Studies pertaining to the function and maintenance of nucleic acids have remained on the forefront of biological research and includes the examination of a wide range of agents capable of cleaving or modifying DNA/RNA, both in vivo and in vitro. This extends to enzymes (e.g., exo- and endonucleases) that hydrolyze nucleic acid polymers in specific or nonspecific fashion, footprinting agents (e.g., EDTA chelators) often used for genetic mapping, and natural metabolites (e.g., bleomycin and the enediyne antibiotics) that are medically useful as anticancer agents (see Fig. 1). Whereas extensive effort has been

From: Methods in Molecular Biology, vol. 335: Fluorescent Energy Transfer Nucleic Acid Probes: Designs and Protocols Edited by: V. V. Didenko © Humana Press Inc., Totowa, NJ



[3] [4] [5]

applied to understanding the mechanisms by which such nucleases and small molecules cleave or modify DNA or RNA, a continuous, simplified, and general assay for quantitatively measuring DNA scission was lacking prior to the availability of the molecular break light (MBL) assay described herein.

An important and versatile tool within nucleic acid analysis has been the use of covalently modified nucleotides for fluorescence resonance energy transfer (FRET). FRET, as reviewed by Klostermeier and Millar (1), is a nonradiative process in which a donor fluorophore transfers its excitation energy to an acceptor fluorophore (i.e., the "quencher") in a distance-dependent manner, resulting in the spatially focused quenching of fluorescence. The power of this phenomenon lies within its ability to scrutinize experimentally accessible distances within a 10-100 A range, the typical intramolecular distances of nucleic acids (1). Using fluorescently labeled nucleotide analogs, such seminal FRET-incorporating molecular biology experiments have been performed toward mapping DNA accessibility in chromatin (2), determining the proximity relationships of substrate binding within RNA polymerase (3,4), and deconvoluting the geometric determinates of DNA recombination (5).

In 1996, Tyagi and Kramer (6) developed the "molecular beacon," a modified oligonucleotide probe characterized by a fluorophore and fluorescence-quencher covalently ligated to the 5'- and 3'-DNA termini, respectively, that contains a short self-complementary "stem" that brings the fluorophore and quencher in close proximity of each other (see Fig. 2A), resulting in low FRET-based fluorescence emission from overlapping emission-absorption spectra. Hybridization of the extended "loop" of the probe with a complementary DNA/ RNA strand affords a spatial separation of the adjacent fluorophore-quencher pair, resulting in a spontaneous fluorescent signal and indicating successful hybridization. We have adapted this model to monitor DNA cleavage wherein the probe, which we call a "molecular break light," is self-complementary through a small T4 loop and contains an extended stem that encodes for the DNA-binding recognition sequence of a specific nuclease (7) (see Fig. 2B). Scission of the probe stem results in the spatial separation of the fluorophore-quencher and results in a spontaneous signal that directly correlates the emerging fluorescence with the extent of DNA degradation. The main advantages to this system, which we have termed the "MBL assay," are as follows:

Fig. 1. (opposite page) DNA-cleaving agents: (A) calicheamicin y1I from Micromonspora echinospora, (B) esperamicin A1 from Actinomadura verrucosospora, (C) bleomycin from Streptomyces verticillus, (D) methidiumpropyl-EDTA-Fe(II), and (E) EDTA-Fe(II).

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