Real-time label-free analysis of the thermostability of DNA structures using GelRed

Real-time label-free analysis of the thermostability of DNA structures using GelRed 1 2 first-author 2 3 2 2 3 2 3 2 corresp Corresponding authors: lijiang@sinap.ac.cn(Jiang Li), Corresponding authors: lijiang@sinap.ac.cn(Jiang Li), lijiang@sinap.ac.cn 1 corresp xujiaqiang@shu.edu.cn (Jia-Qiang Xu), xujiaqiang@shu.edu.cn (Jia-Qiang Xu), xujiaqiang@shu.edu.cn 2 corresp wanglihua@sinap.ac.cn (Li-Hua Wang) wanglihua@sinap.ac.cn (Li-Hua Wang) wanglihua@sinap.ac.cn NEST Lab, Department of Chemistry, College of Science, Shanghai University, Shanghai 200444, China NEST Lab, Department of Chemistry, College of Science, Shanghai University, Shanghai 200444, China Division of Physical Biology and Bioimaging Center, CAS Key Laboratory of Interfacial Physics and Technology, Shanghai Institute of Applied Physics, Chinese Academy of Sciences, Shanghai 201800, China Division of Physical Biology and Bioimaging Center, CAS Key Laboratory of Interfacial Physics and Technology, Shanghai Institute of Applied Physics, Chinese Academy of Sciences, Shanghai 201800, China University of Chinese Academy of Sciences, Beijing 10049, China University of Chinese Academy of Sciences, Beijing 10049, China In biological systems, conformational transformations of nucleic acids play critical roles in genetic regulation. However, it remains a tricky task to design and optimize specific labeling strategies to track these changes. In this study, we exploited an intercalating fluorescent dye, GelRed, to characterize different DNA structures. We studied the correlation between fluorescence intensity and DNA structural properties. We showed that single-stranded DNAs with predicted self-folded secondary structures show much stronger fluorescence than those without such structures. For double-stranded DNAs, we observed that fluorescence intensity is positively correlated to their GC content. We also demonstrated that GelRed can be used to monitor DNA conformational changes upon temperature variations in real time. Based on these findings, we concluded that the fluorescence intensity of a GelRed-stained DNA structure has a good correlation with its thermostability in the form of a change in Gibbs free energy. GelRed Secondary structure GC content Conformational transformation 1 Introduction 1 In living organisms, conformational transformation of nucleic acids, especially genomic DNA, plays a vital role in genetic regulation and, thus, in biological functions such as growth and reproduction [ 1 1 - 6 6 1-6 ] . In the realm of DNA nanotechnology, a variety of nanodevices/nanomachines empowered by DNA conformational transformations have also been developed, showing great promise in smart theranostic applications [ 7 7 - 16 16 7-16 ] . Therefore, it is fundamentally important to develop methods for real-time monitoring of DNA conformational changes. In recent years, much progress has been made to develop fluorescence/luminescence platforms specifically responsive to structural alterations [ 17 17 - 23 23 17-23 ] . For example, dual-labeling strategies based on Förster fluorescence resonance energy transfer (FRET) have been widely exploited [ 24 24 - 27 27 24-27 ] . Because the FRET effect is highly sensitive to the spatial distance between labeled sites, one can obtain real-time information about conformational changes by monitoring the fluorescence variations resulting from FRET [ 28 28 - 30 30 28-30 ] . However, it remains a tricky task to choose proper sites on target structures for FRET labeling; thus, case-by-case optimization is often required to ensure the effectiveness of FRET responses [ 31 31 ] . On the other hand, fluorescent intercalating dyes, which emit fluorescence when intercalated into double-helices of nucleic acids, provide a general and label-free way to characterize nucleic acid structures. For example, ethidium bromide (EtBr), a classic intercalating dye, has been routinely used in nucleic acid analysis applications, such as imaging of electrophoresis, assay of DNA damage in cells, and studies on interactions between DNA and drugs [ 32 32 - 36 36 32-36 ] . More recently, it has been used in detecting DNA mutations with the aid of graphene oxide [ 37 37 ] and in monitoring dynamic structural changes of DNA nanostructures [ 38 38 ] . Nevertheless, EtBr has been proven to be mutagenic by Ames tests [ 39 39 ] and is, thus, potentially harmful to its users and the environment. GelRed is an updated version of EtBr. A GelRed molecule is composed of two EtBr molecules conjugated with a linker [ 40 40 ] . It shows higher sensitivity in nucleic acid characterization, while exhibiting much less genetic toxicity compared to EtBr. Thus, as a substitute to EtBr, GelRed has been widely used in qualitative and semiquantitative analyses of nucleic acids [ 41 41 - 44 44 41-44 ] . However, as far as we know, there have been few studies on tracking conformational changes of DNA structures using GelRed. Here, we used GelRed to characterize different DNA structures, including single-strand (ss-) DNAs with or without self-folded secondary structures and double-strand (ds-) DNA with different base compositions. We analyzed the fluorescence intensity from these GelRed-stained DNA structures. We also monitored the fluorescence dynamics of a DNA structure upon cyclic temperature variations. We found that the florescence intensity of a GelRed-stained DNA structure has a good correlation with its predicted Gibbs free energy, which can be utilized in studying DNA conformational changes. 2 Materials and methods 1 2.1 Materials 2 GelRed was purchased from Biotium. All ssDNAs were purchased from Sangon Biotech (Shanghai, China). Tris, EDTA-2Na•2H 2 O, and Mg(CH 3 COOH) 2 •4H 2 O were all purchased from Sinopharm Chemical Reagent Company (Beijing, China). The 20 bp DNA marker was purchased from Takara (Kusatsu, Japan). 2.2 Preparation and UV-quantification DNA structures 2 The dsDNAs in this study were obtained by annealing complementary ssDNAs of equimolar amounts (1 μM) in 1×TAE buffer, which were then heated to 95 °C and slowly cooled to 25 °C in 5.5 h. All the ssDNA was quantified using a UV-Visible Spectrophotometer (Agilent Cary 100 Bio, PaloAlto, USA) with a dilution ratio of 100 in the 1×TAE buffer (40 mM Tris, 2 mM EDTA-2Na•2H 2 O, and 12.5 mM Mg(CH 3 COOH) 2• 4H 2 O ); then, the molar concentration was calculated using the formula C = ( A 260 - A 330 ) / (ε× M )× D , where C is the molar concentration of ssDNA; A 260 and A 330 are the absorbance of DNA at the wavelength of 260 nm and 330 nm, respectively; ε is the extinction coefficient of DNA; M is the molecular weight of the ssDNA; and D is the dilution ratio. 2.3 Fluorescence spectroscopy of GelRed-stained DNA 2 GelRed was diluted with 1×TAE buffer to a working solution of 20×. Then, 90 μL of DNA structures of different concentrations was mixed with 10 μL 20×GelRed working solution. The fluorescence was read immediately using a Multiscan Spectrum (Bio Tek Synergy MX H1, Gene Company Ltd, Hong Kong, China) and fluorospectrophotometer (Edinburgh FS920, Livingston, Britain) with the excitation wavelength of 303 nm and the emission wavelength of 600 nm. For temperature-dependent fluorescence dynamic analysis, the fluorescence intensity of the GelRed-stained DNA samples was monitored at 600 nm with the excitation wavelength at 520 nm using a real-time PCR instrument (StepOnePlus, Life Technologies, Singapore). The samples were held at 10 °C for 10 min and then heated to 95 °C at a heating rate of 1 °C/min. The melting temperature (Tm) values of the complexes were calculated by obtaining the main peak value from derivative melting curves, which are the first derivatives of the original fluorescence diagram. 2.4 Gel electrophoresis analysis 2 Different volumes (3 μL, 2 μL, 1 μL, 0.5 μL, 0.25 μL, and 0.1 μL) of a 20 bp DNA marker (100 μg/mL) were separately loaded in a 10% polyacrylamide gel (with TAE buffer) and stained with 1×GelRed in 1×TAE buffer for 20 min. Electrophoresis was carried out in 1×TAE buffer under 120 V for 1 h. 3 Results and discussion 1 3.1 Dyeing mechanism of GelRed 2 Fig. 1a Fig. 1a shows the chemical structures of EtBr and GelRed, respectively [ 40 40 ] . GelRed can be regarded as a dimeric EtBr (with Br - replaced by I - ) connected by a hydrocarbon linkage, which has been proven to be a bis-intercalator [ 23 23 , 40 40 , 41 41 , 45 45 ] . The schematic in Fig. 1b Fig. 1b illustrates the possible manner of its intercalation into a DNA double helix. 3.2 Fluorescence quantification of dsDNA 2 First, we tested the capability of GelRed to quantify DNA structures. Here we used the 20 bp DNA ladder as the sample, which can be regarded as a mixture of dsDNAs of varying lengths (ranging from 20 bp to 500 bp). We conducted electrophoresis with a gel loaded with different quantities of the sample. The resulting image ( Fig. 2a Fig. 2a ) shows that along with the decline of DNA quantity (from left to right, 300 ng, 200 ng, 100 ng, 50 ng, 25 ng, and 12.5 ng), the fluorescence intensity dropped correspondingly. We quantified the band intensity from the gel image ( Fig. 2b Fig. 2b ) and found that it linearly correlated to the quantity of DNA ( Fig. 2c Fig. 2c ). The linear regression function is y =141.5 x -10.9 ( R 2 =0.995), where y is the fluorescence intensity (arbitrary unit, or a.u.) obtained from the gel bands and x is the quantity of DNA. These results indicate that the amount of dsDNA structures can be well quantified by measuring their GelRed fluorescence intensity. 3.3 Discrimination of different DNA structures with GelRed 2 We investigated the fluorescence variations among different DNA structures. We designed two ssDNAs with the same length (20 nt) but of different sequences. According to the predictions from NUPACK, one of them can form a self-folded secondary structure (referred to as stem-loop ssDNA, with predicted Gibbs free energy change Δ G =-3.83 kcal/mol, under 25 °C) and the other cannot (referred to as linear ssDNA). The fluorescence spectroscopic measurements ( Fig. 3a Fig. 3a ) showed that the GelRed-stained stem-loop ssDNA showed ~4.8-fold stronger fluorescence compared to the linear one of equal concentration. Meanwhile, the dsDNA of equal length and molar concentration showed much stronger fluorescence compared to both ssDNAs (~3.4-fold stronger than that of the stem-loop ssDNA and ~16.3-fold stronger than that of the linear ssDNA). Considering these results and the mechanism of GelRed staining, we can deduce that DNA structures with more paired bases can be intercalated with more GelRed molecules, thus showing stronger fluorescence. Here, the fully complementary dsDNA has 20 paired bases, the stem-loop ssDNA has 3 paired bases according to the prediction, and the linear ssDNA has no paired bases; their fluorescence intensities indeed matched this relationship. Therefore, for different DNA structures of equal molar concentration, GelRed can be used as an indicator for the extent of base paring in DNA intramolecular self-folding or intermolecular hybridization. We then asked whether GelRed has a bias for the base pair compositions of dsDNAs. We synthesized four dsDNAs (ds1–4) that were 20 bp but had different GC contents (0%, 15%, 30%, and 50%, respectively, as listed in Fig. 3b Fig. 3b ). As we know, a G-C base pair comprises three hydrogen bonds and is thus more thermostable than an A-T base pair with two hydrogen bonds. The fluorescence measurements ( Fig. 3b Fig. 3b ) revealed that the dsDNAs with higher GC content showed higher GelRed fluorescence intensity. The dsDNA with a GC content of 50% (ds4) gave a ~3-fold higher fluorescence intensity compared to that without a G-C base (ds1). We hypothesize that this is because high GC content leads to a more thermostable double-helix structure, and thus benefits the intercalation of GelRed molecules. Based on the above results, we can conclude that the fluorescence intensity of a GelRed-stained DNA structure has a correlation to the thermostability of its secondary structure (including ssDNA self-folding or dsDNA base paring). This thermostability can be quantified in the form of Gibbs free energy change (Δ G ) from the linear ssDNAs to the resulting secondary structures or dsDNAs. In our study, DNA structures with more negative ΔG (more thermostable) indeed showed stronger GelRed fluorescence, which supported this hypothesis. We further analyzed the fluorescence of the component ssDNAs of the four dsDNAs that have different sequences (no predicted secondary structures, predicted ΔG=0). Based on the results (Fig. S1 in Supporting Information), they all showed negligible fluorescence regardless of their sequence, suggesting that the fluorescence intensity is indeed determined by paired bases (thermostability), rather than by different DNA sequences. Next, we compared the fluorescence resulting from EtBr and GelRed staining. According to the results (Fig. S2 in Supporting Information), the GelRed-stained ds4 structure exhibited ~14-fold stronger fluorescence compared to linear ssDNAs, while the EtBr-stained ds4 structure showed only ~7-fold fluorescence enhancement, indicating that GelRed leads to higher resolution in discriminating different DNA structures. Moreover, considering that EtBr has proven to be mutagenic in Ames tests, GelRed would be a good alternative. To test the stability and selectivity of GelRed towards DNA in physiological environments, we incubated DNA structures with GelRed in cell culture media Minimum Essential Medium (MEM) containing 10% (v/v) fetal bovine serum (FBS). By comparing their fluorescence spectra (Fig. S3 in Supporting Information), we found the correlation between GelRed fluorescence intensity and DNA structures was not severely interfered with, suggesting the potential of GelRed to deal with real biological samples. 3.4 Real-time monitoring of DNA conformational transformations 2 Having established the correlation between structural thermostability and GelRed fluorescence, we further demonstrated the use of GelRed in real-time monitoring of DNA conformational changes upon cyclic temperature variations. Here, we treated the GelRed-stained DNA structures with controlled temperature changes and recorded the temperature-dependent dynamics of the fluorescence intensity using a real-time quantitative thermocycler. As the temperature rose, the dsDNA structure (ds4) and the stem-loop ssDNA structure were expected to transform into linear strands ( Fig. 4a Fig. 4a ). The observation ( Fig. 4b Fig. 4b ) showed that, along with the rising temperature, the fluorescence intensity of both structures decreased. When the temperature passed a certain point, the fluorescence dropped more steeply, suggesting the melting of the secondary structures. Therefore, by derivation, we obtained the melting curves ( Fig. 4c Fig. 4c ) of both structures. The main peaks of the melting curves indicated that the measured melting temperature (Tm) of ds3 is ~69 °C, while the Tm of the stem-loop ssDNA structure is ~41 °C. The results were close to those measured using SYBR Green I (71 ˚C and 42 ˚C, respectively, see Table S1 in Supporting Information), another widely used intercalating dye for quantitative thermostability analysis of nucleic acids [ 46 46 ] . These results indicate that GelRed fluorescence can sensitively respond to the conformational changes of DNA structures in a real-time and label-free manner, which shows promise for biological research and DNA nanotechnology. 4. Conclusion 1 In this study, we used an intercalating dye, GelRed, to characterize different DNA structures, including ssDNAs with or without secondary structures and dsDNAs with different base compositions. We showed that GelRed can help discriminate these structures in a quantitative manner. We found that the fluorescence intensity of a GelRed-stained DNA structure is positively correlated to the thermostability (or Gibbs free energy change) of its secondary structure. More thermostable secondary structures (e.g., ssDNAs with stem-loop structures or dsDNAs with higher GC contents) lead to stronger fluorescence intensities. We also demonstrated that by using GelRed, the dynamics of DNA conformational changes can be monitored in real-time. 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(Science Press), Shanghai Institute of Applied Physics, the Chinese Academy of Sciences, ChineseNuclear Society and Springer Nature Singapore Pte Ltd. 2018 This is a post-peer-review, pre-copyedit version of an article published in Nuclear Science and Techniques. The final authenticated version is available online at: http://dx.doi.org/10.1007/s41365-018-0486-x http://dx.doi.org/10.1007/s41365-018-0486-x . 2018 Nuclear Science and Techniques 10 29