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Yen Choo's company: Gendaq


Experimenting with zinc finger constructs
Aaron Klug Scientist
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Three fingers bind to nine base pairs, six fingers will bind to 18 base pairs. Now if you simply go and add another set of six fingers... three fingers to the three fingers, just join them on with the same linker you find it gives you hardly any increase in affinity. Now the reason is very simple and I suspected this from the crystal structures and again from experience. The periodicity of the fingers doesn't quite match the periodicity of the DNA bases. So they get out of register, so you have to restore the register. So when we started making... adding three fingers we put in either some extra amino acids or we left gaps in the DNA and started to do protein engineering, or on the target basically we'd leave a gap and we developed different forms of combination of fingers. Now the most important one came later, where instead of having two times three fingers, we had three times two fingers. And the reason we did that was that the three times two fingers... these pairs of fingers had this cross standard interaction which gave you another tenfold boost in the affinity. So... between them we also... I had a favourite topic which was to say, 'Well, I'm going to find the site which is so rare that a single finger could recognise', and that was to take two DNA sites, that's a distance apart, so ten base pairs apart and you have a finger, two fingers which recognise each of these, linked by a fairly semi-stiff link, it has to be semi-stiff otherwise you loose entropy when you do the binding and in fact we showed that you could get enhanced binding this way if you had something with seven or eight base pairs apart, so it's a very nice laboratory exercise but it turned out to be, and we spent a lot of time on it, but that's the way of things, it turned out to be unnecessary although it's pretty but not useful. But that's what happens when you're doing this kind of research. So we found that when you got... if you put in the correct... did the correct protein engineering putting in things like glycines, serine glycine, things of that... purely empirical. What we found was that putting in a glycine, serine glycine in the zinc finger skipped one base. And that's how we worked out rules of this sort. Putting in a single glycine didn't skip a base, but readjusted, made things better. And so, we had a set of sort of know-how, which we began to build, libraries of six finger proteins.

Born in Lithuania, Aaron Klug (1926-2018) was a British chemist and biophysicist. He was awarded the Nobel Prize in Chemistry in 1982 for developments in electron microscopy and his work on complexes of nucleic acids and proteins. He studied crystallography at the University of Cape Town before moving to England, completing his doctorate in 1953 at Trinity College, Cambridge. In 1981, he was awarded the Louisa Gross Horwitz Prize from Columbia University. His long and influential career led to a knighthood in 1988. He was also elected President of the Royal Society, and served there from 1995-2000.

Listeners: Ken Holmes John Finch

Kenneth Holmes was born in London in 1934 and attended schools in Chiswick. He obtained his BA at St Johns College, Cambridge. He obtained his PhD at Birkbeck College, London working on the structure of tobacco mosaic virus with Rosalind Franklin and Aaron Klug. After a post-doc at Childrens' Hospital, Boston, where he started to work on muscle structure, he joined to the newly opened Laboratory of Molecular Biology in Cambridge where he stayed for six years. He worked with Aaron Klug on virus structure and with Hugh Huxley on muscle. He then moved to Heidelberg to open the Department of Biophysics at the Max Planck Institute for Medical Research where he remained as director until his retirement. During this time he completed the structure of tobacco mosaic virus and solved the structures of a number of protein molecules including the structure of the muscle protein actin and the actin filament. Recently he has worked on the molecular mechanism of muscle contraction. He also initiated the use of synchrotron radiation as a source for X-ray diffraction and founded the EMBL outstation at DESY Hamburg. He was elected to the Royal Society in 1981 and is a member of a number of scientific academies.

John Finch is a retired member of staff of the Medical Research Council Laboratory of Molecular Biology in Cambridge, UK. He began research as a PhD student of Rosalind Franklin's at Birkbeck College, London in 1955 studying the structure of small viruses by x-ray diffraction. He came to Cambridge as part of Aaron Klug's team in 1962 and has continued with the structural study of viruses and other nucleoproteins such as chromatin, using both x-rays and electron microscopy.

Tags: zinc fingers, affinity, experimenting

Duration: 3 minutes, 16 seconds

Date story recorded: July 2005

Date story went live: 24 January 2008