Inhibition of the B to Z transition in poly(dGdC).poly(dGdC) by covalent attachment of ethidium: kinetic studies.
Inhibition of the B to Z transition in poly(dGdC).poly(dGdC) by covalent attachment of ethidium: kinetic studies.
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通过共价连接乙锭抑制聚（dGdC）.聚（dGdC）中的 B 到 Z 转变：动力学研究。
DOI：
10.1021/bi00109a018
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发表时间：
1991
期刊：
影响因子：
2.9
通讯作者：
Chaires,JB
中科院分区：
文献类型：
--
作者：
Gilbert,PL;Graves,DE;Britt,M;Chaires,JB
1991年8月26日收到的修订版摘要：使用光亲和类似物单叠氮乙锭来制备含有共价连接乙锭的聚-（dGdC）-聚（dGdC）样品。用吸收光谱和荧光光谱研究了非共价键和共价键结合的乙锭对NaCl诱导的poly（dGdC）-poly（dGdC）中B → Z跃迁动力学的影响。钴和非共价连接的乙锭在它们降低B到Z转变的速率的程度上是相等的。通过使用荧光来选择性地监测在过渡过程中非共价结合的乙锭的命运，我们发现，乙锭完全解离的反应进行，但在一个速度，滞后于聚合物的转化为Z形式。这些实验提供了证据的重新分配的非共价键结合的乙锭在过程中的B到Z的过渡，导致双相反应动力学的发展。所观察到的动力学表明，共价键和非共价键结合的乙锭的主要效果是在成核步骤的B到Z的过渡。通过非共价或共价结合的乙锭，B向Z转变的速率的降低可以定量地解释为发现足够长的无药物螺旋长度以使成核发生的可能性降低。作为必要的辅助实验，合成了限定长度的脱氧寡核苷酸（dGdC）4、（dGdC）5和（dGdC）6，并将其用于动力学实验，所述动力学实验被设计用于确定B至Z转变的成核长度，发现其为6 bp。证明了B至Z跃迁的活化能与共价结合的乙锭的量无关，并且发现活化能为21.2±1.1 kcal mol-1。观察到乙锭的共价连接增加了反向Z到B转变的速率，推测是通过将聚合物的区域锁定为右手构象，从而提供Z到B转化可以传播的成核位点。
Revised Manuscript Received August 26, 1991 abstract: The photoaffinity analogue ethidium monoazide was used toprepare samples of poly-(dGdC)-poly (dGdC) containing covalently attached ethidium. Theeffects of both noncovalently and covalently bound ethidium on the kinetics of the NaCl-induced B to Z transition in poly (dGdC)-poly (dGdC) was examined using absorbance and fluorescence spectroscopy to monitor the reaction. Covalently and noncovalently attached ethidium were equal in the extent to which they reduce the rate of the B to Z transition. By using fluorescence to selectively monitor the fate of noncovalently bound ethidium over the course of the transition, we found that ethidium completely dissociates as the reaction proceeds, but at a rate that lags behind the conversion of the polymer to the Z form. These experiments provide evidence for the redistribution of noncovalently bound ethidium over the course of the B to Z transition, leading to the development of biphasic reaction kinetics. The observed kinetics suggest that the primary effect of both covalently and noncovalently bound ethidium is on the nucleation step of the B to Z transition. The reduction in the rate of the B to Z transition by noncovalently or covalently bound ethidium may be quantitatively explained as resulting from the reduced probability of finding a drug-free length of helix long enough for nucleation to occur. As necessary ancillary experiments, the defined length deoxyoligonucleotides (dGdC) 4,(dGdC) 5, and (dGdC) 6 were synthesized and used in kinetic experiments designed to determine the nucleation length of the B to Z transition, which was found to be 6 bp. The activation energy of the B to Z transition was demonstrated to be independent of the amount of covalently bound ethidium and was found to be 21.2±1.1 kcal mol" 1. Covalent attachment of ethidium was observed to increase the rate of the reverse Z to B transition, presumablyby locking regions of the polymer into a right-handed conformation and thereby providing nucleation sites from which the Z to B conversion may propagate.