The binding of adenosine to polyuridylic acid in which uracil residues are chemically altered

The binding of adenosine‐ 14 C to polyuridylic acid (poly(U)) and several modified poly(U)s has been studied by equilibrium dialysis. The poly(U) was modified by addition of appropriate reagents across the 5,6‐double bond of the uracil ring to form the photohydrate, photodimer, dihydrouracil, the HOBr addition product and the HSO 3 − addition product. Modification of the uracil rings decreases the amount of adenosine which can be bound to the poly(U); the decrease in binding is a function of the fraction of uracil rings which have been changed. Using the expression S = S 0 (1 − α r ) 2 to relate the fraction of uracil rings modified ( r ) to the number of binding “sites” remaining ( S ), it is found that α is about 1 for all the modifications except photodimer where it is about 2. These observations are taken to mean that the loss of binding capacity of the poly(U) resulting from modifications of the uracil ring is caused by loss of planarity of the uracil rings caused by the modifications, and consequent loss of double helix structure, but that for all modifications except photodimer there is no disruption of the poly(U) double helix on either side of the leison. There does appear to be local melting on either side of the photodimer lesion. The sigmoidal binding isotherms ( A b versus C a ) of modified and unmodified poly(U) can be approximated closely by the following equation: (1)\documentclass{article}\pagestyle{empty}\begin{document}$$ \theta = \frac{{A_{\rm b} }}{S} = \frac{{(K_1 C_{\rm a} )^n \left[ {\frac{n}{{1 - K_1 C_{\rm a} }}} \right] + \frac{{K_1 C_{\rm a} }}{{(1 - K_1 C_{\rm a} )^2 }}}}{{1 + (K_1 C_{\rm a} )^n \left[ {\frac{n}{{1 - K_1 C_{\rm a} }}} \right] + \frac{{K_1 C_{\rm a} }}{{(1 - K_1 C_{\rm a} )^2 }}}} $$\end{document}(1) where A b = bound A , C a = free A , n = minimum number of adjacent A′ s in complex, S = concentration of sites on poly(U), and K 1 = ( K m ) 1/ m for all m ≥ n . The stacking energy of adenosine ( w ) can be calculated accurately using the following equation, where d θ/ d ln C a is obtained from Eq. (1). (2)\documentclass{article}\pagestyle{empty}\begin{document}$$ {{d\theta } \mathord{\left/ {\vphantom {{d\theta } d}} \right. \kern-\nulldelimiterspace} d}\begin{array}{*{20}c} {{\rm }\ln {\rm }C_{\rm a} {\rm } = {\rm }{1 \mathord{\left/ {\vphantom {1 {re^{ - {w \mathord{\left/ {\vphantom {w {2RT}}} \right. \kern-\nulldelimiterspace} {2RT}}} }}} \right. \kern-\nulldelimiterspace} {re^{ - {w \mathord{\left/ {\vphantom {w {2RT}}} \right. \kern-\nulldelimiterspace} {2RT}}} }}12RT} & {at{\rm }\theta {\rm } = {\rm }0.5} \\\end{array} $$\end{document}(2) For unmodified poly(U), w is −2.0 kcal/mole and Δ G ° (−; RT ln K 1 ) is −3.2 kcal/mole. The difference (−1.2 kcal/mole) is attributed to hydrogen bonding. Heavily photohydrated poly(U) does not bind guanosine or guanosine‐5′‐phosphate.