Studies of circular DNA confined to nanofluidic channels are relevant both from a fundamental polymer-physics perspective and due to the importance of circular DNA molecules in vivo. We here observe the unfolding of DNA from the circular to linear configuration as a light-induced double strand break occurs, characterize the dynamics, and compare the equilibrium conformational statistics of linear and circular configurations. This is important because it allows us to determine to which extent existing statistical theories describe the extension of confined circular DNA. We find that the ratio of the extensions of confined linear and circular DNA configurations increases as the buffer concentration decreases. The experimental results fall between theoretical predictions for the extended de Gennes regime at weaker confinement and the Odijk regime at stronger confinement. We show that it is possible to directly distinguish between circular and linear DNA molecules by measuring the emission intensity from the DNA. Finally, we determine the rate of unfolding and show that this rate is larger for more confined DNA, possibly reflecting the corresponding larger difference in entropy between the circular and linear configurations.

We present a nanofluidic device for fluorescence-based detection and characterization of small lipid vesicles on a single particle basis. The device works like a nano flow cytometer where individual vesicles are visualized by fluorescence microscopy while passing through parallel nanochannels in a pressure-driven flow. An experiment requires less than 20 μl sample volume to quantify both the vesicle content and the fluorescence signals emitted by individual vesicles. We show that the device can be used to accurately count the number of fluorescent synthetic lipid vesicles down to a vesicle concentration of 170 fM. We also show that the size-distribution of the vesicles can be resolved from their fluorescence intensity distribution after calibration. We demonstrate the applicability of the assay in two different examples. In the first, we use the nanofluidic device to determine the particle concentration in a sample containing cell-derived extracellular vesicles labelled with a lipophilic dye. In the second, we demonstrate that dual-color detection can be used to probe peptide binding to synthetic lipid vesicles; we identify a positive membrane-curvature sensing behavior of an arginine enriched version of the Antennapedia homeodomain peptide penetratin. Altogether, these results illustrate the potential of this nanofluidic-based methodology for characterization and quantification of small biological vesicles and their interactors without ensemble averaging. The device is therefore likely to find use as a quantitative analytical tool in a variety of fields ranging from diagnostics to fundamental biology research. Moreover, our results have potential to facilitate further development of automated lab-on-a-chip devices for vesicle analysis.

Phylogenetic inference is widely used to investigate the relationships between homologous sequences. RNA molecules have played a key role in these studies because they are present throughout life and tend to evolve slowly. Phylogenetic inference has been shown to be dependent on the substitution model used. A wide range of models have been developed to describe RNA evolution, either with 16 states describing all possible canonical base pairs or with 7 states where the 10 mismatched nucleotides are reduced to a single state. Formal model selection has become a standard practice for choosing an inferential model and works well for comparing models of a specific type, such as comparisons within nucleotide models or within amino acid models. Model selection cannot function across different sized state spaces because the likelihoods are conditioned on different data. Here, we introduce statistical state-space projection methods that allow the direct comparison of likelihoods between nucleotide models and 7-state and 16-state RNA models. To demonstrate the general applicability of our new methods, we extract 287 RNA families from genomic alignments and perform model selection. We find that in 281/287 families, RNA models are selected in preference to nucleotide models, with simple 7-state RNA models selected for more conserved families with shorter stems and more complex 16-state RNA models selected for more divergent families with longer stems. Other factors, such as the function of the RNA molecule or the GC-content, have limited impact on model selection. Our models and model selection methods are freely available in the open-source PHASE 3.0 software.

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Organic and inorganic solid materials form the building blocks for most of today’s high-technological instruments and devices. However, challenges related to dissimilar material properties have hampered the synthesis of thin-film devices comprised of both organic and inorganic films. We here give a detailed description of a carefully optimized processing protocol used for the construction of a three-layered hybrid organic–inorganic waveguide-chip intended for combined scattering and fluorescence evanescent-wave microscopy in aqueous environments using conventional upright microscopes. An inorganic core layer (SiO2 or Si3N4), embedded symmetrically in an organic cladding layer (CYTOP), aids simple, yet efficient in-coupling of light, and since the organic cladding layer is refractive index matched to water, low stray-light (background) scattering of the propagating light is ensured. Another major advantage is that the inorganic core layer makes the chip compatible with multiple well-established surface functionalization schemes that allows for a broad range of applications, including detection of single lipid vesicles, metallic nanoparticles or cells in complex environments, either label-free—by direct detection of scattered light—or by use of fluorescence excitation and emission. Herein, focus is put on a detailed description of the fabrication of the waveguide-chip, together with a fundamental characterization of its optical properties and performance, particularly in comparison with conventional epi illumination. Quantitative analysis of images obtained from both fluorescence and scattering intensities from surface-immobilized polystyrene nanoparticles in suspensions of different concentrations, revealed enhanced signal-to-noise and signal-to-background ratios for the waveguide illumination compared to the epi-illumination.