Studies of DNA dumbbells. IV. Preparation and melting of a DNA dumbbell with the 16 base‐pair sequence 5′G‐T‐A‐T‐C‐C‐C‐T‐C‐T‐G‐G‐A‐T‐A‐C3′ linked on the ends by dodecyl chains

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Abstract
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°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 m MNa+. 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. © 1993 John Wiley & Sons, Inc.