Biology subjects

Stockley, P. G.

Publications and source records attributed to Stockley, P. G..

7 recordsLinked to original sources

bioRxiv2026

Rapid, reversible RNA/DNA precipitation using rare earth ion coordination

Efficient recovery and removal of nucleic acids remain major challenges in molecular biology, diagnostics, and viral-vector manufacturing. Conventional precipitation methods often require organic solvents, low temperatures, and extended centrifugation, while nuclease-based contaminant removal can be incomplete and/or is incompatible with downstream workflows. Here, we evaluate NADA, a trivalent rare earth-based reagent, for rapid, solvent-free precipitation of RNA and DNA, and ALGO-mediated recovery of the full range of properties of unmodified RNA. Using hepatitis B virus pre-genomic RNA, lambda bacteriophage DNA, and the HBV nucleocapsid as model systems, we show that NADA rapidly sequesters both ssRNA and dsDNA from solution, reducing Midori Green nucleic acid fluorescence and UV absorption to baseline levels. The NADA complex prevents HBV core protein binding to the HBV pgRNA, and therefore triggering of nucleocapsid formation, even when added simultaneously with core protein, suggesting that lanthanide ion coordination, out-competes the cognate Cp for viral RNA-binding interactions. Recovery with ALGO restores pgRNA integrity and functionality, enabling subsequent assembly of the expected T=4/T=3 nucleocapsids. In contrast, pre-assembled nucleocapsids resist NADA-ALGO treatment, demonstrating coat protein capsid-mediated protection of encapsidated pgRNA. Similar outcomes for lambda dsDNA establish NADA/ALGO as a rapid, reversible, and selective alternative platform for nucleic acid precipitation, with potential applications in viral particle purification, contaminant removal, and RNA handling workflows.

biochemistry↗

bioRxiv2026

Pandemic Coronavirus Genome Packaging Relies on Multiple Dispersed Packaging Signals

Packaging of viral genomes into progeny virions is a critical step in the viral life cycle. Coronaviruses such as SARS-CoV-2 possess unusually large RNA genomes ([~]30 kb), yet the mechanism by which these genomes are selectively condensed and incorporated into virions remains poorly understood. Here, we demonstrate that the SARS-CoV-2 genome contains multiple dispersed RNA structures, termed packaging signals (PSs), which cooperate with a dominant central PS to direct genomic RNA incorporation into infectious particles. We identify the dominant PS within the coding region of the nsp15 gene, downstream of a packaging signal previously described in Embecoviruses. Using virus-like particles (VLPs), we investigate its role in virion assembly and selective genomic RNA packaging, and show that it promotes the formation of ribonucleoprotein (RNP) complexes with the viral nucleocapsid protein (N), as revealed by mass photometry. Notably, we uncover a unique N-induced conformational rearrangement of the PS RNA, from an extended structure to a double stem-loop architecture. This dominant PS acts together with a nearby stem-loop element to assemble a higher-order RNP complex containing 12 N-protein dimers. Using a SARS-CoV-2 replicon system, we further demonstrate functional cooperativity between the dominant PS, its proximal partner stem-loop, and additional packaging elements located approximately 10 kb upstream. Collectively, our findings support a highly dynamic and cooperative mechanism of SARS-CoV-2 genome packaging that relies on multiple dispersed packaging signals organized around a dominant central PS. These insights provide a mechanistic framework for understanding coronavirus genome packaging and reveal new opportunities for antiviral intervention through disruption of this process. They also have important implications for vaccine development and may enable the design of membrane-based vector systems capable of efficiently delivering large nucleic acid cargoes, expanding the potential of bionanotechnology and genetic medicine.

microbiology↗

bioRxiv2022

An Ultralong Bovine CDRH3 that Targets a Conserved, Cryptic Epitope on SARS-CoV and SARS-CoV-2

The ability of broadly neutralising antibodies to target conserved epitopes gives them huge potential as antibody-based therapeutics, particularly in the face of constant viral antigen evolution. Certain bovine antibodies are highly adept at binding conserved, glycosylated epitopes, courtesy of their ultralong complementarity determining region (CDR)H3. Here, we used a SARS-naive, bovine ultralong CDRH3 library and mammalian cell display, to isolate a bovine paratope that engages the SARS-CoV and SARS-CoV-2 receptor-binding domain (RBD). This neutralises viruses pseudo-typed with SARS-CoV Spike protein but not by competition with RBD binding to ACE2. Instead, using differential hydrogen-deuterium exchange mass spectrometry and site-directed mutagenesis, we demonstrate that this ultralong CDRH3 recognises a rarely identified, conserved, cryptic epitope that overlaps the target of pan-sarbecovirus antibodies (7D6/6D6). The epitope is glycan-shielded and becomes accessible only transiently via inter-domain movements. This represents the first bovine anti-sarbecovirus paratope and highlights the power of this approach in identifying novel tools to combat emerging pathogens.

molecular biology↗

bioRxiv2021

In vitro Functional Analysis of pgRNA Sites Regulating Assembly of Hepatitis B Virus.

The roles of RNA sequence/structure motifs, Packaging Signals (PSs), for regulating assembly of an HBV genome transcript have been investigated in an efficient in vitro assay containing only core protein (Cp) and RNA. Variants of three conserved PSs, within the genome of a strain not used previously, preventing correct presentation of a Cp-recognition loop motif are differentially deleterious for assembly of nucleocapsid-like particles (NCPs). Cryo-electron microscopy reconstruction of the T=4 NCPs formed with the wild-type gRNA transcript, reveal that the interior of the Cp shell is in contact with lower resolution density, potentially encompassing the arginine-rich protein domains and gRNA. Symmetry-relaxation of this reconstruction reveals that such contacts are made at every symmetry axis. We infer from their regulation of assembly that some of these contacts would involve gRNA PSs, and confirmed this by X-ray RNA footprinting. Mutation of the {varepsilon} stem-loop in the gRNA, where polymerase binds in vivo, produces a poor RNA assembly substrate with Cp alone, largely due to alterations in its conformation. The results show that RNA PSs regulate assembly of HBV genomic transcripts in vitro, and therefore may play similar roles in vivo, in concert with other molecular factors.

microbiology↗

bioRxiv2021

RNA X-ray footprinting reveals the consequences of an in vivo acquired determinant of viral infectivity

The secondary structures of the bacteriophage MS2 ssRNA genome, frozen in defined states, were determined with minimal perturbation using constraints from X-ray synchrotron footprinting (XRF). The footprints of the gRNA in the virion and as transcript are consistent with single, dominant but distinct conformations, and reveal the presence of multiple Packaging Signals potentially involved in assembly regulation that have not been detected by other techniques. XRF also reveals the dramatic effect of the unique Maturation Protein (MP) on both the capsid lattice, and the gRNA conformation inside the phage compared with a virus-like-particle composed only of coat protein subunits. Aspects of genome organisation in the phage, their impacts on the capsid shell, and the distortion of lattice geometry by MP, are hallmarks of molecular frustration. Phage assembly therefore appears to prepare the particle for the next step of the infectious cycle.

molecular biology↗

bioRxiv2021

Dysregulation of Hepatitis B Virus Nucleocapsid Assembly with RNA-directed Small Ligands

RNA sequences/motifs dispersed across the pre-genomic copy of the Hepatitis B Virus genome regulate formation of nucleocapsids in vitro in an epsilon/polymerase independent fashion. These multiple RNA Packaging Signals (PSs) form stem-loops presenting in each loop a core protein recognition motif, -RGAG-. Small, drug-like molecules binding these motifs were identified by screening an immobilized library with a fluorescently-labelled RNA oligonucleotide encompassing the most conserved of these sites. This identified 66 "hits", with affinities ranging from low nanomolar to high micromolar in SPR assays. High affinity ligand binding is dependent on the presence of the -RGAG-motif, which also appears to be the common element in cross-binding to other PS sites. Some of these compounds are potent inhibitors of in vitro core protein assembly around the HBV pre-genome. Mathematical modelling confirms the potential of these novel anti-viral drug targets for disrupting replication of this major human pathogen. Preliminary structure-activity relationships of the highest affinity compound reveal critical functional groups for PS-binding. PS-regulated assembly is easily adapted to high-throughput screening allowing future development of pharmacologically active compounds.

microbiology↗

bioRxiv2020

Evolution of a virus-like architecture and packaging mechanism in arepurposed bacterial protein

Viruses are ubiquitous pathogens of global impact. Prompted by the hypothesis that their earliest progenitors recruited host proteins for virion formation, we have used stringent laboratory evolution to convert a bacterial enzyme lacking affinity for nucleic acids into an artificial nucleocapsid that efficiently packages and protects multiple copies of its own encoding mRNA. Revealing remarkable convergence on the molecular hallmarks of natural viruses, the accompanying changes reorganized the protein building blocks into an interlaced 240-subunit icosahedral capsid impermeable to nucleases, while emergence of a robust RNA stem-loop packaging cassette ensured high encapsidation yields and specificity. In addition to evincing a plausible evolutionary pathway for primordial viruses, these findings highlight practical strategies for developing non-viral carriers for diverse vaccine and delivery applications.

biochemistry↗