Computational reprogramming of homing endonuclease specificity at multiple adjacent base pairs

By Justin Ashworth, Gregory K. Taylor, James J. Havranek, S. Quadri, Barry L. Stoddard, David Baker. 2010

111 citations recorded by the source corpus.

Site-specific homing endonucleases are capable of inducing gene conversion via homologous recombination. Reprogramming their cleavage specificities allows the targeting of specific biological sites for gene correction or conversion. We used computational protein design to alter the cleavage specificity of I-MsoI for three contiguous base pair substitutions, resulting in an endonuclease whose activity and specificity for its new site rival that of wild-type I-MsoI for the original site. Concerted design for all simultaneous substitutions was more successful than a modular approach against individual substitutions, highlighting the importance of context-dependent redesign and optimization of protein–DNA interactions. We then used computational design based on the crystal structure of the designed complex, which revealed significant unanticipated shifts in DNA conformation, to create an endonuclease that specifically cleaves a site with four contiguous base pair substitutions. Our results demonstrate that specificity switches for multiple concerted base pair substitutions can be computationally designed, and that iteration between design and structure determination provides a route to large scale reprogramming of specificity.

Subfields: Molecular Biology

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Citations in the corpus

Computationally Designed Bispecific Antibodies Using Negative State Repertoires (2016, background): “…and Kortemme, 2007; Willis et al., 2013), and in designing specificity such as when designing a protein to bind one target but to avoid another (Ashworth et al., 2010; Grigoryan et al., 2009; Zheng et al., 2014) or when organizing multimeric assemblies (Fallas and Hartgerink, 2012; Havranek…”
Massively parallel determination and modeling of endonuclease substrate specificity (2014, methodology): “Base pairs are placed based on the given target sequence using DNA substitution methods described earlier (3,41).”
RosettaRemodel: A Generalized Framework for Flexible Backbone Protein Design (2011, background): “Computational protein design tools to date have been useful for engineering proteins with a wide range of functions, including DNA binding [1,2,3,4], co-factor binding [5], catalysis [6,7,8,9], fluorescence spectral change [10], peptide-protein specificity [11,12], and protein-protein interaction [1”
Improved Modeling of Side-Chain–Base Interactions and Plasticity in Protein–DNA Interface Design (2012, methodology): “Interface positions are identi ﬁ ed using a previously described protocol 17 that builds a set of arginine rotamers at each protein position and checks whether any nucleotide base atom is within 3.8 Å of these arginine side chains.”
A Generic Program for Multistate Protein Design (2011, methodology): “optimized the specificity of the I-Msol1 homoendonuclease by favoring the binding energy for I-Msol1 to the target DNA sequence over alternate DNA sequences [29].”
Reprogramming homing endonuclease specificity through computational design and directed evolution (2013, methodology): “…approaches have been used to design an enzyme cleaving multiple (three) adjacent base-pair substitutions (26), the success rate is still low (12,21,26,27), likely because features of endonuclease binding and catalysis are not yet well understood and therefore cannot be accurately modeled.”
Design of Protein Multi-specificity Using an Independent Sequence Search Reduces the Barrier to Low Energy Sequences (2015, background): “MSD has been successful in engineering proteins when both including [6,7,10,12] and ignoring [9,13,38] these negative design states.”
Achievements and Challenges in Computational Protein Design. (2017, background): “In 2010 the Baker lab altered the cleavage specificity of the I-Msol homing endonuclease for three contiguous base pair substitutions [79].”
RosettaScripts: A Scripting Language Interface to the Rosetta Macromolecular Modeling Suite (2011, methodology): “…including the design of individual changes in the nucleotide specificity of homing endonucleases[29], as well as methods to theoretically maximize specificity[28], introduce small protein backbone shifts[30], and optimize clusters of amino acids for changes in multiple adjacent base pairs[28].”