OptiKinase™

Source: E. coli strain containing a modified clone of T4 Polynucleotide Kinase that has full kinase activity but lacks phosphatase activity.

Applications:

1. 5' phosphorylation of single-stranded oligonucleotides, both DNA and RNA
2. 5' phosphorylation of double-stranded DNA
3. Efficient labeling of DNA without a bias toward the base at the 5' end
4. 5' phosphorylation of oligonucleotides with a 3' phosphate group
5. 5' phosphorylation of mononucleotides with a 3' phosphate group that can generate a substance (pNp) that can be ligated to the 3' end of DNA or RNA by T4 RNA Ligase

Description:
OptiKinase™ is a modified version of T4 Polynucleotide Kinase (T4 PNK)^(1,2,3) which provides greater ³²P or ³³P labeling efficiency of DNA. T4 PNK is a homotetrameric, bifunctional enzyme containing an N-terminal kinase domain which catalyzes the transfer of the -phosphate of ATP to the 5' hydroxyl end of polynucleotides and a C-terminal phosphatase domain which removes the 3' phosphate. In molecular biology applications, T4 PNK is traditionally used for radiolabelling the 5'-ends of DNA and RNA. However, when phosphorylating DNA, T4 PNK displays a severe base bias as the labeling efficiency is variable depending on the nucleotide at the 5'-end. Deoxyguanosine at the 5'end is kinased with teh highest efficiency followed by dA, dT, and then dC which is kinased with the least efficiency⁽⁴⁾. Unlike wild-type T4 PNK, OptiKinase exhibits no such base bias or discrimination (see Figs.1 and 2). Also, since OptiKinase lacks 3' phosphatase activity, it is particularly useful for labeling 3' phosphorylated mononucleotides or RNA with a 3' phosphate. Use OptiKinase for higher levels of phosphorylation and for greater consistency compared to standard T4 PNK.

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Fig. 1. 5'-Phosphorylation of single-stranded oligonucleotides. Test oligonucleotides* (5 pmol), identical except at their 5' ends, and an equimolar amount of 33P-γATP, were treated with 10 units of T4 PNK or OptiKinase in 25 μl at 37°C for 30 min. The specific activities of the resulting 5'-phosphorylated oligonucleotides were determined. *Oliognucleotides corresponded to 31-mers (G, A, T, and C) or 18-mers (GGG and CCC).

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Fig. 2. 5'-Phosphorylation of 5'-C oligo with varying amount of radiolabel. Test 5'-C oligonucleotide 18-mer (5 pmol) and varying amount of 33P-γATP were treated with 10 units of T4 PNK or OptiKinase in 25 μl at 37°C for 30 min. The specific activities of the resulting 5'-phosphorylated oligonucleotides were determined.

Properties:
Molecular Weight: Homotetramer of 140 kDa (4 x 35 kDa)
Optimum pH: 7.6 (Tris-HCI)
Optimum Temperature: 37°C

Purity:
Greater than 95% pure as determined by SDS-PAGE. Tested for contaminating exonucleases, endonucleases and ribonucleases.

Storage Buffer:
20mM Tris-HCl (pH 7.5), 50mM NaCl, 1.0mM DTT, 0.1mM EDTA, 50% glycerol.

Assay Conditions:
The reaction mixture (100 µl) contains 50mM Tris-HCI (pH 7.6), 100µM radiolabeled ATP (~1.5 x 10⁵ cpm/nmol), 10mM MgCl₂, 10mM 2-mercaptoethanol, 20µM spermidine and substrate. Incubation is at 37°C.

Unit Definition:
One unit is the amount of enzyme required to incorporate 1 nmol of phosphate from radiolabeled ATP into DNA substrate in 30 min at 37°C.

Concentration:
10 units/µl

Tested User Friendly^TM Functional Test:
 Greater than 60% phosphorylation of oligonucleotide substrate.

Functionally Tested 10X OptiKinase Reaction Buffer (1 ml included, PN 78336):
0.5M Tris-HCI (pH 7.5), 100mM MgCl₂, 50mM DTT.

References:

1. Wang, L.K. and Shuman S. (2001) J. Biol. Chem. 276, 26868-26874. 
2. Wang, L.K. and Shuman S. (2002) Nucl. Acids Res. 30, 1073-1080.
   
3. Richardson, C. C. (1981) The Enzymes, 3^rd edition, ed. P.D. Boyer, (Academic Press, New York) 14, 299-314.
   
4. Van Houten, V., Denkers, F., Van Dijk, M., Van Den Brekel, M. and Brakenhoff, R. (1998) Anal. Biochemistry 265, 386-389.

Storage:
Shipped on dry ice. Store at -20°C.