Cerofolini Linda, Amato Jussara, Giachetti Andrea, Limongelli Vittorio, Novellino Ettore, Parrinello Michele, Fragai Marco, Randazzo Antonio, Luchinat Claudio
Magnetic Resonance Center (CERM), University of Florence, Sesto Fiorentino, Florence 50019, Italy Giotto Biotech, Via Madonna del Piano 6, Sesto Fiorentino, Florence 50019, Italy.
Department of Pharmacy, University of Naples 'Federico II', Naples I-80131, Italy.
Nucleic Acids Res. 2014 Dec 1;42(21):13393-404. doi: 10.1093/nar/gku1084. Epub 2014 Nov 6.
The occurrence of a G-triplex folding intermediate of thrombin binding aptamer (TBA) has been recently predicted by metadynamics calculations, and experimentally supported by Nuclear Magnetic Resonance (NMR), Circular Dichroism (CD) and Differential Scanning Calorimetry (DSC) data collected on a 3' end TBA-truncated 11-mer oligonucleotide (11-mer-3'-t-TBA). Here we present the solution structure of 11-mer-3'-t-TBA in the presence of potassium ions. This structure is the first experimental example of a G-triplex folding, where a network of Hoogsteen-like hydrogen bonds stabilizes six guanines to form two G:G:G triad planes. The G-triplex folding of 11-mer-3'-t-TBA is stabilized by the potassium ion and destabilized by increasing the temperature. The superimposition of the experimental structure with that predicted by metadynamics shows a great similarity, with only significant differences involving two loops. These new structural data show that 11-mer-3'-t-TBA assumes a G-triplex DNA conformation as its stable form, reinforcing the idea that G-triplex folding intermediates may occur in vivo in human guanine-rich sequences. NMR and CD screening of eight different constructs obtained by removing from one to four bases at either the 3' and the 5' ends show that only the 11-mer-3'-t-TBA yields a relatively stable G-triplex.
凝血酶结合适配体（TBA）的G-四链体折叠中间体的出现最近已通过元动力学计算预测，并得到了核磁共振（NMR）、圆二色性（CD）和差示扫描量热法（DSC）数据的实验支持，这些数据是在3'端TBA截短的11聚体寡核苷酸（11-mer-3'-t-TBA）上收集的。在这里，我们展示了在钾离子存在下11-mer-3'-t-TBA的溶液结构。这种结构是G-四链体折叠的第一个实验实例，其中类似Hoogsteen氢键的网络稳定了六个鸟嘌呤以形成两个G:G:G三联体平面。11-mer-3'-t-TBA的G-四链体折叠由钾离子稳定，并随着温度升高而不稳定。实验结构与元动力学预测的结构叠加显示出极大的相似性，仅在两个环上存在显著差异。这些新的结构数据表明，11-mer-3'-t-TBA以G-四链体DNA构象作为其稳定形式，强化了G-四链体折叠中间体可能在人体内富含鸟嘌呤的序列中体内发生的观点。对通过在3'端和5'端去除一至四个碱基获得的八种不同构建体进行NMR和CD筛选表明，只有11-mer-3'-t-TBA产生相对稳定的G-四链体。
