Z-DNA is one of the many possible double helical structures of DNA . It is a left-handed double helical structure in which the helix winds to the left in a zigzag pattern, instead of to the right, like the more common B-DNA form. Z-DNA is thought to be one of three biologically active double-helical structures along with A-DNA and B-DNA .
155-437: Left-handed DNA was first proposed by Robert Wells and colleagues, as the structure of a repeating polymer of inosine – cytosine . They observed a "reverse" circular dichroism spectrum for such DNAs, and interpreted this incorrectly to mean that the strands wrapped around one another in a left-handed fashion. The relationship between Z-DNA and the more familiar B-DNA was indicated by the work of Pohl and Jovin, who showed that
310-459: A catalyst . Laboratory synthesis of biopolymers, especially of proteins , is an area of intensive research. There are three main classes of biopolymers: polysaccharides , polypeptides , and polynucleotides . In living cells, they may be synthesized by enzyme-mediated processes, such as the formation of DNA catalyzed by DNA polymerase . The synthesis of proteins involves multiple enzyme-mediated processes to transcribe genetic information from
465-591: A charge-coupled device (CCD) camera is explained in the literature. For a two-dye system, the emission signals are then used to calculate the FRET efficiency between the dyes over time. The FRET efficiency is the number of photons emitted from the acceptor dye over the sum of the emissions of the donor and the acceptor dye. The time trajectories produced in every experiment may contain signals from incomplete or incorrect labeling or even aggregates. To account for this multiple research groups have developed sophisticated tools for
620-441: A lower critical solution temperature phase transition (LCST), at which phase separation occurs with heating. In dilute solutions, the properties of the polymer are characterized by the interaction between the solvent and the polymer. In a good solvent, the polymer appears swollen and occupies a large volume. In this scenario, intermolecular forces between the solvent and monomer subunits dominate over intramolecular interactions. In
775-402: A nucleotide are labeled with the donor and acceptor dyes. The change of distance between the two locations changes over time due to the folding and unfolding of the nucleotide plus the random diffusion of the two points over time, within each measuring window and among different windows. Due to the complexity of the folding/unfolding trajectory, it is extremely difficult to measure the process at
930-455: A "frame rate" or a fixed integration time like when using cameras. In fact, unlike cameras, SPADs produce a pulse every time a photon is detected, while additional electronics are needed to "timestamp" each pulse with 10-50 ns resolution. The high time resolution of confocal single-molecule FRET measurements allows observers to potentially detect dynamics on time scales as low as 10 μs. However, detecting "slow" transitions on timescales longer than
1085-536: A B- to Z-DNA junction in 2005 provided a better understanding of the potential role Z-DNA plays in cells. Whenever a segment of Z-DNA forms, there must be B–Z junctions at its two ends, interfacing it to the B-form of DNA found in the rest of the genome . In 2007, the RNA version of Z-DNA, Z-RNA , was described as a transformed version of an A-RNA double helix into a left-handed helix. The transition from A-RNA to Z-RNA, however,
1240-464: A Z-DNA structure. An algorithm for predicting the propensity of DNA to flip from the B-form to the Z-form, ZHunt , was written by P. Shing Ho in 1984 at MIT . This algorithm was later developed by Tracy Camp , P. Christoph Champ , Sandor Maurice , and Jeffrey M. Vargason for genome-wide mapping of Z-DNA (with Ho as the principal investigator). Since the discovery and crystallization of Z-DNA in 1979,
1395-498: A bad solvent or poor solvent, intramolecular forces dominate and the chain contracts. In the theta solvent , or the state of the polymer solution where the value of the second virial coefficient becomes 0, the intermolecular polymer-solvent repulsion balances exactly the intramolecular monomer-monomer attraction. Under the theta condition (also called the Flory condition), the polymer behaves like an ideal random coil . The transition between
1550-807: A clear distinction of signal, FRET overlaps must be small but that also weakens FRET strength. SmFRET corrects its overlap limitations by using band-pass filters and dichroic mirrors which further the signal between two fluorescence acceptors and solve for any bleed-through effects. A major application of smFRET is to analyze the minute biochemical nuances that facilitate protein folding . In recent years, multiple techniques have been developed to investigate single-molecule interactions that are involved in protein folding and unfolding. Force-probe techniques , using atomic force microscopy and laser tweezers , have provided information on protein stability. smFRET allows researchers to investigate molecular interactions using fluorescence. Forster resonance energy transfer (FRET)
1705-408: A degree of crystallinity approaching zero or one will tend to be transparent, while polymers with intermediate degrees of crystallinity will tend to be opaque due to light scattering by crystalline or glassy regions. For many polymers, crystallinity may also be associated with decreased transparency. The space occupied by a polymer molecule is generally expressed in terms of radius of gyration , which
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#17328581277431860-587: A deviation from a simple linear chain. A branched polymer molecule is composed of a main chain with one or more substituent side chains or branches. Types of branched polymers include star polymers , comb polymers , polymer brushes , dendronized polymers , ladder polymers , and dendrimers . There exist also two-dimensional polymers (2DP) which are composed of topologically planar repeat units. A polymer's architecture affects many of its physical properties including solution viscosity, melt viscosity, solubility in various solvents, glass-transition temperature and
2015-469: A different noise distribution due to the different sources. The real-sample noise comes from the thermal disturbance of the system that results in the FRET distance broadening, uneven dye orientation distribution, dye emission variations, fast blinking, and faster-than-integration-time kinetics. Slow variations of the dye emission are likely considered false-positive states that should be experimentally avoided by choosing different dyes. The other noises are from
2170-421: A distinguishable set of states or set of rate constants. The uncertainty level of the rate analysis can be estimated from multiple experimental trials, bootstrapping analysis, and fitting error analysis. State misassignment is a common error source during the data analysis, which originates from state broadening, noise, and camera blurring. Data binning , moving average , and wavelet transform can help reduce
2325-412: A faster time resolution then bin it to the measuring time resolution (integration time) to simulate the camera blurring effect. The time trajectories also contain the photoblinking and photobleaching information of the two dyes. This information has to be removed which creates gaps in the time trajectory where the FRET information is lost. The photoblinking and photobleaching information can be removed for
2480-464: A flexible quality. Plasticizers are also put in some types of cling film to make the polymer more flexible. The attractive forces between polymer chains play a large part in determining the polymer's properties. Because polymer chains are so long, they have many such interchain interactions per molecule, amplifying the effect of these interactions on the polymer properties in comparison to attractions between conventional molecules. Different side groups on
2635-423: A function of time and thereby it is possible to follow kinetic events on the single molecule scale and to build FRET histograms showing the distribution of states in each molecule. However, data from many FRET pairs must be recorded and combined in order to obtain general information about a sample or a dynamic structure. In surface-immobilized experiments, biomolecules labeled with fluorescent tags are bound to
2790-479: A given application, the properties of a polymer can be tuned or enhanced by combination with other materials, as in composites . Their application allows to save energy (lighter cars and planes, thermally insulated buildings), protect food and drinking water (packaging), save land and lower use of fertilizers (synthetic fibres), preserve other materials (coatings), protect and save lives (hygiene, medical applications). A representative, non-exhaustive list of applications
2945-402: A high surface quality and are also highly transparent so that the laser properties are dominated by the laser dye used to dope the polymer matrix. These type of lasers, that also belong to the class of organic lasers , are known to yield very narrow linewidths which is useful for spectroscopy and analytical applications. An important optical parameter in the polymer used in laser applications
3100-419: A junction with B-DNA (called a "B-to-Z junction box") in a structure which involves the extrusion of a base pair. The Z-DNA conformation has been difficult to study because it does not exist as a stable feature of the double helix. Instead, it is a transient structure that is occasionally induced by biological activity and then quickly disappears. It is possible to predict the likelihood of a DNA sequence forming
3255-425: A liquid sample. In freely-diffusing smFRET experiments (or diffusion-based smFRET), the same biomolecules are free to diffuse in solution while being excited by a small excitation volume (usually a diffraction-limited spot ). Bursts of photons due to a single-molecule crossing the excitation spot are acquired with SPAD detectors. The confocal spot is usually fixed in a given position (no scanning happens, and no image
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#17328581277433410-433: A major challenge because the fluorescence of the donor and acceptor fluorophores, as well as the energy transfer, is dependent on the environment and how the dyes are oriented, which can vary depending on the flexibility of where the fluorophores are bound. In addition, the actual distance of a given state can be dynamic and the measured value represents the average distance within the collection time frame. This issue, however,
3565-434: A molecule and collects intensity-time data directly, giving the sample drifting is negligible, or an active feedback loop is used to keep track of the molecule. An advantage of surface-immobilized experiments is that many molecules can be observed in parallel for an extended period of time until photobleaching (typically 1-30 s). This allows us to conveniently study transitions taking place on slow time scales. A disadvantage
3720-431: A molecule stays on state one is t 1 = 100 second (s), state two t 2 = 20 s, and state three is t 3 = 10 s. Among the 100 s the molecule stays state one, it transfers to state two 70 times and transfers to state three 30 times at the end of its dwell times, the rate constant of state 1 to state 2 is thus k 12 = 70/100 = 0.7 s , the rate constant of state 1 to state 3 is k 13 = 30/100 = 0.3 s . Typically
3875-401: A polymer behaves as a continuous macroscopic material. They are classified as bulk properties, or intensive properties according to thermodynamics . The bulk properties of a polymer are those most often of end-use interest. These are the properties that dictate how the polymer actually behaves on a macroscopic scale. The tensile strength of a material quantifies how much elongating stress
4030-421: A polymer is its first and most important attribute. Polymer nomenclature is generally based upon the type of monomer residues comprising the polymer. A polymer which contains only a single type of repeat unit is known as a homopolymer , while a polymer containing two or more types of repeat units is known as a copolymer . A terpolymer is a copolymer which contains three types of repeat units. Polystyrene
4185-433: A polymeric material can be described at different length scales, from the sub-nm length scale up to the macroscopic one. There is in fact a hierarchy of structures, in which each stage provides the foundations for the next one. The starting point for the description of the structure of a polymer is the identity of its constituent monomers. Next, the microstructure essentially describes the arrangement of these monomers within
4340-536: A process called reptation in which each chain molecule is constrained by entanglements with neighboring chains to move within a virtual tube. The theory of reptation can explain polymer molecule dynamics and viscoelasticity . Depending on their chemical structures, polymers may be either semi-crystalline or amorphous. Semi-crystalline polymers can undergo crystallization and melting transitions , whereas amorphous polymers do not. In polymers, crystallization and melting do not suggest solid-liquid phase transitions, as in
4495-471: A result of time-resolved observation of protein folding events. These FRET efficiencies can then be used to infer distances between molecules as a function of time. As the protein transitions between the folded and unfolded states, the corresponding distances between molecules can indicate the sequence of molecular interactions that lead to protein folding. Another application of smFRET is for DNA and RNA folding dynamics. Typically, two different locations of
4650-427: A result, they typically have lower melting temperatures than other polymers. When a polymer is dispersed or dissolved in a liquid, such as in commercial products like paints and glues, the chemical properties and molecular interactions influence how the solution flows and can even lead to self-assembly of the polymer into complex structures. When a polymer is applied as a coating, the chemical properties will influence
4805-405: A role to free the labor in data analysis. SmFRET allows for a more precise analysis of heterogeneous populations and has a few advantages when compared to ensemble FRET. One benefit of studying distances in single molecules is that heterogeneous populations can be studied more accurately with values specific for each molecule rather than computing an average based on an ensemble. This allows for
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4960-456: A statistical distribution of chain lengths, the molecular weight is expressed in terms of weighted averages. The number-average molecular weight ( M n ) and weight-average molecular weight ( M w ) are most commonly reported. The ratio of these two values ( M w / M n ) is the dispersity ( Đ ), which is commonly used to express the width of the molecular weight distribution. The physical properties of polymer strongly depend on
5115-451: A system under equilibrium is lost at the ensemble level because none of the concentrations of the reactants and products change over time. However, at the single-molecule level, the transfer between the reactants and products can happen at a measurable high rate and be balanced over time stochastically. Thus, tracing the time trajectory of a particular molecule enables the direct measurement of the rate constant of each transition step, including
5270-537: A tendency to form amorphous and semicrystalline structures rather than crystals . Polymers are studied in the fields of polymer science (which includes polymer chemistry and polymer physics ), biophysics and materials science and engineering . Historically, products arising from the linkage of repeating units by covalent chemical bonds have been the primary focus of polymer science. An emerging important area now focuses on supramolecular polymers formed by non-covalent links. Polyisoprene of latex rubber
5425-484: A transition happens over the time trajectory using statistical analysis. For example, CPA based on Fisher information theory and the Student's t-test method (STaSI, open-source, GitHub link ) identifies state transitions and minimizes description length by selecting the optimum number of states, i.e. balancing the penalty of noise and the total number of states. Once the states are identified, they can be used to calculate
5580-731: A type of poxvirus . Two critical components to the E3L protein that determine virulence are the N-terminus and the C-terminus . The N-terminus is made of up a sequence similar to that of the Zα domain, also called Adenosine deaminase z-alpha domain , while the C-terminus is composed of a double stranded RNA binding motif. Through research done by Kim, Y. et al. at the Massachusetts Institute of Technology, it
5735-509: A typical dye system with relatively long photoblinking intervals and photobleaching lifetimes that have been chosen in the measurement. Thus, they are less of a problem for data analysis. However, it will become a big problem if a dye with short blinking intervals or a long dark-state lifetime is used. Specific chemical solutions have been developed to mitigate these two problems such as oxygen scavenger solutions or triplet state quenchers. Several data analysis methods have been developed to analyze
5890-432: A variety of different but structurally related monomer residues; for example, polynucleotides such as DNA are composed of four types of nucleotide subunits. A polymer containing ionizable subunits (e.g., pendant carboxylic groups ) is known as a polyelectrolyte or ionomer , when the fraction of ionizable units is large or small respectively. The microstructure of a polymer (sometimes called configuration) relates to
6045-469: A very short period in each camera collection cycle to avoid averaging of the signal in each cycle. A two-step data analysis method has also been reported to increase the analysis accuracy for camera blurred data. The idea is to simulate a trajectory with the Monte Carlo simulation method and compare it to the experimental data. At the right condition, both the simulation and the experimental data will contain
6200-405: A wide-meshed cross-linking between the "main chains". Close-meshed crosslinking, on the other hand, leads to thermosets . Cross-links and branches are shown as red dots in the figures. Highly branched polymers are amorphous and the molecules in the solid interact randomly. An important microstructural feature of a polymer is its architecture and shape, which relates to the way branch points lead to
6355-441: Is r = N nf / 𝛿 t = k nf c n . Thus k nf = N nf / t n . One can see that the single molecule measurement of the rate constant is only dependent on the ergodic hypothesis, which can be judged if a statistically enough number of single molecules are measured and expected well-behaved distributions of dwell times are observed. Heterogeneity among single molecules can be observed as well if each molecule has
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6510-479: Is a crucial physical parameter for polymer manufacturing, processing, and use. Below T g , molecular motions are frozen and polymers are brittle and glassy. Above T g , molecular motions are activated and polymers are rubbery and viscous. The glass-transition temperature may be engineered by altering the degree of branching or crosslinking in the polymer or by the addition of plasticizers . Whereas crystallization and melting are first-order phase transitions ,
6665-410: Is a long-chain n -alkane. There are also branched macromolecules with a main chain and side chains, in the case of polyethylene the side chains would be alkyl groups . In particular unbranched macromolecules can be in the solid state semi-crystalline, crystalline chain sections highlighted red in the figure below. While branched and unbranched polymers are usually thermoplastics, many elastomers have
6820-1131: Is a substance composed of macromolecules. A macromolecule is a molecule of high relative molecular mass, the structure of which essentially comprises the multiple repetition of units derived, actually or conceptually, from molecules of low relative molecular mass. A polymer ( / ˈ p ɒ l ɪ m ər / ) is a substance or material that consists of very large molecules, or macromolecules , that are constituted by many repeating subunits derived from one or more species of monomers . Due to their broad spectrum of properties, both synthetic and natural polymers play essential and ubiquitous roles in everyday life. Polymers range from familiar synthetic plastics such as polystyrene to natural biopolymers such as DNA and proteins that are fundamental to biological structure and function. Polymers, both natural and synthetic, are created via polymerization of many small molecules, known as monomers . Their consequently large molecular mass , relative to small molecule compounds , produces unique physical properties including toughness , high elasticity , viscoelasticity , and
6975-416: Is acquired). Instead, the fluorescence photons emitted by individual molecules crossing the excitation volume are recorded and accumulated in order to build a distribution of different populations present in the sample. Depending on the complexity of this distribution, acquisition times vary from ~5 min to several hours. A distinctive advantage of setups employing SPAD detectors is that they are not limited by
7130-444: Is addressed by smFRET which offers a direct way to observe the intermediates of single molecules regardless of accumulation. When you observe a long enough time on a molecule, there will be events of the transition state that last long enough to distinguish it from noise or the broadening of other states. Therefore, smFRET demonstrates the ability to capture transient subpopulations in a heterogeneous environment. Kinetic information in
7285-400: Is also commonly present in polymer backbones, such as those of polyethylene glycol , polysaccharides (in glycosidic bonds ), and DNA (in phosphodiester bonds ). Polymerization is the process of combining many small molecules known as monomers into a covalently bonded chain or network. During the polymerization process, some chemical groups may be lost from each monomer. This happens in
7440-512: Is an average distance from the center of mass of the chain to the chain itself. Alternatively, it may be expressed in terms of pervaded volume , which is the volume spanned by the polymer chain and scales with the cube of the radius of gyration. The simplest theoretical models for polymers in the molten, amorphous state are ideal chains . Polymer properties depend of their structure and they are divided into classes according to their physical bases. Many physical and chemical properties describe how
7595-481: Is an example of a natural polymer, and the polystyrene of styrofoam is an example of a synthetic polymer. In biological contexts, essentially all biological macromolecules —i.e., proteins (polyamides), nucleic acids (polynucleotides), and polysaccharides —are purely polymeric, or are composed in large part of polymeric components. The term "polymer" derives from Greek πολύς (polus) 'many, much' and μέρος (meros) 'part'. The term
7750-506: Is associated with both DNA transcription and replication, Z-DNA formation is primarily linked to the rate of transcription . A study of human chromosome 22 showed a correlation between Z-DNA forming regions and promoter regions for nuclear factor I . This suggests that transcription in some human genes may be regulated by Z-DNA formation and nuclear factor I activation. Z-DNA sequences upstream of promoter regions have been shown to stimulate transcription. The greatest increase in activity
7905-448: Is associated with systemic lupus erythematosus (SLE) through the presence of naturally occurring antibodies. Significant amounts of anti Z-DNA antibodies were found in SLE patients and were not present in other rheumatic diseases. There are two types of these antibodies. Through radioimmunoassay, it was found that one interacts with the bases exposed on the surface of Z-DNA and denatured DNA, while
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#17328581277438060-424: Is characterized by a macro-time (i.e. a coarse 10-50 ns timestamp) and a micro-time (i.e. delay with respect to the last laser pulse). The latter can be used to extract lifetime information and obtain the FRET signal. Typical smFRET data of a two-dye system are time trajectories of the fluorescent emission intensities of the donor dye and the acceptor dye, called the two channels. Mainly two methods are used to obtain
8215-428: Is composed only of styrene -based repeat units, and is classified as a homopolymer. Polyethylene terephthalate , even though produced from two different monomers ( ethylene glycol and terephthalic acid ), is usually regarded as a homopolymer because only one type of repeat unit is formed. Ethylene-vinyl acetate contains more than one variety of repeat unit and is a copolymer. Some biological polymers are composed of
8370-409: Is defined, for small strains , as the ratio of rate of change of stress to strain. Like tensile strength, this is highly relevant in polymer applications involving the physical properties of polymers, such as rubber bands. The modulus is strongly dependent on temperature. Viscoelasticity describes a complex time-dependent elastic response, which will exhibit hysteresis in the stress-strain curve when
8525-618: Is encoded in DNA by both shape and sequence. A role in regulating type I interferon responses in cancer is also supported by findings that 40% of a panel of tumors were dependent on the ADAR enzyme for survival. In previous studies, Z-DNA was linked to both Alzheimer's disease and systemic lupus erythematosus . To showcase this, a study was conducted on the DNA found in the hippocampus of brains that were normal, moderately affected with Alzheimer's disease, and severely affected with Alzheimer's disease. Through
8680-750: Is formed, but instead they actually promote the formation of the Z-DNA directly from the B* conformation, which is formed by the B-DNA structure being bound by high affinity proteins. A biological role for Z-DNA in the regulation of type I interferon responses has been confirmed in studies of three well-characterized rare Mendelian Diseases: Dyschromatosis Symmetrica Hereditaria (OMIM: 127400), Aicardi-Goutières syndrome (OMIM: 615010) and Bilateral Striatal Necrosis/Dystonia. Families with haploid ADAR transcriptome enabled mapping of Zα variants directly to disease, showing that genetic information
8835-449: Is given below. Single-molecule FRET Single-molecule fluorescence (or Förster) resonance energy transfer (or smFRET ) is a biophysical technique used to measure distances at the 1-10 nanometer scale in single molecules , typically biomolecules . It is an application of FRET wherein a pair of donor and acceptor fluorophores are excited and detected at a single molecule level. In contrast to "ensemble FRET" which provides
8990-492: Is intuitive and has a long history of applications. An example source code can be found in the software postFRET. HMMs are base on algorithms that statistically calculate probability functions of each state assignment, i.e. add penalties to a less probable assignment. The typical open source-code software packages can be found online such as HaMMy, vbFRET, ebFRET, SMART, SMACKS, MASH-FRET, etc. Transition-point analysis or change-point analysis (CPA) uses algorithms to identify when
9145-506: Is less of a problem when the measuring frequency is much faster than the transition rate but becomes a real problem when they approach each other (figure on the right). In order to reflect the camera blurring effect in the simulated smFRET trajectories, one must simulate the data in a higher time resolution (e.g. 10 times faster) than the data collection time and then bin the data into the final trajectories. See GitHub (postFRET) for an example MATLAB codes to simulate smFRET time trajectories with
9300-485: Is necessary for virulence. Overall, these findings show that the similar Z-DNA binding residues within the N-terminus of the E3L protein and the Zα domain are the most important structural factors determining virulence caused by the vaccinia virus, while amino acid residues not involved in Z-DNA binding have little to no effect. A future implication of these findings includes reducing Z-DNA binding of E3L in vaccines containing
9455-454: Is not interested, one can set the two η {\displaystyle \eta } = 1, and the two quantum yields to 1 as well, i.e. γ = 1 {\displaystyle \gamma =1} . For the accumulative emission smFRET data, the time trajectories contain mainly the following information: (1) state transitions, (2) noise, (3) camera blurring (analog of motion blur ), (4) photoblinking and photobleaching of
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#17328581277439610-411: Is not particularly relevant when the distance estimation of the two fluorophores does not need to be determined with exact and absolute precision. Extracting kinetic information from a complicated biological system with a transition rate of around a few milliseconds or below remains challenging. The current time resolutions of such measurements are typically at the millisecond level with a few reports at
9765-419: Is observed when the Z-DNA sequence is placed three helical turns after the promoter sequence . Furthermore, using micrococcal nuclease-crosslinking technique, Z-DNA is unlikely to form nucleosomes , which are often located before and/or after a Z-DNA forming sequence. Because of this property, Z-DNA is hypothesized to code for the boundary in nucleosome positioning. Since the placement of nucleosomes influences
9920-410: Is represented by the additional biochemical modifications needed to link molecules to the surface and the perturbations that the surface can potentially exert on the molecular activity. In addition, the maximum time resolution of single-molecule intensities is limited by the camera acquisition time (typically >1 ms). SmFRET can also be used to study the conformations of molecules freely diffusing in
10075-477: Is shown in the research on the Holliday Junction . SmFRET with the three-color system offers insights into synchronized movements of the junction's three helical sites and the near non-existence of its parallel states. Ensemble FRET can use a three-color system as well. However, any obvious advantages are outweighed by the three-color system's requirements, including a clear separation of fluorophore signals. For
10230-435: Is the change in refractive index with temperature also known as dn/dT. For the polymers mentioned here the (dn/dT) ~ −1.4 × 10 in units of K in the 297 ≤ T ≤ 337 K range. Most conventional polymers such as polyethylene are electrical insulators , but the development of polymers containing π-conjugated bonds has led to a wealth of polymer-based semiconductors , such as polythiophenes . This has led to many applications in
10385-563: Is the main constituent of wood and paper. Hemoglycin (previously termed hemolithin ) is a space polymer that is the first polymer of amino acids found in meteorites . The list of synthetic polymers , roughly in order of worldwide demand, includes polyethylene , polypropylene , polystyrene , polyvinyl chloride , synthetic rubber , phenol formaldehyde resin (or Bakelite ), neoprene , nylon , polyacrylonitrile , PVB , silicone , and many more. More than 330 million tons of these polymers are made every year (2015). Most commonly,
10540-465: Is typically combined with total internal reflection fluorescence microscope (TIRF) . This selectively excites FRET pairs on the surface of the measurement chamber and rejects noise from the bulk of the sample. Alternatively, confocal microscopy minimizes background by focusing the fluorescence light onto a pinhole to reject out-of-focus light. The confocal volume has a diameter of around 220 nm, and therefore it must be scanned across if an image of
10695-475: The Förster resonance energy transfer distances and transition rate constants between the states. For a parallel reaction matrix among the states, the rate constants of each transition cannot be pulled out from the average lifetimes of each transition, which is fixed as the inverse sum of the rate constants. The lifetimes of transitions from one state to all other states are all the "same" for a single molecule. However,
10850-468: The ultraviolet circular dichroism of poly(dG-dC) was nearly inverted in 4 M sodium chloride solution and that the structure of poly d(I–C)·poly d(I–C) was in fact a right-handed D-DNA conformation. The suspicion that this was the result of a conversion from B-DNA to Z-DNA was confirmed by examining the Raman spectra of these solutions and the Z-DNA crystals. Subsequently, a crystal structure of "Z-DNA"
11005-499: The B–Z junction. Biological studies suggested that the Z-DNA binding domain of ADAR1 may localize this enzyme that modifies the sequence of the newly formed RNA to sites of active transcription. A role for Zα, Z-DNA and Z-RNA in defense of the genome against the invasion of Alu retro-elements in humans has evolved into a mechanism for the regulation of innate immune responses to dsRNA. Mutations in Zα are causal for human interferonopathies such as
11160-496: The DNA to RNA and subsequently translate that information to synthesize the specified protein from amino acids . The protein may be modified further following translation in order to provide appropriate structure and functioning. There are other biopolymers such as rubber , suberin , melanin , and lignin . Naturally occurring polymers such as cotton , starch , and rubber were familiar materials for years before synthetic polymers such as polyethene and perspex appeared on
11315-414: The DNA. A Z-DNA high affinity binding protein , hZαADAR1, was used at varying concentrations to induce the transformation from B-DNA to Z-DNA. The smFRET assays revealed a B* transition state, which formed as the binding of hZαADAR1 accumulated on the B-DNA structure and stabilized it. This step occurs to avoid high junction energy, in which the B-DNA structure is allowed to undergo a conformational change to
11470-1073: The FRET signal of a high number of molecules, single-molecule FRET is able to resolve the FRET signal of each individual molecule. The variation of the smFRET signal is useful to reveal kinetic information that an ensemble measurement cannot provide, especially when the system is under equilibrium with no ensemble/bulk signal change. Heterogeneity among different molecules can also be observed. This method has been applied in many measurements of intramolecular dynamics such as DNA/RNA/protein folding/unfolding and other conformational changes, and intermolecular dynamics such as reaction, binding, adsorption, and desorption that are particularly useful in chemical sensing, bioassays, and biosensing. Single-molecule FRET measurements are typically performed on fluorescence microscopes , either using surface-immobilized or freely-diffusing molecules. Single FRET pairs are illuminated using intense light sources, typically lasers , in order to generate sufficient fluorescence signals to enable single-molecule detection. Wide-field multiphoton microscopy
11625-504: The Gaussian function) and a Poisson background noise (signal-count-independent). The latter noise dominates when the channel is in the OFF or low-intensity state (right figure). The noises can be approximated as Gaussian distribution, especially at relatively high signal counts. The noises in the two channels then are combined into the non-linear equation listed above to calculate the FRET values. Thus,
11780-488: The Mendelian Aicardi-Goutières Syndrome.Additionally, Zα domains are demonstrated to localize at the stress granules because of their innate ability in binding nucleic acid. Furthermore, different Zα domains bind to the Z conformation of nucleic acid differently providing important avenues for specific targeting in drug discovery. As Z-DNA has been researched more thoroughly, it has been discovered that
11935-413: The Z-DNA structure without a major, disruptive change in energy. This result coincides with the computational results of Lee et al. proving the mechanism to be step-wise and its purpose being that it provides a lower energy barrier for the conformational change from the B-DNA to Z-DNA configuration. Contrary to the previous notion, the binding proteins do not actually stabilize the Z-DNA conformation after it
12090-437: The above equation is simply based on the assumption that each molecule is the same, the ergodic hypothesis . The existence of each state is just represented by its total time which is its "concentration". Thus, The rate of transition to any other state is the number of transitions normalized by this concentration. Numerically, the time concentration can be converted to number concentration to mimic an ensemble measurement. Because
12245-438: The acceptor and donor channel respectively at the same period of time, η A {\displaystyle \eta _{A}} and η D {\displaystyle \eta _{D}} are the photon collection efficiencies of the two channels, Q A {\displaystyle Q_{A}} and Q D {\displaystyle Q_{D}} are quantum yield of
12400-441: The adhesion of the coating and how it interacts with external materials, such as superhydrophobic polymer coatings leading to water resistance. Overall the chemical properties of a polymer are important elements for designing new polymeric material products. Polymers such as PMMA and HEMA:MMA are used as matrices in the gain medium of solid-state dye lasers , also known as solid-state dye-doped polymer lasers. These polymers have
12555-434: The backbone in a variety of ways. A copolymer containing a controlled arrangement of monomers is called a sequence-controlled polymer . Alternating, periodic and block copolymers are simple examples of sequence-controlled polymers . Tacticity describes the relative stereochemistry of chiral centers in neighboring structural units within a macromolecule. There are three types of tacticity: isotactic (all substituents on
12710-480: The binding of transcription factors , Z-DNA is thought to regulate the rate of transcription. Developed behind the pathway of RNA polymerase through negative supercoiling, Z-DNA formed via active transcription has been shown to increase genetic instability, creating a propensity towards mutagenesis near promoters. A study on Escherichia coli found that gene deletions spontaneously occur in plasmid regions containing Z-DNA-forming sequences. In mammalian cells,
12865-421: The case of water or other molecular fluids. Instead, crystallization and melting refer to the phase transitions between two solid states ( i.e. , semi-crystalline and amorphous). Crystallization occurs above the glass-transition temperature ( T g ) and below the melting temperature ( T m ). All polymers (amorphous or semi-crystalline) go through glass transitions . The glass-transition temperature ( T g )
13020-422: The configuration has left scientists puzzled about the pathway and mechanism from the B-DNA configuration to the Z-DNA configuration. The conformational change from B-DNA to the Z-DNA structure was unknown at the atomic level, but in 2010, computer simulations conducted by Lee et al. were able to computationally determine that the step-wise propagation of a B-to-Z transition would provide a lower energy barrier than
13175-469: The conformational changes within, allowing us to generalize similar dynamics for similar motifs in other eukaryotic channels. The structural dynamics of the KirBac channel has been thoroughly analyzed in both the open and closed states, dependent on the presence of the ligand PIP2 . Part of the results based on smFRET demonstrated the structural rigidity of the extracellular region. The selectivity filter and
13330-469: The continuously linked backbone of a polymer used for the preparation of plastics consists mainly of carbon atoms. A simple example is polyethylene ('polythene' in British English), whose repeat unit or monomer is ethylene . Many other structures do exist; for example, elements such as silicon form familiar materials such as silicones, examples being Silly Putty and waterproof plumbing sealant. Oxygen
13485-440: The data, such as thresholding methods, Hidden Markov Model (HMM) methods, and transition point identification methods. Thresholding methods simply set a threshold between two adjacent states on the smFRET trajectories. The FRET values above the threshold belong to one state and the values below belong to another. This method works for the data with a very high signal-to-noise ratio thus having distinguishable FRET states. This method
13640-536: The degree of crystallinity may be expressed in terms of a weight fraction or volume fraction of crystalline material. Few synthetic polymers are entirely crystalline. The crystallinity of polymers is characterized by their degree of crystallinity, ranging from zero for a completely non-crystalline polymer to one for a theoretical completely crystalline polymer. Polymers with microcrystalline regions are generally tougher (can be bent more without breaking) and more impact-resistant than totally amorphous polymers. Polymers with
13795-559: The diffusion time (typically ~1 ms) is more difficult than in surface-immobilized experiments and generally requires much longer acquisitions. Normally, the fluorescent emission of both donor and acceptor fluorophores is detected by two independent detectors and the FRET signal is computed from the ratio of intensities in the two channels. Some setup configurations further split each spectral channel (donor or acceptor) in two orthogonal polarizations (therefore requiring 4 detectors) and are able to measure both FRET and fluorescence anisotropy at
13950-413: The discrete nature of the measurements. The emission signal has a mismatch with the real transition signal because one or both are stochastic (random). When a state transition happens between the readout of two emission reading intervals, the signal is the average of the two parts in the same measuring window, which then affects the state identification accuracy and eventually the rate constant analysis. This
14105-400: The driving force for mixing is usually entropy , not interaction energy. In other words, miscible materials usually form a solution not because their interaction with each other is more favorable than their self-interaction, but because of an increase in entropy and hence free energy associated with increasing the amount of volume available to each component. This increase in entropy scales with
14260-552: The dyes. The state transition information is the information a typical measurement wants. However, the rest signals interfere with the data analysis and thus have to be addressed. A list of software packages can be found on the KinSoftChallenge website. A report on the comparison results among these software packages can be found here. The noise signal of the dye emission typically contains camera readout noise, shot noise and white noise , and real-sample noise, each following
14415-462: The effect of state broadening and noise but will enhance the camera blurring effect. The camera blurring effect can be reduced via faster sampling frequency relies on the development of a more sensitive camera, special data analysis, or both. Traditionally in HMMs, the data point before and after the transition is specially assigned to reduce the wrong assignment rate of these data points to states in between
14570-413: The emission of the two dyes: (1) accumulative measurement uses a fixed exposure time of the cameras for each frame, such as PMT, APD, EMCCD, and CMOS camera; (2) single-photon arriving time sequence measured using PMT or APD detectors. The principle is to use optical filters or dichroic mirrors to separate the emissions of the two dyes and measure them in two channels. For example, a setup using two halves of
14725-547: The ensemble level. Thus, smFRET becomes a key technique in this field. On top of the challenges of smFRET data analysis, one challenge is to label multiple positions of interest, another is from the two-point dynamics to calculate the overall folding pathways. Single-molecule FRET can also be applied to study the conformational changes of the relevant channel motifs in certain channels . For example, labeled tetrameric KirBac potassium channels were labeled with donor and acceptor fluorophores at particular sites in order to understand
14880-483: The excitation path and the detection path, especially the camera. In the end, the noise of the raw emission data is a combination of noises with Poisson distribution and Gaussian distribution . The noises in each channel sometimes (e.g. for single-photon detectors) can be simplified to the summation of an intensity-dependent Gaussian noise (the higher the count the larger the noise; it is a combination of Gaussian and larger-amplitude Poisson noise that can be represented by
15035-425: The exponential decay function of the accumulated histogram of the state dwell times. The latter only works for well-behaved dwell-time distributions. A system under equilibrium with each state parallel transfers to all others in first-order reactions is a special case for easier understanding. When the system is not under equilibrium, this equation still holds but care should be applied to use it. The interpretation of
15190-417: The field of organic electronics . Nowadays, synthetic polymers are used in almost all walks of life. Modern society would look very different without them. The spreading of polymer use is connected to their unique properties: low density, low cost, good thermal/electrical insulation properties, high resistance to corrosion, low-energy demanding polymer manufacture and facile processing into final products. For
15345-527: The free energy of mixing for polymer solutions and thereby making solvation less favorable, and thereby making the availability of concentrated solutions of polymers far rarer than those of small molecules. Furthermore, the phase behavior of polymer solutions and mixtures is more complex than that of small molecule mixtures. Whereas most small molecule solutions exhibit only an upper critical solution temperature phase transition (UCST), at which phase separation occurs with cooling, polymer mixtures commonly exhibit
15500-401: The glass transition is not. The glass transition shares features of second-order phase transitions (such as discontinuity in the heat capacity, as shown in the figure), but it is generally not considered a thermodynamic transition between equilibrium states. In general, polymeric mixtures are far less miscible than mixtures of small molecule materials. This effect results from the fact that
15655-415: The individual chains more strongly in position and resist deformations and matrix breakup, both at higher stresses and higher temperatures. Copolymers are classified either as statistical copolymers, alternating copolymers, block copolymers, graft copolymers or gradient copolymers. In the schematic figure below, Ⓐ and Ⓑ symbolize the two repeat units . Monomers within a copolymer may be organized along
15810-564: The intermediates that are hidden at the ensemble level due to their low concentrations. This allows smFRET to be used to study DNA , RNA , and protein ’s folding dynamics. Like protein folding , DNA and RNA folding go through multiple interactions, folding pathways, and intermediates before reaching their native states. SmFRET is also shown to utilize a three-color system better than ensemble FRET. Using two acceptor fluorophores rather than one, FRET can observe multiple sites for correlated movements and spatial changes in any complex molecule. This
15965-489: The larger deletions associated with mammalian cells are caused by non-homologous end-joining repair, which is known to be prone to error. The toxic effect of ethidium bromide (EtBr) on trypanosomas is caused by shift of their kinetoplastid DNA to Z-form. The shift is caused by intercalation of EtBr and subsequent loosening of DNA structure that leads to unwinding of DNA, shift to Z-form and inhibition of DNA replication. The first domain to bind Z-DNA with high affinity
16120-451: The latter case, increasing the polymer chain length 10-fold would increase the viscosity over 1000 times. Increasing chain length furthermore tends to decrease chain mobility, increase strength and toughness, and increase the glass-transition temperature (T g ). This is a result of the increase in chain interactions such as van der Waals attractions and entanglements that come with increased chain length. These interactions tend to fix
16275-436: The length (or equivalently, the molecular weight) of the polymer chain. One important example of the physical consequences of the molecular weight is the scaling of the viscosity (resistance to flow) in the melt. The influence of the weight-average molecular weight ( M w {\displaystyle M_{w}} ) on the melt viscosity ( η {\displaystyle \eta } ) depends on whether
16430-417: The load is removed. Dynamic mechanical analysis or DMA measures this complex modulus by oscillating the load and measuring the resulting strain as a function of time. Transport properties such as diffusivity describe how rapidly molecules move through the polymer matrix. These are very important in many applications of polymers for films and membranes. The movement of individual macromolecules occurs by
16585-483: The low background and high signal-to-noise ratio observed with the single-molecule FRET technique leads to ultra-sensitivity (detection limit in the femtomolar range) These days, different types of signal amplification steps are incorporated in order to push down the detection limit. Despite making approximate estimates, a limitation of smFRET is the difficulty of obtaining the correct distance involved in energy transfer. Requiring an accurate distance estimate gives rise to
16740-436: The market. Many commercially important polymers are synthesized by chemical modification of naturally occurring polymers. Prominent examples include the reaction of nitric acid and cellulose to form nitrocellulose and the formation of vulcanized rubber by heating natural rubber in the presence of sulfur . Ways in which polymers can be modified include oxidation , cross-linking , and end-capping . The structure of
16895-413: The material will endure before failure. This is very important in applications that rely upon a polymer's physical strength or durability. For example, a rubber band with a higher tensile strength will hold a greater weight before snapping. In general, tensile strength increases with polymer chain length and crosslinking of polymer chains. Young's modulus quantifies the elasticity of the polymer. It
17050-425: The microsecond level. There is a theoretical limitation to dye photophysics. The lifetime of the excited state of a typical organic dye molecule is about 1 nanosecond. In order to obtain statistical confidence of the FRET values, tens to hundreds of photons are required, which put the best possible time resolution to the order of 1 microsecond. In order to reach this limit, very strong light is required (power density in
17205-523: The noise on the smFRET trajectories is very complicated. The noise is asymmetric above and below the mean FRET values and its magnitude changes towards the two ends (0 and 1) of the FRET values due to the changing uncertainties of the A and D channels. Most noise can be reduced by binning the data (see Figure) with the cost of losing time resolution. See GitHub (postFRET) for an example of MATLAB codes to simulate smFRET time trajectories without and with noise (GitHub link ). The camera blurring signal comes from
17360-444: The number of particles (or moles) being mixed. Since polymeric molecules are much larger and hence generally have much higher specific volumes than small molecules, the number of molecules involved in a polymeric mixture is far smaller than the number in a small molecule mixture of equal volume. The energetics of mixing, on the other hand, is comparable on a per volume basis for polymeric and small molecule mixtures. This tends to increase
17515-422: The order of 1×10 W m , 10 sun typically achieved by focusing a laser beam using a microscope), which often cause photodamage to the organic molecules. Another limitation is the photobleaching lifetime of the dye, which is a function of light intensity and oxidation/reduction stress of the environment. The photobleaching lifetime of a typical organic dye under typical experimental conditions (laser power density just
17670-456: The other exclusively interacts with the zig-zag backbone of only Z-DNA. Similar to that found in Alzheimer's disease, the antibodies vary depending on the stage of the disease, with maximal antibodies in the most active stages of SLE. Z-DNA is commonly believed to provide torsional strain relief during transcription , and it is associated with negative supercoiling . However, while supercoiling
17825-522: The outer loop of the selectivity filter region were labeled with fluorophores and conformational coupling was observed. The individual smFRET trajectories strongly demonstrated a FRET efficiency of around 0.8 with no fluctuations, regardless of the state of the channel. Recently, single-molecule FRET has been applied to quantitatively detect target DNA and to distinguish single nucleotide polymorphism. Unlike ensemble FRET, single-molecule FRET allows real-time monitoring of target binding events. Additionally,
17980-713: The partially negatively charged oxygen atoms in C=O groups on another. These strong hydrogen bonds, for example, result in the high tensile strength and melting point of polymers containing urethane or urea linkages. Polyesters have dipole-dipole bonding between the oxygen atoms in C=O groups and the hydrogen atoms in H-C groups. Dipole bonding is not as strong as hydrogen bonding, so a polyester's melting point and strength are lower than Kevlar 's ( Twaron ), but polyesters have greater flexibility. Polymers with non-polar units such as polyethylene interact only through weak Van der Waals forces . As
18135-422: The physical arrangement of monomer residues along the backbone of the chain. These are the elements of polymer structure that require the breaking of a covalent bond in order to change. Various polymer structures can be produced depending on the monomers and reaction conditions: A polymer may consist of linear macromolecules containing each only one unbranched chain. In the case of unbranched polyethylene, this chain
18290-399: The polymer and create gaps between polymer chains for greater mobility and fewer interchain interactions. A good example of the action of plasticizers is related to polyvinylchlorides or PVCs. A uPVC, or unplasticized polyvinylchloride, is used for things such as pipes. A pipe has no plasticizers in it, because it needs to remain strong and heat-resistant. Plasticized PVC is used in clothing for
18445-426: The polymer at the scale of a single chain. The microstructure determines the possibility for the polymer to form phases with different arrangements, for example through crystallization , the glass transition or microphase separation . These features play a major role in determining the physical and chemical properties of a polymer. The identity of the repeat units (monomer residues, also known as "mers") comprising
18600-540: The polymer can lend the polymer to ionic bonding or hydrogen bonding between its own chains. These stronger forces typically result in higher tensile strength and higher crystalline melting points. The intermolecular forces in polymers can be affected by dipoles in the monomer units. Polymers containing amide or carbonyl groups can form hydrogen bonds between adjacent chains; the partially positively charged hydrogen atoms in N-H groups of one chain are strongly attracted to
18755-403: The polymer is above or below the onset of entanglements . Below the entanglement molecular weight , η ∼ M w 1 {\displaystyle \eta \sim {M_{w}}^{1}} , whereas above the entanglement molecular weight, η ∼ M w 3.4 {\displaystyle \eta \sim {M_{w}}^{3.4}} . In
18910-583: The polymerization of PET polyester . The monomers are terephthalic acid (HOOC—C 6 H 4 —COOH) and ethylene glycol (HO—CH 2 —CH 2 —OH) but the repeating unit is —OC—C 6 H 4 —COO—CH 2 —CH 2 —O—, which corresponds to the combination of the two monomers with the loss of two water molecules. The distinct piece of each monomer that is incorporated into the polymer is known as a repeat unit or monomer residue. Synthetic methods are generally divided into two categories, step-growth polymerization and chain polymerization . The essential difference between
19065-460: The presence of such sequences was found to produce large genomic fragment deletions due to chromosomal double-strand breaks . Both of these genetic modifications have been linked to the gene translocations found in cancers such as leukemia and lymphoma , since breakage regions in tumor cells have been plotted around Z-DNA-forming sequences. However, the smaller deletions in bacterial plasmids have been associated with replication slippage , while
19220-416: The previously hypothesized concerted mechanism. Since this was computationally proven, the pathway would still need to be tested experimentally in the lab for further confirmation and validity, in which Lee et al. specifically states in their journal article, "The current [computational] result could be tested by Single-molecule FRET (smFRET) experiments in the future." In 2018, the pathway from B-DNA to Z-DNA
19375-406: The previously mentioned genes, as a result of loss of hydrogen bonding and london dispersion forces between E3L and the Z-DNA. Overall, these results show that decreasing the bonds and interactions between Z-DNA and Z-DNA binding proteins decreases both virulence and gene expression, hence showing the importance of having bonds between Z-DNA and the E3L binding protein. Polymer A polymer
19530-416: The probabilities of the lengths of the 70 times or the 30 times transition (dwell times) are exponentially distributed (right figure). The average dwell times of the two distributions of state one, i.e. the two lifetimes, τ 12 and τ 13 , are both the same at 1 s for these two transitions (right figure). The number of transitions N can be the total number of state transitions or the fitted amplitude of
19685-439: The protein structure that can be inferred from the ensemble of data only provides a rudimentary structural model of protein folding. However, a true understanding of protein folding requires deciphering the sequence of structural events along the folding pathways between the folded and unfolded states. It is this particular branch of research that smFRET is highly applicable. FRET studies calculate corresponding FRET efficiencies as
19840-503: The rate constants can be calculated from the probability functions, the number of each transition over the total time of the state it transfers from. where i is the initial state, f is the final state of the transition, N is the number of these transitions in the time trajectories, n is the total number of states, k is the rate constant, t is the time of each state before the transition happens. For example, one measures 130 seconds (s) of smFRET time trajectories. The total time of
19995-549: The same degree of blurring information and noise. This simulated trajectory is a better answer than the raw experimental data because its ground truth is "known". This method has open-source codes available as postFRET (GitHub link ) and MASH-FRET. This method can also slightly correct the effect of the non-Gaussian noise that has caused trouble to accurately identify the states using the statistical methods. The current data analysis for smFRET still requires great care and special training, calling for deep-learning algorithms to play
20150-437: The same side), atactic (random placement of substituents), and syndiotactic (alternating placement of substituents). Polymer morphology generally describes the arrangement and microscale ordering of polymer chains in space. The macroscopic physical properties of a polymer are related to the interactions between the polymer chains. When applied to polymers, the term crystalline has a somewhat ambiguous usage. In some cases,
20305-486: The same time. In other configurations, 3 or 4 spectral channels are acquired at the same time in order to measure multiple FRET pairs at the same time. Both CW or pulsed lasers can be used as excitation sources. When using pulsed lasers, suitable acquisition hardware can measure the photon arrival time with respect to the last laser pulse with picosecond resolution, in the so-called time-correlated single photon counting (TCSPC) acquisition. In this configuration, each photon
20460-404: The sample is needed. With confocal excitation, it is possible to measure much deeper into the sample than when using TIRF. The fluorescence signal is detected either using ultra-sensitive CCD or scientific CMOS cameras for wide-field microscopy or SPADs for confocal microscopy. Once the single molecule intensities vs. time are available the FRET efficiency can be computed for each FRET pair as
20615-418: The single molecules are the same as all other molecules, we can assume that the time trajectory is a random combination of a lot of molecules each only occupying a very short period of time, say 𝛿 t . Thus, the "concentration" of the molecule at state n is c n = t n / 𝛿 t . Among all these "molecules", if N nf transfer to state f during this measuring time 𝛿 t , the rate of transfer by definition
20770-512: The size of individual polymer coils in solution. A variety of techniques may be employed for the synthesis of a polymeric material with a range of architectures, for example living polymerization . A common means of expressing the length of a chain is the degree of polymerization , which quantifies the number of monomers incorporated into the chain. As with other molecules, a polymer's size may also be expressed in terms of molecular weight . Since synthetic polymerization techniques typically yield
20925-448: The states is known as a coil–globule transition . Inclusion of plasticizers tends to lower T g and increase polymer flexibility. Addition of the plasticizer will also modify dependence of the glass-transition temperature T g on the cooling rate. The mobility of the chain can further change if the molecules of plasticizer give rise to hydrogen bonding formation. Plasticizers are generally small molecules that are chemically similar to
21080-444: The structural dynamics within the lipid membrane , thus allowing them to generalize similar dynamics for similar motifs in other eukaryotic Kir channels or even cation channels in general. The use of smFRET in this experiment allows for visualization of the conformational changes that cannot be seen if the macroscopic measurements are simply averaged. This will lead to ensemble analysis rather than analysis of individual molecules and
21235-429: The structure of Z-DNA can bind to Z-DNA binding proteins through van der Waal forces and hydrogen bonding . One example of a Z-DNA binding protein is the vaccinia E3L protein, which is a product of the E3L gene and mimics a mammalian protein that binds Z-DNA. Not only does the E3L protein have affinity to Z-DNA, it has also been found to play a role in the level of severity of virulence in mice caused by vaccinia virus,
21390-448: The study of specific homogeneous populations within a heterogeneous population. For example, if two existing homologous populations within a heterogeneous population have different FRET values, an ensemble FRET analysis will produce a weighted averaged FRET value to represent the population as a whole. Thus, the obtained FRET value does not produce data on the two distinct populations. In contrast, smFRET would be able to differentiate between
21545-411: The surface of the coverglass and images of fluorescence are acquired (typically by a CCD or scientific CMOS cameras). Data collection with cameras will produce movies of the specimen which must be processed to derive the single-molecule intensities with time. If a confocal microscope with a SPAD detector is used, molecule searching is done first by scanning the sample. Then the confocal point sits on
21700-686: The term crystalline finds identical usage to that used in conventional crystallography . For example, the structure of a crystalline protein or polynucleotide, such as a sample prepared for x-ray crystallography , may be defined in terms of a conventional unit cell composed of one or more polymer molecules with cell dimensions of hundreds of angstroms or more. A synthetic polymer may be loosely described as crystalline if it contains regions of three-dimensional ordering on atomic (rather than macromolecular) length scales, usually arising from intramolecular folding or stacking of adjacent chains. Synthetic polymers may consist of both crystalline and amorphous regions;
21855-411: The time trace selection based on multiple metrics, or using deep neural network. Usually, only the donor dye is excited, but even more accurate information can be obtained if donor and acceptor dye are alternatingly excited. In a single excitation scheme, the emission of the donor and acceptor dyes is just the number of photons collected for the two dyes divided by the photon collection efficiencies of
22010-405: The transition. However, there is a limitation to this method to work. When the transition frequency is approaching the sampling frequency, too much data are blurred for this method to work (see above the figure of the camera blur). An experimental approach using a pulsed laser has been reported to partially overcome the camera blurring effect without a very fast camera. The idea is to only illuminate
22165-481: The two channels respectively which are the functions of the collection efficiency, the filter and optical efficiency, and the camera efficiency of the two wavelength bands. These efficiencies can be calibrated for a given instrumental setup. where FRET is the FRET efficiency of the two-dye system at a period of time, I A {\displaystyle I_{A}} and I D {\displaystyle I_{D}} are measured photon counts of
22320-415: The two dyes, and γ {\displaystyle \gamma } is a correction to the different intrinsic brightness of the two dyes to the microscope setting. Thus, I / η {\displaystyle I/\eta } calculates the actual number of photons emitted from the dye. If the photon collection efficiencies of the two channels are similar and the actual FRET distance
22475-555: The two is that in chain polymerization, monomers are added to the chain one at a time only, such as in polystyrene , whereas in step-growth polymerization chains of monomers may combine with one another directly, such as in polyester . Step-growth polymerization can be divided into polycondensation , in which low-molar-mass by-product is formed in every reaction step, and polyaddition . Newer methods, such as plasma polymerization do not fit neatly into either category. Synthetic polymerization reactions may be carried out with or without
22630-449: The two populations and would allow an analysis of the existing homologous populations. SmFRET also provides dynamic temporal resolution of an individual molecule that cannot be accomplished through ensemble FRET measurements. Ensemble FRET has the ability to detect well-populated transition states that accumulate in a population, but it lacks the ability to characterize intermediates that are short-lived and do not accumulate. This limit
22785-463: The use of circular dichroism , this study showed the presence of Z-DNA in the DNA of those severely affected. In this study it was also found that major portions of the moderately affected DNA was in the B-Z intermediate conformation. This is significant because from these findings it was concluded that the transition from B-DNA to Z-DNA is dependent on the progression of Alzheimer's disease. Additionally, Z-DNA
22940-556: The vaccinia virus so negative reactions to the virus can be minimized in humans. Furthermore, Alexander Rich and Jin-Ah Kwon found that E3L acts as a transactivator for human IL-6, NF-AT, and p53 genes. Their results show that HeLa cells containing E3L had increased expression of human IL-6, NF-AT, and p53 genes and point mutations or deletions of certain Z-DNA binding amino acid residues decreased that expression. Specifically, mutations in Tyr 48 and Pro 63 were found to reduce transactivation of
23095-669: Was already described in 1984. Z-DNA is quite different from the right-handed forms. In fact, Z-DNA is often compared against B-DNA in order to illustrate the major differences. The Z-DNA helix is left-handed and has a structure that repeats every other base pair. The major and minor grooves, unlike A- and B-DNA, show little difference in width. Formation of this structure is generally unfavourable, although certain conditions can promote it; such as alternating purine – pyrimidine sequence (especially poly(dGC) 2 ), negative DNA supercoiling or high salt and some cations (all at physiological temperature, 37 °C, and pH 7.3–7.4). Z-DNA can form
23250-605: Was coined in 1833 by Jöns Jacob Berzelius , though with a definition distinct from the modern IUPAC definition. The modern concept of polymers as covalently bonded macromolecular structures was proposed in 1920 by Hermann Staudinger , who spent the next decade finding experimental evidence for this hypothesis. Polymers are of two types: naturally occurring and synthetic or man made . Natural polymeric materials such as hemp , shellac , amber , wool , silk , and natural rubber have been used for centuries. A variety of other natural polymers exist, such as cellulose , which
23405-478: Was discovered in ADAR1 using an approach developed by Alan Herbert. Crystallographic and NMR studies confirmed the biochemical findings that this domain bound Z-DNA in a non-sequence-specific manner. Related domains were identified in a number of other proteins through sequence homology . The identification of the Zα domain provided a tool for other crystallographic studies that lead to the characterization of Z-RNA and
23560-414: Was experimentally proven using smFRET assays. This was performed by measuring the intensity values between the donor and acceptor fluorescent dyes, also known as Fluorophores , in relation to each other as they exchange electrons, while tagged onto a DNA molecule. The distances between the fluorophores could be used to quantitatively calculate the changes in proximity of the dyes and conformational changes in
23715-508: Was first applied to single molecules by Ha et al. and applied to protein folding in work by Hochstrasser, Weiss, et al. The benefit that smFRET as a whole has afforded to analyze molecular interactions is the ability to test single molecule interactions directly without having to average ensembles of data. In protein folding analysis, ensemble experiments involve taking measurements of multiple proteins that are in various states of transition between their folded and unfolded state . When averaged,
23870-414: Was published which turned out to be the first single-crystal X-ray structure of a DNA fragment (a self-complementary DNA hexamer d(CG) 3 ). It was resolved as a left-handed double helix with two antiparallel chains that were held together by Watson–Crick base pairs (see X-ray crystallography ). It was solved by Andrew H. J. Wang , Alexander Rich , and coworkers in 1979 at MIT . The crystallisation of
24025-466: Was shown that replacing the N-terminus of the E3L protein with a Zα domain sequence, containing 14 Z-DNA binding residues similar to E3L, had little to no effect on pathogenicity of the virus in mice. In Contrast, Kim, Y. et al. also found that deleting all 83 residues of the E3L N-terminus resulted in decreased virulence. This supports their claim that the N-terminus containing the Z-DNA binding residues
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