B-DNA is a right-handed double-stranded DNA (dsDNA) helix. It is believed that most DNA adopts B-DNA conformation in biological systems. However, DNA can adopt alternative conformations of dsDNA. In Z-DNA, a double helix assumes left-handed conformation that contains a zigzag backbone. 1 Z-DNA formation can be stabilized under certain circumstances such as high salts and negative supercoiling. 2 The formation of thermodynamically disfavored Z-DNA is an interesting subject in the field of nucleic acid research. Much research has been carried out on how B conformation of DNA is structurally transformed into Z conformation (B-Z transition). To date, mainly two mechanisms have been suggested to describe how B-Z transition occurs. The Wang model proposed that the B-Z transition undergoes base pair opening prior to base pair rotation. 1 In contrast, in the Harvey model, the B-Z transition contains base pair rotation without breaking of base pairs. 3 Surely, the identification of Z-DNA binding proteins can provide clues to biological relevance of Z-DNA. The wellcharacterized Z-DNA binding domain named Zα is initially found in human ADAR1 (double-stranded RNA adenosine deaminase 1) and known to be existed in several proteins. 4 Since Zα binds to Z-DNA strongly, it can promote relatively fast B-Z transition through stabilizing Z-DNA and inhibiting re-formation of B-DNA in the B-Z equilibrium. hZα ADAR1 is the first Zα domain present in two tandem Zα domains of human ADAR1, which has been widely used for structural and biochemical studies of Z-DNA/Z-RNA. For example, hZα ADAR1 as a Z-DNA inducer enables to stabilize A-Z, B-Z, and Z-Z junction, 5-8 and Z-RNA by help of hZα ADAR1 successfully forms in physiological salt concentrations so as to solve its structure. 9,10 In addition, hZα ADAR1 has been used to elucidate kinetic behavior of Z-conformer formation of various nucleic acid duplexes by single-molecule studies. 11-13 Here, we reported the effect of the unpaired single-stranded region present in DNA duplexes such as overhangs and loops on the B-Z transition induced by hZα ADAR1 .For this study, we used d(CG) 6 sequence as a core Zforming DNA sequence (ZFD) that forms self-complementary dsDNA with overhangs, which may contain either 5 0 -or 3 0 -overhangs composed of 1-6 nucleotides (Supporting Information Figure S1 and Table S1). First, dT-overhang ZFDs with different overhang-lengths placed at the 5 0 -ends of ZFD (referred to as T1-, T2-, T3-, T4-, and T6-ZFD, respectively) were designed and tested (Supporting Information Table S1). The B-Z transition in the presence of hZα ADAR1 was monitored under the physiological pH condition (pH 7.4) with a relatively low salt concentration (10 mM NaCl). All of 5 0 -dToverhang ZFD substrates readily form Z conformation when hZα ADAR1 is added (Supporting Information Figure S2).For the kinetic analysis, CD measurements performed at 5 C to observe fast hZα ADAR1 -induced B-Z transition. The CD signals at 255 nm were collected immediately after adding hZα ADAR1 . As shown in Figur...