Biology subjects

Sasmal, R.

Publications and source records attributed to Sasmal, R..

4 recordsLinked to original sources

bioRxiv2025

Small amphiphilic DNA for programmable transmembrane signaling and amplification

Interrogating the sequence-specific DNA/RNA content of a living cell without destroying it, is an elusive goal in biology and diagnostics with transformative potential. A DNA/RNA-based approach, however, must relay the recognition event across the intact lipid bilayer, a step that has long been considered impossible because hydrophilic, massively charged DNA/RNA is incompatible with the hydrophobic membrane interior. Here, we show that DNA branch migration can be driven across a lipid bilayer, converting an intracellular nucleic-acid sequence into an amplified, extracellular optical signal while preserving cell viability and compartmentalization, a synthetic transmembrane signal transduction analogous to natural receptors, such as G-protein-coupled receptors (GPCRs), but operating in reverse. We demonstrate this principle with Hybridization Across Lipid for Oligonucleotide Sensing (HALOS), an amphiphilic DNA hairpin comprising a toehold for recognition, a stem for stability, a loop, and cholesterols for transmembrane anchoring. Upon binding to a sequence-specific nucleic acid target, toehold-mediated strand invasion drives DNA branch migration across the bilayer, switching HALOS from a closed to an open conformation that relays the signal to the opposite face. Molecular dynamics simulations revealed that a cholesterol belt stabilizes the DNA stem within the membrane, preserving the hairpin structure necessary for transmembrane signaling. Notably, this branch migration proceeds despite the kinetic barrier of hybridization through the hydrophobic bilayer interior. By combining HALOS with an isothermal hybridization chain reaction (HCR), we established a platform that enables intracellular nucleic acid target detection and amplified fluorescent reporting from outside synthetic vesicles and live mammalian cells, achieving nanomolar-range sensitivity. These results establish DNA branch migration across lipid membranes as a design principle for programmable lysis-free molecular sensing in intact cells.

biophysics↗

bioRxiv2025

DNA origami-based platform for multi-axial single-molecule force spectroscopy reveals hidden dynamics in Holliday junctions

Biomolecules in living cells experience complex multi-directional mechanical forces that regulate their structure, dynamics, and function. However, most single-molecule techniques primarily exert force along a single axis, thereby failing to emulate the mechanical environment of cells. Here, we demonstrate that single-axis force application fundamentally restricts conformational dynamics by kinetically trapping molecules within distinct dynamic classes, preventing interconversion and exploration of the full conformational landscape. We developed MAESTRO (Multi-Axial Entropic Spring Tweezer along a Rigid Origami), a molecular platform that applies up to 9 pN forces from up to 4 directions simultaneously using programmable ssDNA entropic springs anchored to a DNA origami scaffold. We applied MAESTRO to Holliday junctions (HJs), 4-way DNA intermediates that experience multi-directional tension during homologous recombination. Counterintuitively, we discovered >5x slower kinetics of the HJ conformations under multi-axial tension than under tension-free conditions, enabling direct observations of previously hidden HJ conformational intermediates. Most remarkably, we discovered that multi-axial forces unlock conformational dynamics, enabling interconversion between 5 distinct kinetic classes that remain kinetically inaccessible under zero force or single-axial tension. Furthermore, we demonstrated that tension regulates T7 endonuclease I cleavage site selection, directly linking mechanical environments and molecular mechanics to enzymatic function. By overcoming single-axis limitations, MAESTRO opens new frontiers in molecular mechanobiology, revealing how multi-directional cellular force environments are essential for unlocking the full conformational landscape of biomolecules, and that these complex force patterns serve as master regulators of biological function through mechanisms hidden from conventional approaches.

biophysics↗

bioRxiv2023

Supramolecular catch-and-release strategy for bioorthogonal fluorogenic imaging across the visible spectrum

Fluorogenic probes that unmask fluorescence signals in response to a bioorthogonal reaction are a powerful new addition to biological imaging. They can provide significantly reduced background fluorescence and minimize non-specific signals, potentially allowing real-time high-contrast imaging without washing out excess fluorophores. While diverse classes of highly refined synthetic fluorophores are readily available now, their integration into a bioorthogonal fluorogenic scheme still necessitates another level of extensive design efforts and customized structural alterations to optimize quenching mechanisms for each given fluorophore scaffold. Herein, we present an easy-to-implement and highly generalizable supramolecular catch-and-release strategy for generating an efficient bioorthogonal fluorogenic response from essentially any readily available fluorophores without further structural alterations. We designed this distinct strategy based on the macrocyclic cucurbit[7]uril (CB7) host, where a fluorogenic response is achieved by programming a guest displacement reaction from the macrocycle cavity. We used this strategy to rapidly generate fluorogenic probes across the visible spectrum from structurally diverse classes of fluorophore scaffolds, including coumarin, bodipy, rhodamine, and cyanine. These probes were applied to no-wash fluorogenic imaging of various target molecules in live cells and tissue with minimal background and no appreciable non-specific signal. Notably, the orthogonal reactivity profile of the system allowed us to pair this host-guest fluorogenic probe with the covalently clickable fluorogenic probe to achieve high-contrast super-resolution and multiplexed fluorogenic imaging in cells and tissue.

bioengineering↗

bioRxiv2019

Measuring mRNA translation in neuronal processes and somata by tRNA-FRET

In neurons, the specific spatial and temporal localization of protein synthesis is of great importance for function and survival. In this work, we visualized tRNA and protein synthesis events in fixed and live mouse primary cortical culture using fluorescently-labeled tRNAs. We were able to characterize the distribution and movement of tRNAs in different neuronal sub-compartments and to study their association with the ribosome. We found that tRNA motion in neural processes is lower than in somata and corresponds to patterns of slow transport mechanisms, and that larger tRNA puncta co-localize with translational machinery components and are likely the functional fraction. Furthermore, chemical induction of LTP in culture revealed GluR-dependent biphasic up-regulation of mRNA translation with a similar effect in dendrites and somata. Importantly, measurement of protein synthesis in neurons with high resolutions offers new insights into neuronal function in health and disease states.

neuroscience↗