Micronomicin/tobramycin binding with DNA: fluorescence studies using of ethidium bromide as a probe and molecular docking analysis

Two aminoglycosides, micronomicin (MN), and tobramycin (TB), binding with DNA were studied using various spectroscopic techniques including fluorescence, UV–Vis, FT-IR, and CD spectroscopy coupled with relative viscosity and molecular docking. Studies of fluorescence quenching and time-resolved fluorescence spectroscopy all revealed that MN/TB quenching the fluorescence of DNA–EB belonged to static quenching. The binding constants and binding sites were obtained. The values of ΔH, ΔS, and ΔG suggested that van der Waals force or hydrogen bond might be the main binding force. FT-IR and CD spectroscopy revealed that the binding of MN/TB with DNA had an effect on the secondary structure of DNA. Binding mode of MN/TB with DNA was groove binding which was ascertained by viscosity measurements, CD spectroscopy, ionic strength, melting temperature (T_m), contrast experiments with single stranded (ssDNA), and double stranded DNA (dsDNA). Molecular docking analysis further confirmed that the groove binding was more acceptable result.     

The influence of rotor speed on the sedimentation behavior in sucrose gradients of high molecular weight DNA's

Anomalous sedimentation behavior has been observed for high molecular weight duplex DNA's in sucrose gradients. The sedimentation rate of DNA's having molecular weights of 10⁸ or higher is influenced by high centrifugal fields. The change in the sucrose sedimentation coefficient due to this effect, S^RPM_suc-S⁰_suc, is equal to 1 × 10^?48M^3.65( The anomalous behavior is not influenced by DNA concentration at sufficiently low concentrations. Because of the smallness of the coefficient this effect has not been previously detected for DNA's the size of T2 or smaller at rotor speeds below 40000 RPM. For example, the relative sedimentation coefficient of T2 DNA at 65 000 RPM is only 9% less than at 10000 RPM. However, the sedimentation profile of heterogeneous high molecular weight [(100 – 350) × 10⁶] E. coli DNA is severely altered even at moderate rotor speeds (37000 RPM). Therefore, it seems advisable to use low rotor speeds when sedimenting high molecular weight DNA's.     

Use of alkaline sucrose gradients in a zonal rotor to detect integrated and unintegrated avian sarcoma virus-specific DNA in cells.

We have attempted to distinguish integrated and unintegrated forms of avian sarcoma virus-specific DNA in cells by sedimentaton through an alkaline sucrose gradient in a slowly reorienting zonal rotor. Results obtained with this procedure are similar to those obtained by the more convenient analysis of networks of high-molecular-weight cell DNA. Most, if not all, viral DNA appears completely integrated into the host cell genome in an avian sarcoma virus-transformed mammalian cell and in normal chicken cells (in which viral DNA is genetically transmitted). Fully transformed duck cells and duck embryo fibroblasts infected for 20 to 72 h contain both integrated and unintegrated viral DNA; up to one copy per cell is integrated within 20 h after infection, and four to eight copies are integrated in fully transformed cells. The amount of unintegrated DNA varies but may comprise over 75% of the viral DNA in acutely infected cells and from 20 to 70% of the viral DNA in fully transformed cells. The unintegrated DNA in either case consists principally of duplexes with "minus" strands the length of a subunit of the viral genome (2.5 X 10(6) to 3 X 10(6) daltons) and relatively short "plus" strands (0.5 X 10(6) to 1.0 X 10(6) daltons).     

Utility of ethidium bromide in the extraction from whole plants of high molecular weight maize DNA

A procedure is reported for the isolation of high molecular weight maize DNA from whole plant tissue. Nuclei are isolated in the presence of ethidium bromide from leaf, node, and tassel or endosperm tissues and the DNA is extracted and purified. The resulting DNA has a double-strand molecular weight of about 125 kilobase pairs and a single-strand molecular weight of about 125 kilobases. The DNA is cleavable by a number of common restriction endonucleases.     

Parallel and antiparallel DNA: fluorescence quenching of ethidium bromide by 7-deazapurines

The fluorescence quenching of ethidium bromide caused by 7-deaza-2'-deoxyguanosine or 7-deaza2'-deoxyisoguanosine is observed in DNA with parallel or anti-parallel chain orientation.     

Sedimentation velocity of DNA in isokinetic sucrose gradients: calibration against molecular weight using fragments of defined length.

The relationship between sedimentation coefficient and molecular weight for DNA sedimenting in preformed alkaline and neutral sucrose gradients was determined using absolute molecular weight standards (restriction fragments of plasmid pBR322 and phage lambda DNA). The range of calibration for alkaline gradients was extended to small DNA fragments (652 base-pairs) for the first time. The exponent b in the equation S20 degrees, w = aMb was found to be 0.380 in neutral gradients and 0.410 in alkali. The latter value differs significantly from previous estimates. The gradients were isokinetic, and the distance sedimented was shown to be directly proportional to the sedimentation coefficient at all times.     

Covalent binding of ethidium azide analogs to Salmonella DNA in vivo: competition by ethidium bromide.

The photoreactive analogs of ethidium bromide (ethidium mono- and diazide) have been developed as drug probes to determine the actual molecular details of ethidium bromide interactions with DNA. In an effort to demonstrate that the analogs in fact mimic the parent ethidium, competition experiments were designed using ³H thymidine-labeled DNA in intact $\text{Salmonella}$ TA1538, which is reverted by the azide analogs. ¹⁴C-labeled ethidium azide analogs were used in combination with the non-labeled ethidium bromide. The results presented here demonstrate that the parent ethidium competes with the azide analogs as a DNA intercalating drug using CsCl density gradient ultracentrifugation.     

Stacking interactions of ethidium bromide bound to a polyphosphate and phage DNA in situ

Ethidium bromide forms spectroscopically detectable aggregates in aqueous solution and at a high dye concentration larger than 1 × 10^?3 moles/ρ. At moderate concentration in the order of 1 × 10^?4 moles/ρ the dye interacts with inorganic polyphosphate Graham salt and with phage sd DNA in situ by formation of stacking complexes. Maximal stacking was found at a phosphate to dye ratio, P/D, of approximately 1 for Graham salt and 1.5–2 for phages. In going to a higher P/D ratio Graham salt dye complex dissociates again and free dye reappears, while phage dye binding changes from stacking (type II complex) to intercalation (type I complex). Stacking is accompanied by a decrease and intercalation by an increase of relative fluorescence intensity with respect to free dye. However, both binding types lead to hypechromism and a red shift of the dye absorption band in the visible spectral region. Thus spectral behavior of ethidium aggregates deviate clearly from that known for other dyes, i.e., acridines.     

Decreased rate of uridine and glycerin uptake in L cells resistant to ethidium bromide

A comparison was made among rates of uptake of 3H-uridine, 3H-glycerol and 3H-D-xylose into mouse fibroblasts of line L sensitive to ethidium bromide (EB), and into EB-resistant cells obtained from this line by selection. Constants of uridine transport and phosphorylation were determined. For EB sensitive L cells Kt was 162 +/- 27 microM, Vt was 7.5 +/- 0.7 microM/sec. For EB resistant cells Kt was 178 +/- 27 microM, and Vt was 4.6 +/- 0.2 microM/sec. Thus, the transport rate of uridine in resistant cells was twice lower than in EB sensitive cells. The rate of uridine phosphorilation in EB resistant cells was by three times lower than in EB sensitive ones. The uptake of glycerol into resistant cells was also lowered. There was no difference in transport of 3H-D-xylose between sensitive and resistant cells. The data obtained may suggest some changes in plasma membrane in the EB resistant cells.     

The circular dichroism in the ultraviolet of aminoacridines and ethidium bromide bound to DNA

The circular dicrosim (CD) spectra of complexes of DNA with ethidiun bromnide, profiavine, 9-aminoacridine and 4-etliyl-9-amino-acridine have been determined between 220 and 450 nm, the range lieing extended to 600 nm for ethidiufm bromide. The variation of the magnitude of the visible and near—ultraviolet CD spectra of ethidium bromide—DNA complexes with the amount of ligand bound (r) suggests a common binding position with profiavine. On the other hand, 4-ethyl-9-aminoacndine complexed to DNA shows CD spectra not distinguishable from those of 9-aminnoacnidmc in both the visible and ultraviolet. The interpretation of these results with respect to the stereochemistry of the DNA-ligand complexes is discussed.     

Strand breakage in solutions of DNA and ethidium bromide exposed to visible light.

Breaks are introduced into DNA strands when DNA solutions containing ethidium bromide (EB) are exposed to incandescent light. The nicking rate is sensitive to the concentration of EB and the light intensity. At short exposure times, this rate is limited by photon capture and formation of an intermediate capable of nicking DNA and zero-order nicking kinetics are observed. If the EB is pre-irradiated, the nicking rate is limited by DNA concentration and first-order nicking kinetics are observed. The nicking rate is not greatly affected by the presence of a low frequency of ribonucleotides in the duplex structure. The nicking reaction produces neither double-strand breaks nor interstrand crosslinks. The nicks produced cannot be closed by DNA ligase. The fluorescent light intensities under normal laboratory conditions are insufficient to induce significant nicking.     

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

Two photoaffinity analogs of ethidium, 8-azido-3-amino, 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 the binding data determined from spectrophotometric titrations showed that the analogs interacted with both nucleic acids in a manner similar to the parent compound. The 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. The 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 bound drug at high drug concentrations than those particles having intact spacer DNA.     

Significant enhancement of fluorescence on hybridization of a molecular beacon to a target DNA in the presence of a site-specific DNA nickase

We have developed a simple isothermal (55 degrees C) reaction that permits detection of DNA targets using only two components: a molecular beacon and a site-specific DNA nickase without deoxyribonucleotide triphosphates and primers. The loop sequence of the molecular beacon should contain a DNA nickase recognition site. The nickase-molecular beacon (NMB) combination permits a 100-fold increase in fluorescent signal. The applications of the NMB assay for enhancement of fluorescent signal in some isothermal methods are discussed.     

The DNA and RNA specificity of eilatin Ru(II) complexes as compared to eilatin and ethidium bromide

Eilatin-containing ruthenium complexes bind to a broad range of different nucleic acids including: calf thymus (CT) DNA, tRNA(Phe), polymeric RNAs and DNAs, and viral RNAs including the HIV-1 RRE and TAR. The nucleic acid specificity of Lambda- and Delta-[Ru(bpy)2eilatin]2+ have been compared to that of the 'free' eilatin ligand, and to the classic intercalating agent ethidium bromide. Interestingly, all four compounds appear to bind to nucleic acids by intercalation, but the trends in nucleic acid binding specificity are highly diverse. Unlike ethidium bromide, both eilatin and the eilatin-containing coordination complexes bind to certain single-stranded RNAs with high affinity (K(d) < or = 1 microM). Eilatin itself is selective for electron-poor polymeric purines, while the eilatin-coordination complexes exhibit preference for the polypyrimidine r(U). These results show how the binding specificity of an intercalating ligand can change upon its incorporation into an octahedral metal complex.     

Aphidicolin and ethidium bromide as tools to verify dependence of early post-germinative growth on nuclear and organellar DNA synthesis

Embryos of Haplopappus gracilis (Nutt.) Gray were sown in aphidicolin (inhibitor of nuclear DNA synthesis) used alone or in combination with ethidium bromide (inhibitor of organellar DNA synthesis). In both cases germination and the early processes of post-germinative growth, including enlargement and greening of cotyledons, some elongation of radicle, and formation of root hairs, occurred at the same rate as in the controls. Comparable results were obtained using fluorodeoxyuridine. The effect of aphidicolin is, unlike fluorodeoxyuridine, completely reversible and the side effect on protein and RNA synthesis are negligible. Total non-dependence on nuclear and organellar DNA synthesis during early post-germinative growth may not be a generalized situation in all seeds, as was shown by some observations on lettuce and watermelon.