We first synthesized FITC-conjugated LANA-DBM and examined the binding affinity to nucleosomes (Figure ). As a negative control, a mutant LANA without the acidic patchbinding ability, was conjugated with DBM and the Gly linker (16). The electrophilic mobility shift assay (EMSA) showed that LANA-DBM 15 bound to recombinant nucleosome much stronger than LANA (14) or mutant LANA-DBM 16 (Figure ); the fluorescence signal of FITC was observed at the nucleosome band only for 15, but not for 14 and 16. This result suggests that LANA-DBM bivalently bound to nucleosome. Next, we synthesized LANA-DBM-BAHA catalyst 20, in which BAHA was conjugated to the N-terminus of LANA-DBM (Figure ). We examined the nucleosome binding affinity of the catalyst 20 with catalyst 18 or 19, which lacks DBM motif, by a competition assay using FITC-conjugated LANA 17. Nucleosome-bound FITC signals in the presence of 20 was significantly lower than those in the presence of 18 or 19 (Figure ), confirming that the LANA-DBM catalyst more tightly bound to nucleosome than the LANA catalyst. We then addressed histone acylation activity of the catalysts. Recombinant nucleosomes were incubated with the catalysts and acetyl donor 13 for 2 hours and H2BK120 acetylation was detected by immunoblotting. LANA-DBM-BAHA catalyst 20, but not LANA-BAHA catalysts 18 and 19, efficiently acetylated H2BK120 at 0.1 µM (Figure ), indicating that 20 exhibits higher histone acylation activity. Together, these results demonstrated that the bivalent nucleosome ligand LANA-DBM furnished an enhanced binding affinity to nucleosome and increased histone acylation activity when conjugated with BAHA.