The influence of GC/AT composition on intercalating and semi-intercalating binding of ethidium bromide to DNA

Abstract

Os parâmetros de ligação de brometo de etídio (EtBr) com DNA em diversas proporções GC/AT foram determinados usando espectroscopia de absorção e fluorescência. Nossos dados experimentais demonstram claramente a co-existência de tipos de ligação "forte" fluorescente e não-fluorescente em baixas concentrações de EtBr. O complexo fluorescente corresponde ao modelo normal intercalante. O complexo não-fluorescente refere-se à ligação semi-intercalante de EtBr. A constante de ligação (K) e o número de pares de bases correspondentes ao sítio de ligação (n) dos tipos fluorescente (K f e n f ) e não-fluorescente (K nf , n nf ) de interações foram determinados. O tamanho médio do sítio de ligação (n) é igual a 1,5 bp (espectroscopia de absorção), e n f ca. 2 bp (espectroscopia de fluorescência). Mostrou-se que n nf é dependente da quantidade de GC e n total é independente.The binding parameters of ethidium bromide (EtBr) with DNA of various GC/AT ratios were determined using absorption and fluorescence spectroscopy. Our experimental data clearly demonstrate the co-existence of fluorescing and non-fluorescing types of "strong" binding at low concentration of EtBr. The fluorescent complex corresponds to the ordinary intercalative model. The non-fluorescent complex is referred to semi-intercalative binding of EtBr. The binding constant (K) and the number of base pairs corresponding to a binding site (n) of the fluorescent (K f and n f ) and non-fluorescent (K nf , n nf ) types of interactions were determined. The average size of binding site (n) is equal to 1.5 bp (absorption spectroscopy), and n f ca. 2 bp (fluorescence spectroscopy). It was shown that n nf is dependent on GC-content and total n is independent of it.

Real-time Monitoring and Detection of Primer Generation-Rolling Circle Amplification of DNA Using an Ethidium Ion-selective Electrode

“…32,35 The latter is caused by hydrogen bonding and electrostatic interaction between the negatively charged sugar-phosphate DNA backbone and the positively charged Et + cation. 32,34,35 In this way, EtBr binds to the free DNA, leading to the inhibition of hybridization between the target DNA or the primer and the circular probe, as well as the inhibition of DNA polymerase binding to the target DNA. Thus, increasing the EtBr concentrations above a certain extent causes a decrease of the amplification efficiency.…”

“…DNA amplification is performed by PG-RCA in the presence of ethidium bromide (EtBr), which is a cationic fluorochromic dye that inserts into double-stranded DNA (dsDNA). [29][30][31][32][33][34][35][36] A potentiometric system consisting of a small ethidium ion-selective electrode (Et + ISE) as a working electrode and a small reference electrode has been developed. ISEs can directly determine the target ionic species selectively in complex sample solutions.…”

Exploring the Interaction of Ethidium Bromide and HOECHST 33258 with DNA by Means of Electrochemical Approach

“…For this purpose, comparative studies of interaction of both classic intercalator EtBr and groove binder H33258 with DNA by the same method are appropriate, since these ligands bind to DNA by several and principally various modes. [18][19][20][21] Being a stabilizer of DNA double-stranded (ds-) structure EtBr speci¯cally binds to single-stranded (ss-) oligonucleotides and polynucleotides as well. 21 Interacting with DNA H33258 is localized in minor groove of DNA and shows high speci¯city to AT-sequences.…”

“…[11][12][13][14][15][16][17] For investigation of DNA structural and reaction alterations, the temporary electrochemical methods acquire a wide analytic, scienti¯c and applicative value due to their high sensitivity and precision. 18,19 The voltammetry methods of investigations of DNA binding to various molecules can provide a useful complement to spectroscopic methods, e.g., for nonabsorbing species, and yield in information about the mechanism of binding. [21][22][23][24] Among voltammetry methods, square wave voltammetry (SWV) is one of the most sensitive ones, which permits carrying out measurements at low concentrations of reagents and more precisely¯nding out various types of interactions of ligands with DNA.…”

Measuring Fluorescence Anisotropy as One of Very Useful Analytical Methods to Obtain Detailed Information of the Complex Binding Interaction

“…Ethidium bromide (EtBr) has been known to intercalate between base pairs of double helical DNA, causing minor disturbances in DNA structure . Some studies reported additional binding types of EtBr to the DNA double helix, such as binding in the major groove, semi‐intercalation, and electrostatic binding at the phosphate groups of the backbone chain, depending on the ionic strength, the base sequence of DNA, and the ratio of [DNA] to [EtBr] . From all studies, the primary binding mode of EtBr to DNA is thought to be intercalation, but a few different kinds of binding modes could be possible, depending on the environmental conditions.…”