Melting Behavior of a Covalently Closed, Single-Stranded, Circular DNA

Dorothy A. Erie, Roger A. Jones, Wilma K. Olson, Navin K. Sinha, Kenneth J. Breslauer

Research output: Contribution to journalArticle

64 Scopus citations

Abstract

We synthesized the 26-residue deoxynucleotide sequence d(TTCCT5GGAATTCCT5GGAA) which folds intramolecularly to form a dumbbell-shaped, double-hairpin structure with a gap between the 3' and the 5' ends. We used T4 polynucleotide kinase to phosphorylate the 5' end followed by T4 DNA ligase to close the 3' and 5' ends. Melting of the dumbbell structure formed by this ligated sequence produces a covalently closed, single-stranded, circular final state. We employed calorimetric and spectroscopic techniques to characterize thermodynamically the melting behavior of the ligated molecule and compared it with the corresponding melting behavior of its unligated precursor. This comparison allowed us to characterize uniquely the influence of single-stranded ring closure on intramolecular duplex melting. The data reveal that ring closure produces a thermally more stable structure which exhibits significantly altered melting thermodynamics. We rationalize these thermodynamic differences in terms of differential solvation and differential counterion association between the ligated and unligated molecules. We also note the importance of such constrained dumbbell structures as models for hairpins, cruciforms, and locally melted domains within naturally occurring DNA polymers.

Original languageEnglish (US)
Pages (from-to)268-273
Number of pages6
JournalBiochemistry
Volume28
Issue number1
DOIs
StatePublished - Jan 1 1989

All Science Journal Classification (ASJC) codes

  • Biochemistry

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