Lee M, Krowicki K, Shea R G, Lown J W, Pon R T
Department of Chemistry, University of Alberta, Edmonton, Canada.
J Mol Recognit. 1989 Sep;2(2):84-93. doi: 10.1002/jmr.300020206.
MPE-Fe(EDTA) footprinting of a novel monocationic bis-furan lexitropsin 6 on a HindIII/EcoRI restriction fragment of pBR322 DNA revealed a series of four-base binding sites (all 5'----3') of (primary) TGTA, TGAA, AAAT, ACAA, TTAT, and (secondary) CTAA, TCGT, TGTA, GTCA, and GGTT. Thus 6 can accept a GC pair at positions 1, 2 or 3 of the binding site with a strict 3' (4 position) AT requirement. Marked enhancement of cleavage, particularly at GC rich sequences, is observed at regions flanking or even up to 18 base pairs remote from a given binding site. The non-exchangeable and imino 1H NMR resonances of the 1:1 complex and d-[CATGGCCATG]2 were assigned using a combination of NOE differences, NOESY and COSY techniques. 1H NMR studies (ligand induced chemical shifts and NOE differences) of Lexitropsin 6 with d-[CATGGCCATG]2 show unambiguously the location and orientation of the N to C termini of 6 on the sequence 5'-G5C6C7A8-3', with the C terminus oriented to A8. This orientation of 6 in the minor groove of 5'-GCCA is confirmed by an NOE observed between H1 2a of 6 and AH8(8). This preference for binding of 6 to the sequence 5'-GCCA when challenged with d-[CATGGCCATG]2 is in accord with the conclusions of the footprinting experiments wherein GC base pairs can be accepted in the first three positions and with a strict 3' terminus AT reading requirement. Collectively the data support the inference of a GC recognizing capacity for a 2,5-substituted furan moiety within a lexitropsin. The 1H NMR data indicate that the decadeoxyribonucleotide duplex exists in the B conformation in both the 1:1 complex and the free form. The apparent binding constant of 6 to calf thymus DNA is 1.68 X 10(5) M-1 whereas netropsin under similar conditions gives a value of 1.85 X 10(7) M-1. This suggests that if advantage is to be taken of the GC recognizing property of a 2,5-substituted furan in longer lexitropsins it should be flanked by more strongly bound moieties.
用新型单阳离子双呋喃勒克西特罗辛6对pBR322 DNA的HindIII/EcoRI限制性片段进行MPE-Fe(EDTA)足迹分析，揭示了一系列四碱基结合位点（均为5'→3'），主要有TGTA、TGAA、AAAT、ACAA、TTAT，次要的有CTAA、TCGT、TGTA、GTCA和GGTT。因此，6在结合位点的第1、2或3位可接受GC对，但对3'（第4位）有严格的AT要求。在给定结合位点侧翼甚至相距18个碱基对的区域，观察到切割显著增强，尤其是在富含GC的序列处。使用NOE差异、NOESY和COSY技术相结合的方法，对1:1复合物和d-[CATGGCCATG]2的不可交换和亚氨基1H NMR共振进行了归属。勒克西特罗辛6与d-[CATGGCCATG]2的1H NMR研究（配体诱导化学位移和NOE差异）明确显示了6的N端到C端在序列5'-G5C6C7A8-3'上的位置和方向，C端朝向A8。6在5'-GCCA小沟中的这种方向通过6的H1 2a与AH8(8)之间观察到的NOE得到证实。当用d-[CATGGCCATG]2进行挑战时，6对5'-GCCA序列的这种结合偏好与足迹实验的结论一致，即在前三位置可接受GC碱基对且对3'端有严格的AT读取要求。总体而言，这些数据支持了勒克西特罗辛中2,5-取代呋喃部分具有识别GC能力的推断。1H NMR数据表明，在1:1复合物和游离形式中，十脱氧核糖核苷酸双链体均以B构象存在。6与小牛胸腺DNA的表观结合常数为1.68×10(5) M-1，而在类似条件下新诺菌素的值为1.85×10(7) M-1。这表明，如果要在更长的勒克西特罗辛中利用2,5-取代呋喃的GC识别特性，它应该两侧有结合更强有力的部分。
