Biology subjects

Ferguson, A. L.

Publications and source records attributed to Ferguson, A. L..

4 recordsLinked to original sources

bioRxiv2023

Direct monitoring of the thermodynamics and kinetics of DNA and RNA dinucleotide dehybridization from gaps and overhangs

Hybridization of short nucleic acid segments (<4 nucleotides) to single-strand templates occurs as a critical intermediate in processes such as non-enzymatic nucleic acid replication and toehold-mediated strand displacement. These templates often contain adjacent duplex segments that stabilize base pairing with single-strand gaps or overhangs, but the thermodynamics and kinetics of hybridization in such contexts are poorly understood due to experimental challenges of probing weak binding and rapid structural dynamics. Here we develop an approach to directly measure the thermodynamics and kinetics of DNA and RNA dinucleotide dehybridization using steady-state and temperature-jump infrared spectroscopy. Our results suggest that dinucleotide binding is stabilized through coaxial stacking interactions with the adjacent duplex segments as well as from potential non-canonical base pairing configurations and structural dynamics of gap and overhang templates revealed using molecular dynamics simulations. We measure timescales for dissociation ranging from 0.2 to 40 {micro}s depending on the template and temperature. Dinucleotide hybridization and dehybridization involves a significant free energy barrier with characteristics resembling that of canonical oligonucleotides. Together, our work provides an initial step for predicting the stability and kinetics of hybridization between short nucleic acid segments and various templates.

biophysics↗

bioRxiv2023

ProT-VAE: Protein Transformer Variational AutoEncoder for Functional Protein Design

The data-driven design of protein sequences with desired function is challenged by the absence of good theoretical models for the sequence-function mapping and the vast size of protein sequence space. Deep generative models have demonstrated success in learning the sequence to function relationship over natural training data and sampling from this distribution to design synthetic sequences with engineered functionality. We introduce a deep generative model termed the Protein Transformer Variational AutoEncoder (ProT-VAE) that furnishes an accurate, generative, fast, and transferable model of the sequence-function relationship for data-driven protein engineering by blending the merits of variational autoencoders to learn interpretable, low-dimensional latent embeddings and fully generative decoding for conditional sequence design with the expressive, alignment-free featurization offered by transformers. The model sandwiches a lightweight, task-specific variational autoencoder between generic, pre-trained transformer encoder and decoder stacks to admit alignment-free training in an unsupervised or semi-supervised fashion, and interpretable low-dimensional latent spaces that facilitate understanding, optimization, and generative design of functional synthetic sequences. We implement the model using NVIDIAs BioNeMo framework and validate its performance in retrospective functional prediction and prospective design of novel protein sequences subjected to experimental synthesis and testing. The ProT-VAE latent space exposes ancestral and functional relationships that enable conditional generation of novel sequences with high functionality and substantial sequence diversity. We anticipate that the model can offer an extensible and generic platform for machine learning-guided directed evolution campaigns for the data-driven design of novel synthetic proteins with "super-natural" function.

synthetic biology↗

bioRxiv2022

Deep learning-enabled design of synthetic orthologs of a signaling protein

Evolution-based deep generative models represent an exciting direction in understanding and designing proteins. An open question is whether such models can represent the constraints underlying specialized functions that are necessary for organismal fitness in specific biological contexts. Here, we examine the ability of three different models to produce synthetic versions of SH3 domains that can support function in a yeast stress signaling pathway. Using a select-seq assay, we show that one form of a variational autoencoder (VAE) recapitulates the functional characteristics of natural SH3 domains and classifies fungal SH3 homologs hierarchically by function and phylogeny. Locality in the latent space of the model predicts and extends the function of natural orthologs and exposes amino acid constraints distributed near and far from the SH3 ligand-binding site. The ability of deep generative models to specify orthologous function in vivo opens new avenues for probing and engineering protein function in specific cellular environments.

molecular biology↗

bioRxiv2022

High-dimensional and spatial analysis reveals immune landscape dependent progression in cutaneous squamous cell carcinoma

PurposeThe tumour immune microenvironment impacts the biological behaviour of the tumour but its effect on clinical outcomes in head and neck cutaneous squamous cell carcinomas (HNcSCC) is largely unknown. Experimental DesignWe compared the immune milieu of high-risk HNcSCC that never progressed to metastasis with those that metastasised using multi-parameter imaging mass cytometry. The cohort included both immunosuppressed patients (IS) and patients with an absence of clinical immune-suppression (ACIS). Spatial analyses were used to identify cellular interactions that were associated with tumour behaviour. ResultsNon-progressing primary HNcSCC were characterised by higher CD8+ and CD4+ T cell responses, including numerically increased Regulatory T cells. By contrast, primary lesions from HNcSCC patients who progressed were largely devoid of T cells with lower numbers of innate immune cells and increased expression of checkpoint receptors and in the metastatic lesions were characterised by an accumulation of B cells. Spatial analysis reveals multiple cellular interactions associated with non-progressing primary tumours that were distinct in primary tumours of disease progressing patients. Cellular regional analysis of the tumour microenvironment also shows squamous cell-enriched tumour regions associated with primary non-progressing tumours. ConclusionsEffective responses from both CD8+ and CD4+ T cells in the tumour microenvironment are essential for immune control of primary HNcSCC. Our findings indicate that the early events that shape the immune responses in primary tumours dictate progression and disease outcomes in HNcSCC. Translational RelevanceThe ability to predict metastatic tumour progression at the time of initial diagnosis of primary HNcSCC could tailor personalised medical care including disease surveillance strategies and identifying patients who will benefit most from adjuvant therapy. One Sentence SummaryThe immune landscape of high-risk cutaneous squamous cell carcinoma differs in tumours that never progress compared to those that progress to metastasis.

pathology↗