Discrimination between Different DNA Lesions by Monitoring Single-Molecule Polymerase Stalling Kinetics during Nanopore Sequencing

Abstract

O6-Carboxymethylguanosine (O6-CMG), O6-methylguanosine (O6-MeG), and abasic site (AP site) are DNA lesions induced by alkylating agents. Identification of these lesions in DNA may aid in understanding their relevance to carcinogenesis and may be used for diagnosis. Nanopore sequencing (NPS) may directly report nucleotide modifications solely from the nanopore readout. However, the conventional NPS strategy still suffers from interferences from neighboring sequences. Instead, by observation of the enzymatic stalling kinetics caused by the O6-CMG, O6-MeG, or AP site, discrimination between different DNA lesions is directly achieved. This strategy is not interfered with by the sequence context around the lesion. The lesion, which retards the movement of the DNA through the pore, efficiently prohibits misreading of the DNA lesion. These results suggest a new strategy in the identification of DNA lesions or DNA modifications. It also provides a high-resolution biophysical tool to investigate enzymatic kinetics caused by DNA lesions and the corresponding enzymes.

Nanopore Deciphering Single Digital Polymers Towards High‐Density Data Storage

“…With the usage of phi29 DNA polymerase (DNAP) or Hel308 helicase, MspA nanopores have been applied to sequence DNAs that containing naturally modified nucleotides, including O 6 ‐carboxymethylguanosine (O 6 ‐CMG) and O 6 ‐methylguanosine (O 6 ‐MeG) (Figure 2b). [24] Besides, the MspA nanopore‐enzyme motor sequencing system has also been revealed availability to determinate the incorporation of unnatural nucleotides such as d5SICS (Q), dNaM (Z) and dTPT3 in ssDNA (Figure 2c). [10a,25] This indicates high possibility of extending nanopore approaches to direct identification of the unnatural DNA bases and analogues.…”

“…Figure 2a redrawn from Ref. [15a] Copyright (2015), with permission from Springer Nature Ltd. MspA nanopore sequencing of (b) the naturally modified DNAs [24] and (c) the unnatural DNAs ratcheted by phi29 DNAP, [25] respectively. Upper: chemical structures of the standard, modified and unnatural nucleotides.…”

“…Figure 2b adapted with permission from Ref. [24]. Copyright (2022) American Chemical Society; Figure 2c adapted from Ref.…”

“…Lower: the consensus current patterns of the control, modified and unnatural DNAs, where the non-standard nucleotides generated obviously different current variations in the substitute positions. Figure2badapted with permission from Ref [24]…”