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