Studies on the formation and stability of triplex DNA using fluorescence correlation spectroscopy

Triplex DNA has become one of the most useful recognition motifs in the design of new molecular biology tools, therapeutic agents and sophisticated DNA‐based nanomaterials because of its direct recognition of natural double‐stranded DNA. In this paper, we developed a sensitive and microscale method to study the formation and stability characterization of triplex DNA using fluorescence correlation spectroscopy (FCS). The principle of this method is mainly based on the excellent capacity of FCS for sensitively distinguishing between free single‐strand DNA (ssDNA) fluorescent probes and fluorescent probe–double‐strand DNA (dsDNA) hybridized complexes. First, we systematically investigated the experimental conditions of triplex DNA formation. Then, we evaluated the equilibrium association constants (K_a) under different ssDNA probe lengths, composition and pH. Finally, we used FCS to measure the hybridization fraction of a 20‐mer perfectly matched ssDNA probe and three single‐base mismatched ssDNA probes with 146‐mer dsDNA. Our data illustrated that FCS is a useful tool for the direct determination of the thermodynamic parameters of triplex DNA formation and discrimination of a single‐base mismatch of triplex DNA without denaturation. Compared with current methods, our method is characterized by high sensitivity, good universality and small sample and reagent requirements. More importantly, our method has the potential to become a platform for triplex DNA research in vitro. Copyright © 2015 John Wiley & Sons, Ltd.     

Detection method for quantifying global DNA methylation by fluorescence correlation spectroscopy

A method for quantifying global DNA methylation using fluorescence correlation spectroscopy (FCS) has been established. The single-molecule methylation assay (SMMA) is based on two methodologies. One methodology, FCS, estimates the translational diffusion coefficient of molecules in solution, whereas the other methodology uses the high affinity of methyl-CpG-binding domain protein 2 (MBD2) to bind specifically to methylated DNA. We studied the specific binding rates of fluorescence-labeled MBD2 and methylated DNA from biological samples using the automated FCS system. Using a standard curve with methylated control DNA, we developed the SMMA index to assess the global DNA methylation level of the biological samples. A marked decrease in the SMMA index was observed when human leukemia cell lines (U937 and K562) were cultured with DNA demethylating agents. Our findings clearly indicate the applicability of SMMA as a simple and rapid tool for quantifying global DNA methylation. SMMA may prove useful for genome-wide comparative methylation analyses of malignancies and as an indicator of the demethylation effects of epigenetic drugs.     

Polyamide recognition-mass spectrometry for distinguishing hairpin DNA from coil DNA

The discrimination between hairpin DNA and coil DNA has been well achieved through polyamides as probes by electrospray ionization (ESI) mass spectrometry. ESI mass spectra showed that polyamides bind to hairpin DNA with high selectivity, and almost no binding with coil DNA. In addition, the noncovalent interaction between polyamides and hairpin DNA was also studied; the results show that hairpin DNA with longer stem and polyamides with more heterocycles have higher binding affinity and stability in gas phase.     

DNA binding activity of Anabaena sensory rhodopsin transducer probed by fluorescence correlation spectroscopy

Anabaena sensory rhodopsin transducer (ASRT) is believed to be a major player in the photo-signal transduction cascade, which is triggered by Anabaena sensory rhodopsin. Here, we characterized DNA binding activity of ASRT probed by using fluorescence correlation spectroscopy. We observed clear decrease of diffusion coefficient of DNA upon binding of ASRT. The dissociation constant, K_D, of ASRT to 20?bp-long DNA fragments lied in micro-molar range and varied moderately with DNA sequence. Our results suggest that ASRT may interact with several different regions of DNA with different binding affinity for global regulation of several genes that need to be activated depending on the light illumination.     

Detection of oxidative stress-induced mitochondrial DNA damage using fluorescence correlation spectroscopy

Using fluorescence correlation spectroscopy (FCS), we tested the feasibility of rapid detection of oxidative damage of mitochondrial DNA (mtDNA) in a small volume. The complete mtDNA genome was amplified by long polymerase chain reaction (LPCR), and the product was fluorescently labeled with an intercalating dye, YOYO-1. The fluorescence autocorrelation function was analyzed using a simple two-component model with the diffusion time of 0.21 ms for the LPCR primer and 18 ms for the mtDNA LPCR product. When human embryonic kidney 293 (HEK-293) cells were exposed to 0.4 mM H2O2, the fraction of the mtDNA LPCR product decreased significantly. In contrast, the fraction of the nuclear-encoded beta-globin LPCR product remained unchanged. The analysis time of FCS measurement was very short (5 min) compared with that of gel electrophoresis (3 h). Thus, FCS allowed the rapid detection of the vulnerability of mtDNA to oxidative stress within a small volume element at the subfemtoliter level in solution. These results suggest that the LPCR-FCS method can be used for epidemiological studies of diseases caused by mtDNA damage.     

The initial step of DNA hairpin folding: a kinetic analysis using fluorescence correlation spectroscopy

Conformational fluctuations of single-stranded DNA (ssDNA) oligonucleotides were studied in aqueous solution by monitoring contact-induced fluorescence quenching of the oxazine fluorophore MR121 by intrinsic guanosine residues (dG). We applied fluorescence correlation spectroscopy as well as steady-state and time-resolved fluorescence spectroscopy to analyze kinetics of DNA hairpin folding. We first characterized the reporter system by investigating bimolecular quenching interactions between MR121 and guanosine monophosphate in aqueous solution estimating rate constants, efficiency and stability for formation of quenched complexes. We then studied the kinetics of complex formation between MR121 and dG residues site-specifically incorporated in DNA hairpins. To uncover the initial steps of DNA hairpin folding we investigated complex formation in ssDNA carrying one or two complementary base pairs (dC–dG pairs) that could hybridize to form a short stem. Our data show that incorporation of a single dC–dG pair leads to non-exponential decays for opening and closing kinetics and reduces rate constants by one to two orders of magnitude. We found positive activation enthalpies independent of the number of dC–dG pairs. These results imply that the rate limiting step of DNA hairpin folding is not determined by loop dynamics, or by mismatches in the stem, but rather by interactions between stem and loop nucleotides.     

Enzymatic conversion of long DNA to small DNA fragments for the construction of short hairpin RNA expression libraries

Several techniques to enzymatically construct a short hairpin RNA (shRNA) expression library have been reported as tools for comprehensive genetic analyses by RNA interference. Our technique constructs an shRNA expression library from 25- to 35-bp DNA fragments by fragmenting given double-stranded DNA (dsDNA). We compared the following two procedures to efficiently prepare such small DNA fragments: one is the cleavage of dsDNA with deoxyribonuclease I (DNase I) in the presence of Mn²⁺ followed by blunting with T4 DNA polymerase, and the other is the introduction of nicks with DNase I in the presence of Mg²⁺ followed by blunting with the Klenow fragment. Consequently, the latter yielded the DNA fragments more efficiently. However, these DNA fragments were contaminated with fused DNA fragments that had originated from two regions of original dsDNA. Therefore, we used single-strand-specific exonucleases and succeeded in suppressing the production of such fused DNA fragments. Our technique allows the efficient conversion of given dsDNA to small DNA fragments.     

Intermolecular disulfide bond formation promotes immunoglobulin aggregation: Investigation by fluorescence correlation spectroscopy

Protein aggregation generally results from association between hydrophobic regions of individual monomers. However, additional mechanisms arising from specific interactions, such as intermolecular disulfide bond formation, may also contribute to the process. The latter is proposed to be the initiating pathway for aggregation of immunoglobulin (IgG), which is essential for triggering its immune response. To test the veracity of this hypothesis, we have employed fluorescence correlation spectroscopy to measure the kinetics of aggregation of IgG in separate experiments either allowing or inhibiting disulfide formation. Fluorescence correlation spectroscopy measurements yielded a diffusion time (τ_D) of ～200 µsec for Rhodamine‐labeled IgG, corresponding to a hydrodynamic radius (R_H) of 56 Å for the IgG monomer. The aggregation kinetics of the protein was followed by monitoring the time evolution of τ_D under conditions in which its cysteine residues were either free or blocked. In both cases, the progress curves confirmed that aggregation proceeded via the nucleation‐dependent polymerization pathway. However, for aggregation in the presence of free cysteines, the lag times were shorter, and the aggregate sizes bigger, than their respective counterparts for aggregation in the presence of blocked cysteines. This result clearly demonstrates that formation of intermolecular disulfide bonds represents a preferred pathway in the aggregation process of IgG. Fluorescence spectroscopy showed that aggregates formed in experiments where disulfide formation was prevented denatured at lower concentration of guanidine hydrochloride than those obtained in experiments where the disulfides were free to form, indicating that intermolecular disulfide bridging is a valid pathway for IgG aggregation. Proteins 2015; 83:169–177. © 2014 Wiley Periodicals, Inc.     

Analysis of intranuclear binding process of glucocorticoid receptor using fluorescence correlation spectroscopy

The diffusion properties of EGFP-hGRalpha and mutants C421G, A458T and I566 in living cells were analyzed. The wild type and mutants C421G and A458T translocated from the cytoplasm to the nucleus after addition of Dex; however, the Brownian motions of the proteins were different. The diffusion constant of wild-type GRalpha after addition of Dex slowed to 15.6% of that in the absence of Dex, whereas those of A458T and C421G slowed to 34.8% and 61.7%, respectively. This is the first report that dimer formation is less important than the binding activity of GRalpha to GRE in the living cell.     

Investigating the kinetics of DNA–DNA and PNA–DNA interactions using surface plasmon resonance-enhanced fluorescence spectroscopy

Plasmon surface polaritons, resonantly excited in the Kretschmann format, are used to enhance the fluorescence emission of chromophore-labeled oligonucleotides (15mers) binding to surface-attached (via biotin–streptavidin linkages) complement catcher probes. A detailed analysis of the association and dissociation kinetics as well as the affinity constants is given for a mismatch 1 hybrid, emphasizing, in particular, the experimental conditions that are required to allow for an artifact-free determination of rate constants. A first comparison between DNA- and peptide nucleic acid (PNA-) probes shows similar affinities, however, significant deviations from single-exponential kinetics predicted by a simple Langmuir model for the PNA case are found.     

Asymmetric structure of five and six membered DNA hairpin loops

The tertiary structure of nucleic acid hairpins was elucidated by means of the accessibility of the single-strand-specific nuclease from mung bean. This molecular probe has proven especially useful in determining details of the structural arrangement of the nucleotides within a loop. In this study 3'-labeling is introduced to complement previously used 5'-labeling in order to assess and to exclude possible artifacts of the method. Both labeling procedures result in mutually consistent cleavage patterns. Therefore, methodological artifacts can be excluded and the potential of the nuclease as structural probe is increased. DNA hairpins with five and six membered loops reveal an asymmetric loop structure with a sharp bend of the phosphate-ribose backbone between the second and third nucleotide on the 3'-side of a loop. These hairpin structures differ from smaller loops with 3 or 4 members, which reveal this type of bend between the first and second 3' nucleotide, and resemble with respect to the asymmetry anticodon loops of tRNA.Abbreviations The hairpin oligonucleotides are indicated by hp hairpin followed by the loop sequence, starting at the 5'-end, in parenthesis; d for deoxy is omitted for clarity     

Detection of ligand-induced CNTF receptor dimers in living cells by fluorescence cross correlation spectroscopy

Ciliary neurotrophic factor (CNTF) signals via a receptor complex consisting of the specific CNTF receptor (CNTFR) and two promiscuous signal transducers, gp130 and leukemia inhibitory factor receptor (LIFR). Whereas earlier studies suggested that the signaling complex is a hexamer, more recent analyses strongly support a tetrameric structure. However, all studies so far analyzed the stoichiometry of the CNTF receptor complex in vitro and not in the context of living cells. We generated and expressed in mammalian cells acyl carrier protein-tagged versions of both CNTF and CNTFR. After labeling CNTF and CNTFR with different dyes we analyzed their diffusion behavior at the cell surface. Fluorescence (cross) correlation spectroscopy (FCS/FCCS) measurements reveal that CNTFR diffuses with a diffusion constant of about 2 × 10^− 9 cm² s^− 1 independent of whether CNTF is bound or not. FCS and FCCS measurements detect the formation of receptor complexes containing at least two CNTFs and CNTFRs. In addition, we measured Förster-type fluorescence resonance energy transfer between two differently labeled CNTFs within a receptor complex indicating a distance of 5-7 nm between the two. These findings are not consistent with a tetrameric structure of the CNTFR complex suggesting that either hexamers and or even higher-order structures (e.g. an octamer containing two tetramers) are formed.     

Recent applications of fluorescence correlation spectroscopy in live systems

Fluorescence correlation spectroscopy (FCS) is a widely used technique in biophysics and has helped address many questions in the life sciences. It provides important advantages compared to other fluorescence and biophysical methods. Its single molecule sensitivity allows measuring proteins within biological samples at physiological concentrations without the need of overexpression. It provides quantitative data on concentrations, diffusion coefficients, molecular transport and interactions even in live organisms. And its reliance on simple fluorescence intensity and its fluctuations makes it widely applicable. In this review we focus on applications of FCS in live samples, with an emphasis on work in the last 5 years, in the hope to provide an overview of the present capabilities of FCS to address biologically relevant questions.     

Melting studies of short DNA hairpins: Influence of loop sequence and adjoining base pair identity on hairpin thermodynamic stability

Spectroscopic and calorimetric melting studies of 28 DNA hairpins were performed. These hairpins form by intramolecular folding of 16 base self‐complementary DNA oligomer sequences. Sequence design dictated that the hairpin structures have a six base pair duplex linked by a four base loop and that the first five base pairs in the stem are the same in every molecule. Only loop sequence and identity of the duplex base pair closing the loop vary for the set of hairpins. For these DNA samples, melting studies were carried out to investigate effects of the variables on hairpin stability. Stability of the 28 oligomers was ascertained from their temperature‐induced melting transitions in buffered 115 mM Na⁺ solvent, monitored by ultraviolet absorbance and differential scanning calorimetry (DSC). Experiments revealed the melting temperatures of these molecules range from 32.4 to 60.5°C and are concentration independent over strand concentrations of 0.5 to 260 μM; thus, as expected for hairpins, the melting transitions are apparently unimolecular. Model independent thermodynamic transition parameters, ΔH_cal, ΔS_cal, and ΔG_cal, were determined from DSC measurements. Model dependent transition parameters, ΔH_vH, ΔS_vH, and ΔG_vH were estimated from a van't Hoff (two‐state) analysis of optical melting transitions. Results of these studies reveal a significant sequence dependence to DNA hairpin stability. Thermodynamic parameters evaluated by either procedure reveal the transition enthalpy, ΔH_cal (ΔH_vH) can differ by as much as 20 kcal/mol depending on sequence. Similarly, values of the transition entropy ΔS_cal (ΔS_vH) can differ by as much as 60 cal/Kmol (eu) for different molecules. Differences in free energies ΔG_cal (ΔG_vH) are as large as 4 kcal/mol for hairpins with different sequences. Comparisons between the model independent calorimetric values and the thermodynamic parameters evaluated assuming a two‐state model reveal that 10 of the 28 hairpins display non‐two‐state melting behavior. The database of sequence‐dependent melting free energies obtained for the hairpins was employed to extract a set of n‐n (nearest‐neighbor) sequence dependent loop parameters that were able to reproduce the input data within error (with only two exceptions). Surprisingly, this suggests that the thermodynamic stability of the DNA hairpins can in large part be reasonably represented in terms of sums of appropriate nearest‐neighbor loop sequence parameters. © 1999 John Wiley & Sons, Inc. Biopoly 50: 425–442, 1999     

Exonuclease III action on microarrays: Observation of DNA degradation by fluorescence correlation spectroscopy

DNA with all cytosines, thymines, or all pyrimidines of one strand substituted by fluorescently labeled analogs shows diminished solubility in aqueous media and a strong tendency to aggregation that hampers enzymatic downstream processing. In this study, immobilization of fully fluorescently labeled DNA on microarrays was shown to resolve the named problems and to enable successive DNA degradation by exonuclease III. Fluorescence correlation spectroscopy and single-molecule counting for monitoring the course of DNA hydrolysis in real time revealed the virtually processive degradation of labeled DNA that occurred at an average rate of approximately 4 nt/s.     

Monitoring of the formation and dissociation of polyethylenimine/DNA complexes by two photon fluorescence correlation spectroscopy

Polyethylenimines (PEI) constitute efficient nonviral vectors for gene transfer. However, because free PEI shows some cytotoxicity and because intracellular dissociation of PEI/DNA complexes seems to be required for efficient transfection, it is important to monitor the concentrations of free and bound partners in the mixtures of DNA and PEI used for transfection. To reach this objective, we used fluorescence correlation spectroscopy with two-photon excitation to characterize the complexes formed with either rhodamine-labeled 25 kDa PEI or DNA plasmid molecules. At the molar ratios of PEI nitrogen atoms to DNA phosphate usually used for transfection, we found that approximately 86% of the PEI molecules were in a free form. The PEI/DNA complexes are composed on the average by 3.5 (+/-1) DNA plasmids and approximately 30 PEI molecules. From this composition and the pK(a) of PEI, it could be inferred that in contrast to DNA condensation by small multivalent cations, only a limited neutralization of the DNA phosphate groups is required for DNA condensation by PEI. Moreover, DNA appears only poorly compacted in the PEI/DNA complexes. As an application, fluorescence correlation spectroscopy was used to monitor the purification of PEI/DNA complexes by ultrafiltration as well as the heparin-induced dissociation of the complexes.     

Characterizing diffusion dynamics of a membrane protein associated with nanolipoproteins using fluorescence correlation spectroscopy

Nanolipoprotein particles (NLPs) represent a unique nanometer-sized scaffold for supporting membrane proteins (MP). Characterization of their dynamic shape and association with MP in solution remains a challenge. Here, we present a rapid method of analysis by fluorescence correlation spectroscopy (FCS) to characterize bacteriorhodopsin (bR), a membrane protein capable of forming a NLP complex. By selectively labeling individual components of NLPs during cell-free synthesis, FCS enabled us to measure specific NLP diffusion times and infer size information for different NLP species. The resulting bR-loaded NLPs were shown to be dynamically discoidal in solution with a mean diameter of 7.8 nm. The insertion rate of bR in the complex was ～55% based on a fit model incorporating two separate diffusion properties to best approximate the FCS data. More importantly, based on these data, we infer that membrane protein associated NLPs are thermodynamically constrained as discs in solution, while empty NLPs appear to be less constrained and dynamically spherical.