DOI: 10.1002/chem.201002745 2’-O,4’-C-Aminomethylene-Bridged Nucleic Acid Modification with Enhancement of Nuclease Resistance Promotes Pyrimidine Motif Triplex Nucleic Acid Formation at Physiological pH Hidetaka Torigoe,* [a] S. M. Abdur Rahman, [b] Hiroko Takuma, [b] Norihiro Sato, [a] Takeshi Imanishi, [b] Satoshi Obika, [b] and Kiyomi Sasaki [a] Introduction In recent years, triplex nucleic acid has attracted considera- ble interest because of its possible biological functions in vivo and its wide variety of potential applications in vivo, such as regulation of gene expression by antigene technolo- gy, mapping of genomic DNA, and gene-targeted mutagene- sis. [1] A triplex nucleic acid is usually formed through the se- quence-specific interaction between a single-stranded homo- pyrimidine or homopurine triplex-forming oligonucleotide (TFO) and the major groove of homopurine–homopyrimi- dine stretch in duplex DNA. [1c,d] In the pyrimidine motif tri- plex, a homopyrimidine TFO binds parallel to the homopur- ine strand of the target duplex by Hoogsteen hydrogen bonding to form T·A:T and C + ·G:C base triplets. [1c,d] On the other hand, in the purine motif triplex, a homopurine TFO binds antiparallel to the homopurine strand of the target duplex by reverse Hoogsteen hydrogen bonding to form A·A:T (or T·A:T) and G·G:C base triplets. [1c,d] Because protonation of the cytosine bases in a homopyri- midine TFO is required to bind with the guanine bases of the G:C target duplex, the formation of the pyrimidine motif triplex needs acidic pH conditions and is thus ex- tremely unstable at physiological neutral pH. [2] On the other hand, the pH-independent formation of the purine motif tri- plex is available at neutral pH. However, the purine motif triplex formation is severely inhibited by physiological con- centrations of certain monovalent cations, especially K + . [3] Undefined association between K + and the guanine-rich ho- mopurine TFO has been applied to explain the inhibitory effect. [3] Thus, stabilization of the pyrimidine motif triplex at neutral pH is quite necessary for improving the potential of the triplex in various triplex-formation-based strategies in vivo. Replacement of the cytosine bases in a homopyrimi- dine TFO with 5-methylcytosine [2b, 4] or other chemically modified base analogues [5] and conjugation of different Abstract: Due to the instability of pyri- midine motif triplex DNA at physio- logical pH, triplex stabilization at phys- iological pH is crucial in improving its potential in various triplex-formation- based strategies in vivo, such as gene expression regulation, genomic DNA mapping, and gene-targeted mutagene- sis. To this end, we investigated the thermodynamic and kinetic effects of our previously reported chemical modi- fication, 2’-O,4’-C-aminomethylene- bridged nucleic acid (2’,4’-BNA NC ) modification of triplex-forming oligo- nucleotide (TFO), on triplex formation at physiological pH. The thermody- namic analyses indicated that the 2’,4’- BNA NC modification of TFO increased the binding constant of the triplex for- mation at physiological pH by more than 10-fold. The number and position of the 2’,4’-BNA NC modification in TFO did not significantly affect the magnitude of the increase in the bind- ing constant. The consideration of the observed thermodynamic parameters suggested that the increased rigidity and the increased degree of hydration of the 2’,4’-BNA NC -modified TFO in the free state relative to the unmodi- fied TFO may enable the significant in- crease in the binding constant. Kinetic data demonstrated that the observed increase in the binding constant by the 2’,4’-BNA NC modification resulted mainly from the considerable decrease in the dissociation rate constant. The TFO stability in human serum showed that the 2’,4’-BNA NC modification sig- nificantly increased the nuclease resist- ance of TFO. Our results support the idea that the 2’,4’-BNA NC modification of TFO could be a key chemical modi- fication to achieve higher binding affin- ity and higher nuclease resistance in the triplex formation under physiologi- cal conditions, and may lead to prog- ress in various triplex-formation-based strategies in vivo. Keywords: BNA · kinetics · nucle- ase resistance · thermodynamics · triplex [a] Prof. H. Torigoe, N. Sato, K. Sasaki Departmentof Applied Chemistry, Faculty of Science Tokyo University of Science, 1–3 Kagurazaka Shinjuku-ku, Tokyo 162-8601 (Japan) Fax: (+ 81) 3-5261-4631 E-mail: htorigoe@rs.kagu.tus.ac.jp [b] Dr. S. M. A. Rahman, H. Takuma, Prof. T. Imanishi, Prof. S. Obika Graduate School of Pharmaceutical Sciences Osaka University, 1–6 Yamadaoka Suita, Osaka 565-0871 (Japan) Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/chem.201002745.  2011 Wiley-VCH Verlag GmbH&Co. KGaA, Weinheim Chem. Eur. J. 2011, 17, 2742 – 2751 2742