Antitrypanosomal Action Enhanced by Photoaffinity Labeling with Ethidium Analogs

Ba, Cox; Wj, Firth; Hickman, Scott; Fb, Klotz; Lw, Yielding; Kl, Yielding

doi:10.2307/3280565

Cited by 14 publications

(5 citation statements)

References 17 publications

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“…Ethidium monoazide (8-azido-3-amino-5-ethyl-6phenylphenanthridium-chloride) seems to be an appropriate wotoaffinity label of ethidium bromide since the equilibrium interactions and biological properties of the photoreactive derivative are very similar to the parent compound (Hixon etal., 1975;Garland et al, 1980;Fukunaga etal. , 1980;Graves et al, 1981;Laugaa et a/., 1983;Cox et a / . .…”

Section: Introductionmentioning

confidence: 99%

Efficiency of Photoaffinity Labeling Dna Homopolymers and Copolymers With Ethidium Monoazide *

Dannelley¹,

Boyce²,

Gaubatz³

1986

Photochem & Photobiology

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Abstract— Photoaffinity labeling of synthetic DN As with ethidium monoazide was studied to determine if the efficiency of adduct formation was related to DNA sequence. Equilibrium drug binding to DNA homopolymers and copolymers was quanitified by phase partition techniques. The amount of drug bound to a deoxypolymer at equilibrium was then compared to the fraction of ethidium analog covalently‐linked following photoactivation at the same drug/DNA input ratio. There were significant sequence‐related differences in the ability of the photoaffinity probe to label DNA covalently. The efficiency of covalent‐adduct formation decreased in the order poly(dG‐dC). poly(dG‐dC)> poly‐(dG). poly(dC)poly(dA‐dT). poly(dA‐dT)poly(dA). poly(dT). Ethidium monoazide was about 2‐fold more efficient in labeling deoxyhomopolymers and deoxycopolymers composed of G‐C pairs than the A‐T base counterparts. In low ionic buffers (0.015 M Na+), the efficiency of photoactivation decreased with increasing ethidium monoazide concentrations. However. the base sequence effect was observed over a 40‐fold range of drug concentrations. Therefore, the amount of ethidium monoazide bound to a DNA site after irradiation does not appear to represent the true affinity of the drug for that site.

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Section: Introductionmentioning

confidence: 99%

Efficiency of Photoaffinity Labeling Dna Homopolymers and Copolymers With Ethidium Monoazide *

Dannelley¹,

Boyce²,

Gaubatz³

1986

Photochem & Photobiology

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“…It is also possible that the nitrene also reacts with the deoxyribose, and the reaction with the sugar causes chain breakage. Cox et al (6) reported anti-trypanosomal activity of EMA, and Rueckert et al (27) reported the bactericidal effect of EMA on Anoxybacillus flavithermus. However, the mechanism had not been extensively analyzed.…”

Section: Discussionmentioning

confidence: 99%

“…The intensity of OC band was strongest at the concentration of 50 ng/ml of EMA, and OC bands became weaker again at 75 and 100 ng/ml of EMA compared with that of 50 ng/ml of EMA (lanes 7-9). This result shows that a single-stranded breakage occurred at the range of 10 to 100 ng/ml of EMA, and the shape of DNA changed from SC to OC (lanes [3][4][5][6][7][8][9]. At the range of 500 ng/ml to 10 µg/ml, both of SC and OC bands of pBR322 DNA disappeared, which shows that cleavage of double- stranded DNA took place (lanes 10-13).…”

Section: Effect Of Concentrations Of Ema On the Cleavage Of Negativelmentioning

confidence: 94%

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Photoactivated Ethidium Monoazide Directly Cleaves Bacterial DNA and Is Applied to PCR for Discrimination of Live and Dead Bacteria

Soejima

Iida

Qin

et al. 2007

Microbiology and Immunology

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Ethidium monoazide (EMA) is a DNA intercalating agent and a eukaryotic topoisomerase II poison. We found that EMA treatment and subsequent visible light irradiation (photoactivation or photolysis) shows a bactericidal effect, hence the mechanism was analyzed. When bacterial cells were treated with more than 10 µ µg/ml of EMA for 1 hr plus photoactivation for 20 min, cleavage of bacterial DNA was confirmed by agarose gel electrophoresis and electron microscopic studies. The cleavage of chromosomal DNA was seen when it was treated in vitro with EMA and photolysis, which showed that the cleavage directly took place without the assistance of DNA gyrase/topoisomerase IV and the DNA repair enzymes of bacteria. It was also verified, by using negatively supercoiled pBR322 DNA, that medium/high concentrations of EMA (1 to 100 µ µg/ml) led to breaks of double-stranded DNA and that low concentrations of EMA (10 to 100 ng/ml) generated a single-stranded break. EMA is known to easily penetrate dead but not live bacteria. After treatment of 10 µ µg/ml of EMA for 30 min and photoactivation for 5 min, EMA cleaved the DNA of dead but not live Klebsiella oxytoca. When the cleaved DNA was used for templates in PCR targeting 16S rDNA, PCR product from the dead bacteria was completely suppressed. We demonstrated that EMA and photolysis directly cleaved bacterial DNA and are effective tools for discriminating live from dead bacteria by PCR.

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“…36 The monoazido analogs of ethidium have been established as appropriate probes for understanding ethidium-DNA interactions. The noncovalent DNA-binding properties of the monoazido analogs were found previously to mimic those of the parent ethidium compound when they were examined and compared by uv-visible and fluorescence spectroscopic m e t h o d~.~O %~~J~…”

Section: Ethidium As a Probementioning

confidence: 99%

Monoazido analog of ethidium as a chromatin probe: Binding to DNA

Coffman

Yielding

1984

Biopolymers

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SynopsisTwo photoaffinity analogs of ethidium, 8-azido-3-amin0, and 3-azido-8-amino-5-ethyl-6-phenylphenanthridinium chloride, have been used to probe the structure of mammalian chromatin and its interactions with the ethidium moiety. The monoazido analogs were established as suitable probes by comparing their interactions with chromatin and pure DNA prepared from chromatin to those of the parent ethidium bromide. Scatchard analysis of t h e binding data determined from spectrophotometric titrations showed that the analogs interacted with both nucleic acids in a manner similar to the parent compound. T h e effect of chromatin proteins on the interaction of the ethidium moiety with intact chromatin was investigated directly. By exposing the noncovalent complex to visible light, the monoazido analog was attached covalently in its interaction sites within chromatin, and the amount of drug bound covalently to DNA was determined for both protein-free DNA and chromatin. Using saturating concentrations of drug, DNA within intact chromatin was found to be associated with only half as much drug as DNA extracted from its protein prior to drug exposure. T h e distribution of drug bound within chromatin was determined following the attachment of the monoazido analog (by photoactivation) to chromatin that had undergone limited nuclease digestion. Several distinct populations isolated by size fractionation and quantitative measurements revealed that (1 ) both the core particles and the spacer-containing particles contained bound drug, reflecting high-affinity binding sites; and (2) chromatin particles containing 150 DNA base pairs (putatively nucleosome core structures) contained less total hound drug a t high drug concentrations than those particles having intact spacer DNA.

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Antitrypanosomal Action Enhanced by Photoaffinity Labeling with Ethidium Analogs

Cited by 14 publications

References 17 publications

Efficiency of Photoaffinity Labeling Dna Homopolymers and Copolymers With Ethidium Monoazide *

Efficiency of Photoaffinity Labeling Dna Homopolymers and Copolymers With Ethidium Monoazide *

Photoactivated Ethidium Monoazide Directly Cleaves Bacterial DNA and Is Applied to PCR for Discrimination of Live and Dead Bacteria

Monoazido analog of ethidium as a chromatin probe: Binding to DNA

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