Triplex formation at physiological pH by 5-Me-dC-N-4-(spermine) [X] oligodeoxynucleotides: Non protonation of N3 in X of X*G:C triad and effect of base mismatch ionic strength on triplex stabilities

Oligodeoxynucleotide (ODN) directed tripler formation has therapeutic importance and depends on Hoogsteen hydrogen bonds between a duplex DNA and a third DNA strand, T*A:T triplets are formed at neutral pH and C(+*)G:C are favoured at acidic pH, It is demonstrated that spermine conjugation at N-4 Of 5-Me-dC in ODNs 1-5 (sp-ODNs) imparts zwitterionic character, thus reducing the net negative charge of ODNs 1-5, sp-ODNs form triplexes with complementary 24mer duplex 8:9 show foremost stability at neutral pH 7.3 and decrease in stability towards lower pH, unlike the normal ODNs where optimal stability is found at an acidic pH 5.5, At pH 7.3, control ODNs 6 and 7 carrying dC or 5-Me-dC, respectively, do not show any triple helix formation, The stability order of tripler containing 5-Me-dC-N-4-(spermine) with normal and mismatched duplex was found to be X*G:C similar to X*A:T > X*C:G > X*T:A. The hysteresis curve of sp-ODN tripler 3*8:9 indicated a better association with complementary duplex 8:9 as compared to unmodified ODN 6 in tripler 6*8:9, pH-dependent UV difference spectra suggest that N3 protonation is not a requirement for tripler formation by sp-ODN and interstrand interaction of conjugated spermine more than compensates for loss in stability due to absence of a single Hoogsteen hydrogen bond, These results may have importance in designing oligonucleotides for antigene applications.