Biology subjects

Dickman, M. J.

Publications and source records attributed to Dickman, M. J..

13 recordsLinked to original sources

bioRxiv2026

Multi-attribute characterisation of mRNA via MazF endoribonuclease and LC-MS workflows

The rapid expansion of mRNA-based medicines has driven demand for robust, high-resolution analytical methods capable of characterising critical quality attributes including sequence identity, 5[prime] capping efficiency, and poly(A) tail length and heterogeneity. Here, we present two complementary liquid chromatography-mass spectrometry based workflows for mRNA characterisation, both based on site-specific digestion with the endoribonuclease MazF from Escherichia coli. A middle-up approach, optimised via specific 5[prime]-ACA cleavage enables rapid characterisation via oligoribonucleotide mass mapping providing complete sequence coverage and simultaneous assessment of 5[prime]-capping efficiency and 3[prime]-poly(A) tail length and heterogeneity. This workflow was successfully applied to three different mRNA constructs, NLuc, eGFP and FLuc mRNAs, achieving sequence coverages of 100%, 87%, and 74%, respectively. A complementary bottom-up approach, employing less specific 5[prime]-ACX cleavage enables tandem mass spectrometry-based sequencing of shorter oligoribonucleotides and detailed characterisation via mRNA sequence mapping. Applied to SARS-CoV-2 Spike Protein mRNA, this method yielded 50% sequence coverage based on unique oligoribonucleotides with MazF alone, extended to 90% by combining MazF, partial RNase T1, and partial RNase U2 digests. Together, these workflows provide a flexible, orthogonal platform for both high-throughput quality assessment and in-depth primary sequence characterisation of mRNA vaccines and therapeutics.

biochemistry↗

bioRxiv2026

DNA template heterogeneity and in vitro transcription reaction conditions impact the poly(A) tail length and heterogeneity of mRNA

mRNA technology has emerged as a powerful new class of medicines. Importantly, this RNA-based approach holds promise for treatments beyond vaccines and infectious diseases, including treatments for cancer, metabolic disorders, cardiovascular conditions and autoimmune diseases. The 3-polyadenylated (poly(A)) tail of mRNA is required for ribosome initiation, translation, and mRNA stability and is considered a critical quality attribute. In this study, novel direct mass spectrometry approaches were used for the analysis of both the DNA template and corresponding mRNA generated via in vitro transcription. Nucleotide resolution of the poly(A/T) sequence of the DNA template and mRNA poly(A) tail was achieved. The results show that the mRNA poly(A) tail length and heterogeneity is impacted by the heterogeneity of the DNA template, the DNA template design and RNA manufacturing conditions, including relative NTP concentrations. These results provide further important mechanistic insight into the poly(A) tail length and heterogeneity of mRNAs synthesised in vitro, including the identification of 3-end additions of cytidine to mRNA poly(A) tails. The ability to rapidly assess DNA template quality, combined with monitoring mRNA poly(A) tail length and heterogeneity, is important as part of the characterisation of mRNA precision medicines and ensuring consistent quality of mRNA from manufacturing processes.

biochemistry↗

bioRxiv2025

Comparative analysis of convergent and divergent T7 RNA polymerase promoters for the synthesis of dsRNA in vivo and in vitro

Double-stranded RNA plays a key role in various biological processes. The discovery of RNA interference, a gene-silencing mechanism, revolutionised the study of gene function. dsRNA has since been used in novel therapeutics and as an agricultural biocontrol alternative to chemical pesticides. Microbial production typically involves expression systems with convergent T7 promoters. However, convergent transcription from DNA-dependent RNA polymerases can lead to transcriptional interference. In this study, we designed multiple plasmid DNA constructs to investigate the effect of convergent and divergent T7 RNA polymerase production of dsRNA via in vitro transcription and in vivo in E. coli, prior to dsRNA yield quantification and analysis of product quality. We demonstrate that higher yields of larger dsRNA are typically obtained using convergent promoters during in vivo production. A typical fold increase of 2.1 was obtained for dsRNA > 400 bp. However, production of smaller dsRNA (< 250 bp) by divergent promoters resulted in increased yields (2.2 fold). Furthermore, our data demonstrates that in vitro transcription production of dsRNA using divergent T7 promoters results in significantly higher yields of dsRNA, with a maximum fold increase of 6.46. Finally, independent of size, we demonstrate that dsRNA synthesised from DNA templates with multiple transcriptional terminators, improved the quality and purity of dsRNA due to decreased formation of dsRNA multimers or aggregates, compared to run-off transcription. This study highlights alternative optimal strategies for the production of a wide range of different sized dsRNA both in vitro and in microbial systems.

synthetic biology↗

bioRxiv2024

The evolution and developmental dynamics of histone-based chromatin regulation in Annelida

Eukaryotic histones protect and package nuclear DNA into nucleosomes. The dynamic addition and removal of posttranslational modifications on these proteins define regulatory regions that play a central role in genome and chromatin biology. However, our understanding of these regulatory mechanisms in animals is largely based on a few model systems, which prevents a general understanding of how histone-based regulation unfolds and promotes phenotypic variation during animal embryogenesis. Here, we apply a comprehensive multi-omics approach to dissect the histone-based regulatory complement in Annelida, one of the largest invertebrate phyla. Annelids exhibit a conserved histone repertoire organised in clusters of dynamically regulated, hyperaccessible chromatin. However, unlike other animals with reduced genomes, the worm Dimorphilus gyrociliatus shows a dramatically streamlined histone repertoire, revealing that genome compaction has lineage-specific effects on histone-based regulation. Notably, the annelid Owenia fusiformis has two H2A.X variants that co-occur in other animals, whose functional implications are unclear but represent a unique case of widespread parallel evolution of a histone variant in Eukarya. Histone-modifying enzyme complements are largely conserved amongst annelids. Yet, temporal differences in the expression of a reduced set of histone modifiers correlate with distinct ontogenetic traits and variation in the adult landscapes of histone modifications, as revealed by quantitative mass spectrometry in O. fusiformis and Capitella teleta. Collectively, our unparalleled analysis of histone-based epigenetics within a non-model phylum informs the evolution of histone-based regulation, presenting a framework to explore how this fundamental genome regulatory layer contributes to developmental and morphological diversification in annelids and animals generally.

evolutionary biology↗

bioRxiv2024

GRASPS: a simple-to-operate translatome technology reveals omics-hidden disease-associated pathways in TDP-43-related amyotrophic lateral sclerosis

Transcriptomes and translatomes measure genome-wide levels of total and ribosome-associated RNAs. A few hundred translatomes were reported over >250,000 transcriptomes highlighting the challenges of identifying translating RNAs. Here, we used a human isogenic inducible model of TDP-43-linked amyotrophic lateral sclerosis, which exhibits altered expression of thousands of transcripts, as a paradigm for the direct comparison of whole-cell, cytoplasmic and translating RNAs, showing broad uncoupling and poor correlation between disease-altered transcripts. Moreover, based on precipitation of endogenous ribosomes, we developed GRASPS (Genome-wide RNA Analysis of Stalled Protein Synthesis), a simple-to-operate translatome technology. Remarkably, GRASPS identified three times more differentially-expressed transcripts with higher fold changes and statistical significance, providing unprecedented opportunities for data modeling at stringent filtering and discovery of previously omics-missed disease-relevant pathways, which functionally map on dense gene regulatory networks of protein-protein interactions. Based on its simplicity and robustness, GRASPS is widely applicable across disciplines in the biotechnologies and biomedical sciences.

molecular biology↗

bioRxiv2024

Optimising the production of dsRNA biocontrols in microbial systems using multiple transcriptional terminators.

Crop pests and pathogens annually cause over $100 billion in global crop damage, with insects consuming 5-20% of major grain crops. Current crop pest and disease control strategies rely on insecticidal and fungicidal sprays, plant genetic resistance, transgenes and agricultural practices. dsRNA is emerging as a novel sustainable method of plant protection as an alternative to traditional chemical pesticides. Successful commercialisation of dsRNA based biocontrols requires the economical production of large quantities of dsRNA combined with suitable delivery methods to ensure RNAi efficacy against the target pest. In this study, we have optimised the design of plasmid DNA constructs to produce dsRNA biocontrols in E. coli, by employing a wide range of alternative synthetic transcriptional terminators prior to measurement of dsRNA yield. We demonstrate that a 7.8-fold increase of dsRNA was achieved using triple synthetic transcriptional terminators within a dual T7 dsRNA production system compared to the absence of transcriptional terminators. Moreover, our data demonstrates that batch fermentation production dsRNA using multiple transcriptional terminators is scalable and generates significantly higher yields of dsRNA generated in the absence of transcriptional terminators at both small-scale batch culture and large-scale fermentation. In addition, we show that application of these dsRNA biocontrols expressed in E. coli cells results in increased insect mortality. Finally, novel mass spectrometry analysis was performed to determine the precise sites of transcriptional termination at the different transcriptional terminators providing important further mechanistic insight.

synthetic biology↗

bioRxiv2023

Unusual 1-3 peptidoglycan cross-links in Acetobacteriaceae are made by L,D-transpeptidases with a catalytic domain distantly related to YkuD domains

Peptidoglycan is an essential component of the bacterial cell envelope that contains glycan chains substituted by short peptide stems. Peptide stems are polymerized by D,D-transpeptidases, which make bonds between the amino acid in position 4 of a donor stem and the third residue of an acceptor stem (4-3 cross-links). Some bacterial peptidoglycans also contain 3-3 cross-links that are formed by another class of enzymes called L,D-transpeptidases. In this work, we investigate the formation of unusual bacterial 1-3 peptidoglycan cross-links. We describe a version of the PGFinder software which can identify 1-3 cross-links and report the high-resolution peptidoglycan structure of Gluconobacter oxydans (a model organism within the Acetobacteraceae family). We reveal that G. oxydans peptidoglycan contains peptide stems made of a single alanine as well as several dipeptide stems with unusual amino acids at their C-terminus. Using a Sudoku transposon library, we identified a G. oxydans mutant with a drastic reduction in 1-3 cross-links. Through complementation experiments in G. oxydans and recombinant protein production in a heterologous host, we identify an L,D-transpeptidase enzyme with a domain distantly related to the YkuD domain responsible for these non-canonical reactions. This work revisits the enzymatic capabilities of L,D-transpeptidases, a versatile family of enzymes that play a key role in bacterial peptidoglycan remodelling.

microbiology↗

bioRxiv2023

hnRNPUL1 ensures efficient Integrator-mediated cleavage of snRNAs and is mutated in amyotrophic lateral sclerosis

Integrator cleaves nascent RNA, triggering RNA polymerase II transcription termination, but how cleavage is regulated is poorly understood. Here we show hnRNPUL1 ensures efficient Integrator-mediated cleavage of nascent RNA downstream of snRNA genes and, in the case of U2 snRNA, binds a terminal stem-loop involved in this process. In the nucleoplasm, hnRNPUL1 binds U4 snRNA and SART3 and enables efficient reformation of the U4:U6 di-snRNP for further rounds of pre-mRNA splicing. Sustained hnRNPUL1 loss leads to reduced levels of snRNAs, defects in histone mRNA 3' end processing and loss of Cajal bodies. hnRNPUL1 binds RNA through multiple domains, including a globular central domain comprising tightly juxtaposed SPRY and dead polynucleotide kinase folds. This latter fold allows binding to 5'-monophosphorylated RNAs in a mutually exclusive manner with ATP binding and functions as an XRN2 antagonist when overexpressed. We identify a cohort of amyotrophic lateral sclerosis patients harbouring disruptive mutations in hnRNPUL1. SMN loss in spinal muscular atrophy and hnRNPUL1 loss both disrupt snRNP biogenesis, leading to motor neuron death, suggesting a common aetiology.

molecular biology↗

bioRxiv2022

Characterisation and sequence mapping of large RNA and mRNA therapeutics using mass spectrometry

Large RNA including messenger RNA (mRNA) has emerged as an important new class of therapeutic. Recently this has been demonstrated by two highly efficacious vaccines based on mRNA sequences encoding for a modified version of the SARS-CoV-2 spike protein. There is currently significant demand for the development of new and improved analytical methods for the characterization of large RNA including mRNA therapeutics. In this study we have developed an automated, high throughput workflow for the rapid characterisation and direct sequence mapping of large RNA and mRNA therapeutics. Partial RNase digestions using RNase T1 immobilised on magnetic particles was performed in conjunction with high resolution liquid chromatography mass spectrometry analysis. Sequence mapping was performed using automated oligoribonucleotide annotation and identifications based on MS/MS spectra. Using this approach >80% sequence of coverage of a range of large RNAs and mRNA therapeutics including the SARS Co-V2 spike protein was obtained in a single analysis. The analytical workflow, including automated sample preparation can be completed within 90 minutes. The ability to rapidly identify, characterise and sequence map large mRNA therapeutics with high sequence coverage provides important information for identity testing, sequence validation and impurity analysis.

biochemistry↗

bioRxiv2022

Chemically modified dsRNA induces RNAi effects in insects in vitro and in vivo: A potential new tool for improving RNA-based plant protection

Global agriculture loses over $100 billion of produce annually to crop pests such as insects. Many of these crop pests either have no current means of control or have developed resistance against chemical pesticides. Long dsRNAs are capable of inducing RNA interference (RNAi) in insects and are emerging as novel highly selective alternatives for sustainable insect management strategies. However, there are significant challenges associated with RNAi efficacy in insects. In this study, we synthesised a range of chemically modified long dsRNA in an approach to improve nuclease resistance and RNAi efficacy in insects. The results showed that dsRNA containing phosphorothioate modifications demonstrated increased resistance to southern green stink bug saliva nucleases. Phosphorothioate and 2-fluoro modified dsRNA also demonstrated increased resistance to degradation by soil nucleases and increased RNAi efficacy in Drosophila melanogaster cell cultures. In live insects, chemically modified long dsRNA successfully resulted in mortality in both stink bug and corn rootworm. The results provide further mechanistic insight of RNAi efficacy dependence on modifications in the sense or antisense strand of the dsRNA in insects and demonstrate for the first time that RNAi can successfully be triggered by chemically modified long dsRNA in insect cells or live insects.

bioengineering↗

bioRxiv2021

The master energy homeostasis regulator PGC-1alpha couples transcriptional co-activation and mRNA nuclear export

PGC-1 plays a central role in maintaining the mitochondrial and energy metabolism homeostasis, linking external stimuli to the transcriptional co-activation of genes involved in adaptive and age-related pathways. The carboxyl-terminus encodes a serine/arginine-rich (RS) region and a putative RNA recognition motif, however potential RNA-processing role(s) have remained elusive for the past 20 years. Here, we show that the RS domain of human PGC-1 directly interacts with RNA and the nuclear RNA export factor NXF1. Inducible depletion of endogenous PGC-1 and expression of RNAi-resistant RS-deleted PGC-1 further demonstrate that the RNA-binding activity is required for nuclear export of co-activated transcripts and mitochondrial homeostasis. Moreover, a quantitative proteomics approach confirmed PGC-1-dependent RNA transport and mitochondrial-related functions, identifying also novel mRNA nuclear export targets in age-related telomere maintenance. Discovering a novel function for a major cellular homeostasis regulator provides new directions to further elucidate the roles of PGC-1 in gene expression, metabolic disorders, ageing and neurodegenerative diseases.

molecular biology↗

bioRxiv2020

Purification of recombinant SARS-CoV-2 spike, its receptor binding domain, and CR3022 mAb for serological assay

Serology testing for COVID-19 is highly attractive because of the relatively short diagnosis time and the ability to test for an active immune response against the SARS-CoV-2. In many types of serology tests, the sensitivity and the specificity are directly influenced by the quality of the antigens manufactured. Protein purification of these recombinantly expressed viral antigens [e.g., spike and its receptor binding domain (RBD)] is an important step in the manufacturing process. Simple and high-capacity protein purification schemes for spike, RBD, and CR3022 mAb, recombinantly expressed in CHO and HEK293 cells, are reported in this article. The schemes consist of an affinity chromatography step and a desalting step. Purified proteins were validated in ELISA-based serological tests. Interestingly, extracellular matrix proteins [most notably heparan sulfate proteoglycan (HSPG)] were co-purified from spike-expressing CHO culture with a long cultivation time. HSPG-spike interaction could play a functional role in the pathology and the pathogenesis of SARS-CoV-2 and other coronaviruses.

biochemistry↗

bioRxiv2020

Pneumolysin is responsible for differential gene expression and modifications in the epigenetic landscape of primary monocyte derived macrophages.

Epigenetic modifications regulate gene expression in the host response to a diverse range of pathogens. The extent and consequences of epigenetic modification during macrophage responses to Streptococcus pneumoniae, and the role of pneumolysin, a key Streptococcus pneumoniae virulence factor, in influencing these responses, are currently unknown. To investigate this, we infected human monocyte derived macrophages (MDMs) with Streptococcus pneumoniae and addressed whether pneumolysin altered the epigenetic landscape and the associated acute macrophage transcriptional response using a combined transcriptomic and proteomic approach. Transcriptomic analysis identified 503 genes that were differentially expressed in a pneumolysin-dependent manner in these samples. Pathway analysis highlighted the involvement of transcriptional responses to core innate responses to pneumococci including modules associated with metabolic pathways activated in response to infection, oxidative stress responses and NF{kappa}B, NOD-like receptor and TNF signalling pathways. Quantitative proteomic analysis confirmed pneumolysin-regulated protein expression, early after bacterial challenge, in representative transcriptional modules associated with innate immune responses. In parallel, quantitative mass spectrometry identified global changes in the relative abundance of histone post translational modifications (PTMs) upon pneumococcal challenge. We identified an increase in the relative abundance of H3K4me1, H4K16ac and a decrease in H3K9me2 and H3K79me2 in a PLY-dependent fashion. We confirmed that pneumolysin blunted early transcriptional responses involving TNF- and IL-6 expression. Vorinostat, a histone deacetylase inhibitor, similarly downregulated TNF production, reprising the pattern observed with pneumolysin. In conclusion, widespread changes in the macrophage transcriptional response are regulated by pneumolysin and are associated with global changes in histone PTMs. Modulating histone PTMs can reverse pneumolysin-associated transcriptional changes influencing innate immune responses, suggesting that epigenetic modification by pneumolysin plays a role in dampening the innate responses to pneumococci. Author summaryPneumolysin is a toxin that contributes to how Streptococcus pneumoniae, the leading cause of pneumonia, causes disease. In this study, the toxin alters gene expression in immune cells called macrophages, one of the first lines of defence against bacteria at sites of infection. Modulation involved multiple immune responses, including generation of chemical signals coordinating responses in immune cells termed cytokines. In addition, changes were observed in histone proteins that are involved in controlling gene expression in the cell. Pneumolysin reduced early production of the cytokine TNF- and a medicine vorinostat that modifies these epigenetic histone modifications had a similar affect, suggesting epigenetic mechanisms contribute to the ability of pneumolysin to reduce immune responses.

molecular biology↗