Engineering Site-Specific Endonucleases

Creative Biolabs scientists are the experts in engineering site-specific endonucleases in the world. We work closely with our customers to design each project, and produce site-specific endonucleases tailored to your specific requirements.

Site-specific endonucleases, also typically called restriction endonucleases or restriction enzymes, are enzymes that recognize and cleave specific phosphodiester bonds within a polynucleotide chain, usually 4 to 8 basepairs (bp) in length. In the presence of Mg2+, the DNA within or adjacent to the recognition site in both strands are cleaved to produce blunt or sticky ends. These enzymes are extremely specific: 1 bp difference can mean a factor of 106 more slowly than the canonical recognition sequence. They are involved in many aspects of the biochemistry of nucleic acids and have become the key members of DNA editing enzymes. Numerous efforts have been devoted to changing their specificity but with little success, presumably because the recognition process is highly redundant and tightly coupled to the subsequent catalysis.

Due to the many hydrogen bonds and numerous van der Waals contacts involved, it is hardly possible to change the specificity of restriction enzymes by rational protein design (i.e., mutation of one or two amino acids). In addition, in most restriction enzymes, amino acid residues involved in recognition are located nearby residues responsible for catalysis, evolutionary approaches have been used to change the specificity.

Figure 1. The crystal structure of a post-reactive cognate DNA-Eco RI complex. (Based on the PDB crystallographic coordinates of 1QPS.)

Amino acid residues in MutH that are likely candidates for sensing the methylation status of d(GATC) sites have been identified, via multiple sequence alignment of the homologs from E. coli, Haemophilus influenzae, Vibrio cholerae, Shewanella oneidensis, and Colwellia sp. Similar to MutH in sequence, restriction enzymes like Sau3AI from Staphylococcus aureus also recognize d(GATC) sites but with cleaving activity on unmethylated, hemimethylated, and fully methylated DNA in both strands. Sequence comparison revealed the residues important for sequence recognition and methylation sensing. Superimposing the structures of MutH and restriction enzyme-DNA complexes could provide more details on important residues responsible for proper protein functions. Combining methods like rational protein design, structure analysis and candidate site-directed mutagenesis, we have successfully engineered the site-specific endonucleases with altered specificities.

We can work on your restriction enzymes, identify key residues and apply carefully designed mutations. The altered functions will be confirmed using various biochemical and biophysical methods. 3D structures of the mutation enzymes can be determined as well for better functional studies.

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