Design of Modular Protein Tags for Orthogonal Covalent Bond Formation at Specific DNA Sequences
journal contributionposted on 18.05.2017, 00:00 by Thang Minh Nguyen, Eiji Nakata, Masayuki Saimura, Huyen Dinh, Takashi Morii
Simultaneous formation of specific covalent linkages at nucleotides in given DNA sequences demand distinct orthogonal reactivity of DNA modification agents. Such highly specific reactions require well-balanced reactivity and affinity of the DNA modification agents. Conjugation of a sequence-specific DNA binding zinc finger protein and a self-ligating protein tag provides a modular adaptor that expedites formation of a covalent bond between the protein tag and a substrate-modified nucleotide at a specific DNA sequence. The modular adaptor stably locates a protein of interest fused to it at the target position on DNA scaffold in its functional form. Modular adaptors with orthogonal selectivity and fast reaction kinetics to specific DNA sequences enable site-specific location of different protein molecules simultaneously. Three different modular adaptors consisting of zinc finger proteins with distinct DNA sequence specificities and self-ligating protein tags with different substrate specificities achieved orthogonal covalent bond formation at respective sequences on the same DNA scaffold with an overall coassembly yield over 90%. Application of this unique set of orthogonal modular adaptors enabled construction of a cascade reaction of three enzymes from xylose metabolic pathway on DNA scaffold.
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self-ligating protein tagsOrthogonal Covalent Bond Formationsequence-specific DNA binding zinc finger proteinDNA sequence specificitiesDNA scaffoldorthogonal covalent bond formationModular Protein TagsadaptorDNA modification agentsDNA sequences demandSpecific DNA Sequencesself-ligating protein tagzinc finger proteins