Li Y, Geyer C R, Sen D
Institute of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, British Columbia, Canada.
Biochemistry. 1996 May 28;35(21):6911-22. doi: 10.1021/bi960038h.
DNA sequences were isolated by in vitro selection for binding to N-methylmesoporphyrin IX (NMM), a molecule that behaves as a stable transition-state analogue for porphyrin chelatases. Clones approximately 280 and approximately 120 nucleotides long were obtained, which bound to NMM with sub-micromolar affinity but bound mesoporphyrin IX (MPIX), as well as various metalloderivatives of MPIX, with lower affinity. Footprinting experiments with dimethyl sulfate, DNase I, and bound hemin molecules activated by superoxide identified a series of short guanine-rich motifs to be the binding sites for the various porphyrins. One clone, PS2, examined in depth, gave a methylation footprint with bound NMM but not with bound MPIX nor with a number of metalloporphyrins. The binding domain PS2, synthesized as a short oligonucleotide, itself showed high-affinity binding to NMM. The binding sequences from different clones were loosely homologous, and the footprinting data were consistent with their folding to form one or more guanine quartets in the presence of NMM. Ultraviolet--visible absorption and circular dichroism spectroscopy of the DNA--NMM complexes indicates, however, that the interaction is not primarily intercalative in nature. The preferential binding of NMM by these aptamers raises the possibility of their being able to catalyze the chelation of metal ions by the porphyrin MPIX.
通过体外筛选与N - 甲基中卟啉IX（NMM）结合来分离DNA序列，NMM是一种对卟啉螯合酶起稳定过渡态类似物作用的分子。获得了长度约为280和120个核苷酸的克隆，它们以亚微摩尔亲和力与NMM结合，但与中卟啉IX（MPIX）以及MPIX的各种金属衍生物结合的亲和力较低。用硫酸二甲酯、DNase I以及由超氧化物激活的结合血红素分子进行的足迹实验确定了一系列富含鸟嘌呤的短基序是各种卟啉的结合位点。深入研究的一个克隆PS2，与结合的NMM产生甲基化足迹，但与结合的MPIX以及多种金属卟啉均未产生。作为短寡核苷酸合成的结合结构域PS2本身对NMM表现出高亲和力结合。来自不同克隆的结合序列具有松散的同源性，足迹数据与它们在NMM存在下折叠形成一个或多个鸟嘌呤四联体一致。然而，DNA - NMM复合物的紫外 - 可见吸收光谱和圆二色光谱表明这种相互作用本质上并非主要是嵌入性的。这些适体对NMM的优先结合增加了它们能够催化卟啉MPIX螯合金属离子的可能性。
