Kinetics and mechanisms of oxidative cleavage of HIV RRE RNA by Rev-coupled transition metal-chelates
Kinetics and mechanisms of oxidative cleavage of HIV RRE RNA by Rev-coupled transition metal-chelates
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DOI：
10.1039/c3sc22135k
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发表时间：
2013-01-01
期刊：
影响因子：
8.4
通讯作者：
Cowan, J. A.
中科院分区：
文献类型：
--
作者：
Joyner, Jeff C.;Keuper, Kevin D.;Cowan, J. A.
催化金属药物用于氧化切割HIV-1 Rev反应元件RNA（RRE RNA），并使用基质辅助激光解吸/电离飞行时间质谱（MALDI-TOF MS），荧光光谱和凝胶电泳相结合的RNA切割的机制进行了研究。含有过渡金属Fe 2+、Co 2+、Ni 2+和Cu 2+与Rev偶联螯合剂DOTA、DTPA、EDTA、NTA、三肽GGH和四肽KGHK的组合的金属药物通过迄今未知的氧化机制结合并切割HIV RRE RNA。广谱的金属催化剂和共试剂提供了一种系统的氧化反应性的变化，而没有显着的干扰催化剂和RNA之间的结合的手段。使用详细的MS分析来监测在切割位点含有末端2 '，3'-环状磷酸（2 '，3'-cPO（4））、3 '-磷酸（3'-PO 4）、3 '-磷酸乙醇酸（3'-PG）、5 '-羟基（5'-OH）、5 '-磷酸（5'-PO 4）和其它新生突出端的RNA片段的形成。不同的突出端对应于不同的机制，氧化氢提取（H提取），水解，和/或核酸内切，允许解剖的各种机制的氧化裂解的贡献。含有末端3 '-PG、3'-PO 4和5 '-PO 4突出端的片段的快速共反应物和催化剂依赖性形成似乎主要由H提取事件引发。标准硫代巴比妥酸（TBA）测定在本文中以新的用途用于监测由RNA中的4 ′-H提取产生的碱2-羟基丙烯醛产物的形成。通过荧光监测与TBA的加合物的形成，并且其定量与通过MALDI-TOF MS监测的3 '-PG的形成相关，证实氧化4'-H提取是快速催化剂介导的RRE RNA切割的主要机制。3 '-PO 4突出端的快速形成最可能是5'-H提取的结果。在RNA内不同核苷酸位置形成3 '-PG（一种独特的提取产物）的表观速率用于对金属中心的可能3D位置进行三角测量，并建立某些催化金属药物的4'-H提取的距离依赖性。
Catalytic metallodrugs were used to oxidatively cleave HIV-1 Rev Response Element RNA (RRE RNA), and the mechanisms of RNA cleavage were studied using a combination of matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS), fluorescence spectroscopy, and gel electrophoresis. The metallodrugs, which contained combinations of the transition metals Fe2+, Co2+, Ni2+, and Cu2+ and the Rev-coupled chelators DOTA, DTPA, EDTA, NTA, tripeptide GGH, and tetrapeptide KGHK, bind to and cleave HIV RRE RNA through heretofore unknown oxidative mechanisms. The broad spectrum of metal catalysts and co-reagents provided a means for systematic variation of oxidative reactivity without significant perturbation of binding between catalyst and RNA. Detailed MS analyses were used to monitor formation of RNA fragments containing terminal 2',3'-cyclic phosphate (2',3'-cPO(4)), 3'-phosphate (3'-PO4), 3'-phosphoglycolate (3'-PG), 5'-hydroxyl (5'-OH), 5'-phosphate (5'-PO4) and other nascent overhangs at sites of cleavage. The distinct overhangs corresponded to distinct mechanisms of oxidative hydrogen-abstraction (H abstraction), hydrolysis, and/or endonucleolysis, allowing a dissection of the contributions of various mechanisms of oxidative cleavage. Rapid co-reactant-and catalyst-dependent formation of fragments containing terminal 3'-PG, 3'-PO4 and 5'-PO4 overhangs appeared to be initiated primarily by H abstraction events. The standard thiobarbituric acid (TBA) assay was employed herein in a novel usage to monitor the formation of base 2-hydroxypropenal products produced by 4'-H abstraction in RNA. Formation of an adduct with TBA was monitored by fluorescence, and its quantification correlated with the formation of 3'-PG monitored by MALDI-TOF MS, confirming oxidative 4'-H abstraction as a major mechanism of rapid catalyst-mediated cleavage of RRE RNA. Rapid formation of 3'-PO4 overhangs was most likely a result of 5'-H abstraction. Apparent rates of formation of 3'-PG (a unique product of abstraction) at differing nucleotide positions within the RNA were used to triangulate probable 3D positions of metal centers and establish the distance-dependence of 4'-H abstraction for certain catalytic metallodrugs.