Lown J W, Hanstock C C, Lobe C G, Bleackley C
Department of Chemistry, University of Alberta, Edmonton, Canada.
J Biomol Struct Dyn. 1985 Jun;2(6):1107-24. doi: 10.1080/07391102.1985.10507627.
The non self complementary heptadeoxyribonucleotides d(GTCGTCA) and d(TGACGAC) were synthesized by the phosphotriester method. While complete 1H-NMR assignments of the former were obtained by a combination of one and two-dimensional techniques at room temperature, extensive stacking of the latter under these conditions dictated analysis at 50 degrees C when the lines were sharply resolved. The duplex form of the annealed strands under the conditions of the 1H-NMR experiment was established independently of the NMR evidence by 32P end labeling with T4 polynucleotide kinase followed by butt end joining using the absolute specificity of T4 ligase for double strand DNA. Analysis of the resulting ladder of polymers was performed using gel electrophoresis and autoradiography. Complete 1H-NMR assignments of the non-exchangeable protons in the self complementary heptamer was achieved. The assignments were confirmed using NOE differences, and two-dimensional COSY, and HH-INADEQUATE experiments at 400 and 500 MHz. The assignments are in accord with a conformation for the heptamer belonging to the B family of structures.
非自互补的十七脱氧核糖核苷酸d(GTCGTCA)和d(TGACGAC)通过磷酸三酯法合成。虽然前者的完整1H-NMR归属在室温下通过一维和二维技术的结合获得，但后者在这些条件下大量堆积，这决定了在50℃进行分析，此时谱线清晰分辨。在1H-NMR实验条件下，通过用T4多核苷酸激酶进行32P末端标记，然后利用T4连接酶对双链DNA的绝对特异性进行对接末端连接，独立于NMR证据确定了退火链的双链形式。使用凝胶电泳和放射自显影对所得聚合物梯进行分析。实现了自互补七聚体中不可交换质子的完整1H-NMR归属。使用NOE差异以及在400和500 MHz下的二维COSY和HH-INADEQUATE实验对归属进行了确认。这些归属与属于B结构家族的七聚体构象一致。
