Microfluidics can be used to trap a single DNA-enzyme complex in its native state for real-time analysis without having to immobilise the DNA or the enzyme, claim US researchers.
Enzymes called restriction enzymes are used to chop up DNA at specific points called recogition sites, making them useful tools in biochemistry. To anayse how they recognise and cleave DNA, the enzyme or DNA needs to be immobilised on a glass slide, but this can modify their properties, and make it difficult to analyse the products. To combat this, Susan Muller and Weilin Xu at the University of California, Berkeley, pre-bound a restriction enzyme to DNA, and fed it through a microfluidic system. This trapped the complex, and then stretched it out. Adding Mg2+ then activated the enzyme, cleaving the DNA, and permitting analysis of the products.
Ron Larson, a chemical engineering expert at the University of Michigan, Ann Arbor, US, says: 'this work represents a novel and elegant use of fluidics to trap and stretch single DNA molecules without interference by surfaces.' He adds that 'the "look Ma, no hands" approach pursued by Xu and Muller has a number of advantages, not least of which is the ability to recover cleavage products for further study.'
Molecular configuration image showing the trapping, stretching and subsequent cleavage of DNA
The technique can give information about how restriction enzymes act to determine their mechanism of action, which holds promise for a broad range of DNA-protein interaction studies, says Muller. This has been difficult in the past, but is being addressed by single-molecule studies, which avoid the problem of properties being averaged over a range of DNA molecules.
The main benefit of the current work, says Muller, is that the complex does not need to be tethered or modified, making it simpler than previous methods that used labelled enzymes. 'We were able to follow the cleavage process in real time at the single-molecule level,' she adds. The accuracy in determining the location of the recognition site on the DNA, she says, is comparable to or better than other single-molecule techniques. Ultimately, she concludes, 'one may be able to extract subtle differences in binding and cleavage frequencies among multiple recognition sites on a single DNA substrate'.
David Barden
RSC
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