Biology subjects

Griffiths, A. D.

Publications and source records attributed to Griffiths, A. D..

7 recordsLinked to original sources

bioRxiv2025

DNA barcoding for parallelised single-molecule characterisation of kinetic phenotypes

Single-molecule techniques provide exceptional resolution of biomolecular dynamics but are limited by low-throughput. We present a barcoding strategy that enables simultaneous kinetic profiling of multiple protein variants at the single-molecule level. Each protein is covalently linked to a unique DNA barcode, decoded via transient hybridisation of fluorescent probes distinguished by colour and binding duration. We applied this method to a library of 16 ClpS variants to systematically explore evolutionary trajectories towards a variant adapted to recognise N-terminal amino acids with binding kinetics optimised for single-molecule peptide sequencing. The approach uncovered [~]5-fold variation in median dissociation rates across variants. Several variants displayed bimodal kinetics, likely reflecting structural subpopulations, and differences in kinetic properties arose primarily from the relative abundance of these phenotypes. This approach offers a general framework for screening and optimising proteins for single-molecule applications, while revealing mechanistic insights that are inaccessible to traditional ensemble methods.

biophysics↗

bioRxiv2025

Parallelization of single-molecule binding kinetic measurements via protein barcode sequencing

Screening protein variants for desired functions has long relied on coupling of genotype (gene sequence) to phenotype (protein function), limiting the use of powerful single-molecule (SM) techniques. Here, we introduce a scalable SM screening method that bypasses this constraint by linking SM functional analysis to protein identity through SM protein sequencing. Protein variants are tagged with unique C-terminal peptide barcodes and loaded onto a semiconductor chip containing millions of nanowells. Protein-ligand interactions are monitored in real time at the SM level, and a dye-cycling strategy extends the measurable dynamic range, enabling quantification of slow dissociation rates typical of high-affinity interactions. After functional analysis, each protein molecule is identified by sequencing its barcode. We apply this method to 20 barcoded nanobodies spanning over 1,000-fold in affinity, yielding results consistent with published values and individual SM measurements. Our approach should accelerate protein engineering by enabling rapid, multiplexed SM screening of protein libraries.

biochemistry↗

bioRxiv2024

Development of a generalisable tryptophan-optimised quenchbody biosensor based on a synthetic nanobody library

Quenchbodies, antibodies labelled with fluorophores that increase in intensity upon antigen binding, offer great promise for biosensor development. Nanobody-based quenchbodies are particularly attractive due to their small size, ease of expression, high stability, rapid evolvability, and amenability to protein engineering. However, existing designs for protein detection show limited dynamic range, with fluorescence increases of only 1.1-1.7 fold. Here we identify the tryptophan residues in the nanobody complementarity-determining regions (CDRs) that are critical to quenchbody performance. Using a combination of rational design and molecular dynamics simulations, we developed an optimised nanobody scaffold with tryptophans introduced at key positions. We used this scaffold in an in vitro directed-evolution screen against human inflammatory cytokine interleukin-6 (IL-6). This yielded quenchbodies with 1.5-2.4-fold fluorescence increases, enabling IL-6 detection down to 2 nM. Our scaffold provides a valuable platform for developing biosensors for diverse protein targets, with applications in research, diagnostics, and environmental monitoring.

bioengineering↗

bioRxiv2023

Adaptation to a phenotypic trade-off requires an interplay between local and global regulators

Organisms depend on their ability to balance multiple phenotypes by evolutionary adaptation. For instance, cellular growth and movement jointly enable critical processes including microbial colonization and cancer invasiveness. Growth and movement are known to be controlled by local regulators that target single operons, as well as by global regulators that impact hundreds of genes. However, how these different levels of regulation interplay during evolution is unclear. Using Escherichia coli growth and motility as a model system, we show that global regulators enable the adaptation of two phenotypes bound by a trade-off, where improvement in one causes deterioration in the other. We measured how CRISPR-mediated knockdowns of global and local transcription factors impact growth and motility in different environments. We find that local regulators mostly modulate motility, while global regulators jointly modulate growth and motility. Genetic perturbations display complex high order interactions between genes and environments. Nevertheless, gene perturbations display consistent patterns in the growth-motility space when grouped by their position in the regulatory hierarchy. These patterns constrain evolutionary scenarios, where local regulators are typically mutated first to optimize motility, then global regulators allow cells to adjust the trade-off between growth and motility. These findings overall highlight the role of pleiotropic regulators for coordinating phenotypic responses in complex environments.

systems biology↗

bioRxiv2021

The establishment of variant surface glycoprotein monoallelic expression revealed by single-cell RNA-seq of Trypanosoma brucei in the tsetse fly salivary glands.

The long and complex Trypanosoma brucei development in the tsetse fly vector culminates when parasites gain mammalian infectivity in the salivary glands. A key step in this process is the establishment of monoallelic variant surface glycoprotein (VSG) expression and the formation of the VSG coat. The establishment of VSG monoallelic expression is complex and poorly understood, due to the multiple parasite stages present in the salivary glands. Therefore, we sought to further our understanding of this phenomenon by performing single-cell RNA-sequencing (scRNA-seq) on these trypanosome populations. We were able to capture the developmental program of trypanosomes in the salivary glands, identifying populations of epimastigote, gamete, pre-metacyclic and metacyclic cells. Our results show that parasite metabolism is dramatically remodeled during development in the salivary glands, with a shift in transcript abundance from tricarboxylic acid metabolism to glycolytic metabolism. Analysis of VSG gene expression in pre-metacyclic and metacyclic cells revealed a dynamic VSG gene activation program. Strikingly, we found that pre-metacyclic cells contain transcripts from multiple VSG genes, which resolves to singular VSG gene expression in mature metacyclic cells. Single molecule RNA fluorescence in situ hybridisation (smRNA-FISH) of VSG gene expression following in vitro metacyclogenesis confirmed this finding. Our data demonstrate that multiple VSG genes are transcribed before a single gene is chosen. We propose a transcriptional race model governs the initiation of monoallelic expression.

molecular biology↗

bioRxiv2019

Darwinian properties and their trade-offs in autocatalytic RNA networks

Discovering autocatalytic chemistries that can evolve is a major goal in systems chemistry and a critical step towards understanding the origin of life. Autocatalytic networks have been discovered in various chemistries, but we lack a general understanding of how network topology controls the Darwinian properties of variation, differential reproduction, and heredity, which are mediated by the chemical composition. Using barcoded sequencing and droplet microfluidics, we establish a landscape of thousands of networks of RNAs that catalyze their own formation from fragments, and derive relationships between network topology and chemical composition. We find that strong variations arise from catalytic innovations perturbing weakly connected networks, and that reproduction increases with global connectivity. These rules imply trade-offs between reproduction and variation, and between compositional persistence and variation along trajectories of network complexification. Overall, connectivity in reaction networks provides a lever to balance variation (to explore chemical states) with reproduction and heredity (persistence being necessary for selection to act), as required for chemical evolution.

evolutionary biology↗

bioRxiv2019

Metabolic Cost of Rapid Adaptation of Single Yeast Cells Under Unforeseen Challenge

Cells can rapidly adapt to changing environments through non-genetic processes; however, the metabolic cost of such adaptation has never been considered. Here we demonstrate metabolic coupling in a remarkable, rapid adaptation process (10-3cells/hour) by simultaneously measuring metabolism and division of thousands of individual Saccharomyces cerevisiae cells using a droplet microfluidic system. Following a severe challenge, most cells, while not dividing, continue to metabolize, displaying a remarkably wide diversity of metabolic trajectories from which adaptation events can be anticipated. Adaptation requires the consumption of a characteristic amount of energy, indicating that it is an active process. The demonstration that metabolic trajectories predict a priori adaptation events provides the first evidence of tight energetic coupling between metabolism and regulatory reorganization in adaptation.\n\nOne Sentence SummaryDemonstration of the tight coupling between metabolic activity and regulatory processes during rapid adaptation at the single-cell level.

systems biology↗