Silver-Coordinated
Watson–Crick Pairing-Driven
Three-Dimensional DNA Walker for Locus-Specific Detection of Genomic N6‑Methyladenine and N4‑Methylcytosine at the Single-Molecule Level
posted on 2024-01-29, 05:43authored byLi-Juan Wang, Qian Liu, Ying-Ying Lu, Le Liang, Chun-Yang Zhang
N6-Methyladenine (6mdA) and N4-methylcytosine (4mdC) are the two most dominant
DNA modifications in both prokaryotes and eukaryotes, but standard
hybridization-based techniques cannot be applied for the 6mdA/4mdC
assay. Herein, we demonstrate the silver-coordinated Watson–Crick
pairing-driven three-dimensional (3D) DNA walker for locus-specific
detection of genomic 6mdA/4mdC at the single-molecule level. 6mdA-DNA
and 4mdC-DNA can selectively hybridize with the binding probes (BP1
and BP2) to form 6mdA-DNA-BP1 and 4mdC-DNA-BP2 duplexes. The 6mdA-C/4mdC-A
mismatches cannot be stabilized by AgI, and thus, 18-nt
BP1/BP2 cannot be extended by the catalysis of KF exonuclease. Through
toehold-mediated strand displacement (TMSD), the signal probe (SP1/SP2)
functionalized on the gold nanoparticles (AuNPs) can competitively
bind to BP1/BP2 in 6mdA-DNA-BP1/4mdC-DNA-BP2 duplex to obtain SP1–18-nt
BP1 and SP2–18-nt BP2 duplexes. The resulting DNA duplexes
can act as the substrates of lambda exonuclease, leading to the cleavage
of SP1/SP2 and the release of Cy3/Cy5 and 18-nt BP1/BP2. The released
18-nt BP1/BP2 can subsequently serve as the walker DNA, moving along
the surface of the AuNP to activate dynamic 3D DNA walking and releasing
abundant Cy3/Cy5. The released Cy3/Cy5 can be quantified by single-molecule
imaging. This nanosensor exhibits high sensitivity with a limit of
detection (LOD) of 9.80 × 10–15 M for 6mdA-DNA
and 9.97 × 10–15 M for 4mdC-DNA. It can discriminate
6mdA-/4mdC-DNA from unmodified genomic DNAs, distinguish 0.01% 6mdA-/4mdC-DNA
from excess unmethylated DNAs, and quantify 6mdA-/4mdC-DNA at specific
sites in genomic DNAs of liver cancer cells and Escherichia
coli plasmid cloning vector, providing a new platform
for locus-specific analysis of 6mdA/4mdC in genomic DNAs.