Characterizing the interaction between DNA and GelRed fluorescent stain

Abstract

We have performed single molecule stretching experiments and dynamic light scattering (DLS) in order to characterize the interaction between the DNA molecule and the fluorescent stain GelRed. The results from single molecule stretching show that the persistence length of the DNA-GelRed complexes increases as the ligand concentration increases up to some critical concentration, then decreasing for higher concentrations. The contour length of the complexes, on the other hand, increases monotonically as a function of GelRed concentration, suggesting that intercalation is the main binding mechanism. In order to characterize the physical chemistry of the interaction, we use the McGhee-von Hippel binding isotherm to extract the physicochemical parameters of the interaction from the contour length data. Such analysis has allowed us to conclude that the GelRed stain is in fact a bis-intercalator. In addition, DLS experiments were performed to study the changes of the effective size of the DNA-GelRed complexes, measured by the hydrodynamic radius, as a function of ligand concentration. We found a qualitative agreement between the results obtained from the two techniques by comparing the behaviors of the hydrodynamics radius and the radius of gyration, since this last quantity can be expressed as a function of mechanical parameters determined from the stretching experiments.

Extracting physical chemistry from mechanics: a new approach to investigate DNA interactions with drugs and proteins in single molecule experiments

“…This feature is particularly important in the analysis of DNA interactions with intercalators, a class of ligands in which neighbor-exclusion effects is extremely important [70][71][72] . In fact, NEM has become in the past years the standard binding isotherm used to analyze DNA interactions with intercalators [73][74][75][76][77] .…”

“…B. Chaires, D. M. Crothers and coworkers in the 80's 60,73,137,139 . On the other hand, the DNA-EtBr interaction was characterized in various aspects by many authors, but even today one can found somewhat contradictory results about the mechanical behavior of such complexes 23,56,75,77,[140][141][142] , and also for different complexes formed between DNA and other intercalators, especially when comparing results obtained from different experimental techniques 56,[75][76][77]115,[140][141][142][143][144][145][146] .…”

A comparison of DNA stains and staining methods for Agarose Gel Electrophoresis

“…Biotium's website states "because GelRed™ and GelGreen™ are high affinity dyes designed to be larger dyes to improve their safety, they can affect the migration of DNA" (Biotium 2018). Crisafuli et al (2015) suggest GelRed™ to be a bis-intercalator, formed from two cross-linked Ethidium Bromide molecules. If this is so, they state GelRed should be twice as sensitive as EthBr since "the GelRed assay will have approximately twice as many DNAbound sites".…”

The changing landscape of membrane protein structural biology through developments in electron microscopy

“…Indeed, the function of proteins is known to undergo large changes upon purification within a detergent (Postis et al., 2015) casting some doubt on the physiological relevance of some of the membrane protein structures currently available from both EM and crystal studies. Several groups have been developing methodology to combat this lack of native lipids, of particular interest are the efforts to introduce a more native environment through the use of SMALPs and the progress being made in imaging proteins in situ , within a vesicle (Dörr et al., 2015; Frauenfeld et al., 2016; Lee et al., 2016; Wang & Sigworth, 2009, 2010). These in situ studies would not only allow protein activity to be measured in the imaged sample, but also potentially allow different conformations of the protein to be observed by inducing a membrane potential or a chemical gradient through the addition of additives to the sample.…”