Observing the Helical Geometry of Double-Stranded DNA in Solution by Fluorescence Resonance Energy Transfer

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 90; no. 7; pp. 2994 - 2998
• Main Authors: Clegg, Robert M., Alastair I. H. Murchie, Zechel, Annelies, David M. J. Lilley
• Format: Journal Article
• Language: English
• Published: Washington, DC National Academy of Sciences of the United States of America 01.04.1993; National Acad Sciences; National Academy of Sciences
• Subjects: Analytical, structural and metabolic biochemistry; Base Sequence; Biochemistry; Biological and medical sciences; Biology; Deoxyribonucleic acid; DNA; DNA - chemistry; Dyes; Emission spectra; Energy transfer; Fluorescence; Fundamental and applied biological sciences. Psychology; General aspects, investigation methods; Geometry; Mathematics; Melting; Models, Structural; Molecular Sequence Data; Molecules; Nucleic Acid Conformation; Nucleic Acid Denaturation; Nucleic acids; Oligodeoxyribonucleotides - chemistry; Osmolar Concentration; Physics; Sodium Chloride; Solutions; Spectrometry, Fluorescence - methods; Spectrophotometry - methods; Thermodynamics; Double stranded DNA; Double helical structure; Fluorescent labelling; Fluorescence; Resonance energy; Method; Oligodeoxyribonucleotide; Energy transfer; Structural analysis; Conformational transition
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.90.7.2994

The efficiency of fluorescence resonance energy transfer (FRET) between fluorescein and rhodamine covalently attached to both 5' termini of a series of double-stranded DNA species (ranging from 8 to 20 bp) was measured. FRET efficiency varied with a dependence compatible with dye-to-dye distances (R) calculated on the basis of double-stranded B-DNA structure; the helical geometry of double-stranded DNA in solution is clearly evident. The experimental data were consistent with a 1/[1 + (R/R0)6] dependence of FRET efficiencycharacteristic for the Forster dipole-dipole mechanism. The thermal dissociation of the strands of the duplex DNA species can be followed by using FRET, and from these data we have been able to obtain enthalpies of duplex formation in good agreement with earlier measurements using alternative techniques. FRET measurements at very different salt concentrations can be accurately compared. We conclude that FRET is a reliable and valuable method for studying structure and conformational transitions in nucleic acids.