The synthesis of N4-(6-aminopyridin-2-yl)-2′-deoxycytidine for recognizing the CG base pair at neutral pH by oligodeoxyribonucleotide-directed triple helix formation

The sequence-specific recognition of double-helical DNA by oligodeoxyribonucleotide-directed triple helix (triplex) formation is limited mostly to purine tracts. To interrupt the purine tract in a target sequence, a non-natural deoxyribonucleoside N-4-(6-aminopyridin-2-yl)-2'-deoxycytidine (C-p) was designed to interact with the C base in the CG base pair. The protected phosphoramidite synthon of C-p was synthesized in seven steps and then was incorporated into an oligodeoxyribonucleotide by an automatic DNA synthesizer. Two 22-mers, designated as C2 and P, with a common sequence of 5'-d-T(m)CTXT(m)CTTCTGTCTCCAGACAG were synthesized in this study. C-m is 5-methyl-2'-deoxycytidine and X is either 2'-deoxycytidine (C) or C-p for C2 and P, respectively. C2 is able to form a paper clip type tripler with one C . CG mismatched base triad in slightly acidic conditions but not at the neutral pH. On the other hand, P forms a stable tripler under both acidic and neutral conditions. This indicates that C-p is able to form a C-p . CG base triad in the tripler. Their physical properties were studied by UV thermal melting experiments and circular dichorism spectroscopy (CD). The thermal melting results imply that the C-p . CG base triad is as stable as the C+ . GC triad at pH 6.0, and C-p helps the tripler formation preferably at neutral to acidic pH. In addition to the hydrogen bonding interaction with the CG base pair, the hydrophobic interaction of C-p may also play an important role in stabilizing the triplex formation of oligodeoxyribonucleotides. In the presence of spermine at either pH 5.0 or pH 6.0, the melting temperature of the third strand of P was elevated about 30 and 21 degrees C, respectively. Thus, spermine can enhance the stability of the triple-helical structure.