Journal: Nature Communications

Article Title: Quantifying replication fidelity of unnatural base pairs using nanopore sequencing

doi: 10.1038/s41467-026-76964-9

Figure Lengend Snippet: a Nucleobase structures of the standard DNA base pairs (G ≡ C, A = T). Dashed arrows indicate functional groups that participate in hydrogen bonding, drawn from the hydrogen-bond donor to the acceptor. b Nucleobase structures of two hydrogen-bonding UBPs (B ≡ S, P ≡ Z) studied in this work. The structure of S shown is S n (5-methyl-isocytosine). Putative replication error modes of each UBP are shown by red equilibrium arrows. During replication, keto-enol tautomerization (Taut.) of B can rearrange the H-bonding patterns (shown in red), leading to a Watson-Crick-Franklin mismatch with T, resulting in B → A and S → T DNA transitions. Similarly, deprotonation of the Z nucleobase can lead to a mismatch with G, resulting in P → G and Z → C DNA transitions. c Nucleobase structures of the non-hydrogen-bonding UBP, Ds:Diol-Px. Self-pairing of the Ds base can lead to UBP strand inversions (i.e, Ds→Diol-Px and Diol-Px→ Ds).

Article Snippet: Beads were washed twice using freshly prepared 70% ethanol and clean samples were ligated with ligation adapters (LA) in ligation buffer (LNB) using either Quick T4 DNA Ligase (NEB) or Salt T4 DNA Ligase (NEB).

Techniques: Functional Assay

Journal: Nature Communications

Article Title: Quantifying replication fidelity of unnatural base pairs using nanopore sequencing

doi: 10.1038/s41467-026-76964-9

Figure Lengend Snippet: a Segmented and normalized (norm.) nanopore signal traces for DNA sequences containing UBs (blue: B, S; orange: P, Z; red: Ds (D), Diol-Px (X)) compared with identical sequences containing only standard bases (black traces). The colored shade highlights the UB position in the sequence. The median signal is plotted ( n = 500 reads per class). b Overview of comparative basecalling model architecture. c Confusion matrices generated from benchmarking comparative models (BA1, ST1) to detect BS error modes in the 5′-GBC-3′ context (left: B vs A; n = 10,000 reads per class, right: S vs T; n = 10,000 reads per class). The quantified UB fraction B ( \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${f}_{B}$$\end{document} f B ; blue) and fraction S ( \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${f}_{S}$$\end{document} f S ; gray) when the same models are applied to demultiplexed synthetic DNA standards containing an AT (BA1:ST1) or BS (B1:S1) base pair across five independent sequencing experiments. The mean ± SD of experimental replicates ( n = 5) is plotted with replicate values overlaid as points. d Specificity (TNR; true negative rate) versus recall (TPR; true positive rate) for all comparative basecalling models generated in this work. Colors correspond to the UB each model is trained to detect (blue: B; light blue: S; orange: P; light orange: Z; red: Ds; light red: Px). Dashed lines show 98% thresholds for recall and specificity. Raw data are tabulated in the Source Data file.

Article Snippet: Beads were washed twice using freshly prepared 70% ethanol and clean samples were ligated with ligation adapters (LA) in ligation buffer (LNB) using either Quick T4 DNA Ligase (NEB) or Salt T4 DNA Ligase (NEB).

Techniques: Sequencing, Generated

Journal: Nature Communications

Article Title: Quantifying replication fidelity of unnatural base pairs using nanopore sequencing

doi: 10.1038/s41467-026-76964-9

Figure Lengend Snippet: a Synthetic template containing the PZ UBP is PCR amplified at pH 8.0 or pH 8.8. b UB fractions for P ( \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${f}_{P}$$\end{document} f P ; darker shades) and Z ( \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${f}_{Z}$$\end{document} f Z ; lighter shades), following PCR amplification with Takara Taq polymerase at pH 8.0 (orange) or pH 8.8 (gray). UB fractions measured for demultiplexed internal standards (PZ, GC) are shown in orange. c -Log 2 -transformed, normalized average UB fractions ( \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${f}_{\left\langle P,Z\right\rangle }$$\end{document} f P , Z ), plotted as mean ± SD of experimental replicates ( n = 3). Data series include reaction pH 8.8 (gray) and pH 8.0 (orange), at either standard dPTP concentration (0.2 mM; circles) or optimized (opt) dPTP concentration (0.5 mM; diamonds). WLS linear regression fits are shown using a dashed line. The average replication fidelity, \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\varphi }_{\left\langle P,Z\right\rangle }$$\end{document} φ P , Z (95% CI; n = 3) for each condition is displayed on the plot. d 8-letter PCR performed on 6-letter DNA (ATGCBP) ssDNA template. e Normalized species fractions for the various 4-letter (gray; \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${f}_{{GA}}$$\end{document} f G A , \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${f}_{{TC}}$$\end{document} f T C ), 5-letter (blue: \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${f}_{{GB}}$$\end{document} f G B , \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${f}_{{SC}}$$\end{document} f S C ; orange: \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${f}_{{PA}}$$\end{document} f P A , \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${f}_{{TZ}}$$\end{document} f T Z ), and 6-letter (purple: \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${f}_{{PB}}$$\end{document} f P B , \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${f}_{{SZ}}$$\end{document} f S Z ) DNA molecular species. Darker shades represent sense-strand (+) species, and lighter shades represent antisense-strand (-) species. Mean ± SD is plotted with experimental replicate values overlaid as points ( n = 3). Modeled species fractions (Supplementary Equation (41)) are plotted using dashed lines, with the fidelities used to constrain the model ( \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\varphi }_{\left\langle B,S\right\rangle }$$\end{document} φ B , S and \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\varphi }_{\left\langle P,Z\right\rangle }$$\end{document} φ P , Z ) included on the plot. f -Log 2 -transformed average fraction of 6-letter species ( \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${f}_{\left\langle {PB},{SZ}\right\rangle }$$\end{document} f P B , S Z ). Mean ± SD is plotted with experimental replicate values overlaid ( n = 3). The average joint replication fidelity for the 8-letter species, \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\varphi }_{\left\langle B,{S|P},Z\right\rangle }$$\end{document} φ B , S ∣ P , Z (95% CI; n = 3) is shown on the plot. Average replication fidelities for each individual UBP, \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\varphi }_{\left\langle B,S\right\rangle }$$\end{document} φ B , S and \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\varphi }_{\left\langle P,Z\right\rangle }$$\end{document} φ P , Z (95% CI; n = 3) are also shown (gray font). The raw data used to generate these plots are tabulated in the Source Data file.

Article Snippet: Beads were washed twice using freshly prepared 70% ethanol and clean samples were ligated with ligation adapters (LA) in ligation buffer (LNB) using either Quick T4 DNA Ligase (NEB) or Salt T4 DNA Ligase (NEB).

Techniques: Amplification, Transformation Assay, Concentration Assay

Journal: Nature Communications

Article Title: Quantifying replication fidelity of unnatural base pairs using nanopore sequencing

doi: 10.1038/s41467-026-76964-9

Figure Lengend Snippet: a Synthetic ssDNA template containing the Ds base is PCR amplified using AccuPrime Pfx polymerase, resulting in products that may contain DNA transversions or UBP inversions. b Schematic of the DsPx (Px = Diol-Px) PCR model (Supplementary Equation (46)), with arrows representing replication events drawn from the template to the inserted base. Included are UB → N transversion errors (gray), UBP inversion errors (red), and correct UB incorporations (black). Epsilon ( ɛ ε ) is the error rate and mu ( μ μ ) is the correct UB incorporation rate, with the superscript sign denoting the template strand ( + for sense, - for antisense). Normalized fraction of Ds ( \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${f}_{{Ds}}$$\end{document} f D s ), Px ( \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${f}_{{Px}}$$\end{document} f P x ), and N ( \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${f}_{N}$$\end{document} f N ; N = A,T,G,C) located on the sense (+, lighter shades) or antisense (-, darker shades) strands are shown following PCR amplification of ssDNA templates c D1 or d D2. Retained UBs (Sense: Ds; Antisense: Px) are shown in blue, inverted UBs (Sense: Px; Antisense: Ds) are shown in red, and DNA transversions (N; either strand) are shown in gray. On each plot, the mean value ± SD is plotted with experimental replicate values overlaid as points ( n = 3). e -Log 2 -linear transform of the fraction of UB-containing (inversion-agnostic) species ( \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${f}_{{Ds}}$$\end{document} f D s + \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${f}_{{Px}}$$\end{document} f P x ), averaged across strands ( \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${f}_{\left\langle {Ds}+{Px},{Px}+{Ds}\right\rangle }$$\end{document} f D s + P x , P x + D s ) for templates D1 (blue) and D2 (black). The mean value ± SD of experimental replicates is plotted ( n = 3). The DNA transversion fidelities ( \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\varphi }_{\left\langle {Ds},{Px}\right\rangle }^{{{\rm{trnv}}}}$$\end{document} φ D s , P x trnv ) for PCR amplification of templates D1 and D2 are shown on the plot (95% CI). f Fraction Ds ( \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${f}_{{Ds}}$$\end{document} f D s ) relative to the fraction of the UB-containing species ( \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${f}_{{Ds}}$$\end{document} f D s + \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${f}_{{Px}}$$\end{document} f P x ) on sense (+, black) and antisense (-, red) strands for template D2. The mean value ± SD is plotted with experimental replicate values overlaid as points ( n = 3). The dashed lines represent the nonlinear fit of a reduced ( \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\varepsilon }_{{YY}}^{+}={\varepsilon }_{{XX}}^{-}=0$$\end{document} ε Y Y + = ε X X − = 0 ) state-transition model for the effective inversion rate ( \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\varepsilon }_{{inv}}={\varepsilon }_{{XX}}^{+}={\varepsilon }_{{YY}}^{-}$$\end{document} ε i n v = ε X X + = ε Y Y − ) on the sense (black) and antisense (red) strands. The root-mean-square error (RMSE) of the nonlinear model fit is shown. Plotted values are the mean of experimental triplicates ± standard deviation ( n = 3). The raw data used to generate each plot are tabulated in the Source Data file.

Article Snippet: Beads were washed twice using freshly prepared 70% ethanol and clean samples were ligated with ligation adapters (LA) in ligation buffer (LNB) using either Quick T4 DNA Ligase (NEB) or Salt T4 DNA Ligase (NEB).

Techniques: Amplification, Standard Deviation

Journal: Cell Death & Disease

Article Title: ACSL3 inhibits ferroptosis in gastric cancer through the activation of unfolded protein response following endoplasmic reticulum stress

doi: 10.1038/s41419-026-09068-3

Figure Lengend Snippet: A Analysis of the TCGA dataset showed that ACSL3 was significantly up-regulated in GC tissues. B Compared with normal gastric mucosal tissues, ACSL3 was significantly highly expressed in GC tissues. C RT-qPCR results showed that ACSL3 mRNA expression in GC tissues was significantly higher than that in normal gastric mucosal tissues. D Hematoxylin and eosin stains of GC tissue and normal gastric tissue. E The expression of ACSL3 in different T-stage GC tissues and normal gastric tissues was detected by immunohistochemical staining. F Evaluation of ACSL3 expression in early GC tissues and progressive gastric tissues using the H-score method. G CT images of GC patients receiving the same cycle of L-OHP plus S-1 (Tegafur/ Gimeracil/ Oteracil Potassium) regimen (SOX regimen) showed that patients with high ACSL3 expression are less sensitive to chemotherapy. Statistical analysis was performed using the chi-square test (χ² = 7.38, p = 0.007). H –J Aplan-Meier Plotter analysis showed that high expression of ACSL3 led to poor prognosis in GC patients. K Overall survival follow-up of GC patients showed that high expression of ACSL3 leads to poor prognosis in GC patients. Data are represented as the mean ± SD. Statistical analysis was performed using Student’s t-test and survival analysis employed the Log-rank (Mantel-Cox) test, * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Tissues were then deparaffinized, antigenically repaired and blocked, and incubated overnight at 4 °C with the specific primary antibody, and the secondary antibody incubated at room temperature and then stained using diaminobenzidine, while the nuclei of the cells were stained with hematoxylin (M&R HRP/DAB Detection IHC Kit, HC301-01, Vazyme).

Techniques: Analysis, Quantitative RT-PCR, Expressing, Tissue, Immunohistochemical staining, Staining, Chi-Square