Biology subjects

Bocanegra, R.

Publications and source records attributed to Bocanegra, R..

4 recordsLinked to original sources

bioRxiv2025

Hairpin-Functionalized Gold Nanoparticles as an Adaptable Platform for Detecting MicroRNA Signatures

Early, accurate, and fast diagnosis is essential to ensure positive health outcomes, through effective treatment interventions and disease control, as well as the study of physiological changes related to gene expression. Liquid biopsies and point-of-care (PoC) detection are valuable tools to achieve this goal, allowing the study and monitoring of a patients molecular profile in a timely and simple manner. MicroRNAs (or miRNAs) have been proposed as biomarkers for detection in liquid biopsies, as they are stable in body fluids and their dysregulation is associated with many diseases. In this work, a sensor based on gold nanoparticles (AuNPs) functionalized with oligonucleotides is reported, aiming for the detection of nucleic acids, in particular miRNAs. This sensor is based on the recognition of target sequences by hairpin-shaped oligonucleotide probes, which allows the modulation of the colloidal stability of the AuNPs, producing color changes detectable with the naked eye. The system is used to detect a panel of miRNAs, demonstrating its versatility for the detection of relevant nucleic acid signatures. The sensor detects single miRNAs with good sensitivity and selectivity and, what is more, it can be used to recognize several miRNAs simultaneously at picomolar concentrations. The system was further adapted to a lateral flow assay (LFA) format, producing a visible colored line on lateral flow test strips, and coupled with isothermal amplification to reach femtomolar detection levels. Its properties make it suitable for use in the point-of-care (PoC), contributing to fast and early detection of pathological and physiological molecular profiles.

molecular biology↗

bioRxiv2021

GTP and lipids control self-assembly and functional promiscuity of Dynamin2 molecular machinery

Dynamin2 GTPase (Dyn2) is a crucial player in clathrin-mediated endocytosis. Dyn2 is tetrameric in cytoplasm and self-assembles into functional units upon membrane binding. How the curvature activities and functionality of Dyn2 emerge during self-assembly and are regulated by lipids remains unknown. Here we reconstituted the Dyn2 self-assembly process using membrane nanotubes (NT) and vesicles and characterized it using single- molecule fluorescence microscopy, optical tweezers force spectroscopy and cryo-electron microscopy. On NTs, Dyn2 first forms small subhelical oligomers, which are already curvature active and display pronounced curvature sensing properties. Conical lipids and GTP promote their further self-assembly into helical machinery mediating the NT scission. In the presence of large unilamellar vesicles (LUVs), an alternative self- assembly pathway emerges where the subhelical oligomers form membrane tethering complexes mediating LUV-NT binding. Reconstitution of tethering in the LUV system revealed that lipid mixing is controlled by conical lipid species, divalents, GTP, and SH3 binding partners of Dyn2. In membranes with a high content of lipids with negative intrinsic curvature, cryo-EM revealed putative membrane contact sites made by Dyn2 clusters. On such membranes, with GTP lowered to 0.2 mM, both membrane fission and tethering activities become possible, indicating functional promiscuity of Dyn2. We conclude that GTP and lipids control both extent and topology of Dyn2 functional self-assembly. The function of Dyn2 oligomers evolves from curvature sensing, seen in subhelical Dyn2 oligomers, to curvature creation and fission, seen in Dyn2 helices. Under specific circumstances, such as downregulation of SH3 partners of Dyn2 and GTP depletion, membrane tethering activity can emerge in membrane systems enriched with conical lipids. Hence the Dyn2 functionality is actively adapted to lipidome, explaining its large habitat in the cells and tissues.

biophysics↗

bioRxiv2019

Understanding the paradoxical mechanical response of in-phase A-tracts at different force regimes

A-tracts are A:T rich DNA sequences that exhibit unique structural and mechanical properties associated with several functions in vivo. The crystallographic structure of A-tracts has been well characterized. However, their response to forces remains unknown and the variability of their flexibility reported for different length scales has precluded a comprehensive description of the mechanical properties of these molecules. Here, we rationalize the mechanical properties of A-tracts across multiple length scales using a combination of single-molecule experiments and theoretical polymer models applied to DNA sequences present in the C. elegans genome. Atomic Force Microscopy imaging shows that phased A-tracts induce long-range ([~]200 nm) bending. Moreover, the enhanced bending originates from an intrinsically bent structure rather than as a consequence of larger flexibility. In support of this, our data were well described with a theoretical model based on the worm-like chain model that includes intrinsic bending. Magnetic tweezers experiments confirm that the observed bent is intrinsic to the sequence and does not rely on particular ionic conditions. Using optical tweezers, we assess the local rigidity of A-tracts at high forces and unravel an unusually stiff character of these sequences, as quantified by their large stretch modulus. Our work rationalizes the complex multiscale flexibility of A-tracts, shedding light on the cryptic character of these sequences.

biophysics↗

bioRxiv2018

Dynamic constriction and fission of ER membranes by reticulon

The endoplasmic reticulum (ER) is a continuous cell-wide membrane network. Network formation has been widely associated with proteins producing membrane curvature and fusion, such as reticulons and atlastin. Regulated network fragmentation, occurring in different physiological contexts, is less understood. We found that the ER network has an embedded fragmentation mechanism based upon the ability of reticulons to produce fission of elongating network branches. In Drosophila, fission is counterbalanced by atlastin-driven fusion, with their imbalance leading to ER fragmentation. Live imaging of ER network dynamics upon ectopic expression of Drosophila reticulon linked fission to augmented membrane friction. Consistently, in vitro analysis revealed that purified reticulon produced velocity-dependent constriction and fission of lipid nanotubes pulled from a flat reservoir membrane. Fission occurred at elongation rates and pulling force ranges intrinsic to the ER network, thus suggesting a novel principle of organelle morphology regulation where the dynamic balance between fusion and fission is governed by membrane motility.

cell biology↗