Sha F, Chen F M
Department of Chemistry, Tennessee State University, Nashville, Tennessee 37209-1561, USA.
Biophys J. 2000 Oct;79(4):2095-104. doi: 10.1016/S0006-3495(00)76457-5.
Earlier calorimetric studies had indicated that despite the absence of a GpC sequence, the self-complementary octamer d(CGTCGACG) binds strongly to actinomycin D (ACTD) with high cooperativity and a 2:1 drug/duplex ratio. A subsequent optical spectral study with related oligomers led us to suggest that ACTD may likely stack at the G. C basepairs of the duplex termini. New findings are reported herein to indicate that despite the lack of complete self-complementarity, oligomers of d(CGXCGXCG) [X = A or T] motif exhibit unusually strong ACTD affinities with binding constants of roughly 2 x 10(7) M(-1) and binding densities of 1 drug molecule per strand. The ACTD binding affinity for the corresponding heteroduplex obtained by annealing these two oligomers is, however, considerably reduced. Although spectroscopic results with related oligomers obtained by removing, replacing, or appending bases at the termini appear to be consistent with the end-stacking model, capillary electrophoretic (CE) evidence provides additional insights into the binding mode. CE experiments with the self-complementary oligomers d(CGAGCTCG) and d(CGTCGACG) revealed contrasting migration patterns in the presence of ACTD, with mobility retardation and acceleration exhibited by the GpC- and non-GpC-containing octamers, respectively, whereas the X/X-mismatched d(CGXCGXCG) experienced retardation. These results, along with those of related oligomers, suggest that ACTD may in fact stack at the duplex stem end of a monomeric hairpin or at the 3'-end of dG as a single strand. The seemingly cooperative ACTD binding and the curved Scatchard plot for the self-complementary d(CGTCGACG) may thus be attributed to the drug-induced duplex denaturation resulting from strong binding to single strands of d(CGXCGYCG) motif. Detailed structural information on the ACTD-DNA complexes, however, must await further NMR investigations.
早期的量热研究表明，尽管不存在GpC序列，但自我互补的八聚体d(CGTCGACG)仍以高协同性和2:1的药物/双链体比例与放线菌素D（ACTD）强烈结合。随后对相关寡聚物的光谱研究使我们推测，ACTD可能堆叠在双链体末端的G·C碱基对处。本文报道了新的研究结果，表明尽管缺乏完全的自我互补性，但d(CGXCGXCG) [X = A或T]基序的寡聚物表现出异常强的ACTD亲和力，结合常数约为2×10(7) M(-1)，每条链的结合密度为1个药物分子。然而，通过将这两种寡聚物退火得到的相应异源双链体的ACTD结合亲和力却大大降低。尽管通过在末端去除、替换或添加碱基得到的相关寡聚物的光谱结果似乎与末端堆叠模型一致，但毛细管电泳（CE）证据为结合模式提供了更多见解。对自我互补寡聚物d(CGAGCTCG)和d(CGTCGACG)进行的CE实验显示，在ACTD存在下迁移模式形成对比，含GpC和不含GpC的八聚体分别表现出迁移率减慢和加快，而X/X错配的d(CGXCGXCG)则出现迁移率减慢。这些结果以及相关寡聚物的结果表明，ACTD实际上可能堆叠在单体发夹的双链体茎末端或作为单链的dG的3'末端。因此，自我互补的d(CGTCGACG)看似协同的ACTD结合以及弯曲的Scatchard图可能归因于与d(CGXCGYCG)基序单链的强结合导致的药物诱导双链体变性。然而，关于ACTD-DNA复合物的详细结构信息必须等待进一步的核磁共振研究。
