The goal of this proposal is to engineer universal restriction endonucleases with tailored cleavage specificities. The universal endonucleases will contain two functional components: 1) a specificity determinant and 2) a cleavage domain. The DNA binding specificity will be contributed by a modified singlestranded oligonucleotide (ss oligo) or a peptide nucleic acid oligomer (PNA). The cleavage domain will be derived from the cleavage domain of a type IIS restriction endonuclease (REase) or a non-specific nuclease. The first approach involves fusion of streptavidin and a nuclease cleavage domain. The chimeric nuclease, streptavidin-nuclease fusion protein, is linked to a biotinylated ss oligo through non-covalent binding. The cleavage activity of the chimeric nuclease will be examined on appropriate DNA substrates-. The second approach will explore the covalent attachment of an ss oligo or a PNA to the nuclease domain using inteinmediated protein ligation (IPL). The IPL reaction requires a cysteine which will be incorporated into the oligo or PNA during chemical synthesis. For in vivo applications of the universal nuclease, the oligonucleotide-Cys will be modified to contain locked nucleotides (LNA). Such LNA-oligos are resistant to endogeneous endonuclease and exonuclease attack. Accessory proteins such as RecA, helicase, and SSB will be evaluated to facilitate target DNA binding and cleavage. For isothermal applications of the universal nuclease at 50oC-70oC, the nuclease domain will be derived from thermophilic endonucleases.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Small Business Innovation Research Grants (SBIR) - Phase I (R43)
Project #
1R43GM073345-01
Application #
6875448
Study Section
Special Emphasis Panel (ZRG1-SSS-Y (10))
Program Officer
Portnoy, Matthew
Project Start
2005-03-01
Project End
2006-02-28
Budget Start
2005-03-01
Budget End
2006-02-28
Support Year
1
Fiscal Year
2005
Total Cost
$100,000
Indirect Cost
Name
New England Biolabs, Inc.
Department
Type
DUNS #
066605403
City
Ipswich
State
MA
Country
United States
Zip Code
01938
Bao, Yongming; Higgins, Lauren; Zhang, Penghua et al. (2008) Expression and purification of BmrI restriction endonuclease and its N-terminal cleavage domain variants. Protein Expr Purif 58:42-52
Zhang, Penghua; Bao, Yongming; Higgins, Lauren et al. (2007) Rational design of a chimeric endonuclease targeted to NotI recognition site. Protein Eng Des Sel 20:497-504
Chan, Siu-hong; Bao, Yongming; Ciszak, Ewa et al. (2007) Catalytic domain of restriction endonuclease BmrI as a cleavage module for engineering endonucleases with novel substrate specificities. Nucleic Acids Res 35:6238-48