Journal: bioRxiv

Article Title: Click-chemistry-aided quantitation and sequencing of oxidized guanines and apurinic sites uncovers their transcription-linked strand bias in human cells

doi: 10.1101/2024.07.21.604463

Figure Lengend Snippet: a Click-code-seq: clicking DNA-modification sites with a code sequence followed by sequencing; click-fluoro-quant: clicking DNA-modification sites with a fluorophore followed by rapid fluorescence-based quantitation. 8-oxoguanine (8-oxoG), abasic (AP) sites or phosphorylated breaks are converted to 3′-OH-ended breaks via the indicated enzymes (FPG, formamidopyrimidine DNA glycosylase; ENDOIV, endonuclease IV; T4 PNK, T4 polynucleotide kinase), or pre-existing 3′-OH-ended breaks are used. For subsequent ligation, alkyne-modified nucleotides 3’-( O -propargyl)-dNTPs (prop-dNTPs) are added to these 3′-OH sites. The alkyne is conjugated via copper click chemistry (CuAAC) with an azide-fluorophore in click-fluoro-quant or a sequencing adapter called Modified DNA Identifier Sequence (MoDIS, red oligonucleotide with b, a biotin group) in click-code-seq. Three directions of genome-wide DNA-modification analysis performed in this work are indicated. b Endogenous DNA modifications in HAP1-cell gDNA measured using click-fluoro-quant. c Click-fluoro-quant assessment of DNA modifications in HAP1 cells exposed to 50 mM KBrO3 for 30 min. d HPLC-MS/MS quantification of 8-oxoG in gDNA of HAP1 cells exposed to 50 mM KBrO3 for 30 min. e Click-fluoro-quant assessment of DNA modifications in BJ-5ta cells exposed to 10 J/cm 2 UVA. T control: cells always stayed in T=37 °C incubator. f Click-fluoro-quant assessment of induced AP sites in U2OS cells after 4 h exposure to irofulven. Markers: biological replicates (n=3), each of which included three drug concentrations and a vehicle control by which the plotted values are normalized. r and p: Pearson’s correlation coefficient with respective p-value. g Click-fluoro-quant assessment of repair and blocking strategies to remove background DNA modifications in HAP1 gDNA. Repair: nucleotide addition and ligation. Blocking: ddNTP insertion. Before both repair and blocking, 3’-OH groups were released from AP sites and phosphorylated breaks using ENDOIV. h Click-fluoro-quant assessment of sonication-induced artifactual DNA modifications with and without ddNTP blocking in HAP1 gDNA. In panels b-e and g-h , bar: mean of n=3 (click-fluoro-quant) or n=2 (HPLC-MS/MS) biological replicates (markers); p: p-value of the one-tailed (in c-e , HA: greater; in g-h , HA: less) paired t-test between the exposed and unexposed samples treated with the same set of enzymes ( c-e ) or between the arrow-connected samples ( g-h ); fc: fold change of a specific group of DNA modifications relative to the indicated condition.

Article Snippet: To convert DNA modifications to 3’-OH sites, a respective combination of 1 µL FPG (NEB, 8 U/µL), 1 µL ENDOIV (NEB, 10 U/µL) and 1 µL T4 PNK (NEB, 10 U/µL) was mixed with 4 µg gDNA (or a sample from previous blocking/repair step) in 1x NEBuffer 2 (NEB) in a final volume of 20 µL.

Techniques: Modification, Sequencing, Fluorescence, Quantitation Assay, Ligation, Genome Wide, Tandem Mass Spectroscopy, Control, Blocking Assay, Sonication, One-tailed Test