Researchers at the University of Sheffield have developed a new way of monitoring biological processes through a collaboration with scientists at Imperial College London.
Professor Jim Thomas develops ruthenium-based compounds with luminescent properties. These Ru(II) complexes are used to “light up” molecules so they can be studied using advanced microscopy techniques.
Normally, however, small luminescence molecules of this kind can’t be used to stain DNA for single molecule experiments as they do not bind strongly enough, so more complicated techniques are used instead.
But Professor David Rueda from Imperial’s Faculty of Medicine, Department of Infectious Disease has successfully performed a single molecule experiment using one of the dinuclear Ru(II) complexes developed by the Thomas Group at the University of Sheffield.
The Imperial Single-Molecule Imaging Group used a C-Trap, which combines the techniques of optical tweezers and confocal microscopy, to thread the Sheffield complex onto DNA.
Jim said: “Because it has bulky [Ru(bpy)2]2+ at each end of a flat polyaromatic ligand, the complex resembles a ‘molecular dumbbell’. We had previously shown that it could thread through short, and hence unstable, sequences of double stranded DNA.
“Using an optical tweezer to stretch bits of long double stranded DNA, we found we could thread the complex onto normally stable DNA. Once the stretching force is removed, the threaded complex is ‘stapled’ into DNA.
“This process is stereospecific: the complex can be isolated as three different stereoisomers (lambda, lambda; delta, delta; or lambda, delta) and the binding affinity we measure for the lambda, lambda interaction is the highest for any single site DNA intercalator. In fact it is essentially irreversibly bound to DNA once it is threaded through and the duplex stretching force is removed
“Normally you can't use small luminescence molecules to stain DNA for single molecule experiments as they wash out too easily. However, our complex is only luminescent when it binds to DNA and this new procedure described it can be ‘loaded and locked’ onto DNA.
“So, we have a convenient way of staining and visualizing duplex DNA for single molecule experiments. For example, we could potentially use it to directly watch DNA as it is processed in complex biological processes like transcription.”
The study has been published in Angewandte Chemie, International Edition, one of the world’s leading chemistry journals.
