zinc finger technology
Post on 25-May-2015
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Zinc Finger Technology
(Review)
Made By:Munish Chhabra
Roll no-2K10/BT/008IVth Semester
Zinc Finger – Protein motif that forms a compact globular
structure that coordinates one or more Zn ions.
– Consensus Sequence-
– 2 Cys held in beta sheet and 2 His in alpha helix tetrahedrally coordinated to Zn ion.
“zinc finger” describe
finger-like appearance
of the diagram of Xenopus laevis
transcription factor IIIA discovered
In 1985.
• Results-• 5 Zn fingers bound to
21 bp of DNA.• Finger 2-5 fit in major
groove. • The alpha helix of the
finger with N terminal closest to base.
• Conserved regions recognized by finger 4 and 5.
• Finger 1 and 2 held by protein-protein interaction via His linkers.
PCR and fusion????
• CP-QDR and Sp1-QNR Zn fingerProteins were taken.• Sp1-QNR cleavedLambda DNA(48.5 kb)to 9.5kb and 39kb.• CP-QDR cleaved into 5.5 kb and 43 kb.• Kinetics of cleavage1) Optimum T=22 o C2)Efficiency decrease with
increase in KCl concentration.
3)Efficiency increases with in crease in MgCl2
Concentration but leading to nonspecific cleavage.
• 300bp-Lambda DNA of major cleavage site isolated.
• Substrate labeled with 32P at top and bottom .
• Both cleave near binding sit at top and bottom at two distinct locations.
And Then….• Sangamo Biosciences was found in
1995 with a vision to engineer Zn finger proteins to fit desired base sequence like Lego blocks.
• The sequences were overlapped.
• Built library of two finger proteins.
• Individual fingers of different target sites mixed and matched in order to create a protein with a novel target sequence --“Modular Assembly” (MA).
• ZFN creating double stranded breaks (DSB) leads to Homology directed Repair (HDR).
• ZFN invoked DSB increases the rate of homologous recombination (HR).
• Maximal HR frequency when DSB evoked closer to mutated site.
And Now….
• Keith Joung gave a new technique-Oligomerized
Pool Engineering (OPEN).
• Trancriptor activator like effectors (TALENs) on
their way!!!!!!!!!
T he End
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