SUNY at Albany
June 19-23, 2001
Paper-Clip Type Triple Helix Formation by 5'-d-(TC)3Ta(CT)3Cb(AG)3 (a, b = 0 - 4): as a Function of Loop Size And with/without the Pseudoisocytosine Base in the Hoogsteen Strand
The formation of a DNA "paper-clip" type triple helix (triplex) with a common sequence 5'-d-(TC)3Ta(CT)3Cb(AG)3 (a, b = 0 - 4 ) was studied by UV thermal melting experiments and CD spectra. These DNA oligomers form triplexes and duplexes in slightly acidic and neutral conditions, respectively. The stability of the formed triplexes (at pH 4.5) or duplexes (at pH 7.0 or 8.0) does not vary significantly with the size of loops (a, b = 1 - 4). At pH 6.0, the triplex stability is, however, a function of a and b. It is also interesting to note that the oligomer 5'-d-(TC)3(CT)3(AG)3 (a, b = 0) forms a stable triplex at pH 4.5 with a slight lower Tm values, due to dissociation of a base triad at one end and a distorted base triad at the other, observed by 1H NMR. Thus, we have here a model system, 5'-d-(TC)3Ta(CT)3Cb(AG)3, that could form a triplex effectively with (a, b = 1 - 4) and without (a, b = 0) loops in acidic conditions. In addition, the triplex formation of oligomers with replacement of one, two, or three 2'-deoxycytidine in the Hoogsteen strand by either 2'-deoxypseudoisocytidine (D) or 2'-O-methylpseudoisocytidine (M) was also studied in the sequence 5'-d-(TX)3T2(CT)3C2(AG)3 (X = C, D or M). Both CD spectra and UV melting results showed that only D3 ((TX)3 = (TD)3) and M3 ((TX)3 = (TM)3) were able to form the "paper-clip" structure in both neutral and acidic conditions. This is because the N3H of a pseudoisocytosine base can serve as a proton donor without protonation. We hereby proved that the 2'-deoxypseudoisocytidine, similar to 2'-O-methylpseudoisocytidine, could replace 2'-deoxycytidine in the Hoogsteen strand to provide triplex formation at neutral pH.
Lou-sing Kan (1), Tsung-Mei Chin (2), Shwu-Bin Lin (3), Szu-Yin Lee (2), Mu-Li Chang (2), Alice Yu-Yu Cheng (1, 2), Fu-Chao Chang (1, 2) Laura Pasternack (4), and Dee-Hua Huang (4)
(1) Chemistry, Academia Sinica, Taipei, TW 11529