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A quantitative method for evaluating the stabilities of nucleic acids.

C A Gelfand1, G E Plum, S Mielewczyk

  • 1Department of Chemistry, Rutgers, The State University of New Jersey, 610 Taylor Road, Piscataway, NJ 08854, USA.

Proceedings of the National Academy of Sciences of the United States of America
|May 26, 1999
PubMed
Summary

This study introduces a novel fluorescence resonance energy transfer (FRET) method to screen nucleic acid duplex stability changes caused by alterations. The technique quantizes free energy differences, enabling sensitive detection of variations like mismatches and modified bases.

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Biophysics

Background:

  • Assessing nucleic acid duplex stability is crucial for understanding genetic variations and disease.
  • Existing methods for measuring thermodynamic stability often require large sample quantities and are limited in scope.

Purpose of the Study:

  • To develop a general, solution-based method for screening the impact of deviations from canonical Watson-Crick composition on nucleic acid duplex thermodynamic stability.
  • To utilize fluorescence resonance energy transfer (FRET) for direct detection of free energy differences between reference and altered nucleic acid duplexes.

Main Methods:

  • A FRET-based competitive binding assay was employed, involving a labeled reference duplex and an unlabeled test strand.
  • Strand displacement of the reference duplex by the test strand, leading to a loss of FRET signal, was monitored.

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  • Titration curves were analyzed to determine free energy differences between the initial and newly formed duplexes.

    • Main Results:

    • The method directly quantifies free energy differences between reference and test nucleic acid duplexes.
    • It enables measurement of equilibrium association constants beyond the range of conventional techniques.
    • The assay requires significantly less material compared to other solution-based methods due to fluorescence sensitivity.

    Conclusions:

    • This FRET-based approach provides a sensitive and versatile tool for detecting and characterizing modifications affecting nucleic acid duplex stability.
    • The method is applicable to a wide range of nucleic acid variations, including single nucleotide polymorphisms and mutagenic lesions.
    • The technique holds potential for high-throughput screening of nucleic acid alterations.