Biology subjects

Cowieson, N.

Publications and source records attributed to Cowieson, N..

3 recordsLinked to original sources

bioRxiv2025

Fractionation-Free Protein Corona Quantification Through Synchrotron-Based Small-Angle X-ray Scattering

When nanoparticles (NPs) enter biological environments, they are rapidly coated by biomolecules, forming the protein corona (PC) that defines their biological identity and dictates how NPs are recognized, distributed, and processed by living systems. Capturing the authentic features of the PC demands experimental conditions that preserve its native state, which are difficult to achieve once NPs are removed from their biological milieu. Despite significant progress, current PC quantification methods still rely on separating the NP-PC complex from its native environment, which compromises the coronas integrity and prevents accurate evaluation of its physicochemical properties. Here, we introduce a fractionation-free approach based on synchrotron small-angle X-ray scattering (SAXS) to quantitatively determine the amount of protein adsorbed onto silica NPs under native conditions. By modeling the scattering contribution of free versus bound proteins, we directly extracted the adsorbed mass in both single-protein (BSA) and complex proteomic (human serum) systems. The resulting adsorption isotherms enabled the determination of thermodynamic parameters such as binding constants and cooperativity, distinguishing between monolayer and multilayer adsorption regimes. Together, these findings establish SAXS as a robust, non-invasive, and quantitative technique for probing the protein corona in situ, without perturbing the native equilibrium. This methodology paves the way for the development of new in situ analytical frameworks across diverse nanomaterial and proteomic systems, advancing SAXS toward quantitative characterization of the protein corona. Table of Contents Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=108 SRC="FIGDIR/small/695217v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@eea1f1org.highwire.dtl.DTLVardef@c9dcdforg.highwire.dtl.DTLVardef@c3dff6org.highwire.dtl.DTLVardef@1a49813_HPS_FORMAT_FIGEXP M_FIG C_FIG High-throughput synchrotron SAXS enables in situ, quantitative tracking of protein corona formation on nanoparticles.

biophysics↗

bioRxiv2025

Investigating the structural effects of anti-thrombin anticoagulant aptamers on activation of human prothrombin

Disorders of the blood coagulation remain a leading cause of death and disability worldwide raising the search for therapeutic agents able to modulate the coagulation cascade. Different oligonucleotide aptamers have been selected against different coagulation factors and some of them are in preclinical or clinical studies. In particular, anti-thrombin aptamers are promising drugs as they inhibit the activity of the -thrombin and, simultaneously, limit thrombin production via prothrombinase by binding its precursor prothrombin. To investigate the interaction of these aptamers with prothrombin, we performed extensive analyses using calorimetric and spectroscopic techniques, which suggested that they recognize proexosite I of prothrombin and exosite I of thrombin with comparable affinity. SAXS experiments performed on the complex formed by the protein and NU172, the only anti-thrombin aptamer in advanced clinical trials, provided structural insights into aptamer-prothrombin recognition. Interestingly, the aptamer binding to proexosite I shifts the open-closed equilibrium of prothrombin toward the open conformation. A reasonable mechanism underlying the effects of anti-thrombin aptamers towards prothrombin conversion into thrombin has been proposed. Altogether, these results definitively qualify these aptamers as bitargeted drugs, being able to modulate both thrombin function and generation, and supply structural bases to design new anticoagulants, which lack health side effects.

biophysics↗

bioRxiv2022

Biophysical characterisation of the structure of a SARS-CoV-2 self-amplifying - RNA (saRNA) vaccine

The current SARS-Covid-2 pandemic has led to an acceleration of messenger - ribonucleic acid (mRNA) vaccine technology. The development of production processes for these large mRNA molecules, especially self-amplifying mRNA (saRNA) has required concomitant development of analytical characterisation techniques. Characterising the purity, shape and structure of these biomolecules is key to their successful performance as drug products. This paper describes the biophysical characterisation of the Imperial College London Self-amplifying viral RNA vaccine (IMP-1) developed for SARS-CoV-2. A variety of analytical techniques have been used to characterise the IMP-1 RNA molecule. In this paper we use UV spectroscopy, dynamic light scattering (DLS), size-exclusion chromatography small angle scattering (SEC-SAXS) and circular dichroism (CD) to determine key biophysical attributes of IMP-1. Each technique provides important information about the concentration, size, shape, structure and purity of the molecule. Statement of significanceThis paper is highly significant as it provides a prescient biophysical characterisation of an efficacious Sars-Cov-2 vaccine self-amplifying (sa)RNA molecule. RNA vaccines have been a major scientific breakthrough of the Covid-19 pandemic. saRNA is a further development of conventional mRNA vaccines, amplifying the RNA of interest in the cell, allowing the vaccine to be administered at lower dosages. These new biologics are distinct from previous biologics and have required distinct analytical characterisation. The analytics described herein provide detailed information on the size, shape, and structure of the RNA molecule. This paper is therefore an important step in characterising large saRNA biological relevant molecules.

biophysics↗