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Artificial nucleic acids are widely used in various technologies, such as nucleic acid therapeutics and DNA nanotechnologies requiring excellent duplex‐forming abilities and enhanced nuclease resistance. 2′‐ O ,4′‐ C ‐Methylene‐bridged nucleic acid/locked nucleic acid (2′,4′‐BNA/LNA) with 1,3‐diaza‐2‐oxophenoxazine (BNAP ( B H  )) was previously reported. Herein, a novel  B H   analogue, 2′,4′‐BNA/LNA with 9‐(2‐aminoethoxy)‐1,3‐diaza‐2‐oxophenoxazine (G‐clamp), named BNAP‐AEO ( B AEO  ), was designed. The  B AEO   nucleoside was successfully synthesized and incorporated into oligodeoxynucleotides (ODNs). ODNs containing  B AEO   possessed up to 10 4 ‐, 152‐, and 11‐fold higher binding affinities for complementary (c) RNA than those of ODNs containing 2′‐deoxycytidine ( C ), 2′,4′‐BNA/LNA with 5‐methylcytosine ( L ), or 2′‐deoxyribonucleoside with G‐clamp ( P AEO  ), respectively. Moreover, duplexes formed by ODN bearing  B AEO   with cDNA and cRNA were thermally stable, even under molecular crowding conditions induced by the addition of polyethylene glycol. Furthermore, ODN bearing  B AEO   was more resistant to 3′‐exonuclease than ODNs with phosphorothioate linkages.

2′,4′‐BNA/LNA with 9‐(2‐Aminoethoxy)‐1,3‐diaza‐2‐oxophenoxazine Efficiently Forms Duplexes and Has Enhanced Enzymatic Resistance **