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

Abstract

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.