A three-state mechanism for DNA hairpin folding characterized by multiparameter fluorescence fluctuation spectroscopy

The folding of a dye-quencher labeled DNA hairpin molecule was investigated using fluorescence autocorrelation and cross-correlation spectroscopy (FCS) and photon counting histogram analysis (PCH). The autocorrelation and cross-correlation measurements revealed the flow and diffusion times of the DNA molecules through two spatially offset detection volumes, the relaxation time of the folding reaction, and the total concentration of DNA molecules participating in the reaction. The PCH measurements revealed the equilibrium distribution of DNA molecules in folded and unfolded conformations and the specific brightnesses of the fluorophore in each conformational state. These measurements were carried out over a range of NaCl concentrations, from those that favored the open form of the DNA hairpin to those that favored the closed form. DNA melting curves obtained from each sample were also analyzed for comparison. It was found that the reactant concentrations were depleted as the reaction progressed and that the equilibrium distributions measured by FCS and PCH deviated from those obtained from the melting curve analyses. These observations suggest a three-state mechanism for the DNA hairpin folding reaction that involves a stable intermediate form of the DNA hairpin. The reaction being probed by FCS and PCH is suggested to be a rapid equilibrium between open and intermediate conformations. Formation of the fully closed DNA hairpin is suggested to occur on a much longer time scale than the FCS and PCH measurement time. The closed form of the hairpin thus serves as a sink into which the reactants are depleted as the reaction progresses.     

Facile construction of targeted pH-responsive DNA-conjugated gold nanoparticles for synergistic photothermal-chemotherapy

Recently, stimuli-responsive DNA nanostructure-based nanodevices have been applied for cancer therapy. In this study, pH-responsive i-motif DNA was modified on gold nanoparticles (AuNPs) via a facile, time-saving freeze-thaw method and utilized to construct stimuli-responsive drug nanocarriers. When the environment pH changes from 7.4 to 5.0, the i-motif DNA would be folded into four-stranded (C-quadruplex) that could be characterized by circular dichroism, and the characteristic of acid stimulate was verified by fluorescence resonance energy transfer (FRET). To enhance specifical cellular uptake, MUC1 aptamer was employed as the targeting moiety. Doxorubicin (Dox) is an anticancer drug that can be efficiently intercalated into GC base pairs of DNA nanostructure to form drug-loaded nanovehicles (Dox@AuNP-MUC1). Additionally, owing to the excellent photothermal conversion efficiency of AuNPs, the synergistic effect between chemotherapy and PTT can be readily achieved by 808 nm near-infrared (NIR) irradiation, which exhibits specifically and efficiently anticancer efficiency. Hence, this multifunctional drug carrier shows the potential for synergistic photothermal-chemotherapy.     

Identifying i-motif formation using capillary electrophoresis

Over the past few years, intercalated motifs (i-motifs) have attracted attention due to the direct visualization of their existence in the nuclei of human cells. Traditionally, i-motifs have been studied using expensive and complicated NMR, and/or relatively inexpensive but less common circular dichroism spectrometry. The aim of this study was to investigate the feasibility of using less expensive, less complicated, and more widely available CE as an alternative for i-motif related research. The mobilities of two DNA and RNA i-motifs in CE were determined under different pH conditions. Our results demonstrate that CE is able to identify and differentiate mostly folded, partially folded, and mostly unfolded DNA and RNA i-motifs through changes in peak shape and migration time, thus providing a new method to study both i-motif conformation and the interactions between i-motifs and their ligands.     

DNA-Lipid探针用于细胞膜外pH值的原位成像

将二酰基脂质体自动偶联到两端标有Cy3和Cy5的具有i-motif结构的DNA链上,形成二酰基脂质体-DNA共轭物(DNA-lipid)探针。二酰基脂质体与细胞膜之间强烈的疏水作用可使该探针直接插入细胞膜表面,实现缺氧和常氧条件下细胞外pH值的比率型检测。在高pH值条件下,具有i-motif结构的DNA链两端的荧光基团处于分离状态,无FRET效应;在低pH值条件下,具有i-motif结构的DNA链在细胞膜表面形成四聚体结构,两端的荧光基团相互靠近,产生强的FRET效应。通过测定两种荧光基团的荧光强度比值实现了pH值的定量检测。利用此探针对pH值的灵敏响应实现了对缺氧环境中细胞外pH值的精确测量,在生理病理学上具有重大意义。     

Fluorescent Peptide Beacons for the Selective Ratiometric Detection of Heparin

Heparin is extensively used as an anticoagulant drug during surgery. Two fluorophore‐functionalized cationic oligopeptides HS 1 and HS 2 were developed to monitor heparin ratiometrically in aqueous media. Upon binding to heparin, HS 1 and HS 2 undergo a conformational change from an open form to a folded form, which leads to a distinct change in the fluorescence properties. HS 1 switches from pyrene monomer emission to an excimer emission. For HS 2 , a fluorescence resonance energy transfer (FRET) process is enabled between a naphthalene donor and a dansyl acceptor. This method is highly selective for heparin relative to other similar biological analytes such as hyaluronic acid or chondroitin sulfate. HS 1 and HS 2 could also detect heparin ratiometrically in diluted bovine serum. The strong ratiometric emission color change can also be observed by the naked eye. Addition of the polycationic protein protamine releases both HS 1 and HS 2 from their heparin complex, which simultaneously restores pyrene monomer emission for the first case and decreases the FRET process for the latter case, respectively. Dynamic light scattering (DLS) and AFM studies confirm aggregate formation of heparin with HS 1 and HS 2 .     

Single-molecule quantum-dot fluorescence resonance energy transfer.

Colloidal semiconductor quantum dots are promising for single-molecule biological imaging due to their outstanding brightness and photostability. As a proof of concept for single-molecule fluorescence resonance energy transfer (FRET) applications, we measured FRET between a single quantum dot and a single organic fluorophore Cy5. DNA Holliday junction dynamics measured with the quantum dot/Cy5 pair are identical to those obtained with the conventional Cy3/Cy5 pair, that is, conformational changes of individual molecules can be observed by using the quantum dot as the donor.     

Stability and Existence of Noncanonical I-motif DNA Structures in Computer Simulations Based on Atomistic and Coarse-Grained Force Fields

Cytosine-rich DNA sequences are able to fold into noncanonical structures, in which semi-protonated cytosine pairs develop extra hydrogen bonds, and these bonds are responsible for the overall stability of a structure called the i-motif. The i-motif can be formed in many regions of the genome, but the most representative is the telomeric region in which the CCCTAA sequences are repeated thousands of times. The ability to reverse folding/unfolding in response to pH change makes the above sequence and i-motif very promising components of nanomachines, extended DNA structures, and drug carriers. Molecular dynamics analysis of such structures is highly beneficial due to direct insights into the microscopic structure of the considered systems. We show that Amber force fields for DNA predict the stability of the i-motif over a long timescale; however, these force fields are not able to predict folding of the cytosine-rich sequences into the i-motif. The reason is the kinetic partitioning of the folding process, which makes the transitions between various intermediates too time-consuming in atomistic force field representation. Application of coarse-grained force fields usually highly accelerates complex structural transitions. We, however, found that three of the most popular coarse-grained force fields for DNA (oxDNA, 3SPN, and Martini) were not able to predict the stability of the i-motif structure. Obviously, they were not able to accelerate the folding of unfolded states into an i-motif. This observation must be strongly highlighted, and the need to develop suitable extensions of coarse-grained force fields for DNA is pointed out. However, it will take a great deal of effort to successfully solve these problems.     

Rotational and translational diffusion of peptide-coated CdSe/CdS/ZnS nanorods studied by fluorescence correlation spectroscopy

CdSe/CdS/ZnS nanorods (NRs) of three aspect ratios were coated with phytochelatin-related peptides and studied using fluorescence correlation spectroscopy (FCS). Theoretical predictions of the NRs' rotational diffusion contribution to the correlation curves were experimentally confirmed. We monitored rotational and translational diffusion of NRs and extracted hydrodynamic radii from the extracted diffusion constants. Translational and rotational diffusion constants (D(trans) and D(rot)) for NRs were in good agreement with Tirado and Garcia de la Torre's as well as with Broersma's theories when accounting for the ligand dimensions. NRs fall in the size range where rotational diffusion can be monitored with higher sensitivity than translational diffusion due to a steeper length dependence, D(rot) approximately L(-)(3) versus D(trans) approximately L(-)(1). By titrating peptide-coated NRs with bovine serum albumin, we monitored (nonspecific) binding through rotational diffusion and showed that D(rot) is an advantageous observable for monitoring binding. Monitoring rotational diffusion of bioconjugated NRs using FCS might prove to be useful for observing binding and conformational dynamics in biological systems.     

DNA hydrogels formed of bended DNA scaffolds and properties study

In recent years,DNA supramolecular hydrogels have attracted much attention due to their injectability,biocompatibility,responsiveness and self-healing properties.In this work,we designed a linear DNA brick containing one duplex with two cytosine (C)-rich sequence on both ends.This brick can first assemble to form duplex under pH 8 condition.After adjusting the pH to 5,the C-rich sequence tends to form intermolecular i-motif structure,which joins the linear DNA molecules together to form interlocked cyclic structures and yield the DNA hydrogel.By adjusting the length and bending curvature of the duplex part of the molecule,one can change the basic unit of the hydrogel structure to tune the properties of the DNA hydrogel.     

DNA molecular motor driven micromechanical cantilever arrays

The unique ability of living systems to translate biochemical reactions into mechanical work has inspired the design of synthetic DNA motors which generate nanoscale motion via controlled conformational change. However, while Nature has evolved intricate mechanisms to convert molecular shape change into specific micrometer-scale mechanical cellular responses, the integration of artificial DNA motors with mechanical devices presents a major challenge. Here we report the direct integration between an ensemble of DNA motors and an array of microfabricated silicon cantilevers. The forces exerted by the precise duplex to nonclassical i-motif conformational change were probed via differential measurements using an in-situ reference cantilever coated with a nonspecific sequence of DNA. Fueled by the addition of protons, the open to close stroke of the motor induced 32 +/- 3 mN/m compressive surface stress, which corresponds to a single motor force of approximately 11 pN/m, an order of magnitude larger than previous classical hybridization studies. Furthermore, the surface-tethered conformational change was found to be highly reversible, in contrast to classical DNA motors which typically suffer rapid system poisoning. The direction and amplitude of motor-induced cantilever motion was tuneable via control of buffer pH and ionic strength, indicating that electrostatic forces play an important role in stress generation. Hybrid devices which directly harness the multiple accessible conformational states of dynamic oligonucleotides and aptamers, translating biochemical energy into micromechanical work, present a radical new approach to the construction of "smart" nanoscale machinery and mechano-biosensors.     

Unfolding dynamics of cytochrome c revealed by single-molecule and ensemble-averaged spectroscopy

Denaturant-induced conformational change of yeast iso-1-cytochrome c (Cytc) has been comprehensively investigated in the single-molecule and bulk phases. By fluorescence-quenching experiments with dye-labelled heme-protein (Alexa 488-labelled Cytc, Cytc-A488), we clearly show that the fluorescence quenching observed from folded Cytc-A488 is due mainly to photoinduced electron transfer (PET) between electron-donating amino acids such as tryptophan and the dye attached to the protein. In addition, the unfolding process of Cytc-A488 observed in the single-molecule and bulk phases can be explained well in terms of a three-state model: Cytc unfolds through an intermediate with a native-like compactness. By quantitative analysis of fluorescence correlation spectroscopy (FCS) data, we were able to observe a relaxation time of ～1.5 μs corresponding to segmental motion and fast folding dynamics of 55 μs in the unfolded state of Cytc. The results presented here also suggest that a combination of single-molecule and ensemble-averaged spectroscopy is necessary to provide convincing and comprehensive assignments of protein kinetics.     

Exploring protein structure and dynamics under denaturing conditions by single-molecule FRET analysis

Proteins are highly complex biopolymers, exhibiting a substantial degree of structural variability in their properly folded, native state. In the presence of denaturants, this heterogeneity is greatly enhanced, and fluctuations take place among vast numbers of folded and unfolded conformations via many different pathways. To better understand protein folding it is necessary to explore the structural and energetic properties of the folded and unfolded polypeptide chain, as well as the trajectories along which the chain navigates through its multi-dimensional conformational energy landscape. In recent years, single-molecule fluorescence spectroscopy has been established as a powerful tool in this research area, as it allows one to monitor the structure and dynamics of individual polypeptide chains in real time with atomic scale resolution using F?rster resonance energy transfer (FRET). Consequently, time trajectories of folding transitions can be directly observed, including transient intermediates that may exist along these pathways. Here we illustrate the power of single-molecule fluorescence with our recent work on the structure and dynamics of the small enzyme RNase H in the presence of the chemical denaturant guanidinium chloride (GdmCl). For FRET analysis, a pair of fluorescent dyes was attached to the enzyme at specific locations. In order to observe conformational changes of individual protein molecules for up to several hundred seconds, the proteins were immobilized on nanostructured, polymer coated glass surfaces specially developed to have negligible interactions with folded and unfolded proteins. The single-molecule FRET analysis gave insight into structural changes of the unfolded polypeptide chain in response to varying the denaturant concentration, and the time traces revealed stepwise transitions in the FRET levels, reflecting conformational dynamics. Barriers in the free energy landscape of RNase H were estimated from the kinetics of the transitions.     

Bleaching-resistant,Near-continuous Single-molecule Fluorescence and FRET Based on Fluorogenic and Transient DNA Binding

Photobleaching of fluorescent probes limits the observation span of typical single-molecule fluorescence measurements and hinders observation of dynamics at long timescales. Here, we present a general strategy to circumvent photobleaching by replenishing fluorescent probes via transient binding of fluorogenic DNAs to complementary DNA strands attached to a target molecule. Our strategy allows observation of near-continuous single-molecule fluorescence for more than an hour, a timescale two orders of magnitude longer than the typical photobleaching time of single fluorophores under our conditions. Using two orthogonal sequences, we show that our method is adaptable to Förster Resonance Energy Transfer (FRET) and that can be used to study the conformational dynamics of dynamic structures, such as DNA Holliday junctions, for extended periods. By adjusting the temporal resolution and observation span, our approach enables capturing the conformational dynamics of proteins and nucleic acids over a wide range of timescales.     

Probing single-molecule enzyme active-site conformational state intermittent coherence

The relationship between protein conformational dynamics and enzymatic reactions has been a fundamental focus in modern enzymology. Using single-molecule fluorescence resonance energy transfer (FRET) with a combined statistical data analysis approach, we have identified the intermittently appearing coherence of the enzymatic conformational state from the recorded single-molecule intensity-time trajectories of enzyme 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) in catalytic reaction. The coherent conformational state dynamics suggests that the enzymatic catalysis involves a multistep conformational motion along the coordinates of substrate-enzyme complex formation and product releasing, presenting as an extreme dynamic behavior intrinsically related to the time bunching effect that we have reported previously. The coherence frequency, identified by statistical results of the correlation function analysis from single-molecule FRET trajectories, increases with the increasing substrate concentrations. The intermittent coherence in conformational state changes at the enzymatic reaction active site is likely to be common and exist in other conformation regulated enzymatic reactions. Our results of HPPK interaction with substrate support a multiple-conformational state model, being consistent with a complementary conformation selection and induced-fit enzymatic loop-gated conformational change mechanism in substrate-enzyme active complex formation.     

The oxidation state of a protein observed molecule-by-molecule.

We report the observation of the redox state of the blue copper protein azurin on the single-molecule level. The fluorescence of a small fluorophore attached to the protein is modulated by the change in absorption of the copper center via fluorescence resonance energy transfer (FRET). In our model system, the fluorescence label Cy5 was coupled to azurin from Pseudomonas aeruginosa via cysteine K27C. The Cy5 fluorescence was partially quenched by the absorption of the copper center of azurin in its oxidized state. In the reduced state, absorption is negligible, and thus no quenching occurs. We report on single-molecule measurements, both in solution by using fluorescence correlation spectroscopy (FCS) combined with fluorescence intensity distribution analysis (FIDA), and on surfaces by using wide-field fluorescence microscopy.     

Probing the complete folding trajectory of a DNA hairpin using dual beam fluorescence fluctuation spectroscopy

The conformational fluctuations of dye-quencher labeled DNA hairpin molecules in aqueous solution were investigated using dual probe beam fluorescence fluctuation spectroscopy. The measurements revealed the flow and diffusion times of the DNA molecules through two spatially offset optical probe regions, the absolute and relative concentrations of each conformational substate of the DNA, and the kinetics of the DNA hairpin folding and unfolding reactions in the 1 micros to 10 ms time range. A DNA hairpin containing a 21-nucleotide polythymine loop and a 4-base pair stem exhibited double exponential relaxation kinetics, with time constants of 84 and 393 micros. This confirms that folding and melting of the DNA hairpin structure is not a two state process but proceeds by way of metastable intermediate states. The fast time constant corresponds to formation and unfolding of an intermediate, and the slow time constant is due to formation and disruption of the fully base-paired stem. This is consistent with a previous study of a similar DNA hairpin with a 5-base pair stem, in which the fast reaction was attributed to the fluctuations of an intermediate DNA conformation [J. Am. Chem. Soc. 2006, 128, 1240-1249]. In that case, reactions involving the native conformation could not be observed directly due to the limited observation time range of the fluorescence correlation spectroscopy experiment. The intermediate states of the DNA hairpins are suggested to be due to a collapsed ensemble of folded hairpins containing various partially folded or misfolded conformations.     

Single-molecule FRET with diffusion and conformational dynamics

Under relatively mild conditions, we show how one can extract information about conformational dynamics from F?rster resonance energy transfer (FRET) experiments on diffusing molecules without modeling diffusion. Starting from a rigorous theory that does treat diffusion, we first examine when the single-molecule FRET efficiency distribution can be decomposed into the measured distribution of the total number of photons and the efficiency distribution of an immobilized molecule in the absence of shot noise. If the conformation does not change during the time the molecule spends in the laser spot, this is possible when (I) the efficiency is independent of the location in the laser spot and (II) the total number of photons does not depend on conformation. This decomposition is approximate when the conformation changes during the diffusion time. However, it does provide a simple framework for analyzing data. This is illustrated for a two-state system where the FRET efficiency distribution can be found analytically for all values of the interconversion rates. If the arrival time of each donor and acceptor photon can be monitored, we introduce an alternative procedure that allows one to rigorously extract the rates of conformational changes when the above two conditions hold. In this case, the pattern of colors in the photon trajectory depends solely on conformational dynamics. This can be exploited in the framework of statistical inference because the likelihood function, which must be optimized with respect to the model rate parameters, depends only on how the conformation changes during the interval between photons with specified colors.     

Fluorescent amplifying recognition for DNA G-quadruplex folding with a cationic conjugated polymer: a platform for homogeneous potassium detection

Single-stranded DNA with G-rich sequences can fold into secondary structures, G-quadruplexes, via intramolecular hydrogen-bonding interactions. This conformational change can be detected by a homogeneous assay method based on fluorescence resonance energy transfer (FRET) from a water-soluble cationic conjugated polymer (CCP) to a fluorescein chromophore labeled at the terminus of the G-quadruplex DNA. The space charge density around the DNA controls the efficiency of FRET from the CCP to the fluorescein. The higher FRET efficiency for the CCP/G-quadruplex pair is correlated to the stronger electrostatic interactions between the more condensed G-quadruplex and the CCP in comparison to the CCP/ssDNA pair. Since the potassium ion can specifically bind to the G-quadruplex DNA, the G-quartet-DNA/CCPs assembly can also be used as a platform to sense the potassium ion in water with high selectivity and sensitivity.