Many important cellular events, such as DNA replication, recombination, and repair, involve DNA cleavage. These cleavage reactions are usually catalyzed by enzymes, including the most commonly used restriction nucleases and nonspecific nucleases. DNA cleavage assays are important to study these cleavage reactions and characterize these nucleases. Important information for the enzymatic reaction can be obtained through these assays, including enzyme activity, reaction kinetics, reaction mechanisms, and environmental condition

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

effects. The information gleaned will not only deepen our understanding of various cellular events in which these enzymes are involved, but also help provide better tools for molecular cloning, gene mapping, and other genetic manipulations.

Traditional techniques, including gel electrophoresis, filter binding, and high performance liquid chromatography (1-3), have been used to assay DNA cleavage. Unfortunately, these methods are discontinuous and time-consuming, and usually involve radioisotopes. Thus, there has been a standing need for nonradioactive, continuous, sensitive, and easy assays for DNA cleavage reactions. UV assays are continuous and convenient (4) but suffer from a narrow dynamic range and low sensitivity. In recent years, fluorescence-based methods have been developed for DNA cleavage assays (5). In these methods, fluorescence resonance energy transfer or direct quenching was used to produce an increased fluorescent signal when DNA was cleaved (5-8). These assays are continuous, convenient, and environmentally friendly. However, the sensitivity of these reported methods is low. Usually the signal-to-noise (S/N) ratio is about two or less. In addition, these assays require the synthesis and labeling of two complementary DNA strands and then the annealing of the strands into duplex DNA before the assay can commence.

We have developed a simple and sensitive method for DNA cleavage assays, in which MBs are used to signal the cleavage process (9). MB (Fig. 1A) is a probe originally designed to fluoresce only when hybridized to its complementary DNA. It is a hairpin-shaped oligonucleotide with a fluorescent dye at one end and a quencher at the other end. In the absence of the target DNA, the fluorescent dye and quencher molecule are brought close together by the self-complementary stem of the probe, and the fluorescence signal is suppressed. Because the perfectly matched DNA duplex is more stable than the single-stranded hairpin, the MB readily hybridizes to the target, thereby disrupting the stem structure, separating the fluorophore from the quencher, and restoring the fluorescence signal. Owing to its high sensitivity and excellent selectivity, MBs have been widely used in the detection of DNA (10-13) and RNA (1417) in homogeneous solutions.

Fig. 1. (A) The structure of the molecular beacon (MB) used for single-stranded DNA cleavage assay. The MB is designed as a 19-mer loop sequence flanked on the 5'- and 3'-ends with 5-mer long complementary sequences. The oligonucleotide has a stem-and-loop structure, stabilized by a 5-bp duplex formed by intramolecular hybridization of the complementary ends. The fluorophore (TAMRA) and quencher (dabcyl) are attached to the 3'- and 5'-ends of the oligonucleotide by (CH2)6-NH and (CH2)3-O-(CH2)3-NH linker arms, respectively. (B) Schematic representation of the fluorescence mechanism of the MB during cleavage by single-strand-specific DNA nuclease. The large ball labeled as P represents the nuclease. The stem of the MB is designed to have

a melting temperature lower than the assay temperature (37°C), so that the MB breaks into two shorter fragments after being cleaved by the single-strand-specific DNA nuclease, leading to an increase in fluorescence intensity. (C) The structure of the MB used for double-stranded DNA cleavage assay. The stem of the MB contains palindromic recognition sequences for three endonucleases, EcoR I, BstB I, and Taqa I, as indicated by the bars. (D) Working principle of using MBs for double-stranded DNA cleavage assay. The MB has a stem with recognition sequences for DNA endonu-cleases, and it also holds a fluorophore and quencher pair together at its end, causing fluorescence quenching. After cleavage, two complementary short end fragments are produced, unable to pair again owing to thermodynamic instability. As a consequence, the fluorophore and quencher are separated, resulting in a restoration in fluorescence. (Adapted with permission from ref. 9.)

Like the hybridization of a MB to its target DNA or RNA, the enzymatic cleavage of a MB leads to the distance separation of the quencher and the fluorophore, resulting in an increase in the fluorescent signal. The major difference is that the separation in the latter case is permanent and irreversible. Figure 1B,D schematically shows the principles of MBs for monitoring DNA cleavage. In the presence of a single-stranded specific nuclease (Fig. 1B), the nuclease binds and cleaves the single-stranded loop portion the MB. Cleavage by the nuclease breaks the loop, thereby separating the two stem sequences and producing two DNA fragments. Because the melting temperature of these two DNA fragments is lower than the cleavage temperature (37°C), the result of the cleavage is the separation of the quencher and the fluorophore from each other, yielding irreversible fluorescence enhancement. By incorporating recognition sequences of double-stranded DNA specific nuclease in its stem, an MB can also be used to assay double-stranded DNA enzymatic cleavage reactions (Fig. 1D). When the MB is incubated with the corresponding restriction enzyme, the enzyme will bind to its recognition sequence and cleave at that binding site. The cleavage produces a shorter hairpin structure and two end fragments linked to the fluorophore and quencher, respectively. The end fragments are designed in such a way that they will not be able to pair and form a stable double helix once the enzyme cuts them. As a result, the fluorophore and quencher are separated from each other after cleavage, and the fluorescence is restored.

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