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

Informazioni relative al progetto

CAC-seq.

ID dell’accordo di sovvenzione: 887491

DOI

10.3030/887491

Progetto concluso il 31 Dicembre 2021

Data della firma CE 21 Febbraio 2020

Data di avvio 1 Aprile 2020

Data di completamento 31 Marzo 2022

Finanziato da

• EXCELLENT SCIENCE - Marie Skłodowska-Curie Actions

Costo totale

€ 212 933,76

Contributo UE

€ 212 933,76

Coordinato da

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.)

Periodo di rendicontazione: 2020-04-01 al 2022-03-31

Sintesi del contesto e degli obiettivi generali del progetto

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.

Lavoro eseguito dall’inizio del progetto fino alla fine del periodo coperto dalla relazione e principali risultati finora ottenuti

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.

Progressi oltre lo stato dell’arte e potenziale impatto previsto (incluso l’impatto socioeconomico e le implicazioni sociali più ampie del progetto fino ad ora)

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.

sb-group-photo-2021-l3a1936-taken-by-gabriella-bocchetti-university-of-cambridge-m2-e1632143845189.jpg