Systematic Probing of the Sequence Selectivity of Exonuclease III with a Photosensitization Colorimetric Assay

[Image: see text] Exonuclease III (Exo III) is an important enzymatic tool that is being widely used in molecular biology, biotechnology, and bioassay development. Exo III prefers to cleave double-stranded DNA (dsDNA) with blunt and recessed 3′-termini rather than their protruding counterpart. While it has been accepted that a short 3′-overhang (e.g., >4 nt) is necessary to protect a dsDNA from Exo III cleavage, critical roles of the length and sequence of this 3′-overhang remain unexplored. Herein, we develop a novel light-induced colorimetric assay allowing the systematic probe of the sequence selectivity of Exo III in a rapid and high-throughput manner. Our finding that Exo III is highly specific to 3′-overhang in terms of both length and sequence will be valuable for guiding the design of bioassays and DNA manipulating tools mediated by Exo III.