DNA is the carrier of all cellular genetic information and increasingly used in nanotechnology. Quantitative understanding and optimization of its functions requires precise experimental characterization and accurate modeling of DNA properties. A defining feature of DNA is its helicity. DNA unwinds with increasing temperature, even for temperatures well below the melting temperature. However, accurate quantitation of DNA unwinding under external forces and a microscopic understanding of the corresponding structural changes are currently lacking. Here we combine single-molecule magnetic tweezers measurements with atomistic molecular dynamics and coarse-grained simulations to obtain a comprehensive view of the temperature dependence of DNA twist. Experimentally, we find that DNA twist changes by ΔTw(T) = (−11.0 ± 1.2)°/(°C·kbp), independent of applied force, in the range of forces where torque-induced melting is negligible. Our atomistic simulations predict ΔTw(T) = (−11.1 ± 0.3)°/(°C·kbp), in quantitative agreement with experiments, and suggest that the untwisting of DNA with temperature is predominantly due to changes in DNA structure for defined backbone substates, while the effects of changes in substate populations are minor. Coarse-grained simulations using the oxDNA framework yield a value of ΔTw(T) = (−6.4 ± 0.2)°/(°C·kbp) in semi-quantitative agreement with experiments.

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Adaptive immune responses are initiated by triggering of the T cell receptor. Single-molecule imaging based on total internal reflection fluorescence microscopy at coverslip/basal cell interfaces is commonly used to study this process. These experiments have suggested, unexpectedly, that the diffusional behavior and organization of signaling proteins and receptors may be constrained before activation. However, it is unclear to what extent the molecular behavior and cell state is affected by the imaging conditions, i.e., by the presence of a supporting surface. In this study, we implemented single-molecule light-sheet microscopy, which enables single receptors to be directly visualized at any plane in a cell to study protein dynamics and organization in live, resting T cells. The light sheet enabled the acquisition of high-quality single-molecule fluorescence images that were comparable to those of total internal reflection fluorescence microscopy. By comparing the apical and basal surfaces of surface-contacting T cells using single-molecule light-sheet microscopy, we found that most coated-glass surfaces and supported lipid bilayers profoundly affected the diffusion of membrane proteins (T cell receptor and CD45) and that all the surfaces induced calcium influx to various degrees. Our results suggest that, when studying resting T cells, surfaces are best avoided, which we achieve here by suspending cells in agarose.

The composition of soluble toxic protein aggregates formed in vivo is currently unknown in neurodegenerative diseases, due to their ultra-low concentration in human biofluids and their high degree of heterogeneity. We introduce the structure-specific chemical antibody; a Y shaped, bioinspired small molecule with a dimeric region to mimic avidity, and an attachment region to mimic the Fc region. Our probe, capture molecule for amyloid precipitation (CAP-1), consists of a derivative of Pittsburgh compound B (dimer) to target the cross -sheets of amyloids and a biotin moiety for surface immobilization. By coupling CAP-1 to magnetic beads, we targeted the amyloid structure of protein aggregates in human cerebrospinal fluid, isolated them for analysis and then characterised them using single-molecule fluorescence imaging and mass spectrometry. AP allows unbiased determination of the molecular composition and structural features of the in vivo aggregates, formed in neurodegenerative diseases, that are present in biofluids.