Computational redesign of endonuclease DNA binding and cleavage specificity

Publisher Website
Google Scholar
Abstract
Altering the specificity of DNA-cleaving enzymes could be useful in many medical or biotechnological applications, but it is quite a challenge in terms of computational protein design. Ashwell et al. have used computational redesign to alter the target-site specificity of the I-MsoI homing endonuclease, while maintaining wild-type binding affinity. The redesigned enzyme binds and cleaves the new DNA recognition site about 10,000 times more effectively than the wild-type enzyme, with target discrimination comparable to the original endonuclease. These results suggest that computational protein design methods can be used to create novel and highly specific endonucleases for gene therapy and other applications. Redesign of the I-MsoI endonuclease binds and cleaves the new recognition site ∼10,000-fold more effectively than does the wild-type enzyme, with a level of target discrimination comparable to the original endonuclease. The reprogramming of DNA-binding specificity is an important challenge for computational protein design that tests current understanding of protein–DNA recognition, and has considerable practical relevance for biotechnology and medicine1,2,3,4,5,6. Here we describe the computational redesign of the cleavage specificity of the intron-encoded homing endonuclease I-MsoI7 using a physically realistic atomic-level forcefield8,9. Using an in silico screen, we identified single base-pair substitutions predicted to disrupt binding by the wild-type enzyme, and then optimized the identities and conformations of clusters of amino acids around each of these unfavourable substitutions using Monte Carlo sampling10. A redesigned enzyme that was predicted to display altered target site specificity, while maintaining wild-type binding affinity, was experimentally characterized. The redesigned enzyme binds and cleaves the redesigned recognition site ∼10,000 times more effectively than does the wild-type enzyme, with a level of target discrimination comparable to the original endonuclease. Determination of the structure of the redesigned nuclease-recognition site complex by X-ray crystallography confirms the accuracy of the computationally predicted interface. These results suggest that computational protein design methods can have an important role in the creation of novel highly specific endonucleases for gene therapy and other applications.