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Work on zinc fingers and mitochondria


Making zinc finger archives
Aaron Klug Scientist
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We began to make two kinds of libraries, of archives of zinc fingers. At the beginning, we did two times three, putting in various extender linkers and so on. And they bound with picomolar affinity. But what we found was they didn't discriminate so well, if you had a target of... a target of eighteen base pairs, if you change one-base pairs, it didn't discriminate so well because you had to have even higher sensitivity. Now, I had thought at one time that we should put in, just link individual fingers and have spaces between them. That seems a lot of work, but as I said a moment ago, the... we were making two-finger constructs anyway because of the way of getting in the cross-strand interaction. So I suggested that we ought to distribute the strain, and Yen Choo said, 'Why don't we just combine three lots of two fingers to produce six fingers?' And so, we did that and Michael Moore developed the correct linkers for that and that's when I talked about gylcine serine, things of that sort. And it turned out that these had a great advantage. They bound with picomolar affinity but they were much more sensitive to any mutations or omissions in the DNA sequence and so that, if you... if you introduce a single mutation they loose anything between ten to 100 fold... loss of affinity and sometimes 200 fold. So... so we decided in the end that, and Sangamo's libraries are mostly made now, combining sets of two fingers. So we tend to make twos, fours, sixes and even eights. We did make some nines here, that is three times three, but they were just laboratory exercises and Sangamo, not Sangamo, Gendaq made a few of those, but they didn't really give much gain. You see, because once you got 18 base pairs, it's pretty well unique in the human genome, six base, nine base pairs would occur several hundred thousand times, 18 base pairs would occur an only a very small number of times, in the genomes which is random, as you can calculate that. But the... of course genomes aren't random so you've got to have some reservations about that.

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: Gendaq, Yen Choo, Michael Moore

Duration: 2 minutes, 53 seconds

Date story recorded: July 2005

Date story went live: 24 January 2008