Computational redesign of endonuclease DNA binding and cleavage specificity
By Justin Ashworth, James J. Havranek, Carlos M. Duarte, D. Sussman, R. Monnat, Barry L. Stoddard, David Baker. 2006
78 citations recorded by the source corpus.
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 medicine. Here we describe the computational redesign of the cleavage specificity of the intron-encoded homing endonuclease I-MsoI using a physically realistic atomic-level forcefield. 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 sampling. 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.
Subfields: Genetics, Molecular Biology
How researchers see this paper
The researchers whose trusted networks rank it highest, strongest advocate first. Every researcher gets a different order.
In Genetics
- Tanja Kortemme
- Xingjie Pan
- Jens Meiler
- Eva-Maria Strauch
- Paul Murphy
- David Baker
- Barry L. Stoddard
- Jill Bolduc
In Molecular Biology
- David Baker
- Jens Meiler
- Tanja Kortemme
- I. Samish
- Xingjie Pan
- Jeffrey J. Gray
- Andrew Leaver-Fay
- Sagar D. Khare
Citations in the corpus
- Recent advances in de novo protein design: Principles, methods, and applications (2021, methodology): “Scoring functions for DNA (127) and RNA (128) have been successfully applied to structure prediction and design (129, 130).”
- Alteration of enzyme specificity by computational loop remodeling and design (2009, methodology): “C protein design methodology has been used to optimize properties such as protein stability (1, 2) and to introduce functions such as binding of small molecules (3), proteins (4), and nucleic acids (5), as well as enzymatic activity (6, 7).”
- RosettaScripts: A Scripting Language Interface to the Rosetta Macromolecular Modeling Suite (2011, methodology): “It allows the redesign of protein structure[1] and has been used to generate new protein folds[2], stabilize enzymes[3], generate novel enzymes[4,5], protein-protein interactions and inhibitors[6], and redesign specificities in protein-protein[7] and protein-DNA interactions[8].”