Modeling the effect of intercalators on the high-force stretching behavior of DNA

DNA is structurally and mechanically altered by the binding of intercalator molecules. Intercalation strongly affects the force-extension behavior of DNA, in particular the overstretching transition. We present a statistical model that captures all relevant findings of recent force-extension experiments. Two predictions from our model are presented. The first suggests the existence of a novel hyper-stretching regime in the presence of intercalators and the second, a linear dependence of the overstretching force on intercalator concentration, is verified by re-analyzing available experimental data. Our model pins down the physical principles that govern intercalated DNA mechanics, providing a predictive understanding of its limitations and possibilities.Comment: 5 pages, 4 figure

Revealing in real-time a multistep assembly mechanism for SV40 virus-like particles

Many viruses use their genome as template for self-assembly into an infectious particle. However, this reaction remains elusive because of the transient nature of intermediate structures. To elucidate this process, optical tweezers and acoustic force spectroscopy are used to follow viral assembly in real time. Using Simian virus 40 (SV40) virus-like particles as model system, we reveal a multistep assembly mechanism. Initially, binding of VP1 pentamers to DNA leads to a significantly decreased persistence length. Moreover, the pentamers seem able to stabilize DNA loops. Next, formation of interpentamer interactions results in intermediate structures with reduced contour length. These structures stabilize into objects that permanently decrease the contour length to a degree consistent with DNA compaction in wild-type SV40. These data indicate that a multistep mechanism leads to fully assembled cross-linked SV40 particles. SV40 is studied as drug delivery system. Our insights can help optimize packaging of therapeutic agents in these particles

Duplex DNA and BLM regulate gate opening by the human TopoIIIα-RMI1-RMI2 complex

Topoisomerase IIIα is a type 1A topoisomerase that forms a complex with RMI1 and RMI2 called TRR in human cells. TRR plays an essential role in resolving DNA replication and recombination intermediates, often alongside the helicase BLM. While the TRR catalytic cycle is known to involve a protein-mediated single-stranded (ss)DNA gate, the detailed mechanism is not fully understood. Here, we probe the catalytic steps of TRR using optical tweezers and fluorescence microscopy. We demonstrate that TRR forms an open gate in ssDNA of 8.5 ± 3.8 nm, and directly visualize binding of a second ssDNA or double-stranded (ds)DNA molecule to the open TRR-ssDNA gate, followed by catenation in each case. Strikingly, dsDNA binding increases the gate size (by ~16%), while BLM alters the mechanical flexibility of the gate. These findings reveal an unexpected plasticity of the TRR-ssDNA gate size and suggest that TRR-mediated transfer of dsDNA may be more relevant in vivo than previously believed

Sliding and jumping of single EcoRV restriction enzymes on non-cognate DNA

The restriction endonuclease EcoRV can rapidly locate a short recognition site within long non-cognate DNA using ‘facilitated diffusion’. This process has long been attributed to a sliding mechanism, in which the enzyme first binds to the DNA via nonspecific interaction and then moves along the DNA by 1D diffusion. Recent studies, however, provided evidence that 3D translocations (hopping/jumping) also help EcoRV to locate its target site. Here we report the first direct observation of sliding and jumping of individual EcoRV molecules along nonspecific DNA. Using fluorescence microscopy, we could distinguish between a slow 1D diffusion of the enzyme and a fast translocation mechanism that was demonstrated to stem from 3D jumps. Salt effects on both sliding and jumping were investigated, and we developed numerical simulations to account for both the jump frequency and the jump length distribution. We deduced from our study the 1D diffusion coefficient of EcoRV, and we estimated the number of jumps occurring during an interaction event with nonspecific DNA. Our results substantiate that sliding alternates with hopping/jumping during the facilitated diffusion of EcoRV and, furthermore, set up a framework for the investigation of target site location by other DNA-binding proteins

Entwicklung und Untersuchung von Sonden für Mikroskopie zur hochaufgelösten Kolokalisation

Biebricher A. Development and investigation of probes for high-resolution colocalization microscopy. Bielefeld (Germany): Bielefeld University; 2006.The presented work is centered on the advancement of high-resolution microscopy using spectrally-resolved fluorescence lifetime imaging microscopy (SFLIM). This technique allows to circumvent the intrinsic limitation that due to the wavelike nature of the light no structures below 200nm can be resolved with conventional fluorescence microscopy. This seriously corrupts its application to the study of single biomolecule imaging within a cell since binding events take place at distances below 50nm. The high resolution can be achieved employing different types of luminescent labels which can be distinguished from each other both by their spectral and lifetime characteristics. The work is divided into two parts: In the first part, gold surfaces patterned by electron-beam lithography were tested concerning an application as substrates for a Cellular Positioning System (CPS). This system is analogous to the Globular Positioning System (GPS) in that it uses at least three differently labeled spots positioned with nm-accuracy on a surface to follow the absolute trajectories of single fluorescent molecules. The use of gold as surface for fluorescent probes has the disadvantage that strong quenching of the dyes occurs. Therefore, a modification scheme was developed which enables a controlled modification of the surface with fluorescently labeled proteins. The protein labels used are efficient spacers to reduce the quenching effect of the gold and to facilitate immobilization of further protein layers. Additionally, an organic linker was used to enhance the protein binding efficiency to the gold and to further increase the distance from the gold surface to the dyes. By controlled growing of several layers of organic linker and subsequent layers of protein, it could be demonstrated that via this method high signal to background ratios can be obtained even for structures smaller than 100 nm. In a second set of experiments, semiconductor nanocrystals were investigated concerning an application as probes for high-resolution microscopy. For this purpose, the photophysical fluctuations were observed with the aid of a SFLIM set-up. Three different samples consisting of commercially available NCs were investigated emitting at 605, 655 and 705 nm (denoted QD605, QD655 and QD705), respectively, which are made up of CdSe (QD605, QD655) or CdTe (QD705), covered with a ZnS shell and modified with strepavidin. It could be demonstrated that the set-up allows for the determination of photophysical fluctuations of single NCs with a time resolution down to 1 ms. By calibration and test measurements it could be verified, that in this fashion, photoluminescence intensity, lifetime and spectral fluctuations can be determined with high accuracy. By correlation of the observables an interrelation between spectral diffusion and lifetime fluctuations caused by changes of the radiative rate was uncovered. This novel connection was exploited to calculate radiative rate fluctuations and photoluminescence quantum yields of single NCs. In addition, a novel subpopulation of quenched emission could be uncovered indicating NC transition into a fundamentally different state. The obtained data was furthermore used to construct density plots containing all three observables which are characteristic for a certain type of NC and can also be used for NC assessment. Finally, with the aid of the developed depiction method it could be shown that anti-blinking agents lead to a significant acceleration of photophysical fluctuations. The set-up was also employed in a modified form to investigate the antibunching characteristics of the NC samples. In particular, it was demonstrated that the NC sample of type QD655 display significant biexciton emission. A novel analytical method was presented which allows for the unequivocal extraction of photons stemming from biexciton emission. This was used to calculate the biexciton decays for single NCs which was shown to vary significantly from NC to NC. For a given NC, it was also found for the first time that the relative biexciton quantum yield compared to the exciton is not constant but shows a strong increase for quenched states. This finding could be confirmed by spectrally resolved investigation of triexciton emission, which can also be used to extract the lifetime information of triexciton emission

Quantitative Acoustophoresis

Studying cellular mechanics allows important insights into its cytoskeletal composition, developmental stage, and health. While many force spectroscopy assays exist that allow probing of mechanics of bioparticles, most of them require immobilization of and direct contact with the particle and can only measure a single particle at a time. Here, we introduce quantitative acoustophoresis (QAP) as a simple alternative that uses an acoustic standing wave field to directly determine cellular compressibility and density of many cells simultaneously in a contact-free manner. First, using polymeric spheres of different sizes and materials, we verify that our assay data follow the standard acoustic theory with great accuracy. We furthermore verify that our technique not only is able to measure compressibilities of living cells but can also sense an artificial cytoskeleton inside a biomimetic vesicle. We finally provide a thorough discussion about the expected accuracy our approach provides. To conclude, we show that compared to existing methods, our QAP assay provides a simple yet powerful alternative to study the mechanics of biological and biomimetic particles

Quantitative Acoustophoresis

[Image: see text] Studying cellular mechanics allows important insights into its cytoskeletal composition, developmental stage, and health. While many force spectroscopy assays exist that allow probing of mechanics of bioparticles, most of them require immobilization of and direct contact with the particle and can only measure a single particle at a time. Here, we introduce quantitative acoustophoresis (QAP) as a simple alternative that uses an acoustic standing wave field to directly determine cellular compressibility and density of many cells simultaneously in a contact-free manner. First, using polymeric spheres of different sizes and materials, we verify that our assay data follow the standard acoustic theory with great accuracy. We furthermore verify that our technique not only is able to measure compressibilities of living cells but can also sense an artificial cytoskeleton inside a biomimetic vesicle. We finally provide a thorough discussion about the expected accuracy our approach provides. To conclude, we show that compared to existing methods, our QAP assay provides a simple yet powerful alternative to study the mechanics of biological and biomimetic particles