Extra Adenosine Stacks into the Self-Complementary d(CGCAGAATTCGCG) Duplex in Solution

Dinshaw J. Patel, Sharon A. Kozlowski, Janet A. Rice, Chris Broka, Keiichi Itakura, Luis A. Marky, Kenneth J. Breslauer

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Abstract

The structural and energetic parameters for the helix to coil transition of the duplex formed by the self-complementary d(C1G2C3AG4A5A6T6T5C4G3C2G1) sequence (henceforth called 13-mer duplex) that contains an extra noncomplementary dA residue between positions dC3 and dG4 are compared with the duplex formed by the corresponding self-complementary d(CGCGAATTCGCG) sequence (henceforth called 12-mer duplex) that lacks the extra base [Patel, D.J., Kozlowski, S.A., Marky, L.A., Broka, C., Rice, J.A., Itakuru, K., & Breslauer, K.J. (1981) Biochemistry (first paper of four in this issue)]. The nonexchangeable base proton nuclear magnetic resonance (NMR) resonances from the extra dA residue are identified and monitored through the melting transition of the 13-mer duplex. These markers demonstrate that the adenosine stacks into the 13-mer duplex rather than form a bulge loop. Furthermore, the base pairing on either side of the extra stacked dA residue remains intact. A downfield-shifted phosphorus resonance in the 31P spectrum of the 13-mer duplex is tentatively assigned to the extended phosphodiester linkage opposite the stacked, extra dA residue. The nonexchangeable protons from base pairs 2–6 and the extra dA residue exhibit a duplex to strand transition midpoint of 57 ± 2 °C in 0.1 M phosphate solution. The thermally induced transition of the 13-mer duplex was also monitored by differential scanning calorimetry. This technique reveals that insertion of an extra dA into each strand of the duplex results in a 19 °C reduction in the melting temperature of the 13-mer relative to the 12-mer duplex. A calorimetric enthalpy change of 104 kcal (mol of double strand)−1 and a van't Hoff enthalpy change of 70 kcal were determined for the helix to coil transition of the 13-mer duplex in 0.1 M NaCl solution. Comparison of these enthalpies reveals that the transition is not a two-state process and that the size of the cooperative unit is 9 ± 1 base pairs. The similarity between the calorimetric transition enthalpies for the 12-mer and 13-mer duplexes is consistent with a picture in which the stacking of the extra dA residue into the duplex on one strand compensates for the loss of stacking between dG·dC base pairs 3 and 4 at the site of the helix interruption.

Original languageEnglish (US)
Pages (from-to)445-451
Number of pages7
JournalBiochemistry
Volume21
Issue number3
DOIs
StatePublished - Feb 1 1982

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All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

Patel, D. J., Kozlowski, S. A., Rice, J. A., Broka, C., Itakura, K., Marky, L. A., & Breslauer, K. J. (1982). Extra Adenosine Stacks into the Self-Complementary d(CGCAGAATTCGCG) Duplex in Solution. Biochemistry, 21(3), 445-451. https://doi.org/10.1021/bi00532a004