Nanopore Sequencing Accurately Identifies the Mutagenic DNA Lesion O⁶‐Carboxymethyl Guanine and Reveals Its Behavior in Replication

Abstract

O 6 -carboxymethylguanine (O 6 -CMG) is ah ighly mutagenic alkylation product of DNA, triggering transition mutations relevant to gastrointestinal cancer.However,precise localization of asingle O 6 -CMG with conventional sequencing platforms is challenging.H ere nanopore sequencing (NPS), which directly senses single DNAb ases according to their physiochemical properties,w as employed to detect O 6 -CMG. Au nique O 6 -CMG signal was observed during NPS and asingle-event call accuracy of > 95 %was achieved. Moreover, O 6 -CMG was found to be ar eplication obstacle for Phi29 DNApolymerase (Phi29 DNAP), suggesting this lesion could cause DNAs equencing biases in next generation sequencing (NGS) approaches.

Electrode-free nanopore sensing by DiffusiOptoPhysiology

“…Electrophysiology recordings were performed as previously reported ( 7 , 53 , 54 ). The electrophysiology trace was acquired with 25-kHz sampling rate, low-pass–filtered at 1 kHz (Axopatch 200B, Molecular Devices), digitized, and recorded using the Digidata 1550A digitizer (Molecular Devices).…”

A Chemical Link between Meat Consumption and Colorectal Cancer Development?

“…In the context of a modern epigenetic understanding of genome function, seminal reports have recently emerged concerning strategies for genomic mapping of DNA damage. 41−43 In the case of O 6 -CMG, two significant advances of the last two years, namely, a nanopore-based O 6 -CMG sequencing approach 50 and an artificial nucleoside/ engineered polymerase-based analysis strategy, 51 are anticipated to enable future research involving the identification of the presence and effects of O 6 -CMG in selected DNA regions, such as cancer genes.…”

“…The nanopore-based sequencing data reported for samples of DNA containing O 6 -CMG entailed the preparation of a library of O 6 -CMG-modified oligonucleotides, tagged with a deca(deoxythymidine) sequence. 50 The library was electro-phoretically driven into a customized pore, and the passage of ssDNA through the pore caused a variation in the applied current that enabled the identification of the different bases. A high-current signal with flickering characteristics was recorded with high accuracy (95%) at the passage of O 6 -CMG (Figure 5A).…”

“…A high-current signal with flickering characteristics was recorded with high accuracy (95%) at the passage of O 6 -CMG (Figure 5A). 50 In the same study, the replication of O 6 -CMG by Phi29 DNA Pol, which serves as a motor protein and controls the passage of the DNA through the nanopore, was characterized. O 6 -CMG was a replication blockage for Phi29 DNA Pol.…”

“…Emerging research concerning detection strategies that retain information concerning O 6 -CMG damage location and persistence is expected to further clarify relationships between O 6 -CMG and cancer. 50,51 Herein, we review evidence linking meat consumption with O 6 -CMG occurrence, present mechanistic data elucidating the role of DNA synthesis and repair in O 6 -CMG-induced mutagenicity, and present emerging strategies to better address contributions to carcinogenesis. Focusing on chemical advances in this area, we describe synthetic and analytical strategies that have been instrumental to the elucidation of O 6 -CMG-induced phenotypes.…”