Journal: Analytical sciences : the international journal of the Japan Society for Analytical Chemistry

Article Title: Synthesis of circular double-stranded DNA having single-stranded recognition sequence as molecular-physical probe for nucleic acid hybridization detection based on atomic force microscopy imaging.

doi: 10.2116/analsci.25.993

Figure Lengend Snippet: Fig. 1 Agarose gel electrophoresis profiles for the polymerase reaction products after Hind III digestion. Lane: 1, λDNA; 2, m13L; 3, m13D; 4, M13mp18 RF DNA.

Article Snippet: For the probe DNA synthesis, M13mp18 single strand DNA (7249 base) was used for the template and replication was made by Takara Ex Taq® polymerase using dNTP mixture as the substrate.

Techniques: Agarose Gel Electrophoresis

Journal: Analytical sciences : the international journal of the Japan Society for Analytical Chemistry

Article Title: Synthesis of circular double-stranded DNA having single-stranded recognition sequence as molecular-physical probe for nucleic acid hybridization detection based on atomic force microscopy imaging.

doi: 10.2116/analsci.25.993

Figure Lengend Snippet: Fig. 4 Detection of individual m13L molecule adsorbed through hybridization at the target-DNA confined substrate surface. Results of a series of AFM imaging are shown for the bare Au(111) substrate (a), after the target-DNA/protein adlayer formation (b), and the same substrates treated with the m13L solution (c). For comparison, results for the hybridization experiment of the Au(111) with the target-DNA/protein interfacial structure using the m13L solution with 1000-times higher concentration (d), M13mp18 single strand DNA (e), and m13D (f) are also shown.

Article Snippet: For the probe DNA synthesis, M13mp18 single strand DNA (7249 base) was used for the template and replication was made by Takara Ex Taq® polymerase using dNTP mixture as the substrate.

Techniques: Hybridization, Imaging, Comparison, Concentration Assay

Journal: Analytical sciences : the international journal of the Japan Society for Analytical Chemistry

Article Title: Synthesis of circular double-stranded DNA having single-stranded recognition sequence as molecular-physical probe for nucleic acid hybridization detection based on atomic force microscopy imaging.

doi: 10.2116/analsci.25.993

Figure Lengend Snippet: Fig. 6 Comparison of the value of the image roughness determined for the Au substrate after various surface treatment experiments. Entries: 1, bare Au(111) substrate; 2, after the target-DNA/protein adlayer formation; 3, after treatment with M13mp18 single strand; 4, after treatment with M13D; 5, after treatment with M13L; 6, after treatment with M13L solution of 1000-times higher concentration; 7, control experiments of M13L hybridization-adsorption for the Au(111) substrate with scramble-DNA/protein interfacial structure; 8, control experiment using the m1-DNA/protein/Au substrate; 9, control experiment using the m3-DNA/protein/Au substrate.

Article Snippet: For the probe DNA synthesis, M13mp18 single strand DNA (7249 base) was used for the template and replication was made by Takara Ex Taq® polymerase using dNTP mixture as the substrate.

Techniques: Comparison, Concentration Assay, Control, Hybridization, Adsorption

Journal: eLife

Article Title: Bacterial exonuclease III expands its enzymatic activities on single-stranded DNA

doi: 10.7554/eLife.95648

Figure Lengend Snippet:

Article Snippet: Peptide, recombinant protein , SSB ( E. coli ) , Beyotime Biotech. Inc, China , P7415 , .

Techniques: Recombinant, Plasmid Preparation, Expressing, Chromatography, Protein Purification, Cloning, In Vitro, Amplification, Fluorescence, Sequencing, Software

Journal: iScience

Article Title: SART1 modulates poly-(ADP-ribose) chain accumulation and PARP1 chromatin localization.

doi: 10.1016/j.isci.2024.111252

Figure Lengend Snippet: Figure 2. SART1 interaction with PARP1 and cellular localization are modified by DNA damage (A) SART1-PARP1 interaction is induced by DNA damage as measured by immunoprecipitation in the presence or absence of DNA damage (6 Gy IR). (B) Immunofluorescence analysis of HeLa cells untreated or treated with 4 Gy of IR. Cells were fixed on coverslips 4 h after treatment and blotted with anti-SART1 antibody, DAPI staining was performed to visualize the nuclei of cells. DAPI and SART1 signal were merged to discriminate cytoplasmic and nuclear localization of SART1. One hundred cells were counted and assessed for nuclear or cytoplasmic SART1 signal. Results of the counts are shown as histograms on the right part of the figure. Scale bar, 1 mm. Two independent experiments are shown (Repeat 1 and 2).

Article Snippet: UWB1.289 and UWB1.289+BRCA1 cell lines were used for clonogenic assays to assess sensitivity to DNA damage by ionizing irradiation and to three PARP inhibitors (olaparib (TMO-T3015, TargetMOL), rucaparib (TMO-T4463, TargetMOL) and talazoparib (TMO-T6253, TargetMOL) in SART1-proficient or -deficient (silenced) cells.

Techniques: Immunoprecipitation, Staining

Journal: iScience

Article Title: SART1 modulates poly-(ADP-ribose) chain accumulation and PARP1 chromatin localization.

doi: 10.1016/j.isci.2024.111252

Figure Lengend Snippet: Figure 4. SART1 fragment cellular localization, influence on PARP1 chromatin binding and PARP1 interaction (A) SART1 sequence analysis and fragment design. Left panel, multiple sequence analysis (ClustalW). Residues in yellow are conserved and red boxes indicate RG/RGG-rich motifs. Central panel, schema of the full length SART1 (WT), the three fragments created, the location of RG/RGG-rich motifs, and the cluster PARylated serine residues (F3). Right panel, western blot showing expression of each fragment in 293T cells. (B) SART1 fragments’ cellular localization. UWB cells were transfected with vectors expressing SART1 F1, F2, F3 or empty plasmid. Tubulin, lamin-B1, and histone H2AX have been used as cytoplasm, nuclear, and chromatin references, respectively. (C) Chromatin fraction in UWB cells after expression of SART1 fragments and then untreated or treated with 4 Gy of IR. UWB cells were transfected with vector expressing SART1 F1, F2, F3 or empty plasmid, the day after cells were mock-treated or irradiated with 4 Gy of IR and collected after 4 h. Change in PARP1 chromatin level between treated and untreated sample was measured with ImageJ and normalized on chromatin loading control. (D) SART1 fragments-PARP1 interaction in presence or absence of DNA damage. 293T cells were transfected with plasmids expressing SART1 fragments or empty vector and cells collected untreated or 4 h after 8 Gy irradiation.

Article Snippet: UWB1.289 and UWB1.289+BRCA1 cell lines were used for clonogenic assays to assess sensitivity to DNA damage by ionizing irradiation and to three PARP inhibitors (olaparib (TMO-T3015, TargetMOL), rucaparib (TMO-T4463, TargetMOL) and talazoparib (TMO-T6253, TargetMOL) in SART1-proficient or -deficient (silenced) cells.

Techniques: Binding Assay, Sequencing, Western Blot, Expressing, Transfection, Plasmid Preparation, Irradiation, Control

Journal: iScience

Article Title: SART1 modulates poly-(ADP-ribose) chain accumulation and PARP1 chromatin localization.

doi: 10.1016/j.isci.2024.111252

Figure Lengend Snippet: Figure 5. SART1 fragments effect on chromatin localization and PARylation activity (A) Experimental design to analyze SART1 fragments effect on PARylation. UWB cells silenced for SART1 with a shRNA targeting the 30UTR of the sequence were seeded on day 1. The next day cells were transfected with a plasmid expressing either a SART1 fragment, a silencing-resistant full-length protein, or with an empty plasmid. On day 3, cells were reseeded. On day 4, they were mock-treated or irradiated with 4 Gy IR and collected at 4 h and 8 h after IR. (B) PAR chains in samples transfected with empty plasmid, full-length SART1, or SART1 fragment in presence or absence of DNA damage. PAR levels were evaluated using an anti-PAR antibody and their intensity was measured using ImageJ software. Data are shown as mean G SE of three independent experiments and analyzed with Student’s t test. *p < 0.05, **p < 0.01, ***p < 0.001. If not noted, the difference was not significant. (C) Mutagenesis of first RGG sequence in F1. (D and E) PAR chains in samples transfected with SART1 F1, mutated F1 (F1-ALA) and empty plasmid in presence or absence of DNA damage. UWB cells silenced for SART1 and transfected with SART1 F1, mutated F1 (F1-ALA) and empty plasmid were irradiated with 4 Gy of IR and collected at 4 h and 8 h after IR. PAR levels were evaluated using an anti-PAR antibody. Fragment expression has been evaluated with anti-FLAG antibody and anti-tubulin as loading control. An anti- gH2AX antibody has been used to visualize DNA damage and an anti-H2AX antibody has been used as loading control. Whole cell (D) and chromatin (E) extracts.

Article Snippet: UWB1.289 and UWB1.289+BRCA1 cell lines were used for clonogenic assays to assess sensitivity to DNA damage by ionizing irradiation and to three PARP inhibitors (olaparib (TMO-T3015, TargetMOL), rucaparib (TMO-T4463, TargetMOL) and talazoparib (TMO-T6253, TargetMOL) in SART1-proficient or -deficient (silenced) cells.

Techniques: Activity Assay, shRNA, Sequencing, Transfection, Plasmid Preparation, Expressing, Irradiation, Software, Mutagenesis, Control