Characterization of organelle DNA degradation mediated by DPD1 exonuclease in the rice genome-edited line.

Mitochondria and plastids, originated as ancestral endosymbiotic bacteria, contain their own DNA sequences. These organelle DNAs (orgDNAs) are, despite the limited genetic information they contain, an indispensable part of the genetic systems but exist as multiple copies, making up a substantial amount of total cellular DNA. Given this abundance, orgDNA is known to undergo tissue-specific degradation in plants. Previous studies have shown that the exonuclease DPD1, conserved among seed plants, degrades orgDNAs during pollen maturation and leaf senescence in Arabidopsis. However, tissue-specific orgDNA degradation was shown to differ among species. To extend our knowledge, we characterized DPD1 in rice in this study. We created a genome-edited (GE) mutant in which OsDPD1 and OsDPD1-like were inactivated. Characterization of this GE plant demonstrated that DPD1 was involved in pollen orgDNA degradation, whereas it had no significant effect on orgDNA degradation during leaf senescence. Comparison of transcriptomes from wild-type and GE plants with different phosphate supply levels indicated that orgDNA had little impact on the phosphate starvation response, but instead had a global impact in plant growth. In fact, the GE plant showed lower fitness with reduced grain filling rate and grain weight in natural light conditions. Taken together, the presented data reinforce the important physiological roles of orgDNA degradation mediated by DPD1.

Lysosomal endonuclease RNase T2 and PLD exonucleases cooperatively generate RNA ligands for TLR7 activation.

Toll-like receptor 7 (TLR7) is essential for recognition of RNA viruses and initiation of antiviral immunity. TLR7 contains two ligand-binding pockets that recognize different RNA degradation products: pocket 1 recognizes guanosine, while pocket 2 coordinates pyrimidine-rich RNA fragments. We found that the endonuclease RNase T2, along with 5' exonucleases PLD3 and PLD4, collaboratively generate the ligands for TLR7. Specifically, RNase T2 generated guanosine 2',3'-cyclic monophosphate-terminated RNA fragments. PLD exonuclease activity further released the terminal 2',3'-cyclic guanosine monophosphate (2',3'-cGMP) to engage pocket 1 and was also needed to generate RNA fragments for pocket 2. Loss-of-function studies in cell lines and primary cells confirmed the critical requirement for PLD activity. Biochemical and structural studies showed that PLD enzymes form homodimers with two ligand-binding sites important for activity. Previously identified disease-associated PLD mutants failed to form stable dimers. Together, our data provide a mechanistic basis for the detection of RNA fragments by TLR7.

Portable smartphone-based RecJf exonuclease-modulated enhanced ratiometric fluorescence bioplatform for rapid visual detection of As3.

Arsenite (As3+), a highly carcinogenic heavy metal ion and widely distributed in nature, can have serious health implications even with minimal exposure. Herein, a portable smartphone device-based ratiometric fluorescence platform was established for sensitive detection of As3+. The work relied on the use of metal-organic framework-tagged cDNA (PCN-224-cDNA), with high adsorption capability and fluorescence properties, as an internal reference to quench the fluorescence of FAM-anchored aptamer (FAM-Apt) via hybridization. In the presence of As3+, FAM-Apt specifically bound to As3+ leading to conformational changes, which detached from the PCN-224-cDNA surface. Interestingly, a smartphone-based readout equipment engineered using a 3D-printed hardware device administered the portable detection of As3+. The limit of detection (LOD) for the proposed ratiometric biosensor was calculated to be 0.021 ng/mL, significantly below WHO's safety threshold. Hence, it demonstrates significant potential for large-scale screening of As3+ residues in food and the environment.

Arbitrary Digital DNA Computing: A Programmable Molecular Perceptron Driven by Lambda Exonuclease for Lighting up Concatenated Circuits.

DNA circuits, as a type of biochemical system, have the capability to synchronize the perception of molecular information with a chemical reaction response and directly process the molecular characteristic information in biological activities, making them a crucial area in molecular digital computing and smart bioanalytical applications. Instead of cascading logic gates, the traditional research approach achieves multiple logic operations which limits the scalability of DNA circuits and increases the development costs. Based on the interface reaction mechanism of Lambda exonuclease, the molecular perceptron proposed in this study, with the need for only adjusting weight and bias parameters to alter the corresponding logic expressions, enhances the versatility of the molecular circuits. We also establish a mathematical model and an improved heuristic algorithm for solving weights and bias parameters for arbitrary logic operations. The simulation and FRET experiment results of a series of logic operations demonstrate the universality of molecular perceptron. We hope the proposed molecular perceptron can introduce a new design paradigm for molecular circuits, fostering innovation and development in biomedical research related to biosensing, targeted therapy, and nanomachines.

Structural and biochemical characterization of the mitomycin C repair exonuclease MrfB.

Mitomycin C (MMC) repair factor A (mrfA) and factor B (mrfB), encode a conserved helicase and exonuclease that repair DNA damage in the soil-dwelling bacterium Bacillus subtilis. Here we have focused on the characterization of MrfB, a DEDDh exonuclease in the DnaQ superfamily. We solved the structure of the exonuclease core of MrfB to a resolution of 2.1 Å, in what appears to be an inactive state. In this conformation, a predicted α-helix containing the catalytic DEDDh residue Asp172 adopts a random coil, which moves Asp172 away from the active site and results in the occupancy of only one of the two catalytic Mg2+ ions. We propose that MrfB resides in this inactive state until it interacts with DNA to become activated. By comparing our structure to an AlphaFold prediction as well as other DnaQ-family structures, we located residues hypothesized to be important for exonuclease function. Using exonuclease assays we show that MrfB is a Mg2+-dependent 3'-5' DNA exonuclease. We show that Leu113 aids in coordinating the 3' end of the DNA substrate, and that a basic loop is important for substrate binding. This work provides insight into the function of a recently discovered bacterial exonuclease important for the repair of MMC-induced DNA adducts.

Crystal structure of the 3'→5' exonuclease from Methanocaldococcus jannaschii.

RecJ exonucleases are members of the DHH phosphodiesterase family ancestors of eukaryotic Cdc45, the key component of the CMG (Cdc45-MCM-GINS) complex at the replication fork. They are involved in DNA replication and repair, RNA maturation and Okazaki fragment degradation. Bacterial RecJs resect 5'-end ssDNA. Conversely, archaeal RecJs are more versatile being able to hydrolyse in both directions and acting on ssDNA as well as on RNA. In Methanocaldococcus jannaschii two RecJs were previously characterized: RecJ1 is a 5'→3' DNA exonuclease, MjaRecJ2 works only on 3'-end DNA/RNA with a preference for RNA. Here, I present the crystal structure of MjaRecJ2, solved at a resolution of 2.8 Å, compare it with the other RecJ structures, in particular the 5'→3' TkoGAN and the bidirectional PfuRecJ, and discuss its characteristics in light of the more recent knowledge on RecJs. This work adds new structural data that might improve the knowledge of these class of proteins.

WRN exonuclease imparts high fidelity on translesion synthesis by Y family DNA polymerases.

Purified translesion synthesis (TLS) DNA polymerases (Pols) replicate through DNA lesions with a low fidelity; however, TLS operates in a predominantly error-free manner in normal human cells. To explain this incongruity, here we determine whether Y family Pols, which play an eminent role in replication through a diversity of DNA lesions, are incorporated into a multiprotein ensemble and whether the intrinsically high error rate of the TLS Pol is ameliorated by the components in the ensemble. To this end, we provide evidence for an indispensable role of Werner syndrome protein (WRN) and WRN-interacting protein 1 (WRNIP1) in Rev1-dependent TLS by Y family Polη, Polι, or Polκ and show that WRN, WRNIP1, and Rev1 assemble together with Y family Pols in response to DNA damage. Importantly, we identify a crucial role of WRN's 3' → 5' exonuclease activity in imparting high fidelity on TLS by Y family Pols in human cells, as the Y family Pols that accomplish TLS in an error-free manner manifest high mutagenicity in the absence of WRN's exonuclease function. Thus, by enforcing high fidelity on TLS Pols, TLS mechanisms have been adapted to safeguard against genome instability and tumorigenesis.

[Characterization of a Taq DNA polymerase fused with a DNA binding domain of Escherichia coli colicin].

Taq DNA polymerase, which was discovered from a thermophilic aquatic bacterium (Thermus aquaticus), is an enzyme that possesses both reverse transcriptase activity and DNA polymerase activity. Colicin E (CE) protein belongs to a class of Escherichia coli toxins that utilize the vitamin receptor BtuB as a transmembrane receptor. Among these toxins, CE2, CE7, CE8, and CE9 are classified as non-specific DNase-type colicins. Taq DNA polymerase consists of a 5'→3' exonuclease domain, a 3'→5' exonuclease domain, and a polymerase domain. Taq DNA polymerase lacking the 5'→3' exonuclease domain (ΔTaq) exhibits higher yield but lower processivity, making it unable to amplify long fragments. In this study, we aimed to enhance the processivity of ΔTaq. To this end, we fused dCE with ΔTaq and observed a significant improvement in the processivity of the resulting dCE-ΔTaq compared to Taq DNA polymerase and dCE-Taq. Furthermore, its reverse transcriptase activity was also higher than that of ΔTaq. The most notable improvement was observed in dCE8-ΔTaq, which not only successfully amplified 8 kb DNA fragments within 1 minute, but also yielded higher results compared to other mutants. In summary, this study successfully enhanced the PCR efficiency and reverse transcription activity of Taq DNA polymerase by fusing ΔTaq DNA polymerase with dCE. This approach provides a novel approach for modifying Taq DNA polymerase and holds potential for the development of improved variants of Taq DNA polymerase.

A signal transmission strategy driven by gap-regulated exonuclease hydrolysis for hierarchical molecular networks.

Exonucleases serve as efficient tools for signal processing and play an important role in biochemical reactions. Here, we identify the mechanism of cooperative exonuclease hydrolysis, offering a method to regulate the cooperative hydrolysis driven by exonucleases through the modulation of the number of bases in gap region. A signal transmission strategy capable of producing amplified orthogonal DNA signal is proposed to resolve the polarity of signals and byproducts, which provides a solution to overcome the signal attenuation. The gap-regulated mechanism combined with DNA strand displacement (DSD) reduces the unpredictable secondary structures, allowing for the coexistence of similar structures in hierarchical molecular networks. For the application of the strategy, a molecular computing model is constructed to solve the maximum weight clique problems (MWCP). This work enhances for our knowledge of these important enzymes and promises application prospects in molecular computing, signal detection, and nanomachines.

Dietary restriction fails to extend lifespan of Drosophila model of Werner syndrome.

Werner syndrome (WS) is a rare genetic disease in humans, caused by mutations in the WRN gene that encodes a protein containing helicase and exonuclease domains. WS is characterized by symptoms of accelerated aging in multiple tissues and organs, involving increased risk of cancer, heart failure, and metabolic dysfunction. These conditions ultimately lead to the premature mortality of patients with WS. In this study, using the null mutant flies (WRNexoΔ) for the gene WRNexo (CG7670), homologous to the exonuclease domain of WRN in humans, we examined how diets affect the lifespan, stress resistance, and sleep/wake patterns of a Drosophila model of WS. We observed that dietary restriction (DR), one of the most robust nongenetic interventions to extend lifespan in animal models, failed to extend the lifespan of WRNexoΔ mutant flies and even had a detrimental effect in females. Interestingly, the mean lifespan of WRNexoΔ mutant flies was not reduced on a protein-rich diet compared to that of wild-type (WT) flies. Compared to WT control flies, the mutant flies also exhibited altered responses to DR in their resistance to starvation and oxidative stress, as well as changes in sleep/wake patterns. These findings show that the WRN protein is necessary for mediating the effects of DR and suggest that the exonuclease domain of WRN plays an important role in metabolism in addition to its primary role in DNA-repair and genome stability.

Experimental and analytical pipeline for sub-genomic RNA landscape of coronavirus by Nanopore sequencer.

Coronaviruses (CoVs), including severe acute respiratory syndrome coronavirus 2, can infect a variety of mammalian and avian hosts with significant medical and economic consequences. During the life cycle of CoV, a coordinated series of subgenomic RNAs, including canonical subgenomic messenger RNA and non-canonical defective viral genomes (DVGs), are generated with different biological implications. Studies that adopted the Nanopore sequencer (ONT) to investigate the landscape and dynamics of viral RNA subgenomic transcriptomes applied arbitrary bioinformatics parameters without justification or experimental validation. The current study used bovine coronavirus (BCoV), which can be performed under biosafety level 2 for library construction and experimental validation using traditional colony polymerase chain reaction and Sanger sequencing. Four different ONT protocols, including RNA direct and cDNA direct sequencing with or without exonuclease treatment, were used to generate RNA transcriptomic libraries from BCoV-infected cell lysates. Through rigorously examining the k-mer, gap size, segment size, and bin size, the optimal cutoffs for the bioinformatic pipeline were determined to remove the sequence noise while keeping the informative DVG reads. The sensitivity and specificity of identifying DVG reads using the proposed pipeline can reach 82.6% and 99.6% under the k-mer size cutoff of 15. Exonuclease treatment reduced the abundance of RNA transcripts; however, it was not necessary for future library preparation. Additional recovery of clipped BCoV nucleotide sequences with experimental validation expands the landscape of the CoV discontinuous RNA transcriptome, whose biological function requires future investigation. The results of this study provide the benchmarks for library construction and bioinformatic parameters for studying the discontinuous CoV RNA transcriptome.IMPORTANCEFunctional defective viral genomic RNA, containing all the cis-acting elements required for translation or replication, may play different roles in triggering cell innate immune signaling, interfering with the canonical subgenomic messenger RNA transcription/translation or assisting in establishing persistence infection. This study does not only provide benchmarks for library construction and bioinformatic parameters for studying the discontinuous coronavirus RNA transcriptome but also reveals the complexity of the bovine coronavirus transcriptome, whose functional assays will be critical in future studies.

Biochemical characterization and mutational analysis of the NurA protein from the hyperthermophilic euryarchaeon Thermococcus barophilus Ch5.

Archaeal NurA protein plays a key role in producing 3'-single stranded DNA used for homologous recombination repair, together with HerA, Mre11, and Rad50. Herein, we describe biochemical characteristics and roles of key amino acid residues of the NurA protein from the hyperthermophilic euryarchaeon Thermococcus barophilus Ch5 (Tba-NurA). Tba-NurA possesses 5'-3' exonuclease activity for degrading DNA, displaying maximum efficiency at 45 °C-65 °C and at pH 8.0 in the presence of Mn2+. The thermostable Tba-NurA also possesses endonuclease activity capable of nicking plasmid DNA and circular ssDNA. Mutational data demonstrate that residue D49 of Tba-NurA is essential for exonuclease activity and is involved in binding ssDNA since the D49A mutant lacked exonuclease activity and reduced ssDNA binding. The R96A and R129A mutants had no detectable dsDNA binding, suggesting that residues R96 and R129 are important for binding dsDNA. The abolished degradation activity and reduced dsDNA binding of the D120A mutant suggest that residue D120 is essential for degradation activity and dsDNA binding. Additionally, residues Y392 and H400 are important for exonuclease activity since these mutations resulted in exonuclease activity loss. To our knowledge, it is the first report on biochemical characterization and mutational analysis of the NurA protein from Thermococcus.

Purification of Circular RNAs Using Poly(A) Tailing Followed by RNase R Digestion.

Thousands of eukaryotic protein-coding genes can be alternatively spliced to yield linear mRNAs and circular RNAs (circRNAs). Some circRNAs accumulate to higher levels than their cognate linear mRNAs, but the vast majority are expressed at low levels. Hence, for most circRNAs, only a handful of sequencing reads, if any, that span the backsplicing junction are observed in standard RNA-seq libraries. It thus has become common to use the 3'-5' exonuclease ribonuclease R (RNase R) to selectively degrade linear RNAs when aiming to prove transcript circularity or biochemically enrich circRNAs. However, RNase R fails to degrade linear RNAs with structured 3' ends or internal G-quadruplex structures. To overcome these shortcomings, we describe an improved protocol for circRNA purification from total RNA that employs a poly(A) tailing step prior to RNase R digestion, which is performed in a Li+ containing buffer (rather than K+) to destabilize G-quadruplexes. This biochemical method enables higher enrichment (two- to threefold) of circRNAs to be obtained compared to standard RNase R protocols due to more efficient removal of linear RNAs. By then performing quantitative RT-PCR (RT-qPCR) or generating RNA-seq libraries, the expression of individual circRNAs can be examined or the entire set of expressed circRNAs defined using established annotation algorithms. We describe step-by-step methods for annotating circRNAs using the CIRI2 and CIRCexplorer2 algorithms. In total, this overall approach can be used to enrich for circRNAs from any total RNA sample, thereby enabling one to quickly identify and validate circRNAs of interest for functional studies.

Stable structures or PABP1 loading protects cellular and viral RNAs against ISG20-mediated decay.

ISG20 is an IFN-induced 3'-5' RNA exonuclease that acts as a broad antiviral factor. At present, the features that expose RNA to ISG20 remain unclear, although recent studies have pointed to the modulatory role of epitranscriptomic modifications in the susceptibility of target RNAs to ISG20. These findings raise the question as to how cellular RNAs, on which these modifications are abundant, cope with ISG20. To obtain an unbiased perspective on this topic, we used RNA-seq and biochemical assays to identify elements that regulate the behavior of RNAs against ISG20. RNA-seq analyses not only indicate a general preservation of the cell transcriptome, but they also highlight a small, but detectable, decrease in the levels of histone mRNAs. Contrarily to all other cellular ones, histone mRNAs are non-polyadenylated and possess a short stem-loop at their 3' end, prompting us to examine the relationship between these features and ISG20 degradation. The results we have obtained indicate that poly(A)-binding protein loading on the RNA 3' tail provides a primal protection against ISG20, easily explaining the overall protection of cellular mRNAs observed by RNA-seq. Terminal stem-loop RNA structures have been associated with ISG20 protection before. Here, we re-examined this question and found that the balance between resistance and susceptibility to ISG20 depends on their thermodynamic stability. These results shed new light on the complex interplay that regulates the susceptibility of different classes of viruses against ISG20.

Exonuclease-enhanced prime editors.

Prime editing (PE) is a powerful gene-editing technique based on targeted gRNA-templated reverse transcription and integration of the de novo synthesized single-stranded DNA. To circumvent one of the main bottlenecks of the method, the competition of the reverse-transcribed 3' flap with the original 5' flap DNA, we generated an enhanced fluorescence-activated cell sorting reporter cell line to develop an exonuclease-enhanced PE strategy ('Exo-PE') composed of an improved PE complex and an aptamer-recruited DNA-exonuclease to remove the 5' original DNA flap. Exo-PE achieved better overall editing efficacy than the reference PE2 strategy for insertions ≥30 base pairs in several endogenous loci and cell lines while maintaining the high editing precision of PE2. By enabling the precise incorporation of larger insertions, Exo-PE complements the growing palette of different PE tools and spurs additional refinements of the PE machinery.

Exploring Co-occurring POLE Exonuclease and Non-exonuclease Domain Mutations and Their Impact on Tumor Mutagenicity.

POLE driver mutations in the exonuclease domain (ExoD driver) are prevalent in several cancers, including colorectal cancer and endometrial cancer, leading to dramatically ultra-high tumor mutation burden (TMB). To understand whether POLE mutations that are not classified as drivers (POLE Variant) contribute to mutagenesis, we assessed TMB in 447 POLE-mutated colorectal cancers, endometrial cancers, and ovarian cancers classified as TMB-high ≥10 mutations/Mb (mut/Mb) or TMB-low <10 mut/Mb. TMB was significantly highest in tumors with "POLE ExoD driver plus POLE Variant" (colorectal cancer and endometrial cancer, P < 0.001; ovarian cancer, P < 0.05). TMB increased with additional POLE variants (P < 0.001), but plateaued at 2, suggesting an association between the presence of these variants and TMB. Integrated analysis of AlphaFold2 POLE models and quantitative stability estimates predicted the impact of multiple POLE variants on POLE functionality. The prevalence of immunogenic neoepitopes was notably higher in the "POLE ExoD driver plus POLE Variant" tumors. Overall, this study reveals a novel correlation between POLE variants in POLE ExoD-driven tumors, and ultra-high TMB. Currently, only select pathogenic ExoD mutations with a reliable association with ultra-high TMB inform clinical practice. Thus, these findings are hypothesis-generating, require functional validation, and could potentially inform tumor classification, treatment responses, and clinical outcomes. SIGNIFICANCE: Somatic POLE ExoD driver mutations cause proofreading deficiency that induces high TMB. This study suggests a novel modifier role for POLE variants in POLE ExoD-driven tumors, associated with ultra-high TMB. These data, in addition to future functional studies, may inform tumor classification, therapeutic response, and patient outcomes.

N3-Methyluridine and 2'-O-Alkyl/2'-Fluoro-N3-methyluridine functionalized nucleic acids improve nuclease resistance while maintaining duplex geometry.

Herein, we report the synthesis of 2'-O-alkyl/2'-fluoro-N3-methyluridine (2'-O-alkyl/2'-F-m3U) phosphoramidites and their incorporation in DNA and RNA oligonucleotides. The duplex binding affinity and base discrimination studies showed that all 2'-O-alkyl/2'-F-m3U modifications significantly decreased the thermal stability and base-pairing discrimination ability. Serum stability study of dT20 with 2'-O-alkyl-m3U modification exhibited excellent nuclease resistance when incubated with 3'-exonucleases (SVPD) or 5'-exonucleases (PDE-II) as compared to m3U, 2'-F, 2'-OMe modified oligonucleotides. MD simulation studies with RNA tetradecamer duplexes illustrated that the m3U and 2'-O-methyl-m3U modifications reduce the duplex stabilities by disrupting the Watson-Crick hydrogen bonding and base-stacking interactions. Further molecular modelling investigations demonstrated that the 2'-O-propyl-m3U modification exhibits steric interactions with amino acid residues in the active site of 3'- and 5'-exonuclease, leading to enhanced stability. These combined data indicate that the 2'-modified-m3U nucleotides can be used as a promising tool to enhance the stability, silencing efficiency, and drug-like properties of antisense/siRNA-based therapeutics.

Retroelement decay by the exonuclease XRN1 is a viral mimicry dependency in cancer.

Viral mimicry describes the immune response induced by endogenous stimuli such as double-stranded RNA (dsRNA) from endogenous retroelements. Activation of viral mimicry has the potential to kill cancer cells or augment anti-tumor immune responses. Here, we systematically identify mechanisms of viral mimicry adaptation associated with cancer cell dependencies. Among the top hits is the RNA decay protein XRN1 as an essential gene for the survival of a subset of cancer cell lines. XRN1 dependency is mediated by mitochondrial antiviral signaling protein and protein kinase R activation and is associated with higher levels of cytosolic dsRNA, higher levels of a subset of Alus capable of forming dsRNA, and higher interferon-stimulated gene expression, indicating that cells die due to induction of viral mimicry. Furthermore, dsRNA-inducing drugs such as 5-aza-2'-deoxycytidine and palbociclib can generate a synthetic dependency on XRN1 in cells initially resistant to XRN1 knockout. These results indicate that XRN1 is a promising target for future cancer therapeutics.

Sm complex assembly and 5' cap trimethylation promote selective processing of snRNAs by the 3' exonuclease TOE1.

Competing exonucleases that promote 3' end maturation or degradation direct quality control of small non-coding RNAs, but how these enzymes distinguish normal from aberrant RNAs is poorly understood. The Pontocerebellar Hypoplasia 7 (PCH7)-associated 3' exonuclease TOE1 promotes maturation of canonical small nuclear RNAs (snRNAs). Here, we demonstrate that TOE1 achieves specificity toward canonical snRNAs through their Sm complex assembly and cap trimethylation, two features that distinguish snRNAs undergoing correct biogenesis from other small non-coding RNAs. Indeed, disruption of Sm complex assembly via snRNA mutations or protein depletions obstructs snRNA processing by TOE1, and in vitro snRNA processing by TOE1 is stimulated by a trimethylated cap. An unstable snRNA variant that normally fails to undergo maturation becomes fully processed by TOE1 when its degenerate Sm binding motif is converted into a canonical one. Our findings uncover the molecular basis for how TOE1 distinguishes snRNAs from other small non-coding RNAs and explain how TOE1 promotes maturation specifically of canonical snRNAs undergoing proper processing.

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.

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.