Jack Kennefick

University of Birmingham

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Co-Authors

Robert K. Neely

University of Birmingham

Krystian Ubych

University of Warsaw

Francisco Fernández‐Trillo

University of Birmingham

Elodie Jagu

Institut de Chimie de Clermont-Ferrand

Andrew A. Wilkinson

University of Birmingham

Ashleigh E. Rushton

University of Birmingham

Qiang Su

Guizhou University

Steven Coulthard

University of Birmingham

Luca Tosti

Istituto Nazionale di Fisica Nucleare, Sezione di Perugia

Calum Mould

Cooperative Institutions

University of Birmingham

University of Nottingham

University of Warwick

Istituto Nazionale di Fisica Nucleare, Sezione di Perugia

Instituto de Astrofísica de Canarias

Universidad de La Laguna

University of Perugia

University of Siena

ASST Fatebenefratelli Sacco

Istituto Nazionale di Fisica Nucleare, Sezione di Bari

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

bioRxiv (Cold Spring Harbor Laboratory) (2024)

Luca Tosti Calum Mould Imogen Gatehouse Anyela Camargo Anthony D. Smith

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.

Epigenomics

Bisulfite sequencing

CpG site

Methylated DNA immunoprecipitation

Differentially methylated regions

10.1101/2024.02.16.575381

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Site-selective and Re-writable Labeling of DNA through Enzymatic, Reversible and Click Chemistries

Andrew A. Wilkinson Elodie Jagu Krystian Ubych Steven Coulthard Ashleigh E. Rushton

Current methods for bioconjugation rely on the introduction of stable linkers that lack the required versatility to perform sequential functionalizations. However, sequential manipulations are an increasing requirement in chemical biology because they can underpin multiple analyses of the same sample to provide a wider understanding of cell behavior. Here, we present a new method to site-selectively write , remove and re-write chemical functionality to a biomolecule, DNA in this case. Our method combines the precision and robustness of methyltransferase-directed labeling with the reversibility of acyl hydrazones and the efficiency of click chemistry. Underpinning the method is a new S-adenosyl-l-methionine derivative to site-selectively label DNA with a bifunctional chemical handle containing an acyl hydrazone-linker and a terminal azide. Functional tags are conjugated via the azide, and can be removed (i.e. un-tagged) when needed at the acyl hydrazone via exchange with hydroxyl amine. The formed hydrazide-labeled DNA is a versatile intermediate that can be either re-written to reset the original chemical handle, or covalently reacted with a permanent tag . This ability to write , tag , un-tag and permanently tag DNA is exploited to sequentially introduce two fluorescent dyes on DNA. Finally, we demonstrate the potential of the method by developing a protocol to sort labeled DNA using magnetic beads, with subsequent amplification of the sorted DNA sample for further analysis. The presented method opens new avenues for site-selective bioconjugation and should underpin integrative approaches in chemical biology where sequential functionalizations of the same sample are required.

Bioconjugation

Hydrazone

Chemical Biology

Bioorthogonal Chemistry

Biomolecule

10.26434/chemrxiv.9939008.v2

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Citations (2)

Mutation of M13 Bacteriophage Major Coat Protein for Increased Conjugation to Exogenous Compounds

Bioconjugate Chemistry (2018)

Matthew Tridgett James R. Lloyd Jack Kennefick Charles Moore-Kelly Timothy R. Dafforn

Over the past ten years there has been increasing interest in the conjugation of exogenous compounds to the surface of the M13 bacteriophage. M13 offers a convenient scaffold for the development of nanoassemblies with useful functions, such as highly specific drug delivery and pathogen detection. However, the progress of these technologies has been hindered by the limited efficiency of conjugation to the bacteriophage. Here we generate a mutant version of M13 with an additional lysine residue expressed on the outer surface of the M13 major coat protein, pVIII. We show that this mutation is accommodated by the bacteriophage and that up to an additional 520 exogenous groups can be attached to the bacteriophage surface via amine-directed conjugation. These results could aid the development of high payload drug delivery nanoassemblies and pathogen detection systems with increased sensitivity.

Filamentous bacteriophage

10.1021/acs.bioconjchem.8b00307

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Citations (7)

Site-Selective and Rewritable Labeling of DNA through Enzymatic, Reversible, and Click Chemistries

ACS Central Science (2020)

Andrew A. Wilkinson Elodie Jagu Krystian Ubych Steven Coulthard Ashleigh E. Rushton

Current methods for bioconjugation rely on the introduction of stable linkers that lack the required versatility to perform sequential functionalizations. However, sequential manipulations are an increasing requirement in chemical biology because they can underpin multiple analyses of the same sample to provide a wider understanding of cell behavior. Here, we present a new method to site-selectively write, remove, and rewrite chemical functionality to a biomolecule, DNA in this case. Our method combines the precision and robustness of methyltransferase-directed labeling with the reversibility of acyl hydrazones and the efficiency of click chemistry. Underpinning the method is a new S-adenosyl-l-methionine derivative to site-selectively label DNA with a bifunctional chemical handle containing an acyl hydrazone-linker and a terminal azide. Functional tags are conjugated via the azide and can be removed (i.e., untagged) when needed at the acyl hydrazone via exchange with hydroxyl amine. The formed hydrazide-labeled DNA is a versatile intermediate that can be either rewritten to reset the original chemical handle or covalently reacted with a permanent tag. This ability to write, tag, untag, and permanently tag DNA is exploited to sequentially introduce two fluorescent dyes on DNA. Finally, we demonstrate the potential of the method by developing a protocol to sort labeled DNA using magnetic beads, with subsequent amplification of the sorted DNA sample for further analysis. The presented method opens new avenues for site-selective bioconjugation and should underpin integrative approaches in chemical biology where sequential functionalizations of the same sample are required.

Bioconjugation

Hydrazone

Chemical Biology

Biomolecule

Linker

10.1021/acscentsci.9b01023

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Citations (16)

Site-Selective and Re-Writable Labelling of DNA Through Enzymatic, Dynamic and Click Chemistries

Andrew A. Wilkinson Elodie Jagu Krystian Ubych Ashleigh E. Rushton Jack Kennefick

Current methods for bioconjugation rely on the introduction of stable linkers that lack the required versatility to perform sequential functionalizations. However, sequential manipulations are an increasing requirement in chemical biology because they can underpin multiple analyses of the same sample to provide a wider understanding of cell behavior. Here, we present a new method to site-selec- tive write, erase and re-write chemical functionality to a biomolecule, DNA in this case. Our method com- bines the robustness of methyltransferase-directed labeling with the versatility of dynamic covalent chemistry. Underpinning the method is a new S-adenosyl-L-methionine derivative to site-selectively la- bel DNA with a bifunctional chemical handle containing an acyl hydrazone-linker and a terminal azide. Functional tags are conjugated via the azide, and can be erased when needed at the acyl hydrazone via dynamic exchange. The formed hydrazide-labeled DNA is a versatile intermediate that can be either re- written to reset the original chemical handle, or covalently reacted with a permanent tag. This ability to write, tag, erase and permanently tag DNA is exploited to sequentially introduce two fluorescent dyes on DNA. Finally, we demonstrate the potential of the method by developing a protocol to sort labeled DNA using magnetic beads, with subsequent amplification of the sorted DNA sample for further analysis. The presented method opens new avenues for site-selective bioconjugation, and should underpin integrative approaches in chemical biology where sequential functionalizations of the same sample are required.

Bioconjugation

Hydrazone

Chemical Biology

Bioorthogonal Chemistry

10.26434/chemrxiv.9939008.v1

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Citations (0)

Site-selective and Re-writable Labeling of DNA through Enzymatic, Reversible and Click Chemistries

Andrew A. Wilkinson Elodie Jagu Krystian Ubych Steven Coulthard Ashleigh E. Rushton

Current methods for bioconjugation rely on the introduction of stable linkers that lack the required versatility to perform sequential functionalizations. However, sequential manipulations are an increasing requirement in chemical biology because they can underpin multiple analyses of the same sample to provide a wider understanding of cell behavior. Here, we present a new method to site-selectively write, remove and re-write chemical functionality to a biomolecule, DNA in this case. Our method combines the precision and robustness of methyltransferase-directed labeling with the reversibility of acyl hydrazones and the efficiency of click chemistry. Underpinning the method is a new S-adenosyl-l-methionine derivative to site-selectively label DNA with a bifunctional chemical handle containing an acyl hydrazone-linker and a terminal azide. Functional tags are conjugated via the azide, and can be removed (i.e. un-tagged) when needed at the acyl hydrazone via exchange with hydroxyl amine. The formed hydrazide-labeled DNA is a versatile intermediate that can be either re-written to reset the original chemical handle, or covalently reacted with a permanent tag. This ability to write, tag, un-tag and permanently tag DNA is exploited to sequentially introduce two fluorescent dyes on DNA. Finally, we demonstrate the potential of the method by developing a protocol to sort labeled DNA using magnetic beads, with subsequent amplification of the sorted DNA sample for further analysis. The presented method opens new avenues for site-selective bioconjugation and should underpin integrative approaches in chemical biology where sequential functionalizations of the same sample are required.

Bioconjugation

Hydrazone

Bioorthogonal Chemistry

Linker

10.26434/chemrxiv.9939008

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Citations (0)