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

Project Information

CAC-seq.

Grant agreement ID: 887491

DOI

10.3030/887491

Project terminated on 31 December 2021

EC signature date 21 February 2020

Start date 1 April 2020

End date 31 March 2022

Funded under

• EXCELLENT SCIENCE - Marie Skłodowska-Curie Actions

Total cost

€ 212 933,76

EU contribution

€ 212 933,76

Coordinated by

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.)

Reporting period: 2020-04-01 to 2022-03-31

Summary of the context and overall objectives of the project

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.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

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.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

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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