Ethidium bromide interactions with DNA: an exploration of a classic DNA–ligand complex with unbiased molecular dynamics simulations

Abstract

Visualization of double stranded DNA in gels with the binding of the fluorescent dye ethidium bromide has been a basic experimental technique in any molecular biology laboratory for >40 years. The interaction between ethidium and double stranded DNA has been observed to be an intercalation between base pairs with strong experimental evidence. This presents a unique opportunity for computational chemistry and biomolecular simulation techniques to benchmark and assess their models in order to see if the theory can reproduce experiments and ultimately provide new insights. We present molecular dynamics simulations of the interaction of ethidium with two different double stranded DNA models. The first model system is the classic sequence d(CGCGAATTCGCG)2 also known as the Drew–Dickerson dodecamer. We found that the ethidium ligand binds mainly stacked on, or intercalated between, the terminal base pairs of the DNA with little to no interaction with the inner base pairs. As the intercalation at the terminal CpG steps is relatively rapid, the resultant DNA unwinding, rigidification, and increased stability of the internal base pair steps inhibits further intercalation. In order to reduce these interactions and to provide a larger groove space, a second 18-mer DNA duplex system with the sequence d(GCATGAACGAACGAACGC) was tested. We computed molecular dynamics simulations for 20 independent replicas with this sequence, each with ∼27 μs of sampling time. Results show several spontaneous intercalation and base-pair eversion events that are consistent with experimental observations. The present work suggests that extended MD simulations with modern DNA force fields and optimized simulation codes are allowing the ability to reproduce unbiased intercalation events that we were not able to previously reach due to limits in computing power and the lack of extensively tested force fields and analysis tools.

Protection of Double-Stranded RNA via Complexation with Double Hydrophilic Block Copolymers: Influence of Neutral Block Length in Biologically Relevant Environments

“…It is worth noting that this phenomenon has been shown to have a larger impact on dsRNA than on DNA. 59 , 60 , 65 , 69 The fluorescence intensity of polyplexes formed with dsRNA is plotted as a function of the NaCl concentration in Figure 8 A. To explain the behavior of these systems, we analyze the results alongside size analysis (see Figure S6 in the SI) to determine the decomplexation point.…”

“…Prior to the addition of NaCl to polyplex formulations, the relative fluorescence intensity of the polyplexes is low as a result of the displacement of EB from dsRNA. 61 , 63 , 65 − 67 , 69 …”

Dicationic styryl dyes for colorimetric and fluorescent detection of nucleic acids

“…The DNA models used in this experiment consist of d(AT) 10 and d(GC) 10 to ensure a homogeneous binding mode. In general, EtBr can intercalate to both AT-rich and GC-rich DNAs 56 , while DAPI could only bind to minor grooves of AT-rich DNA 57 . As shown in Fig.…”

Progress on the Physiological Function of Mitochondrial DNA and Its Specific Detection and Therapy

“…Due to the use of agarose gel electrophoresis in 1967, DNA staining has become essential. At the beginning, radiolabeling was the only way to stain DNA in gels, until ethidium bromide was found to intercalate with DNA after gel electrophoresis and became the main colorant from 1972 [42] . It was later discovered that 4,6‐TOTO‐1 stains DNA by binding in small grooves rich in A−T sequences [43] .…”