Endonuclease substrate selectivity characterized with full-length PA of influenza A virus polymerase

► Full-length PA has endonuclease activity against both RNA and DNA. ► The divalent metal cofactor affects both level of activity and cleavage pattern. ► The minimal substrate size for the PA endonuclease is five bases. ► A FRET-based endonuclease assay is well suited for screening novel antivirals.