Chemical assisted enrichment of 5-carboxycytosine that also allows for DNA sequencing at single base resolution.

Información del proyecto

CAC-seq.

Identificador del acuerdo de subvención: 887491

DOI

10.3030/887491

Proyecto finalizado el 31 Diciembre 2021

Fecha de la firma de la CE 21 Febrero 2020

Fecha de inicio 1 Abril 2020

Fecha de finalización 31 Marzo 2022

Financiado con arreglo a

• EXCELLENT SCIENCE - Marie Skłodowska-Curie Actions

Coste total

€ 212 933,76

Aportación de la UE

€ 212 933,76

Coordinado por

THE CHANCELLOR MASTERS AND SCHOLARS OF THE UNIVERSITY OF CAMBRIDGE
United Kingdom

Periodic Reporting for period 1 - CAC-seq. (Chemical assisted enrichment of 5-carboxycytosine that also allows for DNA sequencing at single base resolution.)

Período documentado: 2020-04-01 hasta 2022-03-31

Resumen del contexto y de los objetivos generales del proyecto

Next-generation sequencing constitutes a powerful tool to analyze genomic material at unprecedented depth and scale to detect disease-specific mutations. Besides changes in the canonical base sequence epigenetically modified cytosine derivatives have also been linked to various disease states. Chemical and enzymatic methods that selectively target epigenetic markers are central to study their distribution and function with the help of nucleic acid sequencing. During the course of this action, a photochemical method for the conversion of epigenetic 5-carboxycytosine (5caC) to a T-like base has been developed that allows for base resolution sequencing of 5caC. In addition, the developed chemistry can also be applied to sequence 5-methylcytosine (5mC) in genomic DNA. The methodology could be used in diagnostic applications for the early detection of deadly diseases such as cancer.

Trabajo realizado desde el comienzo del proyecto hasta el final del período abarcado por el informe y los principales resultados hasta la fecha

A reductive decarboxylation of 5-carboxycytosine (caC) by photochemistry has been developed. This allows for the first application of photochemistry to epigenetic sequencing via direct base conversion, and is a rare example of the radical decarboxylation of an aromatic substrate. The work shows that the base conversion is selective for 5-carboxycytosine over other canonical or modified nucleosides, and has been optimised for the treatment of modified oligonucleotides. Finally, in conjunction with enzymatic TET oxidation, the application of the photochemistry to base-resolution sequencing of 5-methylcytosine (mC) in a model genomic system (bacteriophage-lambda) has been demonstrated. A patent application has been filed and is currently in the PCT phase. Further, a manuscript for publication in a scientific journal has been prepared and submitted.

Avances que van más allá del estado de la técnica e impacto potencial esperado (incluida la repercusión socioeconómica y las implicaciones sociales más amplias del proyecto hasta la fecha)

A mild, biocompatible method for sequencing of epigenetic DNA bases has been developed, which represents both the first application of photochemistry to epigenetic sequencing via direct base conversion, and is a rare example of the radical decarboxylation of an aromatic substrate. The base conversion is selective for 5-carboxycytosine (caC) over other canonical or modified nucleosides and allows for sequencing of caC and methyl cytosine. Compared to other base-conversion chemistries, the photochemical reaction takes place within minutes, which might provide advantages for high-throughput detection applications such as diagnostics. Methyl cytosine for example is a candidate for early cancer detection from cell free DNA in blood.

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