Doktycz M J, Paner T M, Benight A S
Department of Chemistry, University of Illinois, Chicago 60680.
Biopolymers. 1993 Dec;33(12):1765-77. doi: 10.1002/bip.360331203.
The preparation and melting of a 16 base-pair duplex DNA linked on both ends by C12H24 (dodecyl) chains is described. Absorbance vs temperature curves (optical melting curves) were measured for the dodecyl-linked molecule and the same duplex molecule linked on the ends instead by T4 loops. Optical melting curves of both molecules were measured in 25, 55, and 85 mM Na+ and revealed, regardless of [Na+], the duplex linked by dodecyl loops is more stable by at least 6 degrees C than the same duplex linked by T4 loops. Experimental curves in each salt environment were analyzed in terms of the two-state and multistate theoretical models. In the two-state, or van't Hoff analysis, the melting transition is assumed to occur in an all-or-none manner. Thus, the only possible states accessible to the molecule throughout the melting transition are the completely intact duplex and the completely melted duplex or minicircle. In the multistate analysis no assumptions regarding the melting transition are required and the statistical occurrence of every possible partially melted state of the duplex is explicitly considered. Results of the analysis revealed the melting transitions of both the dodecyl-linked molecule and the dumbbell with T4 end loops are essentially two state in 25 and 55 mM Na+. In contrast, significant deviations from two-state behavior were observed in 85 mM Na+. From our previously published melting data of DNA dumbbells with Tn end loops where n = 2, 3, 4, 6, 8, 10, 14 [T. M. Paner, M. Amaratunga, and A. S. Benight, (1992) Biopolymers, Vol. 32, pp. 881-892] and the dumbbell with T4 end loops of this study, a plot of d(Tm)/d ln [Na+] was constructed. Extrapolation of this data to n = 1 intersects with the value of d (Tm)/d ln [Na+] obtained for the alkyl-linked dumbbell, suggesting the salt-dependent stability of the alkyl-linked molecule behaves as though the duplex of this molecule were linked by end loops comprised of a single T residue.
本文描述了一种两端通过C12H24（十二烷基）链连接的16个碱基对双链DNA的制备和熔解过程。测量了十二烷基连接分子以及两端通过T4环连接的相同双链分子的吸光度与温度曲线（光学熔解曲线）。在25、55和85 mM Na+条件下测量了两种分子的光学熔解曲线，结果表明，无论[Na+]如何，十二烷基环连接的双链比T4环连接的相同双链至少稳定6摄氏度。在每种盐环境下的实验曲线都根据二态和多态理论模型进行了分析。在二态或范特霍夫分析中，假设熔解转变以全或无的方式发生。因此，在整个熔解转变过程中分子可达到的唯一可能状态是完全完整的双链和完全熔解的双链或小环。在多态分析中，不需要对熔解转变做任何假设，并且明确考虑了双链每一种可能的部分熔解状态的统计出现情况。分析结果表明，在25和55 mM Na+条件下，十二烷基连接分子和带有T4末端环的哑铃状分子的熔解转变基本上都是二态的。相比之下，在85 mM Na+条件下观察到明显偏离二态行为的情况。根据我们之前发表的带有Tn末端环（n = 2、3、4、6、8、10、14）的DNA哑铃状分子的熔解数据[T. M. Paner, M. Amaratunga, and A. S. Benight, (1992) Biopolymers, Vol. 32, pp. 881 - 892]以及本研究中带有T4末端环的哑铃状分子，绘制了d(Tm)/d ln [Na+]的图。将该数据外推至n = 1时与烷基连接哑铃状分子获得的d (Tm)/d ln [Na+]值相交，这表明烷基连接分子的盐依赖性稳定性表现得就好像该分子的双链是由包含单个T残基的末端环连接的一样。
