Characterizing the Interaction Between DNA and GelRed Fluorescent Stain.
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.
Keywords:intercalation single molecule stretching dynamic light scattering binding isotherm
pacs:82.37.Rs 87.14.gk 87.80.Cc
Rational drug design is a process in which a new compound is sketched and synthesized to achieve a specific biological target and/or to perform specific functions. In health sciences, for example, there is much interest in developing new drugs to treat human diseases such as cancer. In biochemistry and molecular biology, new drugs are usually developed to stain DNA and proteins, allowing visualization by fluorescence microscopy and the possibility of following the route of various biological processes.
GelRed is a fluorescent nucleic acid stain designed with the purpose of replacing the highly toxic ethidium bromide (EtBr) in gel electrophoresis and other experimental techniques which depends on the fluorescence of stained DNA. When bound to DNA, GelRed has the same absorption and emission spectra of EtBr and, according to its manufacturer (Biotium Inc., Hayward, CA, USA), the compound can be used in electrophoresis experiments with greater sensitivity than EtBr, with the advantage of being much less toxic and mutagenic GRSafety (), Huang (). This last property, according to the manufacturer, is due to the fact that the chemical structure of the dye was designed in a way such that it is incapable of crossing cell membranes GRSafety (). The chemical structure of the GelRed dye is proprietary and was not officially reported by the manufacturer. Nevertheless, one can find unofficial information in the internet claiming that GelRed is synthesized basically by crosslinking two EtBr molecules WikiGelRed (), which suggests that the dye is maybe a bis-intercalator.
Even though the manufacturer states that GelRed binds to DNA via a combination of intercalation and electrostatic binding GRSafety (), most details of the interaction are so far not reported in the literature. In this work we have performed single molecule stretching experiments and dynamic light scattering in order to gain insights about such interaction. Recently, we have developed a methodology that allows one to easily extract the physical chemistry of the DNA-ligand interaction from pure mechanical DNA parameters such as those easily obtained from single molecule stretching. This methodology allows one to determine the relevant physicochemical parameters of the interaction and to deduce the particular binding mechanism(s) Siman (), Cesconetto (), Silva (). The purpose of the present work is to perform a robust characterization of the DNA-GelRed interaction, determining the changes of the basic mechanical properties of the DNA molecule as GelRed binds, the physicochemical parameters of the interaction and the nature of the binding mechanism.
2 Materials and Methods
2.1 Stretching experiments
In these experiments the samples consist of -DNA molecules end-labeled with biotin in a phosphate-buffered saline (PBS) solution with [NaCl] = 140 mM. One end of the DNA molecule is attached to a streptavidin-coated glass coverslip using the procedure reported in ref. Amitani () while the other end of the molecule is attached to a streptavidin-coated polystyrene bead with 3 m diameter (Bangs Labs). As described earlier CrisafuliIB (), Cesconetto (), in this configuration one can easily trap the polystyrene bead with the optical tweezers and stretch the DNA molecule by moving the microscope stage with a piezoelectric actuator. The sample chamber consists of an o-ring glued in the coverslip, such that one can exchange the buffer and consequently the ligand concentration without affecting the trapped DNA molecule by using micropipettes. The DNA base-pair concentration used in all stretching experiments was C = 2.4 M.
The optical tweezers consist of a 1064 nm ytterbium-doped fiber laser with a maximum output power of 5.8 W (IPG Photonics) mounted on a Nikon Ti-S inverted microscope with a 100 N.A. 1.4 objective. The apparatus is previously calibrated by two independent methods as described earlier CrisafuliIB (). Once calibrated, the optical tweezers are used to trap the polystyrene bead attached to a DNA molecule, allowing one to perform the DNA stretching with high resolution and consequently to obtain the force extension curves of the DNA-ligand complexes for each studied situation. These curves were fitted to the Marko-Siggia WormLike Chain (WLC) model Marko (). Therefore, one can study how the persistence and contour lengths of the DNA molecule vary as a function of ligand concentration. All the stretching experiments were performed in the entropic low-force regime ( 2 pN), in order to avoid enthalpic contributions to the values of the mechanical parameters CrisafuliIB (), Cesconetto (). All the experimental details can be found in our references CrisafuliIB (), Cesconetto (), Silva (), Reis ().
2.2 Dynamic Light Scattering (DLS)
In order to confirm the results obtained from the stretching experiments with a second technique, we have also performed DLS on the DNA-GelRed complexes. DLS performed at a fixed scattering angle allows one to evaluate the hydrodynamic radius of the complexes, a parameter that gives an estimation of the size of the complexes Reis (). This parameter was measured in order to investigate the effect of the ligand on the effective size of the DNA molecule, which depends on its persistence and contour lengths. Thus, the results obtained from two very different experimental techniques can be compared (at least indirectly).
The DLS apparatus used was a ZetaSizer Nano-S (Malvern Instruments Ltd) with a low volume cuvette (ZEN2112, Hellma Analytics). The backscattering angle used was 173 in all experiments. The particle size was determined using the Non-Negative Least Squares (NNLS) algorithm.
The DNA used in these experiments was a 3000 bp molecule (Thermo Scientific) in the same buffer used in the optical tweezers samples (-DNA is difficult to be used in DLS due to the long contour length). The DNA molecules are equilibrated with a certain concentration of GelRed directly in the cuvette used in the DLS apparatus. The DNA concentration used in all DLS experiments was the same one used in the optical tweezers (2.4 M of base-pairs). This concentration is sufficiently low to avoid entanglements and relevant interactions between different DNA molecules Hur ().
3.1 Stretching experiments
In Fig. 1 we show the behavior of the persistence length of DNA-GelRed complexes as a function of ligand total concentration in the sample . Observe that it initially increases from the bare DNA value ( nm) until reaching a maximum value of nm at C = 4.0 M. For higher concentrations, it abruptly decays to nm.
In Fig. 2 we show the behavior of the contour length of DNA-GelRed complexes as a function of ligand total concentration in the sample . Observe that monotonically increases from the bare DNA value (m) up to a saturation value of m.
To achieve these results, the experiments were performed as follows. Firstly, we choose a particular bare DNA molecule and stretch it five times, determining the mean values of the persistence and contour lengths. Then, we change the ligand concentration in the sample using a micropipette. After changing the concentration, we wait minutes for the ligand to equilibrate with DNA. This time scale is sufficient for GelRed to equilibrate with DNA for our experimental conditions. This fact was verified by analyzing the reversibility of the stretching curves and by performing some experiments waiting longer time intervals, finding no significant difference. We then perform again five stretching experiments, obtaining the new values of the persistence and contour lengths for the chosen GelRed concentration. This procedure is then repeated sequentially for each ligand concentration. We have also repeated the entire procedure, scanning all the concentrations, for other DNA molecules using different samples. The error bars presented in Fig. 1 and Fig. 2 for each concentration are the standard errors obtained from this set of measurements.
The behavior of both persistence and contour lengths strongly suggests that the dominant mechanism of interaction between DNA and GelRed is the intercalative binding. In fact, the general behavior of the persistence length shown in Fig. 1 was previously verified by our group for the intercalators ethidium bromide (EtBr), daunomycin, psoralen and diaminobenzidine under nearly similar experimental conditions RochaJCP2 (), RochaPB (), RochaAPLPso (), Reis (), by using optical tweezers in the low-force regime ( 2 pN). Some authors have also reported the same qualitative behavior of the persistence length for intercalators, finding that this parameter increases for low ligand concentrations and decreases for higher concentrations Tessmer (), Cassina (), Kaji (). In refs. RochaPB (), RochaAPLPso () it was proposed that the abrupt decrease of the persistence length shown in Fig. 1 for 4 M is related to partial DNA denaturation, with the formation of denaturing bubbles, probably due to the pulling force RochaJCP2 (), RochaPB (), RochaAPLPso (), Reis ().
In addition, it is well established that intercalators always increase the DNA contour length when binding, by increasing the axial distance between two adjacent base-pairs in the intercalative site Sischka (), Fritzsche (), Chaires (). The other common types of interactions between DNA and ligands, such as groove binding, electrostatic interaction or covalent binding, do not increase the DNA contour length. On the contrary, in some cases these kinds of interaction can cause DNA compaction with a decrease of the “apparent contour length” measured by force spectroscopy in the low-force regime used in our experiments CrisafuliIB (), Silva (). Therefore, due to the strong increase of for the DNA-GelRed complexes, we can conclude that if there exist other binding mode, it is certainly much weaker than intercalation.
The physicochemical properties of the interaction can be extracted from the contour length data. Firstly, we use the data of Fig. 2 to determine the fractional increase of the contour length, = ( - )/, where is the bare DNA contour length, as a function of ligand concentration . This data is plotted in Fig. 3 (circles). For intercalators this fractional increase is proportional to the bound ligand fraction = /, while is the bound ligand concentration and is the DNA base-pair concentration. In other words, , where is the ratio between the increase of the contour length due to a single intercalating molecule () and the mean distance between two consecutive base pairs in the bare DNA ( 0.34 nm) RochaJCP2 (), RochaPB (), Daune (). For typical monointercalating molecules one has 0.34 nm and consequently 1 Sischka (), Fritzsche (). The bound fraction (and consequently the mechanical parameter ) can be linked to the physicochemical properties by using a binding isotherm. The McGhee - von Hippel binding isotherm usually describes very well the physical chemistry of the DNA interactions with intercalators, since it computes in detail the neighbor exclusion effects which are usually associated to this type of interaction McGhee (), RochaNEM (). This binding isotherm reads
where is the exclusion number (a measure of the effective number of base-pairs occupied by a single ligand molecule McGhee ()), is the intrinsinc equilibrium association constant and = - is the free (not bound) ligand concentration in solution.
In Fig. 3 we show a fitting (solid line) of this binding isotherm to the experimental data, by using the computational method described in detail in ref. Cesconetto (). Observe that the model describes well the experimental data. The fitting returns the parameters = 3.8 0.5, = (2.4 0.3) 10 M, and = 1.9 0.1. These results strongly suggest that the GelRed dye is a DNA bis-intercalator. In fact, the exclusion parameter indicates that each bound GelRed molecule effectively occupies 3.8 DNA base-pairs, a value considerably higher than the results found for most monointercalators, and approximately twice the result for EtBr RochaPB (), Chaires (), Gaugain (). The equilibrium constant is also higher than the result obtained for typical monointercalators ( 10 M) RochaJCP2 (), RochaPB (), Chaires (), Gaugain (), and within the range found for most bis-intercalators (10 to 10 M) Gunther (), Berge (), Murade (), Maaloum (), Garbay (). In particular, it is two orders of magnitude higher than the equilibrium constant for EtBr, a situation very similar to what occurs for the bis-intercalator YOYO when compared to its precursor YO, a monointercalator as EtBr Murade (). Finally, the result = 1.9 0.1 is approximately twice the value obtained for typical monointercalators, suggesting that each bound GelRed molecule increases the DNA contour length by 0.65 nm, a result also compatible to typical bis-intercalators Gunther (), Maaloum (). Observe that the bis-intercalators should increase approximately twice the DNA contour length per bound molecule, since each ligand molecule contains two intercalating portions.
These results together are a strong evidence that the GelRed dye is in fact a bis-intercalator probably consisting of two EtBr molecules, as claimed before WikiGelRed (). In fact, if one supposes that GelRed is really a bis-intercalator formed by crosslinking two EtBr molecules, it is straightforward to understand the statement of the dye manufacturer which claims that GelRed presents a higher sensitivity in electrophoresis experiments when compared to EtBr GRSafety (): if one prepares two electrophoresis assays staining one of them with EtBr and the other with GelRed at the same molar concentrations, the GelRed assay will present approximately twice DNA bound sites (at least for concentrations far from saturation), which implies in more fluorescence signal and consequently more contrast. In addition, the fact that the absorption/emission spectra of the two compounds are the same GRSafety () can be easily understood with the discussion above.
3.2 DLS experiments
In Fig. 4 (circles) we show the behavior of the hydrodynamic radius of DNA-GelRed complexes, obtained from the DLS experiments, as a function of GelRed concentration. Observe that increases monotonically with GelRed concentration, from 87 nm measured for bare DNA, up to 207 nm obtained for = 6 M. Each experimental point is the mean value calculated from a set of 100 measurements of 15 seconds long, and the error bars are the standard deviations. In the same figure, we represent an estimation of the radius of gyration for the DNA-GelRed complexes (squares). was calculated as a function of the mechanical parameters obtained from the tweezers experiments as Daune (). We have used the values of the persistence length shown in Fig. 1 and have assumed that the contour length of the 3000 bp DNA will increase with the ligand concentration on the same ratio of that shown in Fig. 3 for the -DNA. As pointed before in ref. Reis (), since the 3000 bp DNA is sufficiently long (it contains 20 persistence lengths), it is reasonable to expect that base-pair sequence and other molecular details do not interfere on large-scale mechanical properties such as the persistence and contour lengths.
The radius of gyration increases with both and . Thus, as shown in Fig. 4, decreases for the two largest concentrations used, due to the abrupt decrease of the persistence length shown in Fig. 1. A similar decrease was not verified for the hydrodynamic radius , indicating that the abrupt transition of the persistence length do not occur in the samples used for the DLS experiments. The same result was previously verified by our group for the intercalator diaminobenzidine Reis (). As discussed in this previous work, such result was expected since the abrupt transition of the persistence length is probably related to the pulling force exerted in the stretching experiments, which may locally denature the previously deformed double-helix structure of the DNA-intercalator complex RochaPB (), RochaAPLPso (). In this way, one can say that the behavior of obtained in DLS experiments qualitatively agrees with the results obtained from the optical tweezers experiments. In addition, our DLS results also agree with the results obtained by most authors who have measured the persistence length of DNA-intercalator complexes with non-stretching techniques (fluorescence microscopy, electron microscopy, viscosimetry, etc) Yoshikawa92 (), Quake (). These authors have found that intercalators, aside increasing the DNA contour length, in general also increase the DNA persistence length under these conditions, thus increasing the effective size of the DNA-ligand complexes. Nevertheless, it is important to mention that some works performed with typical DNA-stretching techniques (optical or magnetic tweezers) have found a monotonic decrease of the DNA persistence length as a function of the intercalator concentration Murade (), Sischka (), Lipfert (). In our opinion such results are due to the force regime used to perform the measurements, since as the forces used to stretch the DNA increase, the probability of forming denaturation bubbles in the highly distorted double-helix of the DNA-intercalator complexes increases accordingly, thus decreasing the persistence length. Other factors that can surely influence the results of such measurements are the salt concentration used in the buffers, the model used to fit force-extension data (which may include DNA stretch modulus if forces are 10 pN), and the ratio of ligand concentration per DNA base pair concentration RochaJCP2 ().
By using two very different experimental techniques (single molecule stretching and dynamic light scattering) we have characterized the interaction of the DNA molecule with the fluorescent stain GelRed, determining the changes of the mechanical properties of DNA-GelRed complexes as a function of ligand concentration and extracting the physical chemistry of the interaction from these data. It was found that GelRed binds strongly to DNA ( 10 M). In addition, we have estimated that each bound GelRed molecule effectively occupies 3.8 DNA base-pairs and increases the contour length by 0.65 nm. These numbers are compatible to the results expected for bis-intercalating molecules, which has allowed us to determine the main binding mechanism of the GelRed dye and to understand the higher sensitivity presented by this compound when compared to ethidium bromide in electrophoresis experiments.
Acknowledgements.This work was supported by the Brazilian agencies: Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). The DLS experiments were performed in “Laboratório de Microfluídica e Fluidos Complexos (LMFFC)” of Universidade Federal de Viçosa.
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