bi0004201_si_001.pdf (443.51 kB)
“Paper-Clip” Type Triple Helix Formation by 5‘-d-(TC)3Ta(CT)3Cb(AG)3 (a and b = 0−4) as a Function of Loop Size with and without the Pseudoisocytosine Base in the Hoogsteen Strand†
journal contributionposted on 2000-09-13, 00:00 authored by Tsung-Mei Chin, Shwu-Bin Lin, Szu-Yin Lee, Mu-Li Chang, Alice Yu-Yu Cheng, Fu-Chao Chang, Laura Pasternack, Dee-Hua Huang, Lou-Sing Kan
The formation of a DNA “paper-clip” type triple helix (triplex) with a common sequence 5‘-d-(TC)3Ta(CT)3Cb(AG)3 (a and b = 0−4) was studied by UV thermal melting experiments and CD spectra. These DNA oligomers form triplexes and duplexes under 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 the loops (a and 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 and b = 0) forms a stable triplex at pH 4.5 with a slightly lower Tm value, 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 and b = 1−4) and without (a and b = 0) loops under 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 (where X is 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 under 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.