Biology subjects

Neely, R. K.

Publications and source records attributed to Neely, R. K..

7 recordsLinked to original sources

bioRxiv2025

A modified methyl transferase cofactor to selectively disable gene expression in E. coli

Artificial control of gene expression in bacteria offers interesting prospects for influencing bacterial pathogenicity and antibiotic resistance. We show that the methyl-transferase cofactor, AdoHcy azide, can disable gene expression in modified plasmids in some strains of E. coli, where ampicillin and kanamycin resistance as well as eGFP genes were selectively and independently disabled. The disabling of transcription is likely due to steric inhibition during transcription initiation, which is further confirmed by Sanger and nanopore sequencing results. Both sequencing methods showed that 3-6 nucleotides were absent from around the modification site, with the post growth, extracted AmpR/ eGFP plasmid showing evidence of restriction, with sections of the plasmid, including the modification site, missing for the AdoHcy azide modified plasmids. Notably, the AdoHcy azide modification on the DNA is resistant against demethylation in the BL21 strain of E. coli.

genomics↗

bioRxiv2024

Expansion microscopy allows quantitative characterisation of structural organisation of platelet aggregates

1Current microscopy approaches applied to platelet aggregates in both haemostatic and thrombotic settings indicate their structure has important implications in efficient haemostasis and in clinical treatment of thrombosis. However, current fluorescence microscopy approaches are not amenable to volumetric imaging of platelet aggregate structures. This is largely due to the small size of individual platelets and the tight packing of platelets within aggregates, resulting in optical opacity. Here we demonstrate that expansion microscopy, applied to platelet aggregates, can reveal multi-scale information about the structure of platelet aggregates. We produced volumetric images at nanoscale resolution of >700 platelet aggregates under normal and perturbed conditions, stained for cytoskeletal and membrane components. We demonstrate our custom analysis workflow provides quantitative description of platelet numbers, volumes and morphology within entire platelet aggregates. Additionally, we quantitatively describe subcellular organisation of F-actin. By comparing these measurements following treatment with the actin inhibitors, cytochalasin D and latrunculin A, we can robustly detect structural disruptions in platelet aggregates. Together these data provide a workflow to qualitatively and quantitatively describe the architecture of platelet aggregates at a range of scales (whole aggregates down to sub-cellular features within individual platelets).

cell biology↗

bioRxiv2024

Epigenomic profiling of active regulatory elements by enrichment of unmodified CpG dinucleotides

Current approaches for the study of DNA methylation and other modified bases focus on the modified fraction of the genome and are particularly well-suited to the detection of DNA hypermethylation. However, the study of hypomethylation (loss of DNA methylation), which is typically associated with markers of active chromatin, has been largely overlooked, in part, due to the lack of a suitable methodology. We present an enrichment-based and bisulfite-free approach for epigenomic profiling named "Active-Seq" (Azide Click Tagging for In Vitro Epigenomic sequencing) that achieves genome-wide profiling of DNA, by enriching for non-modified CpG sites using a mutated methyltransferase enzyme. We show that the genomic regions enriched by Active-Seq overlap with promoters, enhancers and partially methylated domains, all of which have had their methylation status linked to the development and progression of diseases. Active-Seq is a fast epigenetic profiling platform with a simple and streamlined workflow, performed in tandem with sequencing library preparation. The enzymatic chemistry is non-damaging toward the DNA which is critical for working with low concentration DNA input and will facilitate the future development of multiomics assays. We demonstrate robust and reproducible performance of Active-Seq using low DNA input in cell lines, liquid biopsies (cell-free DNA, cfDNA) and formalin-fixed paraffin embedded (FFPE) tissue.

genomics↗

bioRxiv2023

Sterically enhanced control of enzyme-assisted DNA assembly

Traditional methods for the assembly of functionalised DNA structures, involving enzyme restriction and modification, present difficulties when working with small DNA fragments (<100bp), in part due to a lack of control over enzymatic action during the DNA modification process. This limits the design flexibility and range of accessible DNA structures. Here, we show that these limitations can be overcome by introducing chemical modifications into the DNA, which spatially restrict enzymatic activity. This approach, Sterically Controlled Nuclease Enhanced (SCoNE) DNA assembly, thereby circumvents the size limitations of conventional Gibson assembly (GA) and allows for the preparation of well-defined, functionalised DNA structures with multiple probes for specific analytes, such as IL-6, procalcitonin (PCT), and a biotin reporter group. Notably, using the same starting materials conventional GA under typical conditions fails. We demonstrate successful analyte capture based on standard and modified sandwich ELISA and also show how the inclusion of biotin probes provides additional functionality for product isolation.

biochemistry↗

bioRxiv2021

Imaging Nanoscale Nuclear Structures with Expansion Microscopy

Commonly applied super-resolution light microscopies have provided insight into subcellular processes at the nanoscale. However, imaging depth, speed, throughput and cost remain significant challenges, reducing the numbers of three-dimensional, nanoscale processes that can be investigated and the number of laboratories able to undertake such analysis. Expansion microscopy solves many of these limitations but its application to imaging nuclear processes has been constrained by concerns of unequal nuclear expansion. Here we demonstrate the conditions for isotropic expansion of the nucleus. Using DNA damage response proteins, BRCA1, 53BP1 and RAD51 as exemplars we quantitatively describe the three-dimensional nanoscale organisation of over 50,000 DNA damage response structures. We demonstrate the ability to assess chromatin regulated events and show the simultaneous assessment of four elements. This study thus provides the means by which expansion microscopy can contribute to the investigation of nanoscale nuclear processes.

molecular biology↗

bioRxiv2019

Evaluation of direct grafting strategies in Expansion Microscopy

High resolution fluorescence microscopy is a key tool in the elucidation of biological fine-structure, providing insights into the distribution and interactions of biomolecular systems down to the nanometer scale. Expansion microscopy is a recently developed approach to achieving nanoscale resolution in optical imaging. In the experiment, biological samples are embedded in a hydrogel, which is isotropicaly swollen. This physically pulls labels apart, allowing more of them to be resolved. However, in the gelation and swelling process, two factors combine to reduce the signal in the final image; signal dilution and the polymerization reaction, which can damage some fluorophores. Here, we show a chemical linking approach that allows covalent grafting of biomolecular target and reporter in expansion microscopy. Through the combination of a targeting ligand, a reporter moiety and a polymerizable group in a single linker, complex constructs can be prepared in a single, labelling step. We show application of this new series of molecules in the targeting of the cell cytoskeleton, a first example of lipid membranes in expansion microscopy; direct immunostaining with primary and secondary antibodies, and direct grafting of ISH probes and signal amplification initiators (HCR and RollFISH). Our probes allow direct, multiplexed targeting of the cellular blueprint and enable a range of novel imaging approaches in combination with expansion microscopy.

biophysics↗

bioRxiv2018

DNA barcodes for rapid, whole genome, single-molecule analyses

We report an approach for visualizing DNA sequence and using these DNA barcodes to search complex mixtures of genomic material for DNA molecules of interest. We demonstrate three applications of this methodology; by identifying specific molecules of interest from a dataset containing gigabasepairs of genome; by straightforward strain-typing of bacteria from such a dataset and, finally, by locating infecting virus molecules in a background of human genomic material. DNA barcodes enable quantitative understanding of complex genome mixtures, on a large scale. As a result of the dense fluorescent labelling of the DNA, individual barcodes of the order 40 kilobase pairs in length can be reliably identified. This means DNA can be prepared for imaging using standard handling and purification techniques. The recorded dataset provides stable physical and electronic records of the total genomic content of a sample that can be readily searched for a molecule or region of interest.

genomics↗