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• W2023163715 abstract "Extreme instability of pyrimidine motif triplex DNA at physiological pH severely limits its use in an artificial control of gene expression in vivo. Stabilization of the pyrimidine motif triplex at physiological pH is, therefore, crucial in improving its therapeutic potential. To this end, we have investigated the thermodynamic and kinetic effects of our previously reported chemical modification, 2′-O,4′-C-methylene bridged nucleic acid (2′,4′-BNA) modification of triplex-forming oligonucleotide (TFO), on pyrimidine motif triplex formation at physiological pH. The thermodynamic analyses indicated that the 2′,4′-BNA modification of TFO increased the binding constant of the pyrimidine motif triplex formation at neutral pH by ∼20 times. The number and position of the 2′,4′-BNA modification introduced into the TFO did not significantly affect the magnitude of the increase in the binding constant. The consideration of the observed thermodynamic parameters suggested that the increased rigidity itself of the 2′,4′-BNA-modified TFO in the free state relative to the unmodified TFO may enable the significant increase in the binding constant at neutral pH. Kinetic data demonstrated that the observed increase in the binding constant at neutral pH by the 2′,4′-BNA modification of TFO resulted from the considerable decrease in the dissociation rate constant. Our results certainly support the idea that the 2′,4′-BNA modification of TFO could be a key chemical modification and may eventually lead to progress in therapeutic applications of the antigene strategy in vivo. Extreme instability of pyrimidine motif triplex DNA at physiological pH severely limits its use in an artificial control of gene expression in vivo. Stabilization of the pyrimidine motif triplex at physiological pH is, therefore, crucial in improving its therapeutic potential. To this end, we have investigated the thermodynamic and kinetic effects of our previously reported chemical modification, 2′-O,4′-C-methylene bridged nucleic acid (2′,4′-BNA) modification of triplex-forming oligonucleotide (TFO), on pyrimidine motif triplex formation at physiological pH. The thermodynamic analyses indicated that the 2′,4′-BNA modification of TFO increased the binding constant of the pyrimidine motif triplex formation at neutral pH by ∼20 times. The number and position of the 2′,4′-BNA modification introduced into the TFO did not significantly affect the magnitude of the increase in the binding constant. The consideration of the observed thermodynamic parameters suggested that the increased rigidity itself of the 2′,4′-BNA-modified TFO in the free state relative to the unmodified TFO may enable the significant increase in the binding constant at neutral pH. Kinetic data demonstrated that the observed increase in the binding constant at neutral pH by the 2′,4′-BNA modification of TFO resulted from the considerable decrease in the dissociation rate constant. Our results certainly support the idea that the 2′,4′-BNA modification of TFO could be a key chemical modification and may eventually lead to progress in therapeutic applications of the antigene strategy in vivo. triplex-forming oligonucleotide bridged nucleic acid 4′-BNA, 2′-O,4′-C-methylene bridged nucleic acid 4′-BNA, 3′-O,4′-C-methylene bridged nucleic acid electrophoretic mobility shift assay circular dichroism isothermal titration calorimetry interaction analysis system purine pyrimidine biotinylated nonspecific melting temperature on-rate constant association rate constant off-rate constant dissociation rate constant In recent years, triplex DNA has attracted considerable interest because of its possible biological functions in vivo and its wide variety of potential applications, such as regulation of gene expression, site-specific cleavage of duplex DNA, mapping of genomic DNA, and gene-targeted mutagenesis (1Mirkin S.M. Frank-Kamenetskii M.D. Annu. Rev. Biophys. Biomol. Struct. 1994; 23: 541-576Crossref PubMed Scopus (224) Google Scholar, 2Frank-Kamenetskii M.D. Mirkin S.M. Annu. Rev. Biochem. 1995; 64: 65-95Crossref PubMed Scopus (642) Google Scholar, 3Soyfer V.N. Potaman V.N. Triple-Helical Nucleic Acids. Springer-Verlag, New York1996Crossref Google Scholar). A triplex is usually formed through the sequence-specific interaction of a single-stranded homopurine or homopyrimidine triplex-forming oligonucleotide (TFO)1 with the major groove of homopurine-homopyrimidine stretch in duplex DNA (1Mirkin S.M. Frank-Kamenetskii M.D. Annu. Rev. Biophys. Biomol. Struct. 1994; 23: 541-576Crossref PubMed Scopus (224) Google Scholar, 2Frank-Kamenetskii M.D. Mirkin S.M. Annu. Rev. Biochem. 1995; 64: 65-95Crossref PubMed Scopus (642) Google Scholar, 3Soyfer V.N. Potaman V.N. Triple-Helical Nucleic Acids. Springer-Verlag, New York1996Crossref Google Scholar, 4Sun J.-S. Helene C. Curr. Opin. Struct. Biol. 1993; 3: 345-356Crossref Scopus (170) Google Scholar, 5Sun J.-S. Garestier T. Helene C. Curr. Opin. Struct. Biol. 1996; 6: 327-333Crossref PubMed Scopus (115) Google Scholar). In the pyrimidine motif triplex, a homopyrimidine TFO binds parallel to the homopurine strand of the target duplex by Hoogsteen hydrogen bonding to form T·A:T and C+·G:C triplets (1Mirkin S.M. Frank-Kamenetskii M.D. Annu. Rev. Biophys. Biomol. Struct. 1994; 23: 541-576Crossref PubMed Scopus (224) Google Scholar, 2Frank-Kamenetskii M.D. Mirkin S.M. Annu. Rev. Biochem. 1995; 64: 65-95Crossref PubMed Scopus (642) Google Scholar, 3Soyfer V.N. Potaman V.N. Triple-Helical Nucleic Acids. Springer-Verlag, New York1996Crossref Google Scholar, 4Sun J.-S. Helene C. Curr. Opin. Struct. Biol. 1993; 3: 345-356Crossref Scopus (170) Google Scholar, 5Sun J.-S. Garestier T. Helene C. Curr. Opin. Struct. Biol. 1996; 6: 327-333Crossref PubMed Scopus (115) Google Scholar). 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 triplets (1Mirkin S.M. Frank-Kamenetskii M.D. Annu. Rev. Biophys. Biomol. Struct. 1994; 23: 541-576Crossref PubMed Scopus (224) Google Scholar, 2Frank-Kamenetskii M.D. Mirkin S.M. Annu. Rev. Biochem. 1995; 64: 65-95Crossref PubMed Scopus (642) Google Scholar, 3Soyfer V.N. Potaman V.N. Triple-Helical Nucleic Acids. Springer-Verlag, New York1996Crossref Google Scholar, 4Sun J.-S. Helene C. Curr. Opin. Struct. Biol. 1993; 3: 345-356Crossref Scopus (170) Google Scholar, 5Sun J.-S. Garestier T. Helene C. Curr. Opin. Struct. Biol. 1996; 6: 327-333Crossref PubMed Scopus (115) Google Scholar).Because the cytosine bases in a homopyrimidine TFO must be protonated to bind with the guanine bases of the G:C duplex, the formation of the pyrimidine motif triplex needs an acidic pH condition and is thus extremely unstable at physiological pH (6Frank-Kamenetskii M.D. Methods Enzymol. 1992; 211: 180-191Crossref PubMed Scopus (39) Google Scholar, 7Singleton S.F. Dervan P.B. Biochemistry. 1992; 31: 10995-11003Crossref PubMed Scopus (162) Google Scholar, 8Shindo H. Torigoe H. Sarai A. Biochemistry. 1993; 32: 8963-8969Crossref PubMed Scopus (65) Google Scholar). Instead, the pH-independent formation of the purine motif triplex is available at physiological pH. However, the purine motif triplex formation is severely inhibited by physiological concentrations of certain monovalent cations, especially K+. Undefined association between K+ and the guanine-rich homopurine TFO has been applied to explain the inhibitory effect (9Milligan J.F. Krawczyk S.H. Wadwani S. Matteucci M.D. Nucleic Acids Res. 1993; 21: 327-333Crossref PubMed Scopus (105) Google Scholar, 10Cheng A.-J. Van Dyke M.W. Nucleic Acids Res. 1993; 21: 5630-5635Crossref PubMed Scopus (98) Google Scholar). Therefore, stabilization of the pyrimidine motif triplex at physiological pH is of great importance in improving its therapeutic potential to artificially control gene expression in vivo. Numerous efforts such as the replacement of cytosine bases in a homopyrimidine TFO with 5-methylcytosine (7Singleton S.F. Dervan P.B. Biochemistry. 1992; 31: 10995-11003Crossref PubMed Scopus (162) Google Scholar, 11Lee J.S. Woodsworth M.L. Latimer L.J.P. Morgan A.R. Nucleic Acids Res. 1984; 12: 6603-6614Crossref PubMed Scopus (274) Google Scholar, 12Povsic T.J. Dervan P.B. J. Am. Chem. Soc. 1989; 111: 3059-3061Crossref Scopus (280) Google Scholar, 13Xodo L.E. Manzini G. Quadrifoglio F. van der Marel G.A. van Boom J.H. Nucleic Acids Res. 1991; 19: 5625-5631Crossref PubMed Scopus (203) Google Scholar) or other chemically modified bases (14Ono A. Ts'o P.O.P. Kan L.-S. J. Am. Chem. Soc. 1991; 113: 4032-4033Crossref Scopus (170) Google Scholar, 15Krawczyk S.H. Milligan J.F. Wadwani S. Moulds C. Froehler B.C. Matteucci M.D. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 3761-3764Crossref PubMed Scopus (101) Google Scholar, 16Koh J.S. Dervan P.B. J. Am. Chem. Soc. 1992; 114: 1470-1478Crossref Scopus (141) Google Scholar, 17Jetter M.C. Hobbs F.W. Biochemistry. 1993; 32: 3249-3254Crossref PubMed Scopus (67) Google Scholar, 18Ueno Y. Mikawa M. Matsuda A. Bioconj. Chem. 1998; 9: 33-39Crossref PubMed Scopus (44) Google Scholar), the conjugation of different DNA intercalators to TFO (19Sun J.S. Giovannangeli C. Francois J.C. Kurfurst R. Montenay-Garestier T. Asseline U. Saison-Behmoaras T. Thuong N.T. Helene C. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 6023-6027Crossref PubMed Scopus (107) Google Scholar,20Mouscadet J.-F. Ketterle C. Goulaouic H. Carteau S. Subra F. Le Bret M. Auclair C. Biochemistry. 1994; 33: 4187-4196Crossref PubMed Scopus (63) Google Scholar), and the use of polyamines such as spermine or spermidine as triplex stabilizers (21Hampel K.J. Crosson P. Lee J.S. Biochemistry. 1991; 30: 4455-4459Crossref PubMed Scopus (135) Google Scholar) have been made to improve the stability of the pyrimidine motif triplex at physiological pH.We first synthesized and developed a new class of chemical modifications of nucleic acids, bridged nucleic acid (BNA), such as 2′-O,4′-C-methylene BNA (2′,4′-BNA; Fig.1 a; Refs. 22Obika S. Nanbu D. Hari Y. Morio K. In Y. Ishida T. Imanishi T. Tetrahedron Lett. 1997; 38: 8735-8738Crossref Scopus (487) Google Scholar, 23Obika S. Nanbu D. Hari Y. Andoh J. Morio K. Doi T. Imanishi T. Tetrahedron Lett. 1998; 39: 5401-5404Crossref Scopus (601) Google Scholar, 24Obika S. Andoh J. Sugimoto T. Miyashita K. Imanishi T. Tetrahedron Lett. 1999; 40: 6465-6468Crossref Scopus (45) Google Scholar, 25Imanishi T. Obika S. J. Syn. Org. Chem. Jpn. 1999; 57: 969-980Crossref Scopus (41) Google Scholar, 26Obika S. Hari Y. Morio K. Imanishi T. Tetrahedron Lett. 2000; 41: 215-219Crossref Scopus (30) Google Scholar, 27Obika S. Hari Y. Morio K. Imanishi T. Tetrahedron Lett. 2000; 41: 221-224Crossref Scopus (42) Google Scholar) 2After our report on the first synthesis of 2′,4′-BNA monomers, Wengel's group demonstrated some properties of 2′,4′-BNA (28Singh, S. K., Nielsen, P., Koshkin, A. A., and Wengel, J. (1998) Chem. Commun. 455–456Google Scholar, 29Koshkin A.A. Singh S.K. Nielsen P. Rajwanshi V.K. Kumar R. Meldgaard M. Olsen C.E. Wengel J. Tetrahedron. 1998; 54: 3607-3630Crossref Scopus (911) Google Scholar).2After our report on the first synthesis of 2′,4′-BNA monomers, Wengel's group demonstrated some properties of 2′,4′-BNA (28Singh, S. K., Nielsen, P., Koshkin, A. A., and Wengel, J. (1998) Chem. Commun. 455–456Google Scholar, 29Koshkin A.A. Singh S.K. Nielsen P. Rajwanshi V.K. Kumar R. Meldgaard M. Olsen C.E. Wengel J. Tetrahedron. 1998; 54: 3607-3630Crossref Scopus (911) Google Scholar).and 3′-O,4′-C-methylene BNA (3′,4′-BNA; Refs.30Obika, S., Morio, K., Nanbu, D., and Imanishi, T. (1997) Chem. Commun. 1643–1644Google Scholar, 31Obika, S., Morio, K., Hari, Y., and Imanishi, T. (1999) Chem. Commun. 2423–2424Google Scholar, 32Obika S. Morio K. Hari Y. Imanishi T. Bioorg. Med. Chem. Lett. 1999; 9: 515-518Crossref PubMed Scopus (43) Google Scholar). The 2′,4′-BNA modification of TFO increased the thermal stability of the pyrimidine motif triplex DNA at neutral pH using a homopurine-homopyrimidine target duplex and its specific cytosine-rich TFO (25Imanishi T. Obika S. J. Syn. Org. Chem. Jpn. 1999; 57: 969-980Crossref Scopus (41) Google Scholar). However, the mechanistic explanation for the 2′,4′-BNA modification-mediated triplex stabilization was not clearly understood. Here, therefore, we have further extended our previous study to explore thermodynamic and kinetic effects of the 2′,4′-BNA modification on the pyrimidine motif triplex formation at neutral pH. The thermodynamic and kinetic effects of the 2′,4′-BNA modification on the pyrimidine motif triplex formation between a 23-base pair homopurine-homopyrimidine target duplex (Pur23A·Pyr23T; Fig.1 b) and its specific 15-mer unmodified or 2′,4′-BNA-modified homopyrimidine TFO (Pyr15T, Pyr15BNA7-1, Pyr15BNA7-2, Pyr15BNA5-1, and Pyr15BNA5-2; Fig. 1 b) have been analyzed by electrophoretic mobility shift assay (EMSA; Refs. 33Lyamichev V.I. Mirkin S.M. Frank-Kamenetskii M.D. Cantor C.R. Nucleic Acids Res. 1988; 16: 2165-2178Crossref PubMed Scopus (153) Google Scholar, 34Torigoe H. Ferdous A. Watanabe H. Akaike T. Maruyama A. J. Biol. Chem. 1999; 274: 6161-6167Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar), UV melting, isothermal titration calorimetry (ITC; Refs. 34Torigoe H. Ferdous A. Watanabe H. Akaike T. Maruyama A. J. Biol. Chem. 1999; 274: 6161-6167Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 35Langerman N. Biltonen R.L. Methods Enzymol. 1979; 61: 261-286Crossref PubMed Scopus (50) Google Scholar, 36Biltonen R.L. Langerman N. Methods Enzymol. 1979; 61: 287-318Crossref PubMed Scopus (59) Google Scholar, 37Wiseman T. Williston S. Brandts J.F. Lin L.-N. Anal. Biochem. 1989; 179: 131-137Crossref PubMed Scopus (2416) Google Scholar, 38Kamiya M. Torigoe H. Shindo H. Sarai A. J. Am. Chem. Soc. 1996; 118: 4532-4538Crossref Scopus (59) Google Scholar, 39Torigoe H. Shimizume R. Sarai A. Shindo H. Biochemistry. 1999; 38: 14653-14659Crossref PubMed Scopus (25) Google Scholar, 40Torigoe H. Ferdous A. Watanabe H. Akaike T. Maruyama A. Nucleosides, Nucleotides & Nucleic Acids. 1999; 18: 1655-1656Crossref Scopus (4) Google Scholar), and interaction analysis system (IAsys; Refs. 34Torigoe H. Ferdous A. Watanabe H. Akaike T. Maruyama A. J. Biol. Chem. 1999; 274: 6161-6167Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 39Torigoe H. Shimizume R. Sarai A. Shindo H. Biochemistry. 1999; 38: 14653-14659Crossref PubMed Scopus (25) Google Scholar, 40Torigoe H. Ferdous A. Watanabe H. Akaike T. Maruyama A. Nucleosides, Nucleotides & Nucleic Acids. 1999; 18: 1655-1656Crossref Scopus (4) Google Scholar, 41Cush R. Cronin J.M. Stewart W.J. Maule C.H. Molloy J. Goddard N.J. Biosens. Bioelectron. 1993; 8: 347-353Crossref Scopus (384) Google Scholar, 42Edwards P.R. Gill A. Pollard-Knight D.V. Hoare M. Buckle P.E. Lowe P.A. Leatherbarrow R.J. Anal. Biochem. 1995; 231: 210-217Crossref PubMed Scopus (159) Google Scholar, 43Bates P.J. Dosanjh H.S. Kumar S. Jenkins T.C. Laughton C.A. Neidle S. Nucleic Acids Res. 1995; 23: 3627-3632Crossref PubMed Scopus (72) Google Scholar). Results from these independent lines of experiments have clearly indicated the significant effect of the 2′,4′-BNA modification to promote the pyrimidine motif triplex formation at neutral pH. The binding constant at neutral pH for the pyrimidine motif triplex formation with the 2′,4′-BNA-modified TFO was ∼10–20 times larger than that observed with the corresponding unmodified TFO. Kinetic data have also demonstrated that the contribution for the increase in the binding constant by the 2′,4′-BNA modification of TFO resulted from the considerable decrease in the dissociation rate constant. The ability of the 2′,4′-BNA modification of TFO to promote the pyrimidine motif triplex formation at physiological pH would support further progress in therapeutic applications of the antigene strategy in vivo.DISCUSSIONThe K a of the pyrimidine motif triplex formation with Pyr15T at pH 5.8 was 20 times larger than that observed with Pyr15T at pH 6.8 (Table II), which is consistent with the previously reported results that the neutral pH is unfavorable for the pyrimidine motif triplex formation involving C+·GC triads (6Frank-Kamenetskii M.D. Methods Enzymol. 1992; 211: 180-191Crossref PubMed Scopus (39) Google Scholar, 7Singleton S.F. Dervan P.B. Biochemistry. 1992; 31: 10995-11003Crossref PubMed Scopus (162) Google Scholar, 8Shindo H. Torigoe H. Sarai A. Biochemistry. 1993; 32: 8963-8969Crossref PubMed Scopus (65) Google Scholar). The K a of the triplex formation with Pyr15BNA7-1, Pyr15BNA7-2, Pyr15BNA5-1, or Pyr15BNA5-2 at pH 6.8 was 10–20 times larger than that observed with Pyr15T at pH 6.8 (TableII). The increase in K a at pH 6.8 by the 2′,4′-BNA modification of TFO was supported by the results of EMSA (Fig. 2) and IAsys (Table III). In addition, the 2′,4′-BNA modification of TFO increased the thermal stability of the pyrimidine motif triplex at pH 6.8 (Table I). These results indicate that the 2′,4′-BNA modification of TFO considerably promotes the pyrimidine motif triplex formation at neutral pH.The ΔH on the triplex formation measured by ITC reflects a major contribution from the hydrogen bonding and the base stacking involved in the triplex formation (38Kamiya M. Torigoe H. Shindo H. Sarai A. J. Am. Chem. Soc. 1996; 118: 4532-4538Crossref Scopus (59) Google Scholar, 46Edelhoch H. Osborne Jr., J.C. Adv. Protein Chem. 1976; 30: 183-250Crossref PubMed Scopus (134) Google Scholar, 47Cheng Y.- K. Pettitt B.M. Prog. Biophys. Mol. Biol. 1992; 58: 225-257Crossref PubMed Scopus (148) Google Scholar, 48Shafer R.H. Prog. Nucleic Acids Res. Mol. Biol. 1998; 59: 55-94Crossref PubMed Scopus (89) Google Scholar). On the other hand, the ΔS on the triplex formation measured by ITC includes a positive entropy change from release of structured water on the triplex formation and a major contribution of a negative conformational entropy change from the conformational restraint of TFO involved in the triplex formation (38Kamiya M. Torigoe H. Shindo H. Sarai A. J. Am. Chem. Soc. 1996; 118: 4532-4538Crossref Scopus (59) Google Scholar, 46Edelhoch H. Osborne Jr., J.C. Adv. Protein Chem. 1976; 30: 183-250Crossref PubMed Scopus (134) Google Scholar, 47Cheng Y.- K. Pettitt B.M. Prog. Biophys. Mol. Biol. 1992; 58: 225-257Crossref PubMed Scopus (148) Google Scholar, 48Shafer R.H. Prog. Nucleic Acids Res. Mol. Biol. 1998; 59: 55-94Crossref PubMed Scopus (89) Google Scholar). Because the formed triplex structure involving Pyr15T at pH 5.8 and that involving Pyr15T at pH 6.8 is the same, the magnitude of ΔH and ΔS on the triplex formation measured by ITC could be the same between the two conditions. However, unexpectedly, the magnitudes of ΔH and ΔS for Pyr15T at pH 6.8 were significantly smaller than those observed for Pyr15T at pH 5.8 (Table II). When the ΔH and ΔS are calculated from the fitting procedure of ITC, the heat observed by ITC is divided not by the effective concentration really involved in the triplex formation but by the apparent concentration added to the triplex formation (37Wiseman T. Williston S. Brandts J.F. Lin L.-N. Anal. Biochem. 1989; 179: 131-137Crossref PubMed Scopus (2416) Google Scholar). The calculation does not take it into consideration what percentage of the added concentration is really effectively involved in the triplex formation. Thus, if the triplex formation is less stoichiometric under a certain condition, the magnitudes of ΔH and ΔS for the less stoichiometric triplex formation estimated by ITC are smaller than those observed for the more stoichiometric triplex formation under another condition. Therefore, the significantly smaller magnitudes of ΔH and ΔS for Pyr15T at pH 6.8 relative to those for Pyr15T at pH 5.8 (Table II) suggest that the triplex formation with Pyr15T at pH 6.8 was significantly less stoichiometric than that with Pyr15T at pH 5.8, which was also supported by the significantly smaller magnitudes ofK a and ΔG for Pyr15T at pH 6.8 (TableII). In contrast, the K a and ΔG for Pyr15T at pH 5.8 and those for the 2′,4′-BNA modified TFOs at pH 6.8 were quite similar (Table II), suggesting that the triplex formation under the two conditions was similarly quite stoichiometric. We conclude that the triplex formation with Pyr15T at pH 6.8 was significantly less stoichiometric than that with Pyr15T at pH 5.8 and that with the 2′,4′-BNA-modified TFOs at pH 6.8. Thus, to discuss the promotion mechanism of the triplex formation by the 2′,4′-BNA modification, the comparison of the ΔH and ΔSbetween Pyr15T at pH 6.8 and 2′,4′-BNA-modified TFOs at pH 6.8 is not valid because of the significantly reduced stoichiometry for Pyr15T at pH 6.8. The comparison of the ΔH and ΔSbetween Pyr15T at pH 5.8 and 2′,4′-BNA-modified TFOs at pH 6.8 with similar stoichiometry will provide a reasonable promotion mechanism for the triplex formation by the 2′,4′-BNA modification, as discussed below.Although the K a and ΔG for Pyr15T at pH 5.8 and those for the 2′,4′-BNA-modified TFOs at pH 6.8 were quite similar (Table II), the ingredients of ΔG, that is, ΔH and ΔS, were obviously different from each other. The magnitudes of the negative ΔH and ΔS for the 2′,4′-BNA-modified TFOs at pH 6.8 were smaller than those observed for Pyr15T at pH 5.8 (Table II). The hydrogen bonding and the base stacking involved in the triplex formation are usually considered the major sources of the negative ΔH on the triplex formation (38Kamiya M. Torigoe H. Shindo H. Sarai A. J. Am. Chem. Soc. 1996; 118: 4532-4538Crossref Scopus (59) Google Scholar, 46Edelhoch H. Osborne Jr., J.C. Adv. Protein Chem. 1976; 30: 183-250Crossref PubMed Scopus (134) Google Scholar, 47Cheng Y.- K. Pettitt B.M. Prog. Biophys. Mol. Biol. 1992; 58: 225-257Crossref PubMed Scopus (148) Google Scholar, 48Shafer R.H. Prog. Nucleic Acids Res. Mol. Biol. 1998; 59: 55-94Crossref PubMed Scopus (89) Google Scholar). Thus, the difference in ΔH for the stoichiometric triplex formations between Pyr15T at pH 5.8 and the 2′,4′-BNA-modified TFOs at pH 6.8 (Table II) suggests that the hydrogen bonding and the base stacking of the triplex with the 2′,4′-BNA-modified TFOs are significantly different from those with the corresponding unmodified TFO. In fact, the CD spectra show that the triplexes with the 2′,4′-BNA-modified TFO had the A-like conformation (Ref. 45Johnson K.H. Gray D.M. Sutherland J.C. Nucleic Acids Res. 1991; 19: 2275-2280Crossref PubMed Scopus (51) Google Scholar and Fig. 4). The A-like conformation by the 2′,4′-BNA modification of TFO may result in the difference in the negative ΔH between the unmodified and 2′,4′-BNA-modified TFOs. On the other hand, the negative ΔS on the triplex formation is mainly contributed by a negative conformational entropy change attributable to the conformational restraint of TFO involved in the triplex formation (38Kamiya M. Torigoe H. Shindo H. Sarai A. J. Am. Chem. Soc. 1996; 118: 4532-4538Crossref Scopus (59) Google Scholar, 46Edelhoch H. Osborne Jr., J.C. Adv. Protein Chem. 1976; 30: 183-250Crossref PubMed Scopus (134) Google Scholar, 47Cheng Y.- K. Pettitt B.M. Prog. Biophys. Mol. Biol. 1992; 58: 225-257Crossref PubMed Scopus (148) Google Scholar, 48Shafer R.H. Prog. Nucleic Acids Res. Mol. Biol. 1998; 59: 55-94Crossref PubMed Scopus (89) Google Scholar). Therefore, the smaller magnitude of the negative ΔS for the 2′,4′-BNA modified TFOs at pH 6.8 relative to that for Pyr15T at pH 5.8 (Table II) suggests that the 2′,4′-BNA-modified TFO in the free state is more rigid than the corresponding unmodified TFO. The increased rigidity of the 2′,4′-BNA modified TFO in the free state relative to the corresponding unmodified TFO causes the smaller entropic loss on the triplex formation with the 2′,4′-BNA-modified TFO at neutral pH, which provides a favorable component to the ΔG and leads to the increase in theK a of the triplex formation at neutral pH. We conclude that the increased rigidity of the 2′,4′-BNA-modified TFO in the free state may be one of the factors that increases theK a of the pyrimidine motif triplex formation at neutral pH.The increase in the K a by the 2′,4′-BNA modification was similar in magnitude among the four modified TFOs (Fig. 2 and Tables II and III), indicating that the number and position of the 2′,4′-BNA modification did not significantly affect the magnitude of the increase in the K a at neutral pH. The rigidity itself of the 2′,4′-BNA-modified TFO may be more important to achieve the increase in the K a at neutral pH than the variation of the number and position of the 2′,4′-BNA modification. Thus, other modification strategies to gain the increased rigidity of TFO may also be useful to increase the K a at neutral pH.Kinetic data have demonstrated that the 2′,4′-BNA modification of TFO considerably decrease the k dissoc of the pyrimidine motif triplex formation (Table III). The decrease in thek dissoc is a plausible kinetic reason to explain the remarkable gain in the K a at neutral pH by the 2′,4′-BNA modification (Fig. 2 and Tables II and III). Both our group (38Kamiya M. Torigoe H. Shindo H. Sarai A. J. Am. Chem. Soc. 1996; 118: 4532-4538Crossref Scopus (59) Google Scholar) and others (49Rougee M. Faucon B. Mergny J.L. Barcelo F. Giovannangeli C. Garestier T. Helene C. Biochemistry. 1992; 31: 9269-9278Crossref PubMed Scopus (248) Google Scholar) have previously proposed a model that triplexes form along nucleation-elongation processes: in a nucleation step only a few base contacts of the Hoogsteen hydrogen bonds may be formed between TFO and the target duplex, and this may be followed by an elongation step, in which Hoogsteen base pairings progress to complete triplex formation. Both groups (38Kamiya M. Torigoe H. Shindo H. Sarai A. J. Am. Chem. Soc. 1996; 118: 4532-4538Crossref Scopus (59) Google Scholar, 49Rougee M. Faucon B. Mergny J.L. Barcelo F. Giovannangeli C. Garestier T. Helene C. Biochemistry. 1992; 31: 9269-9278Crossref PubMed Scopus (248) Google Scholar) have also suggested that the observedK a , which is the ratio ofk assoc to k dissoc, may mostly reflect rapid equilibrium of the nucleation step, which is probably the rate-limiting process of the triplex formation. In this sense, the 2′,4′-BNA modification of TFO is considered to slow the collapse of the nucleation intermediate using the rigidity of TFO to increase the K a of the pyrimidine motif triplex formation.The present study has clearly demonstrated that the 2′,4′-BNA modification of TFO promotes pyrimidine motif triplex formation at neutral pH. We conclude that the design of TFO to bridge different positions of sugar moiety with the alkyl chain to gain the increased rigidity of TFO is certainly a promising strategy for the promotion of triplex formation under physiological condition and may eventually lead to progress in therapeutic applications of the antigene strategyin vivo. In recent years, triplex DNA has attracted considerable interest because of its possible biological functions in vivo and its wide variety of potential applications, such as regulation of gene expression, site-specific cleavage of duplex DNA, mapping of genomic DNA, and gene-targeted mutagenesis (1Mirkin S.M. Frank-Kamenetskii M.D. Annu. Rev. Biophys. Biomol. Struct. 1994; 23: 541-576Crossref PubMed Scopus (224) Google Scholar, 2Frank-Kamenetskii M.D. Mirkin S.M. Annu. Rev. Biochem. 1995; 64: 65-95Crossref PubMed Scopus (642) Google Scholar, 3Soyfer V.N. Potaman V.N. Triple-Helical Nucleic Acids. Springer-Verlag, New York1996Crossref Google Scholar). A triplex is usually formed through the sequence-specific interaction of a single-stranded homopurine or homopyrimidine triplex-forming oligonucleotide (TFO)1 with the major groove of homopurine-homopyrimidine stretch in duplex DNA (1Mirkin S.M. Frank-Kamenetskii M.D. Annu. Rev. Biophys. Biomol. Struct. 1994; 23: 541-576Crossref PubMed Scopus (224) Google Scholar, 2Frank-Kamenetskii M.D. Mirkin S.M. Annu. Rev. Biochem. 1995; 64: 65-95Crossref PubMed Scopus (642) Google Scholar, 3Soyfer V.N. Potaman V.N. Triple-Helical Nucleic Acids. Springer-Verlag, New York1996Crossref Google Scholar, 4Sun J.-S. Helene C. Curr. Opin. Struct. Biol. 1993; 3: 345-356Crossref Scopus (170) Google Scholar, 5Sun J.-S. Garestier T. Helene C. Curr. Opin. Struct. Biol. 1996; 6: 327-333Crossref PubMed Scopus (115) Google Scholar). In the pyrimidine motif triplex, a homopyrimidine TFO binds parallel to the homopurine strand of the target duplex by Hoogsteen hydrogen bonding to form T·A:T and C+·G:C triplets (1Mirkin S.M. Frank-Kamenetskii M.D. Annu. Rev. Biophys. Biomol. Struct. 1994; 23: 541-576Crossref PubMed Scopus (224) Google Scholar, 2Frank-Kamenetskii M.D. Mirkin S.M. Annu. Rev. Biochem. 1995; 64: 65-95Crossref PubMed Scopus (642) Google Scholar, 3Soyfer V.N. Potaman V.N. Triple-Helical Nucleic Acids. Springer-Verlag, New York1996Crossref Google Scholar, 4Sun J.-S. Helene C. Curr. Opin. Struct. Biol. 1993; 3: 345-356Crossref Scopus (170) Google Scholar, 5Sun J.-S. Garestier T. Helene C. Curr. Opin. Struct. Biol. 1996; 6: 327-333Crossref PubMed Scopus (115) Google Scholar). 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 triplets (1Mirkin S.M. Frank-Kamenetskii M.D. Annu. Rev. Biophys. Biomol. Struct. 1994; 23: 541-576Crossref PubMed Scopus (224) Google Scholar, 2Frank-Kamenetskii M.D. Mirkin S.M. Annu. Rev. Biochem. 1995; 64: 65-95Crossref PubMed Scopus (642) Google Scholar, 3Soyfer V.N. Potaman V.N. Triple-Helical Nucleic Acids. Springer-Verlag, New York1996Crossref" @default.
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• W2023163715 cites W1574255548 @default.
• W2023163715 cites W1629010506 @default.
• W2023163715 cites W1970284439 @default.
• W2023163715 cites W1973712778 @default.
• W2023163715 cites W1976791590 @default.
• W2023163715 cites W1980572462 @default.
• W2023163715 cites W1983157047 @default.
• W2023163715 cites W1986064937 @default.
• W2023163715 cites W1988102395 @default.
• W2023163715 cites W1999222678 @default.
• W2023163715 cites W2000184211 @default.
• W2023163715 cites W2007753720 @default.
• W2023163715 cites W2015014732 @default.
• W2023163715 cites W2017157816 @default.
• W2023163715 cites W2020357919 @default.
• W2023163715 cites W2024236380 @default.
• W2023163715 cites W2028094132 @default.
• W2023163715 cites W2030371865 @default.
• W2023163715 cites W2030707266 @default.
• W2023163715 cites W2035164099 @default.
• W2023163715 cites W2038332844 @default.
• W2023163715 cites W2043593862 @default.
• W2023163715 cites W2044963356 @default.
• W2023163715 cites W2050956642 @default.
• W2023163715 cites W2052333294 @default.
• W2023163715 cites W2055791011 @default.
• W2023163715 cites W2072316309 @default.
• W2023163715 cites W2076746045 @default.
• W2023163715 cites W2080853945 @default.
• W2023163715 cites W2081108249 @default.
• W2023163715 cites W2083771897 @default.
• W2023163715 cites W2086598566 @default.
• W2023163715 cites W2086779091 @default.
• W2023163715 cites W2108185026 @default.
• W2023163715 cites W2111386319 @default.
• W2023163715 cites W2114462755 @default.
• W2023163715 cites W2122117524 @default.
• W2023163715 cites W2123017921 @default.
• W2023163715 cites W2167796192 @default.
• W2023163715 cites W2173267258 @default.
• W2023163715 cites W2949452731 @default.
• W2023163715 cites W2949860727 @default.
• W2023163715 cites W2950476415 @default.
• W2023163715 cites W2952510860 @default.
• W2023163715 cites W330286904 @default.
• W2023163715 cites W4251663802 @default.
• W2023163715 cites W997220336 @default.
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