A Temperature-Jump Optical Trap for Single-Molecule Manipulation

To our knowledge, we have developed a novel temperature-jump optical tweezers setup that changes the temperature locally and rapidly. It uses a heating laser with a wavelength that is highly absorbed by water so it can cover a broad range of temperatures. This instrument can record several force-distance curves for one individual molecule at various temperatures with good thermal and mechanical stability. Our design has features to reduce convection and baseline shifts, which have troubled previous heating-laser instruments. As proof of accuracy, we used the instrument to carry out DNA unzipping experiments in which we derived the average basepair free energy, entropy, and enthalpy of formation of the DNA duplex in a range of temperatures between 5°C and 50°C. We also used the instrument to characterize the temperature-dependent elasticity of single-stranded DNA (ssDNA), where we find a significant condensation plateau at low force and low temperature. Oddly, the persistence length of ssDNA measured at high force seems to increase with temperature, contrary to simple entropic models.     

Dna Damage by Chemically Generated Singlet Oxygen

A naphthalenic endoperoxide was used as a non-photochemical source of singlet oxygen (¹O₂) to examine some interactions between this reactive oxygen species and DNA. High molecular weight DNA (ca. 10⁸ daltons) was exposed to 120 mol m^?31O₂ (cumulative concentration) and analyzed for interstrand crosslinkage by hydroxyl apatite chromatography following formamide denaturation. No evidence for ¹O₂-induced interstrand crosslinking was obtained. The capacity of ¹O₂ to generate strand breaks in single-stranded (ss) and double-stranded (ds) DNA was investigated by sucrose gradient centrifugation analysis of bacteriophage øX174 DNA. No direct strand breaks could be detected at neutral pH, whereas extensive strand breakage was observed after treatment with alkali. Possible biological consequences of ¹O₂ -exposure were assessed by examining the plaque-forming capacity of ss and ds øX 174 DNA molecules using wildtype Escherichia coli spheroplasts as recipients. Without any further treatment with heat or alkali, exposure to the endoperoxide resulted in a time- and dose-dependent inactivation, ss DNA being considerably more sensitive than ds DNA. From the present results and those reported earlier (Nieuwint et al.,²⁰) we infer that ¹O₂-induced inactivation of øX174 DNA is not due to DNA backbone breakage nor to interstrand crosslinking, but rather to some form of damage to the base or sugar moiety of the DNA, the exact nature of which remains to be elucidated.     

Single-Molecule Vibrational Spectroscopy Adds Structural Resolution to the Optical Trap

The ability to apply forces on single molecules with an optical trap is combined with the endogenous structural resolution of Raman spectroscopy in an article in this issue, and applied to measure the Raman spectrum of ds-DNA during force-extension.The resounding success of single-molecule biophysical techniques has encouraged the development of additional tools for more detailed exploration. The unique ability of single-molecule methods to apply force and torque, to disentangle heterogeneity, and to watch equilibrium kinetics would pair beautifully with the ultrafast time resolution and atomistic structural sensitivity of vibrational spectroscopy. However, the weak signal levels endemic to vibrations have left them mostly in the domain of bulk spectroscopy; cross-sections for Raman scattering are typically 10¹⁴ times smaller than for fluorescence emission. In this issue, Rao et al. (1) overcome this gap using surface-enhanced Raman spectroscopy (SERS) (2,3) to add vibrational spectroscopic resolution to their optical trap. In this experiment, a single DNA strand is brought into the near-field vicinity of a silver nanoparticle-coated silica bead that enhances its Raman scattering, and the spectrum is recorded as the DNA is extended in the optical trap. The authors find that applying force shifts the phosphate-stretching vibrational frequency. Molecular dynamics and density functional theory calculations were used to explain these results by showing that external load applied to the DNA backbone induces Ångstrom-level displacements in the P-O bonds.This work is immediately relevant to the communities interested in DNA mechanics and single-molecule Raman spectroscopy. While the authors’ results may refine our structural models for DNA in the low-force regime (1–9 pN), the ongoing debate about the molecular nature of the transition into overstretched DNA (≥65 pN) (4) would be well served by additional structural resolution. For the SERS community, the optical trap provides a fantastic control as it allows one to unambiguously verify that a single-molecule is probed and systematically control its distance and orientation to the metal surface, which may finally resolve long-standing mysteries about the mechanism of SERS. Ideally, both methods will be advanced in concert at the expense of coercing as much information as possible out of a single molecule.While this work is groundbreaking, the real excitement is in its potential. One limitation in most implementations of both single-molecule force and fluorescence spectroscopy is acute sensitivity to distance changes >5 nm, which diminishes upon approaching the subnanometer scale. Raman scattering and infrared absorption vibrational spectroscopies offer a complementary distance sensitivity as molecular oscillators sense their local environment and couple to one another on scales of ∼0.1 nm; see Fig. 1 for a comparison. The optical trap can now be used to initiate specific structural changes to be probed by SERS. In such mechanistic studies, one benefits from the fact that the vibrational spectrum is an endogenous probe, arising from oscillations in all the different bonds present (enzyme as well as substrate), that directly encodes the kinetics and dynamics of structural changes. Such a detailed view of hydrogen-bond rearrangements, covalent-bond formation/breaking, and symmetry changes can offer subtle details that are impossible to tag with fluorophores or directly monitor via a force measurement. As vibrational spectroscopy is rapidly approaching the molecular fingerprinting level with DNA base resolution (5) and protein identification (6), there is an optimistic future for this apparently new multiplexed technique across the various divisions of biophysics.Open in a separate windowFigure 1(A) Examples of mesoscopic structural changes typically underlying single-molecule experiments, such as unfolding of DNA and proteins; translocation of enzymes on a scaffold such as the motor proteins, dynein and kinesin, and replication proteins; and binding of substrates such as ATP and FAD (7,8). (B) Examples of microscopic structural changes probed by bulk vibrational spectroscopy, which may complement single-molecule studies such as hydrogen bonding, isomerization, subtle secondary structural changes such as α-helix rotation and β-sheet reordering, and ligand-binding geometry and kinetics (9–12).     

Singlet Oxygen Is the Major Reactive Oxygen Species Involved in Photooxidative Damage to Plants

Reactive oxygen species act as signaling molecules but can also directly provoke cellular damage by rapidly oxidizing cellular components, including lipids. We developed a high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry-based quantitative method that allowed us to discriminate between free radical (type I)- and singlet oxygen (¹O₂; type II)-mediated lipid peroxidation (LPO) signatures by using hydroxy fatty acids as specific reporters. Using this method, we observed that in nonphotosynthesizing Arabidopsis (Arabidopsis thaliana) tissues, nonenzymatic LPO was almost exclusively catalyzed by free radicals both under normal and oxidative stress conditions. However, in leaf tissues under optimal growth conditions, ¹O₂ was responsible for more than 80% of the nonenzymatic LPO. In Arabidopsis mutants favoring ¹O₂ production, photooxidative stress led to a dramatic increase of ¹O₂ (type II) LPO that preceded cell death. Furthermore, under all conditions and in mutants that favor the production of superoxide and hydrogen peroxide (two sources for type I LPO reactions), plant cell death was nevertheless always preceded by an increase in ¹O₂-dependent (type II) LPO. Thus, besides triggering a genetic cell death program, as demonstrated previously with the Arabidopsis fluorescent mutant, ¹O₂ plays a major destructive role during the execution of reactive oxygen species-induced cell death in leaf tissues.Plant leaves capture sun-derived light energy to drive CO₂ fixation during photosynthesis. During this process, leaves need to cope with photooxidative stress when the balance between energy absorption and consumption is disturbed. Excess excitation energy in the photosystems (PSI and PSII) leads to the inhibition of photosynthesis via the production of various reactive oxygen species (ROS) at different spatial levels of the cell (Apel and Hirt, 2004; Asada, 2006; Van Breusegem and Dat, 2006). Both exposure to high light intensities and decreased CO₂ availability direct linear electron transfer toward the reduction of molecular oxygen, generating superoxide radicals (O₂^−.) at PSI (the Mehler reaction). Superoxide dismutation generates hydrogen peroxide (H₂O₂), which is detoxified in the chloroplast by ascorbate peroxidases. As such, this so-called water-water cycle participates in the dissipation of excess energy (Asada, 2006). Decreased CO₂ availability affects the first step in CO₂ fixation by shifting the carboxylation of Rubisco by the Rubisco carboxylase-oxygenase enzyme toward oxygenation, a process called photorespiration. This leads, through the action of glycolate oxidase, to peroxisomal H₂O₂ production that is counteracted by catalases. Finally, when the intersystem electron carriers are overreduced, triplet excited P680 in the PSII reaction center as well as triplet chlorophylls in the light-harvesting antennae are produced, with the production of singlet oxygen (¹O₂) as a consequence (Krieger-Liszkay, 2005). In photosynthetic membranes, ¹O₂ is quenched by carotenoids and tocopherols. When antioxidant mechanisms are overwhelmed, increased cellular ROS levels trigger signal transduction events related to stress signaling and programmed cell death (Mittler et al., 2004; Van Breusegem and Dat, 2006). On the other hand, excessive ROS accumulation damages pigments, proteins, nucleic acids, and lipids (Halliwell and Gutteridge, 2007), thereby contributing to or executing cell death.Since under environmental stress conditions different ROS are produced simultaneously, a causal link between the accumulation of a specific ROS and its signaling or damaging effects has always been difficult to establish. In recent years, the production of various transgenic Arabidopsis (Arabidopsis thaliana) plants with compromised levels of specific antioxidant enzymes and the identification of the conditional fluorescent (flu) mutant provided important tools to assess the specific effects of O₂^−., H₂O₂, and ¹O₂ within a particular subcellular compartment (Dat et al., 2003; op den Camp et al., 2003; Pnueli et al., 2003; Rizhsky et al. 2003; Vandenabeele et al., 2004; Wagner et al., 2004; Queval et al., 2007). For example, with catalase-deficient [Cat(−)] plants, the signaling effects of increased photorespiratory H₂O₂ levels could be identified (Dat et al., 2003; Vandenabeele et al., 2004; Queval et al., 2007). Similarly, in the conditional flu mutant increased plastid ¹O₂ levels were shown to induce a genetic program leading to cell death (op den Camp et al., 2003; Wagner et al., 2004). Nevertheless, whereas careful monitoring of gene expression on the whole-genome level enables to pinpoint specific signaling capacities for diverse ROS (Mittler et al., 2004; Gadjev et al., 2006), it remained impossible to discriminate between the oxidative damaging effects on cellular components of different ROS.One consequence of ROS formation is lipid peroxidation (LPO; Halliwell and Gutteridge, 2007). Two nonenzymatic reaction types lead to specific patterns of oxidized membrane polyunsaturated fatty acids (PUFAs; Stratton and Liebler, 1997; Montillet et al., 2004; Mueller et al., 2006). Type I reactions are initiated by free radicals (FRs) having high redox potential, such as hydroxyl radicals (^.OH) or organic oxyl and peroxyl radicals, and type II reactions are the result of ¹O₂ action. Notably, O₂^−. and H₂O₂ are not sufficiently reactive to oxidize any PUFA. However, both ROS can be nonenzymatically converted to ^.OH through Fenton-type reactions in the presence of transition metal ions such as Fe²⁺ (Halliwell and Gutteridge, 2007). Both type I and type II reactions lead to the formation of respective oxygenated fatty acids. Here, we propose a novel and quantitative approach to distinguish between FR- and ¹O₂-mediated LPO in plants by quantifying type II oxidation-specific hydroxy fatty acids with HPLC-tandem mass spectrometry (MS/MS), allowing us to monitor the relative contribution of LPO caused by PSI-dependent O₂^−./H₂O₂, photorespiratory H₂O₂, and photosynthetic ¹O₂ during photooxidative stress and cell death. We demonstrate that nonenzymatic LPO in leaves is almost exclusively mediated by ¹O₂ and that photooxidative stress-dependent cell death involves ¹O₂ production in its final stage.     

Oxidative Damage of U937 Human Leukemic Cells Caused by Hydroxyl Radical Results in Singlet Oxygen Formation

The exposure of human cells to oxidative stress leads to the oxidation of biomolecules such as lipids, proteins and nuclei acids. In this study, the oxidation of lipids, proteins and DNA was studied after the addition of hydrogen peroxide and Fenton reagent to cell suspension containing human leukemic monocyte lymphoma cell line U937. EPR spin-trapping data showed that the addition of hydrogen peroxide to the cell suspension formed hydroxyl radical via Fenton reaction mediated by endogenous metals. The malondialdehyde HPLC analysis showed no lipid peroxidation after the addition of hydrogen peroxide, whereas the Fenton reagent caused significant lipid peroxidation. The formation of protein carbonyls monitored by dot blot immunoassay and the DNA fragmentation measured by comet assay occurred after the addition of both hydrogen peroxide and Fenton reagent. Oxidative damage of biomolecules leads to the formation of singlet oxygen as conformed by EPR spin-trapping spectroscopy and the green fluorescence of singlet oxygen sensor green detected by confocal laser scanning microscopy. It is proposed here that singlet oxygen is formed by the decomposition of high-energy intermediates such as dioxetane or tetroxide formed by oxidative damage of biomolecules.     

Involvement of Singlet Oxygen in 5-Aminolevulinic Acid-Induced Photodynamic Damage of Cucumber (Cucumis sativus L.) Chloroplasts

Cucumber (Cucumis sativus L., cv Poinsette) plants were sprayed with 20 millimolar 5-aminolevulinic acid and then incubated in the dark for 14 hours. The intact chloroplasts were isolated from the above plants in the dark and were exposed to weak light (250 micromoles per square meter per second). Within 30 minutes, photosystem II activity was reduced by 50%. The singlet oxygen (¹O₂) scavengers, histidine and sodium azide (NaN₃) significantly protected against the damage caused to photosystem II. The hydroxyl radical scavenger formate failed to protect the thylakoid membranes. The production of ¹O₂ monitored as N,N-dimethyl p-nitrosoaniline bleaching increased as a function of light exposure time of treated chloroplasts and was abolished by the ¹O₂ quencher, NaN₃. Membrane lipid peroxidation monitored as malondialdehyde production was also significantly reduced when chloroplasts were illuminated in the presence of NaN₃ and histidine. Protochlorophyllide was the most abundant pigment accumulated in intact chloroplasts isolated from 5-aminolevulinic acid-treated plants and was probably acting as type II photosensitizer.     

The Ene Reaction of Singlet Oxygen with Olefins

The reactions of singlet oxygen (¹O₂) with cis and trans butenes-1,1,1-d₃, at—80°C in Freon-11, show a product isotope effect (k_H/k_D) of 1.38 and 1.25 respectively. Isomerization of the starting materials or formation of dioxetanes were not observed during the course of the photooxygenation. Together with the isotope effects on the reactions of tetramethylethylene-d₆ isomers with singlet oxygen, these results require the reversible formation of a perepoxide or charge transfer intermediate.     

Increasing the Time Resolution of Single-Molecule Experiments with Bayesian Inference

Many time-resolved single-molecule biophysics experiments seek to characterize the kinetics of biomolecular systems exhibiting dynamics that challenge the time resolution of the given technique. Here, we present a general, computational approach to this problem that employs Bayesian inference to learn the underlying dynamics of such systems, even when they are much faster than the time resolution of the experimental technique being used. By accurately and precisely inferring rate constants, our Bayesian inference for the analysis of subtemporal resolution dynamics approach effectively enables the experimenter to super-resolve the poorly resolved dynamics that are present in their data.     

The Ene Reaction of Singlet Oxygen with Olefins

The reactions of singlet oxygen (¹O₂) with cis and trans butenes-1,1,1-d₃, at—80°C in Freon-11, show a product isotope effect (k_H/k_D) of 1.38 and 1.25 respectively. Isomerization of the starting materials or formation of dioxetanes were not observed during the course of the photooxygenation. Together with the isotope effects on the reactions of tetramethylethylene-d₆ isomers with singlet oxygen, these results require the reversible formation of a perepoxide or charge transfer intermediate.     

Empirical Bayes Methods Enable Advanced Population-Level Analyses of Single-Molecule FRET Experiments

Many single-molecule experiments aim to characterize biomolecular processes in terms of kinetic models that specify the rates of transition between conformational states of the biomolecule. Estimation of these rates often requires analysis of a population of molecules, in which the conformational trajectory of each molecule is represented by a noisy, time-dependent signal trajectory. Although hidden Markov models (HMMs) may be used to infer the conformational trajectories of individual molecules, estimating a consensus kinetic model from the population of inferred conformational trajectories remains a statistically difficult task, as inferred parameters vary widely within a population. Here, we demonstrate how a recently developed empirical Bayesian method for HMMs can be extended to enable a more automated and statistically principled approach to two widely occurring tasks in the analysis of single-molecule fluorescence resonance energy transfer (smFRET) experiments: 1), the characterization of changes in rates across a series of experiments performed under variable conditions; and 2), the detection of degenerate states that exhibit the same FRET efficiency but differ in their rates of transition. We apply this newly developed methodology to two studies of the bacterial ribosome, each exemplary of one of these two analysis tasks. We conclude with a discussion of model-selection techniques for determination of the appropriate number of conformational states. The code used to perform this analysis and a basic graphical user interface front end are available as open source software.     

Empirical Bayes Methods Enable Advanced Population-Level Analyses of Single-Molecule FRET Experiments

Many single-molecule experiments aim to characterize biomolecular processes in terms of kinetic models that specify the rates of transition between conformational states of the biomolecule. Estimation of these rates often requires analysis of a population of molecules, in which the conformational trajectory of each molecule is represented by a noisy, time-dependent signal trajectory. Although hidden Markov models (HMMs) may be used to infer the conformational trajectories of individual molecules, estimating a consensus kinetic model from the population of inferred conformational trajectories remains a statistically difficult task, as inferred parameters vary widely within a population. Here, we demonstrate how a recently developed empirical Bayesian method for HMMs can be extended to enable a more automated and statistically principled approach to two widely occurring tasks in the analysis of single-molecule fluorescence resonance energy transfer (smFRET) experiments: 1), the characterization of changes in rates across a series of experiments performed under variable conditions; and 2), the detection of degenerate states that exhibit the same FRET efficiency but differ in their rates of transition. We apply this newly developed methodology to two studies of the bacterial ribosome, each exemplary of one of these two analysis tasks. We conclude with a discussion of model-selection techniques for determination of the appropriate number of conformational states. The code used to perform this analysis and a basic graphical user interface front end are available as open source software.     

纳米金提高光敏剂单态氧产率的研究

光动力疗法是效果很好的癌症微创治疗方法,主要依靠光敏性药物(也称为光敏剂)进行癌细胞杀伤。在光动力治疗中,光敏剂单态氧产率是影响光动力效果的关键因素。利用纳米金的光学特性来提高光敏剂的单态氧产率为研制新型光敏剂来改进光动力治疗方法提供了一种新的途径。利用绿光LED灯、红光LED灯、氙灯和635 nm连续激光四种光源对混合有光敏剂原卟啉Ⅸ和纳米金的溶液进行光照。用单态氧检测试剂测定了光照后的单态氧产率。     

光动力学疗法的单态氧剂量学研究进展

单态氧(1O2)是光动力学疗法(photodynamic therapy,PDT)过程中Ⅱ型光化学反应的主要细胞毒性物质.通过直接测量1O2发光强度预测PDT疗效已成为PDT剂量学研究的前沿热点,这种方法的最大优点在于能够克服现有其它剂量学方法中的光、光敏剂、氧分子,以及组织光学特性等因素之间的复杂相互影响,将PDT的...     

Hydroxyl Radical Generation Caused by the Reaction of Singlet Oxygen with a Spin Trap,DMPO, Increases Significantly in the Presence of Biological Reductants

Photosensitizers newly developed for photodynamic therapy of cancer need to be assessed using accurate methods of measuring reactive oxygen species (ROS). Little is known about the characteristics of the reaction of singlet oxygen (¹O²) with spin traps, although this knowledge is necessary in electron spin resonance (ESR)/spin trapping. In the present study, we examined the effect of various reductants usually present in biological samples on the reaction of ¹O² with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). The ESR signal of the hydroxyl radical (?OH) adduct of DMPO (DMPO-OH) resulting from ¹O²-dependent generation of ?OH strengthened remarkably in the presence of reduced glutathione (GSH), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), ascorbic acid, NADPH, etc. A similar increase was observed in the photosensitization of uroporphyrin (UP), rose bengal (RB) or methylene blue (MB). Use of 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide (DEPMPO) as a spin trap significantly lessened the production of its ?OH adduct (DEPMPO-OH) in the presence of the reductants. The addition of DMPO to the DEPMPO-spin trapping system remarkably increased the signal intensity of DEPMPO-OH. DMPO-mediated generation of ?OH was also confirmed utilizing the hydroxylation of salicylic acid (SA). These results suggest that biological reductants enhance the ESR signal of DMPO-OH produced by DMPO-mediated generation of ?OH from ¹O², and that spin trap-mediated ?OH generation hardly occurs with DEPMPO.     

利用RNO脱色反应检测类囊体中的单线态氧

光敏剂RB在光照射下与O₂反应产生 ¹O₂, ¹O₂与组氨酸或咪唑反应的中间产物使RNO发生氧化,导致RNO在440 nm处吸光度减小,此即为RNO脱色反应.RNO脱色反应随着光照时间的增加而增大,表明RB受光照射后使 ¹O₂增加;随着组氨酸或咪唑浓度的增加,RNO脱色反应增大;咪唑在RNO脱色反应中的作用更明显. ¹O₂淬灭剂NaN₃或DABCO存在时,RNO脱色反应降低.利用RNO脱色反应检测到莴苣类囊体在强光照射下产生的 ¹O₂,随着光强和照射时间增加,类囊体中 ¹O₂的产生增加.     

Virucidal Nanofiber Textiles Based on Photosensitized Production of Singlet Oxygen

Novel biomaterials based on hydrophilic polycaprolactone and polyurethane (Tecophilic®) nanofibers with an encapsulated 5,10,5,20-tetraphenylporphyrin photosensitizer were prepared by electrospinning. The doped nanofiber textiles efficiently photo-generate O₂(¹Δ_g), which oxidize external chemical and biological substrates/targets. Strong photo-virucidal effects toward non-enveloped polyomaviruses and enveloped baculoviruses were observed on the surface of these textiles. The photo-virucidal effect was confirmed by a decrease in virus infectivity. In contrast, no virucidal effect was detected in the absence of light and/or the encapsulated photosensitizer.     

Developing Single-Molecule TPM Experiments for Direct Observation of Successful RecA-Mediated Strand Exchange Reaction

RecA recombinases play a central role in homologous recombination. Once assembled on single-stranded (ss) DNA, RecA nucleoprotein filaments mediate the pairing of homologous DNA sequences and strand exchange processes. We have designed two experiments based on tethered particle motion (TPM) to investigate the fates of the invading and the outgoing strands during E. coli RecA-mediated pairing and strand exchange at the single-molecule level in the absence of force. TPM experiments measure the tethered bead Brownian motion indicative of the DNA tether length change resulting from RecA binding and dissociation. Experiments with beads labeled on either the invading strand or the outgoing strand showed that DNA pairing and strand exchange occurs successfully in the presence of either ATP or its non-hydrolyzable analog, ATPγS. The strand exchange rates and efficiencies are similar under both ATP and ATPγS conditions. In addition, the Brownian motion time-courses suggest that the strand exchange process progresses uni-directionally in the 5′-to-3′ fashion, using a synapse segment with a wide and continuous size distribution.     

The Ambivalent Role of Glutathione in the Protection of Dna Against Singlet Oxygen

Glutathione (GSH) was examined with respect to its ability to protect DNA against ¹O₂ damage. We have found that GSH protected, at least partly, the DNA against inactivation by ¹O₂. Up to 10 mM the protection increased as a function of GSH concentration. Above 10 mM the protection remained constant and less than expected on the basis of scavenging/quenching of ¹O₂, in contrast to the protection offered by sodium-azide. Especially at the higher concentrations of GSH the protection against the biological inactivation is accompanied by an increase in single-strand breaks and also probably lethal base damage. However, all together the data suggest that at least in the physiologically important range (0.1-10 mM) GSH is able to protect efficiently against ¹O₂-induced inactivating DNA damage.