Overcoming conservation in TALE-DNA interactions: a minimal repeat scaffold enables selective rerognition of an oxidized 5-methylcytosine
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Date
2018
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Abstract
Transcription-activator-like effectors (TALEs) are repeat-based proteins featuring programmable DNA
binding. The repulsion of TALE repeats by 5-methylcytosine (5mC) and its oxidized forms makes TALEs
potential probes for their programmable analysis. However, this potential has been limited by the inability
to engineer repeats capable of actual, fully selective binding of an (oxidized) 5mC: the extremely
conserved and simple nucleobase recognition mode of TALE repeats and their extensive involvement in
inter-repeat interactions that stabilize the TALE fold represent major engineering hurdles. We evaluated
libraries of alternative, strongly truncated repeat scaffolds and discovered a repeat that selectively
recognizes 5-carboxylcytosine (5caC), enabling construction of the first programmable receptors for an
oxidized 5mC. In computational studies, this unusual scaffold executes a dual function via a critical
arginine that provides inter-repeat stabilization and selectively interacts with the 5caC carboxyl group via
a salt-bridge. These findings argue for an unexpected adaptability of TALE repeats and provide a new
impulse for the design of programmable probes for nucleobases beyond A, G, T and C.