A Fluorescent Intercalator Displacement Assay for Establishing DNA Binding Selectivity and Affinity

Tse, Winston C.; Boger, Dale L.

doi:10.1002/0471142700.nc0805s20

Cited by 27 publications

(24 citation statements)

References 44 publications

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“…Alternatively, the peptides may bind to DNA and compete with SYTOX green for binding sites, which will also lead to a decrease in, or quenching of fluorescence. A similar quenching effect was observed by Tse and colleagues with the fluorescent intercalator displacement assay [33]. In this assay a fluorescent dye (ethidium bromide) binds DNA, which leads to an increase in fluorescence.…”

Section: Discussionsupporting

confidence: 75%

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Investigation into the mechanism of action of the antimicrobial peptides Os and Os-C derived from a tick defensin

et al. 2015

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Highlights• Os and Os-C caused altered intracellular morphology of E. coli and B. subtilis.• Membrane effects were observed with fluorescence and TEM studies.• Os and Os-C were able to enter bacterial cells.• The peptides may have intracellular targets such as DNA.• Differences observed between Os and Os-C indicate dissimilar modes of action. AbstractOs and Os-C are two novel antimicrobial peptides, derived from a tick defensin, which have been shown to have a larger range of antimicrobial activity than the parent peptide, OsDef2. The aim of this study was to determine whether the peptides Os and Os-C are mainly membrane acting, or if these peptides have possible additional intracellular targets in Escherichia coli and Bacillus subtilis. Transmission electron microscopy revealed that both peptides adversely affected intracellular structure of both bacteria causing different degrees of granulation of the intracellular contents. At the minimum bactericidal concentrations, permeabilization as determined with the SYTOX green assay seemed not to be the principle mode of killing when compared to melittin. However, fluorescent triple staining indicated that the peptides caused permeabilization of stationary phase bacteria and TEM indicated membrane effects. Studies using fluorescently labeled peptides revealed that the membrane penetrating activity of Os and Os-C was similar to buforin II. Os-C was found to associate with the septa of B. subtilis. Plasmid binding studies showed that Os and Os-C binds E. coli plasmid DNA at a similar charge ratio as melittin. These studies suggest membrane activity for Os and Os-C with possible intracellular targets such as DNA.The differences in permeabilization at lower concentrations and binding to DNA between Os and Os-C, suggest that the two peptides have dissimilar modes of action.

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Section: Discussionsupporting

confidence: 75%

“…In this assay a fluorescent dye (ethidium bromide) binds DNA, which leads to an increase in fluorescence. The addition of a DNA binding compound displaces the bound ethidium bromide, and in turn leads to a decrease in fluorescence [33].…”

Section: Discussionmentioning

confidence: 99%

Investigation into the mechanism of action of the antimicrobial peptides Os and Os-C derived from a tick defensin

et al. 2015

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“…We first performed fluorescent intercalator displacement (FID) assays (23)(24)(25) to probe the interaction and binding preferences of (-)-lomaiviticins A-C (1-3) and (-)-kinamycin C (4) to duplex DNA (Fig. 2).…”

Section: Resultsmentioning

confidence: 99%

Structural basis for DNA cleavage by the potent antiproliferative agent (–)-lomaiviticin A

Woo

Paulson

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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(-)-Lomaiviticin A (1) is a complex antiproliferative metabolite that inhibits the growth of many cultured cancer cell lines at low nanomolar-picomolar concentrations. (-)-Lomaiviticin A (1) possesses a C 2 -symmetric structure that contains two unusual diazotetrahydrobenzo [b]fluorene (diazofluorene) functional groups. Nucleophilic activation of each diazofluorene within 1 produces vinyl radical intermediates that affect hydrogen atom abstraction from DNA, leading to the formation of DNA double-strand breaks (DSBs). Certain DNA DSB repair-deficient cell lines are sensitized toward 1, and 1 is under evaluation in preclinical models of these tumor types. However, the mode of binding of 1 to DNA had not been determined. Here we elucidate the structure of a 1:1 complex between 1 and the duplex d(GCTATAGC) 2 by NMR spectroscopy and computational modeling. Unexpectedly, we show that both diazofluorene residues of 1 penetrate the duplex. This binding disrupts base pairing leading to ejection of the central AT bases, while placing the proreactive centers of 1 in close proximity to each strand. DNA binding may also enhance the reactivity of 1 toward nucleophilic activation through steric compression and conformational restriction (an example of shape-dependent catalysis). This study provides a structural basis for the DNA cleavage activity of 1, will guide the design of synthetic DNA-activated DNA cleavage agents, and underscores the utility of natural products to reveal novel modes of small molecule-DNA association. (-)-Lomaiviticin A (1) possesses half-maximal inhibitory potencies (IC 50 s) in the low nanomolar-picomolar range against several cultured cancer cell lines (1, 2). (-)-Lomaiviticin C (3) and (-)-kinamycin C (4) are several orders of magnitude less potent, whereas (-)-lomaiviticin B (2) is ∼10-100-fold less potent. The cytotoxicity of 1 derives from the induction of double-strand breaks (DSBs) in DNA (18,19). K562 cells exposed to 5 nM of 1 for 30 min accumulated DSB levels that were comparable to 40 Gy of ionizing radiation. This DNA cleavage activity is not recapitulated by 3 or 4, suggesting both diazofluorenes of 1 are essential for cleavage activity. DNA DSBs are exceedingly cytotoxic (20), and these data provide an explanation for the remarkable potency of 1.The molecular mechanism of DNA DSB induction by (-)-lomaiviticin A (1) has been studied (18). Cell-free deuteriumlabeling experiments are consistent with a pathway comprising reductive activation of one diazofluorene (Fig. 1B) to generate the vinyl radical intermediate 1•, followed by hydrogen atom abstraction from the deoxyribose chain, a process known to lead to singlestrand breaks (SSBs) (21). Reductive activation of the remaining diazofluorene, followed by hydrogen atom abstraction, is believed to cleave the complementary DNA (cDNA) strand, leading to the observed DSB.However, the mode of interaction of (-)-lomaiviticin A (1) with DNA has not been elucidated. As C(sp 2 ) radicals are high-energy species, the mechanistic model outlined above pr...

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“…[1][2][3][4][5][6][7] Ever since the discovery of the structure of DNA in the 1950s, DNA has been a target for many therapeutic compounds. Several of these compounds have been found to bind to DNA while interfering with the activity of many vital enzymes and protein factors involved in DNA metabolism.…”

Section: Drug-dna Interactionsmentioning

confidence: 99%

Insights into the Relative DNA Binding Affinity and Preferred Binding Mode of Homologous Compounds Using Isothermal Titration Calorimetry (ITC)

McKnight¹

2013

Applications of Calorimetry in a Wide Context - Differential Scanning Calorimetry, Isothermal Titration Calorimetry and Microca

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A Fluorescent Intercalator Displacement Assay for Establishing DNA Binding Selectivity and Affinity

Abstract: A protocol for a fluorescent intercalator displacement (FID) assay useful for establishing DNA binding selectivity, affinity, stoichiometry, and binding site size, and for distinguishing modes of DNA binding is presented.

Cited by 27 publications

References 44 publications

Investigation into the mechanism of action of the antimicrobial peptides Os and Os-C derived from a tick defensin

Investigation into the mechanism of action of the antimicrobial peptides Os and Os-C derived from a tick defensin

Structural basis for DNA cleavage by the potent antiproliferative agent (–)-lomaiviticin A

Insights into the Relative DNA Binding Affinity and Preferred Binding Mode of Homologous Compounds Using Isothermal Titration Calorimetry (ITC)

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