Aramini J M, Kalisch B W, Pon R T, van de Sande J H, Germann M W
Department of Pharmacology, Kimmel Cancer Institute, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.
Biochemistry. 1996 Jul 23;35(29):9355-65. doi: 10.1021/bi960612p.
We report a comparative spectroscopic study of a novel self-complementary duplex decamer, d(GCGAAT-3'-3'-(alpha T)-5'-5'-CGC)2, in which an alpha-anomeric nucleotide has been inserted into the sequence in a parallel orientation via 3'-3' and 5'-5' phosphodiester bonds, and its unmodified B-DNA analog, d(GCGAATTCGC)2. Plots of the hyperchromicity and circular dichroism of these oligonucleotides are virtually identical, indicating that the overall base stacking and handedness are preserved in the alpha duplex. Thermodynamic parameters extracted from UV melting experiments show that the alpha duplex is only slightly less stable than the control. A near complete set of 1H and 31P nuclear magnetic resonance (NMR) assignments were obtained for both duplexes using classical one- and two-dimensional approaches. Several lines of evidence, in particular, imino 1H, 31P, nuclear Overhauser enhancement, and deoxyribose ring proton spin-spin coupling data, convincingly demonstrate that the overall structural integrity of the alpha and control duplexes are quite comparable, with any perturbations in the former localized to the regions of the construct encompassing the alpha-nucleotide and the unique backbone linkages. Specifically, the alpha duplex exhibits normal Watson-Crick type base pairing, it remains antiparallel except at the inverted nucleotide, all bases are in the anti orientation, and the sugar ring puckering is predominantly "S"-type. However, the J-coupling information for the alpha-nucleotide and the neighboring (3') cytidine are notably different, and reflect a decrease in the amplitude of the sugar pucker in alpha T7, and a significant shift in the conformational equilibrium of the furanose ring in C8 toward the "N"-type pucker. The feasibility of synthesizing oligodeoxynucleotides containing a combination of alpha sugars and short parallel stranded segments, their propensity for forming stable duplexes, and the structural insights into such complexes reported here are of potential importance in the area of antisense therapy.
我们报道了一种新型自互补双链十聚体d(GCGAAT-3'-3'-(αT)-5'-5'-CGC)2的比较光谱研究，其中一个α-异头核苷酸通过3'-3'和5'-5'磷酸二酯键以平行方向插入序列中，以及其未修饰的B-DNA类似物d(GCGAATTCGC)2。这些寡核苷酸的增色性和圆二色性图谱几乎相同，表明α双链体中整体碱基堆积和螺旋方向得以保留。从紫外熔解实验中提取的热力学参数表明，α双链体的稳定性仅略低于对照。使用经典的一维和二维方法，获得了两种双链体几乎完整的1H和31P核磁共振（NMR）归属。几条证据线，特别是亚氨基1H、31P、核Overhauser效应以及脱氧核糖环质子自旋-自旋耦合数据，令人信服地证明了α双链体和对照双链体的整体结构完整性相当，前者的任何扰动都局限于包含α-核苷酸和独特主链连接的构建体区域。具体而言，α双链体表现出正常的沃森-克里克型碱基配对，除了在反向核苷酸处外仍保持反平行，所有碱基均处于反式构象，糖环皱折主要为“S”型。然而，α-核苷酸和相邻（3'）胞嘧啶的J耦合信息明显不同，反映出αT7中糖环皱折幅度减小，以及C8中呋喃糖环的构象平衡显著向“N”型皱折转移。合成包含α糖和短平行链段组合的寡脱氧核苷酸的可行性、它们形成稳定双链体的倾向以及本文报道的对此类复合物的结构见解在反义治疗领域具有潜在重要性。
