for the characterisation of biomolecules with substrates labelled with various markers, such as fluorogenic, chromogenic, or radioactive tags, but it is often hampered by limited throughput and difficult handling. 

Labelling is also used in fluorescence resonance energy transfer (FRET) and this method acts as a spectroscopic 'ruler' for measuring the distance between dye-labelled sites (usually a donor and an acceptor) in a molecule. The donor molecule is detected upon donor excitation when the molecules are dissociated and when the acceptor comes closer to the donor (<7 nm) their interaction allows detection. This method can be used to study conformational changes within proteins as well as protein-protein and protein-DNA interactions and allows for real time kinetic data to be gathered with high throughput. However, FRET suffers from poor labelling, potential fluorophore bleaching and the high cost of dye labelling. 

To overcome these drawbacks, Seong Keun Kim and colleagues at Seoul National University, have used alternating laser excitation FRET (ALEX-FRET), which measures the ratio of probe dyes (effectively distinguishing cleaved substrates from uncleaved substrates) together with their FRET efficiency. Kim's team labelled two ends of a deoxyribozyme substrate with two different fluorophore dyes, and then initiated a cleavage reaction by adding Mg2+ to the enzyme-substrate mixture. Using ALEX-FRET, they were able to detect the fraction of uncleaved substrate as a function of time and calculate the cleavage rate. The detection platform that detects the single molecules is continuously replenished by free diffusion causing little fluorophore bleaching.

ALEX-FRET allows the ratio of cleaved and uncleaved substrates to be measured

'We have demonstrated that the activity of an enzyme can be measured in real time at the single-molecule level, thereby dramatically reducing (by a factor of 103) the amount of sample required for such a measurement,' explains Kim. 'The ALEX-FRET method is far superior to conventional FRET methods, reducing the consumption of expensive FRET samples and removing the constraints of dye labelling between FRET pairs,' he adds.

Yi Lu, an expert in chemical biology at University of Illinois at Urbana-Champaign, US, comments 'This work is a beautiful demonstration of single molecule FRET in advancing our understanding of this new class of metalloenzymes. Prior FRET and single molecule FRET studies have shed light on the metal ion dependent folding and activity in this DNAzyme. This method should be applicable to other DNAzyme systems as well.'

The group are now planning to extend this method to real-time measurements of biological enzymes in vivo and would like to use DNA-chips to measure the activities of hundreds of enzymes simultaneously.

Carl Saxton 

Enjoy this story? Spread the word using the 'tools' menu on the left or add a comment to the Chemistry World blog.