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    Double Helix dna double helix
    (A) Diagram and sequence of CDKN2A region targeted by additional gRNAs. Due to the display of the reverse complement sequence of Fig. 3 , CpGs are numbered differently (black) but Fig. 3 numbering system is shown below in red. gRNA sequences are shown in green, where the entire green sequence represents the S. pyogenes gRNA and the addition 5’ nucleotide in black represents the additional nucleotide needed for S. aureus gRNAs. S. aureus PAM site is shown in blue, with the first 3 nucleotides (5’ to 3’) represent the NGG PAM of the S. pyogenes gRNA. (B) Each horizontal row depicts a heatmap of average <t>DNA</t> methylation at each numbered <t>CpG</t> over 10-20 (except SP-gRNA4, 4 clones) individual strands of DNA (bisulfite-converted clones) where light blue represents 0% methylation and dark red represents 100% methylation. The CpGs within the binding site of the labeled gRNA are labeled and enclosed in dashed lines. gRNAs1-3 interrogate DNA methylation interference of 5’ proximal CpGs and gRNA4 interrogates that of 3’ proximal CpGs. Lowly methylated strands of DNA (poor M.SssI methylation) and strands with unaffected binding sites (unbound by dCas9) were excluded from the analysis because efficacy was not under evaluation. (C-D) Data from (B) transformed into a percent methylation as a function of CpG distance in base pairs from the 5’ (C) or 3’ (D) end of the gRNA sequence (including PAM) and S. aureus (grey) or S. pyogenes (pink) across gRNAs 1-3 (C) or gRNA4 (D).
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    Double Helix dna double helix structure
    The Stern-Volmer plots of the fluorescence quenching of pyrene derivatives by C-myc <t>DNA</t> at 298 K and 310 K (the concentrations of pyrene derivatives are 1 μmol/L, pH = 7.4). ( A ) 1-OHP and ( B ) <t>1-PBO.</t>
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    Double Helix b form dna double helix
    <t>LSS-11</t> interacts with <t>DNA</t> and increases its stability ( A ) UV-Vis spectra and ( B ) fluorescent emission spectra of LSS-11 (50 μM) with increasing concentrations of CT DNA (0 to 200 μM). ( C ) Scatchard plot of the fluorescent intensity of LSS-11 at 570 nm with increasing concentrations of CT DNA [DNA], F stands for fluorescent intensity and F0 refers to fluorescent intensity without CT DNA. ( D ) Increased Tm of a 63 bp DNA fragment in the presence of LSS-11 at indicated concentrations.
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    Double Helix dna damage
    Top view and all structure view of the molecular docking top-ranked poses of the AO derivatives intercalated in the d(ACGTACGT) 2 sequence. ( a ) 125 I-C 3 ; ( b ) 125 I-C 5 ; ( c ) 125 I-C 8 ; ( d ) <t>99m</t> Tc-C 3 ; ( e ) 99m Tc-C 5 and ( f ) 99m Tc-C 8 . Distance between the 125 I or 99m Tc atom relative to the <t>DNA</t> helical axis is displayed in Å.
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    Image Search Results


    (A) Diagram and sequence of CDKN2A region targeted by additional gRNAs. Due to the display of the reverse complement sequence of Fig. 3 , CpGs are numbered differently (black) but Fig. 3 numbering system is shown below in red. gRNA sequences are shown in green, where the entire green sequence represents the S. pyogenes gRNA and the addition 5’ nucleotide in black represents the additional nucleotide needed for S. aureus gRNAs. S. aureus PAM site is shown in blue, with the first 3 nucleotides (5’ to 3’) represent the NGG PAM of the S. pyogenes gRNA. (B) Each horizontal row depicts a heatmap of average DNA methylation at each numbered CpG over 10-20 (except SP-gRNA4, 4 clones) individual strands of DNA (bisulfite-converted clones) where light blue represents 0% methylation and dark red represents 100% methylation. The CpGs within the binding site of the labeled gRNA are labeled and enclosed in dashed lines. gRNAs1-3 interrogate DNA methylation interference of 5’ proximal CpGs and gRNA4 interrogates that of 3’ proximal CpGs. Lowly methylated strands of DNA (poor M.SssI methylation) and strands with unaffected binding sites (unbound by dCas9) were excluded from the analysis because efficacy was not under evaluation. (C-D) Data from (B) transformed into a percent methylation as a function of CpG distance in base pairs from the 5’ (C) or 3’ (D) end of the gRNA sequence (including PAM) and S. aureus (grey) or S. pyogenes (pink) across gRNAs 1-3 (C) or gRNA4 (D).

    Journal: bioRxiv

    Article Title: Unraveling the functional role of DNA methylation using targeted DNA demethylation by steric blockage of DNA methyltransferase with CRISPR/dCas9

    doi: 10.1101/2020.03.28.012518

    Figure Lengend Snippet: (A) Diagram and sequence of CDKN2A region targeted by additional gRNAs. Due to the display of the reverse complement sequence of Fig. 3 , CpGs are numbered differently (black) but Fig. 3 numbering system is shown below in red. gRNA sequences are shown in green, where the entire green sequence represents the S. pyogenes gRNA and the addition 5’ nucleotide in black represents the additional nucleotide needed for S. aureus gRNAs. S. aureus PAM site is shown in blue, with the first 3 nucleotides (5’ to 3’) represent the NGG PAM of the S. pyogenes gRNA. (B) Each horizontal row depicts a heatmap of average DNA methylation at each numbered CpG over 10-20 (except SP-gRNA4, 4 clones) individual strands of DNA (bisulfite-converted clones) where light blue represents 0% methylation and dark red represents 100% methylation. The CpGs within the binding site of the labeled gRNA are labeled and enclosed in dashed lines. gRNAs1-3 interrogate DNA methylation interference of 5’ proximal CpGs and gRNA4 interrogates that of 3’ proximal CpGs. Lowly methylated strands of DNA (poor M.SssI methylation) and strands with unaffected binding sites (unbound by dCas9) were excluded from the analysis because efficacy was not under evaluation. (C-D) Data from (B) transformed into a percent methylation as a function of CpG distance in base pairs from the 5’ (C) or 3’ (D) end of the gRNA sequence (including PAM) and S. aureus (grey) or S. pyogenes (pink) across gRNAs 1-3 (C) or gRNA4 (D).

    Article Snippet: Given that bound dCas9 envelopes nearly the entire DNA double helix [ ], we predicted that both CpG sites would be equally protected.

    Techniques: Sequencing, DNA Methylation Assay, Methylation, Binding Assay, Labeling, Transformation Assay

    (A) Bisulfite-cloning and sanger sequencing analysis of the Il33-002 promoter in NIH-3T3 cells treated with 1 µg/mL poly(I:C) or water control for 8 or 24 hours. Each horizontal row is one strand of DNA. Numbers indicate the CpG in the promoter. Red squares indicate methylated CpGs, blue squares indicate unmethylated CpGs, and white squares indicate a lack of data due to sequencing failure. (B) Il33-002 expression in 50nM TSA or vehicle (DMSO) treated NIH-3T3 cell lines stably expressing gRNAscr, gRNA1, gRNA2, or gRNA3 under high-puromycin conditions in combination with dCas9, followed by dCas9 removal by Cre recombinase as assayed by qRT-PCR and normalized to Actb expression (n=3). (C) Il33-002 expression in 100 ng/mL LPS or vehicle (PBS) treated NIH-3T3 cell lines stably expressing gRNAscr or gRNA3 under high-puromycin conditions in combination with dCas9, followed by dCas9 removal by Cre recombinase, as assayed by qRT-PCR and normalized to Actb expression, either 1 or 3 hours after treatment and displayed relative to expression measured at time=0 (n=3). * indicates statistically significant difference of p

    Journal: bioRxiv

    Article Title: Unraveling the functional role of DNA methylation using targeted DNA demethylation by steric blockage of DNA methyltransferase with CRISPR/dCas9

    doi: 10.1101/2020.03.28.012518

    Figure Lengend Snippet: (A) Bisulfite-cloning and sanger sequencing analysis of the Il33-002 promoter in NIH-3T3 cells treated with 1 µg/mL poly(I:C) or water control for 8 or 24 hours. Each horizontal row is one strand of DNA. Numbers indicate the CpG in the promoter. Red squares indicate methylated CpGs, blue squares indicate unmethylated CpGs, and white squares indicate a lack of data due to sequencing failure. (B) Il33-002 expression in 50nM TSA or vehicle (DMSO) treated NIH-3T3 cell lines stably expressing gRNAscr, gRNA1, gRNA2, or gRNA3 under high-puromycin conditions in combination with dCas9, followed by dCas9 removal by Cre recombinase as assayed by qRT-PCR and normalized to Actb expression (n=3). (C) Il33-002 expression in 100 ng/mL LPS or vehicle (PBS) treated NIH-3T3 cell lines stably expressing gRNAscr or gRNA3 under high-puromycin conditions in combination with dCas9, followed by dCas9 removal by Cre recombinase, as assayed by qRT-PCR and normalized to Actb expression, either 1 or 3 hours after treatment and displayed relative to expression measured at time=0 (n=3). * indicates statistically significant difference of p

    Article Snippet: Given that bound dCas9 envelopes nearly the entire DNA double helix [ ], we predicted that both CpG sites would be equally protected.

    Techniques: Clone Assay, Sequencing, Methylation, Expressing, Stable Transfection, Quantitative RT-PCR

    The footprint of dCas9 (A) Genome browser diagram of the CDKN2A (p16) promoter region, which was used for the methylation assay, showing transcription start site (TSS, marked by black arrow), gRNA position overlapping CpG 17, and surrounding CpGs. Below, DNA sequence is shown in black, gRNA sequence in blue, and PAM site in red, with CpGs bolded, underlined, and numbered according to the figures that follow. (B-E) Methylation of individual strands of the CDKN2A promoter plasmid following standard methylation (B,D) or methylation preceded by incubation with dCas9 and p16 gRNA (C,E). Red squares indicate methylated CpGs and blue squares indicate unmethylated CpGs; white squares indicate no data. Figures (B) and (C) represent the forward strand whereas (D) and (E) represent the reverse strand. Figures generated by BISMA software ( http://services.ibc.uni-stuttgart.de/BDPC/BISMA/ ). Regions below 80% methylation were filtered out as strands that were not effectively methylated by M.SssI.

    Journal: bioRxiv

    Article Title: Unraveling the functional role of DNA methylation using targeted DNA demethylation by steric blockage of DNA methyltransferase with CRISPR/dCas9

    doi: 10.1101/2020.03.28.012518

    Figure Lengend Snippet: The footprint of dCas9 (A) Genome browser diagram of the CDKN2A (p16) promoter region, which was used for the methylation assay, showing transcription start site (TSS, marked by black arrow), gRNA position overlapping CpG 17, and surrounding CpGs. Below, DNA sequence is shown in black, gRNA sequence in blue, and PAM site in red, with CpGs bolded, underlined, and numbered according to the figures that follow. (B-E) Methylation of individual strands of the CDKN2A promoter plasmid following standard methylation (B,D) or methylation preceded by incubation with dCas9 and p16 gRNA (C,E). Red squares indicate methylated CpGs and blue squares indicate unmethylated CpGs; white squares indicate no data. Figures (B) and (C) represent the forward strand whereas (D) and (E) represent the reverse strand. Figures generated by BISMA software ( http://services.ibc.uni-stuttgart.de/BDPC/BISMA/ ). Regions below 80% methylation were filtered out as strands that were not effectively methylated by M.SssI.

    Article Snippet: Given that bound dCas9 envelopes nearly the entire DNA double helix [ ], we predicted that both CpG sites would be equally protected.

    Techniques: Methylation, Sequencing, Plasmid Preparation, Incubation, Generated, Software

    (A) Genome browser view of the murine Tnf locus;(Top) each CG location marked by a blue dash and numbered below, TSS indicated by a black arrow., and the location of gRNA Tnf2 is labeled with a red line and marked accordingly; (Bottom) Two known distal enhancers of Tnf expression indicated with purple boxes, named and marked with distances to Tnf TSS [ 67 ]. (B) Table demonstrating the average methylation of Tnf CpGs numbered in (A) as measured by bisulfite-pyrosequencing a function of six candidate Tnf -targeting gRNAs or gRNAscr control in NIH-3T3 cells also stably expressing dCas9. CpGs within the gRNA binding site are indicated below the gRNA number and their methylation status is highlighted in yellow in the corresponding gRNAs. (C) DNA methylation levels assessed by bisulfite-pyrosequencing of NIH-3T3 cells stably expressing dCas9 and either gRNA Tnf2 (pink) or gRNAscr (grey) (n=3). (D) Tnf expression in NIH-3T3 cell lines subcloned from those in Fig. 6H stably expressing gRNAscr (grey) or gRNA Tnf2 (pink) under high-puromycin conditions in combination with dCas9, followed by dCas9 removal by Cre recombinase as assayed by RT-qPCR and normalized to Actb expression (n=8-9). * indicates statistically significant difference of p

    Journal: bioRxiv

    Article Title: Unraveling the functional role of DNA methylation using targeted DNA demethylation by steric blockage of DNA methyltransferase with CRISPR/dCas9

    doi: 10.1101/2020.03.28.012518

    Figure Lengend Snippet: (A) Genome browser view of the murine Tnf locus;(Top) each CG location marked by a blue dash and numbered below, TSS indicated by a black arrow., and the location of gRNA Tnf2 is labeled with a red line and marked accordingly; (Bottom) Two known distal enhancers of Tnf expression indicated with purple boxes, named and marked with distances to Tnf TSS [ 67 ]. (B) Table demonstrating the average methylation of Tnf CpGs numbered in (A) as measured by bisulfite-pyrosequencing a function of six candidate Tnf -targeting gRNAs or gRNAscr control in NIH-3T3 cells also stably expressing dCas9. CpGs within the gRNA binding site are indicated below the gRNA number and their methylation status is highlighted in yellow in the corresponding gRNAs. (C) DNA methylation levels assessed by bisulfite-pyrosequencing of NIH-3T3 cells stably expressing dCas9 and either gRNA Tnf2 (pink) or gRNAscr (grey) (n=3). (D) Tnf expression in NIH-3T3 cell lines subcloned from those in Fig. 6H stably expressing gRNAscr (grey) or gRNA Tnf2 (pink) under high-puromycin conditions in combination with dCas9, followed by dCas9 removal by Cre recombinase as assayed by RT-qPCR and normalized to Actb expression (n=8-9). * indicates statistically significant difference of p

    Article Snippet: Given that bound dCas9 envelopes nearly the entire DNA double helix [ ], we predicted that both CpG sites would be equally protected.

    Techniques: Labeling, Expressing, Methylation, Stable Transfection, Binding Assay, DNA Methylation Assay, Quantitative RT-PCR

    dCas9 causes demethylation in mammalian cells (A-C) Methylation levels assayed by bisulfite-pyrosequencing at CpGs 1, 2, 3, 5, 9, 10, and 11 of NIH-3T3 cells stably expressing dCas9 and gRNA1 (A, blue), gRNA2 (B, purple), gRNA3 (C, pink) or scrambled gRNA (A-C, grey; identical in all). Data is displayed as mean +/- SEM. (D). Cells from (C) were passaged for an additional 30 days and methylation percentage was assayed as previously (n=3, mean +/- SEM). (E) Cells from (C) were subjected to clonal isolation and expansion. Grey circles represent methylation levels of clones containing dCas9 and scrambled gRNA and various red circles represent methylation levels of randomly selected clones stably expressing dCas9 and gRNA 3 (n=10 per condition). (F) Average DNA methylation at CpGs 9-11, assayed by bisulfite-pyrosequencing, as a function of increasing the selection antibiotic puromycin (lentivirus is expressing puromycin resistance gene) concentration in cell lines (pools) stably expressing dCas9 and gRNA3 (n=1 per puromycin concentration) fitted with a line of best fit. (G). DNA methylation at CpGs 1, 2, 3, 9, 10, and 11 in NIH-3T3 cells stably expressing dCas9 and gRNA3 (pink) or control gRNAscr (grey) and treated with 10 µg/mL puromycin until no antibiotic-associated cell death could be observed and surviving cells were of sufficient quantity for DNA extraction and other procedures (approximately 2 weeks). * indicates statistically significant difference of p

    Journal: bioRxiv

    Article Title: Unraveling the functional role of DNA methylation using targeted DNA demethylation by steric blockage of DNA methyltransferase with CRISPR/dCas9

    doi: 10.1101/2020.03.28.012518

    Figure Lengend Snippet: dCas9 causes demethylation in mammalian cells (A-C) Methylation levels assayed by bisulfite-pyrosequencing at CpGs 1, 2, 3, 5, 9, 10, and 11 of NIH-3T3 cells stably expressing dCas9 and gRNA1 (A, blue), gRNA2 (B, purple), gRNA3 (C, pink) or scrambled gRNA (A-C, grey; identical in all). Data is displayed as mean +/- SEM. (D). Cells from (C) were passaged for an additional 30 days and methylation percentage was assayed as previously (n=3, mean +/- SEM). (E) Cells from (C) were subjected to clonal isolation and expansion. Grey circles represent methylation levels of clones containing dCas9 and scrambled gRNA and various red circles represent methylation levels of randomly selected clones stably expressing dCas9 and gRNA 3 (n=10 per condition). (F) Average DNA methylation at CpGs 9-11, assayed by bisulfite-pyrosequencing, as a function of increasing the selection antibiotic puromycin (lentivirus is expressing puromycin resistance gene) concentration in cell lines (pools) stably expressing dCas9 and gRNA3 (n=1 per puromycin concentration) fitted with a line of best fit. (G). DNA methylation at CpGs 1, 2, 3, 9, 10, and 11 in NIH-3T3 cells stably expressing dCas9 and gRNA3 (pink) or control gRNAscr (grey) and treated with 10 µg/mL puromycin until no antibiotic-associated cell death could be observed and surviving cells were of sufficient quantity for DNA extraction and other procedures (approximately 2 weeks). * indicates statistically significant difference of p

    Article Snippet: Given that bound dCas9 envelopes nearly the entire DNA double helix [ ], we predicted that both CpG sites would be equally protected.

    Techniques: Methylation, Stable Transfection, Expressing, Isolation, Clone Assay, DNA Methylation Assay, Selection, Concentration Assay, DNA Extraction

    Targeting the Il33 promoter with dCas9-TET. (A) Schematic of the murine Il33 genomic locus depicting the two transcriptional isoforms with a highlighted region of an 800bp region of the Il33-002 promoter and the locations of the 11 CpGs as well as 4 gRNAs targeting specific CpGs. The 11 CpGs are numbered sequentially in the 5’ to 3’ direction. The promoter-targeting gRNAs used in these experiments are shown relative to the CpGs and are approximately to scale such that CpGs 1, 2, and 3 are targeted by gRNA1, CpG 5 by gRNA 2, and gRNA 3 targets CpGs 9, 10, 11 – which overlap the transcription start site (TSS), marked by a black arrow. The orientation of the gRNAs is indicated by an arrow, where an arrow pointing to the left indicates a gRNA that binds the plus strand. (B) Percent of DNA methylation assayed by bisulfite-pyrosequencing at the three transcription start site (TSS) CpGs (labeled 9-11) following treatment of NIH-3T3 cells with indicated concentrations of 5-aza-2’-deoxycytidine or water control. (C) Expression of Il33-002 quantified by RT-qPCRand normalized to beta Actin ( Actb ) expression following treatment of NIH-3T3 cells with indicated concentrations of 5-aza-2’-deoxycytidine or water control. (D) Expression of predicted (Transfac) and experimentally validated (Qiagen, ENCODE, Gene Transcription Regulation Database) Il33-002 transcription factors quantified by RT-qPCR and normalized to Actb expression following treatment of NIH-3T3 cells with indicated concentrations of 5-aza-2’-deoxycytidine or water control. (E-G) Percent of DNA methylation assayed by bisulfite-pyrosequencing at 7 targeted CpGs in the Il33-002 promoter following transduction with lentiviruses and antibiotic selection of virally infected cells (gRNAs) or selection by flow cytometry (BFP; dCas9 constructs) of NIH-3T3 cells with dCas9-Tet/dCas9-deadTET (BFP) and gRNA1 (E), gRNA2 (F), or gRNA3 (G) compared to gRNAscr (light and dark grey, data identical in E-G and shown for comparison) (n=4-8). (H-I) Expression of Il33-002 (H) and Il33-001 (I) quantified by RT-qPCR and normalized to Actb expression in NIH-3T3 stably expressing one of 4 gRNAs and dCas9-TET or dCas9-deadTET. n=3 for all data panels, except E-G as indicated, and all data shown as (mean+/-SEM). (J) Relative light units normalized to protein quantity in transfected HEK293 cells. Cells were transiently transfected with methylated or unmethylated SV40-luciferase vector along with mammalian TET2 expression plasmid or empty vector (pcDNA3.1) control. * indicates statistically significant difference of p

    Journal: bioRxiv

    Article Title: Unraveling the functional role of DNA methylation using targeted DNA demethylation by steric blockage of DNA methyltransferase with CRISPR/dCas9

    doi: 10.1101/2020.03.28.012518

    Figure Lengend Snippet: Targeting the Il33 promoter with dCas9-TET. (A) Schematic of the murine Il33 genomic locus depicting the two transcriptional isoforms with a highlighted region of an 800bp region of the Il33-002 promoter and the locations of the 11 CpGs as well as 4 gRNAs targeting specific CpGs. The 11 CpGs are numbered sequentially in the 5’ to 3’ direction. The promoter-targeting gRNAs used in these experiments are shown relative to the CpGs and are approximately to scale such that CpGs 1, 2, and 3 are targeted by gRNA1, CpG 5 by gRNA 2, and gRNA 3 targets CpGs 9, 10, 11 – which overlap the transcription start site (TSS), marked by a black arrow. The orientation of the gRNAs is indicated by an arrow, where an arrow pointing to the left indicates a gRNA that binds the plus strand. (B) Percent of DNA methylation assayed by bisulfite-pyrosequencing at the three transcription start site (TSS) CpGs (labeled 9-11) following treatment of NIH-3T3 cells with indicated concentrations of 5-aza-2’-deoxycytidine or water control. (C) Expression of Il33-002 quantified by RT-qPCRand normalized to beta Actin ( Actb ) expression following treatment of NIH-3T3 cells with indicated concentrations of 5-aza-2’-deoxycytidine or water control. (D) Expression of predicted (Transfac) and experimentally validated (Qiagen, ENCODE, Gene Transcription Regulation Database) Il33-002 transcription factors quantified by RT-qPCR and normalized to Actb expression following treatment of NIH-3T3 cells with indicated concentrations of 5-aza-2’-deoxycytidine or water control. (E-G) Percent of DNA methylation assayed by bisulfite-pyrosequencing at 7 targeted CpGs in the Il33-002 promoter following transduction with lentiviruses and antibiotic selection of virally infected cells (gRNAs) or selection by flow cytometry (BFP; dCas9 constructs) of NIH-3T3 cells with dCas9-Tet/dCas9-deadTET (BFP) and gRNA1 (E), gRNA2 (F), or gRNA3 (G) compared to gRNAscr (light and dark grey, data identical in E-G and shown for comparison) (n=4-8). (H-I) Expression of Il33-002 (H) and Il33-001 (I) quantified by RT-qPCR and normalized to Actb expression in NIH-3T3 stably expressing one of 4 gRNAs and dCas9-TET or dCas9-deadTET. n=3 for all data panels, except E-G as indicated, and all data shown as (mean+/-SEM). (J) Relative light units normalized to protein quantity in transfected HEK293 cells. Cells were transiently transfected with methylated or unmethylated SV40-luciferase vector along with mammalian TET2 expression plasmid or empty vector (pcDNA3.1) control. * indicates statistically significant difference of p

    Article Snippet: Given that bound dCas9 envelopes nearly the entire DNA double helix [ ], we predicted that both CpG sites would be equally protected.

    Techniques: DNA Methylation Assay, Labeling, Expressing, Quantitative RT-PCR, Transduction, Selection, Infection, Flow Cytometry, Construct, Stable Transfection, Transfection, Methylation, Luciferase, Plasmid Preparation

    The effect of targeted promoter DNA demethylation on Il33 expression (A) Diagram illustrating the principle of site-specific demethylation with dCas9 removal in order to facilitate transcription factor binding to the newly demethylated region. First, DNA is endogenously methylated by DNMT1 with every round of replication and RNA-polII is not recruited to the promoter. After the introduction of dCas9 and a promoter-targeting gRNA, DNMT1 is physically occluded from the locus and nascent strands of DNA are unmethylated, facilitating passive demethylation of the bound region. However, RNA-polII is also physically occluded by dCas9. If dCas9 is successfully removed, the unmethylated DNA no longer serves as a substrate for DNMT1 and continues to remain unmethylated and RNA-polII may now be recruited. (B) Methylation of CpGs 9, 10, and 11 which had been previously demethylated by high-puromycin gRNA3:dCas9 in NIH-3T3 cells, after 75 days of passaging following the lentiviral transduction of Cre recombinase (pink) or empty-vector control (red). (C) Il33-002 expression in NIH-3T3 cell lines stably expressing gRNAscr (grey) or gRNA1 (blue), gRNA2 (purple), or gRNA3 (pink) under high-puromycin conditions in combination with dCas9, followed by dCas9 removal by Cre recombinase as assayed by RT-qPCR and normalized to Actb expression (n=3). (D-E) Il33-002 expression (D) or Il33-001 expression (E) in NIH-3T3 cells from (C) following treatment wither water control or 1 µM 5-aza-2’-deoxycytidine, measured by RT-qPCR and normalized to Actb expression (n=4-5). (F) Il33-002 expression measured by RT-qPCR and normalized to Actb expression, in dCas9:gRNAscr (grey) or dCas9:gRNA3 (pink) NIH-3T3 cells following Cre recombinase treatment and then treated with poly(I:C) (1 µg/mL) or water control for 4 or 8 hours. (G) DNA methylation assayed by bisulfite-pyrosequencing in NIH-3T3 cells expressing dCas9, gRNAscr, and Cre treated with 1 µg/mL poly(I:C) or water control for 8 hrs and 24 hrs (n=3). (H) Maximal Il33-002 induction (left y-axis, pink bars; data in log2 scale but axis numbering is not transformed) and maximal promoter demethylation (right y-axis, calculated as percent unmethylated divided by control methylation) under different treatments (x-axis: dCas9, 5-aza-2’-deoxycytidine, dCas9-VP64, dCas9-TET, and dCas9-deadTET). Where relevant, data for maximally inducing/demethylating gRNA is shown. * indicates statistically significant difference of p

    Journal: bioRxiv

    Article Title: Unraveling the functional role of DNA methylation using targeted DNA demethylation by steric blockage of DNA methyltransferase with CRISPR/dCas9

    doi: 10.1101/2020.03.28.012518

    Figure Lengend Snippet: The effect of targeted promoter DNA demethylation on Il33 expression (A) Diagram illustrating the principle of site-specific demethylation with dCas9 removal in order to facilitate transcription factor binding to the newly demethylated region. First, DNA is endogenously methylated by DNMT1 with every round of replication and RNA-polII is not recruited to the promoter. After the introduction of dCas9 and a promoter-targeting gRNA, DNMT1 is physically occluded from the locus and nascent strands of DNA are unmethylated, facilitating passive demethylation of the bound region. However, RNA-polII is also physically occluded by dCas9. If dCas9 is successfully removed, the unmethylated DNA no longer serves as a substrate for DNMT1 and continues to remain unmethylated and RNA-polII may now be recruited. (B) Methylation of CpGs 9, 10, and 11 which had been previously demethylated by high-puromycin gRNA3:dCas9 in NIH-3T3 cells, after 75 days of passaging following the lentiviral transduction of Cre recombinase (pink) or empty-vector control (red). (C) Il33-002 expression in NIH-3T3 cell lines stably expressing gRNAscr (grey) or gRNA1 (blue), gRNA2 (purple), or gRNA3 (pink) under high-puromycin conditions in combination with dCas9, followed by dCas9 removal by Cre recombinase as assayed by RT-qPCR and normalized to Actb expression (n=3). (D-E) Il33-002 expression (D) or Il33-001 expression (E) in NIH-3T3 cells from (C) following treatment wither water control or 1 µM 5-aza-2’-deoxycytidine, measured by RT-qPCR and normalized to Actb expression (n=4-5). (F) Il33-002 expression measured by RT-qPCR and normalized to Actb expression, in dCas9:gRNAscr (grey) or dCas9:gRNA3 (pink) NIH-3T3 cells following Cre recombinase treatment and then treated with poly(I:C) (1 µg/mL) or water control for 4 or 8 hours. (G) DNA methylation assayed by bisulfite-pyrosequencing in NIH-3T3 cells expressing dCas9, gRNAscr, and Cre treated with 1 µg/mL poly(I:C) or water control for 8 hrs and 24 hrs (n=3). (H) Maximal Il33-002 induction (left y-axis, pink bars; data in log2 scale but axis numbering is not transformed) and maximal promoter demethylation (right y-axis, calculated as percent unmethylated divided by control methylation) under different treatments (x-axis: dCas9, 5-aza-2’-deoxycytidine, dCas9-VP64, dCas9-TET, and dCas9-deadTET). Where relevant, data for maximally inducing/demethylating gRNA is shown. * indicates statistically significant difference of p

    Article Snippet: Given that bound dCas9 envelopes nearly the entire DNA double helix [ ], we predicted that both CpG sites would be equally protected.

    Techniques: Expressing, Binding Assay, Methylation, Passaging, Transduction, Plasmid Preparation, Stable Transfection, Quantitative RT-PCR, DNA Methylation Assay, Transformation Assay

    The effect of dCas9-based demethylation of TSS on expression of SerpinB5 , Tnf and FMR1 genes (A) (Top) Schematic of the human SERPINB5 promoter region, including the start site of transcription (marked by black arrow) and the binding site and PAM of the SERPINB5 gRNA. CG sequences are boxed in red. (Bottom) SERPINB5 gene with purple boxes indicating enhancer positions relative to gene body. Enhancer IDs correspond to the GeneHancer database. (B) DNA methylation level of each CpG averaged over 19 gRNAscr (red) and 23 gRNAmaspin (black) MDA-MB-231 clones as assessed by pyrosequencing (mean ± SEM). (C) Same data as (B) except now shown as the calculated methylation fraction for each of the 19 gRNAscr (red) and 23 gRNAmaspin (black) clones, rather than the average of all clones. (D) SERPINB5 expression levels measured by RT-qPCR and normalized to GAPDH expression levels for 5 gRNAscr and 5 lowly-methylated gRNAmaspin clones (mean ± SEM, n=5). (E) SERPINB5 expression levels measured by RT-qPCR and normalized to GAPDH expression levels for 48 (24 for each treatment) MDA-MB-231 clones subcloned from the clones in (D). (F) SERPINB5 expression levels measured by RT-qPCR and normalized to GAPDH expression levels for clones from (D) following treatment with 1 µM 5-aza-2’-deoxycytidine or water control (n=5). (G) Expression fold change of murine Il33-002 (grey) and Tnf (pink), normalized to Actb and water control, following treatment of control NIH-3T3 cells with 1 µM 5-aza-2’-deoxycytidine (n=3). (H) Tnf expression in NIH-3T3 cell lines stably in control (water); grey bars) or 1 µM 5-aza-2’-deoxycytidine (pink bars) expressing either gRNAscr or gRNA Tnf2 :Cre under high-puromycin conditions in combination with dCas9, followed by dCas9 removal by Cre recombinase, as assayed by RT-qPCR and normalized to Actb expression (n=3). (I) Schematic of the human FMR1 repeat region showing the 5’ untranslated region (UTR) that is prone to CGG repeat expansion and methylation in Fragile X syndrome. Sequence of the gRNA targeting this region is shown (gRNA-CGG) and the extent of the available binding sites for this gRNA is represented by purple lines which indicate binding sites, the 13 presented here represent less than 15% of the available binding site in the Fragile X syndrome patient primary fibroblasts used in this study, which have approximately 700 CGG repeats. (J) FMR1 expression quantified by RT-qPCR and normalized to GAPDH expression levels in Fragile X syndrome patient primary fibroblasts that had stably expressed dCas9 (later removed with Cre) and either gRNAscr (grey) or gRNA-CGG (purple) under high-puromycin selection (n=6). * indicates statistically significant difference of p

    Journal: bioRxiv

    Article Title: Unraveling the functional role of DNA methylation using targeted DNA demethylation by steric blockage of DNA methyltransferase with CRISPR/dCas9

    doi: 10.1101/2020.03.28.012518

    Figure Lengend Snippet: The effect of dCas9-based demethylation of TSS on expression of SerpinB5 , Tnf and FMR1 genes (A) (Top) Schematic of the human SERPINB5 promoter region, including the start site of transcription (marked by black arrow) and the binding site and PAM of the SERPINB5 gRNA. CG sequences are boxed in red. (Bottom) SERPINB5 gene with purple boxes indicating enhancer positions relative to gene body. Enhancer IDs correspond to the GeneHancer database. (B) DNA methylation level of each CpG averaged over 19 gRNAscr (red) and 23 gRNAmaspin (black) MDA-MB-231 clones as assessed by pyrosequencing (mean ± SEM). (C) Same data as (B) except now shown as the calculated methylation fraction for each of the 19 gRNAscr (red) and 23 gRNAmaspin (black) clones, rather than the average of all clones. (D) SERPINB5 expression levels measured by RT-qPCR and normalized to GAPDH expression levels for 5 gRNAscr and 5 lowly-methylated gRNAmaspin clones (mean ± SEM, n=5). (E) SERPINB5 expression levels measured by RT-qPCR and normalized to GAPDH expression levels for 48 (24 for each treatment) MDA-MB-231 clones subcloned from the clones in (D). (F) SERPINB5 expression levels measured by RT-qPCR and normalized to GAPDH expression levels for clones from (D) following treatment with 1 µM 5-aza-2’-deoxycytidine or water control (n=5). (G) Expression fold change of murine Il33-002 (grey) and Tnf (pink), normalized to Actb and water control, following treatment of control NIH-3T3 cells with 1 µM 5-aza-2’-deoxycytidine (n=3). (H) Tnf expression in NIH-3T3 cell lines stably in control (water); grey bars) or 1 µM 5-aza-2’-deoxycytidine (pink bars) expressing either gRNAscr or gRNA Tnf2 :Cre under high-puromycin conditions in combination with dCas9, followed by dCas9 removal by Cre recombinase, as assayed by RT-qPCR and normalized to Actb expression (n=3). (I) Schematic of the human FMR1 repeat region showing the 5’ untranslated region (UTR) that is prone to CGG repeat expansion and methylation in Fragile X syndrome. Sequence of the gRNA targeting this region is shown (gRNA-CGG) and the extent of the available binding sites for this gRNA is represented by purple lines which indicate binding sites, the 13 presented here represent less than 15% of the available binding site in the Fragile X syndrome patient primary fibroblasts used in this study, which have approximately 700 CGG repeats. (J) FMR1 expression quantified by RT-qPCR and normalized to GAPDH expression levels in Fragile X syndrome patient primary fibroblasts that had stably expressed dCas9 (later removed with Cre) and either gRNAscr (grey) or gRNA-CGG (purple) under high-puromycin selection (n=6). * indicates statistically significant difference of p

    Article Snippet: Given that bound dCas9 envelopes nearly the entire DNA double helix [ ], we predicted that both CpG sites would be equally protected.

    Techniques: Expressing, Binding Assay, DNA Methylation Assay, Multiple Displacement Amplification, Clone Assay, Methylation, Quantitative RT-PCR, Stable Transfection, Sequencing, Selection

    DNA methylation levels assessed by bisulfite-pyrosequencing of CpGs 1-6 in the SERPINB5 promoter in MDA-MB-231 cell lines stably expressing dCas9 and either gRNAscr (red) or gRNAmaspin (black), averaged across all 6 CpGs and plotted as a function of increasing puromycin concentration (n=1 per puromycin concentration).

    Journal: bioRxiv

    Article Title: Unraveling the functional role of DNA methylation using targeted DNA demethylation by steric blockage of DNA methyltransferase with CRISPR/dCas9

    doi: 10.1101/2020.03.28.012518

    Figure Lengend Snippet: DNA methylation levels assessed by bisulfite-pyrosequencing of CpGs 1-6 in the SERPINB5 promoter in MDA-MB-231 cell lines stably expressing dCas9 and either gRNAscr (red) or gRNAmaspin (black), averaged across all 6 CpGs and plotted as a function of increasing puromycin concentration (n=1 per puromycin concentration).

    Article Snippet: Given that bound dCas9 envelopes nearly the entire DNA double helix [ ], we predicted that both CpG sites would be equally protected.

    Techniques: DNA Methylation Assay, Multiple Displacement Amplification, Stable Transfection, Expressing, Concentration Assay

    The Stern-Volmer plots of the fluorescence quenching of pyrene derivatives by C-myc DNA at 298 K and 310 K (the concentrations of pyrene derivatives are 1 μmol/L, pH = 7.4). ( A ) 1-OHP and ( B ) 1-PBO.

    Journal: Molecules

    Article Title: Studies on the Interaction Mechanism of Pyrene Derivatives with Human Tumor-Related DNA

    doi: 10.3390/molecules171214159

    Figure Lengend Snippet: The Stern-Volmer plots of the fluorescence quenching of pyrene derivatives by C-myc DNA at 298 K and 310 K (the concentrations of pyrene derivatives are 1 μmol/L, pH = 7.4). ( A ) 1-OHP and ( B ) 1-PBO.

    Article Snippet: The average lifetime of 1-PBO with DNA interaction is shorter than that of unbound 1-PBO, suggesting that 1-PBO does affect the DNA double helix structure, similar with some groove binding and intercalation agents in previous studies [ , ].

    Techniques: Fluorescence

    Transient fluorescence spectra of pyrene derivatives (1 μmol/L) in the absence and presence of DNA (10 μmol/L).( A ) 1-OHP and ( B ) 1-PBO.

    Journal: Molecules

    Article Title: Studies on the Interaction Mechanism of Pyrene Derivatives with Human Tumor-Related DNA

    doi: 10.3390/molecules171214159

    Figure Lengend Snippet: Transient fluorescence spectra of pyrene derivatives (1 μmol/L) in the absence and presence of DNA (10 μmol/L).( A ) 1-OHP and ( B ) 1-PBO.

    Article Snippet: The average lifetime of 1-PBO with DNA interaction is shorter than that of unbound 1-PBO, suggesting that 1-PBO does affect the DNA double helix structure, similar with some groove binding and intercalation agents in previous studies [ , ].

    Techniques: Fluorescence

    CD and ICD spectra of DNA solutions (5 μmol/L) with the addition of 1-PBO (0–20 μmol/L). ( A ) CDspectra of p53 DNA; ( B ) CDspectra of C-myc DNA; ( C ) ICDspectra of p53 DNA and ( D ) ICDspectra of C-myc DNA.

    Journal: Molecules

    Article Title: Studies on the Interaction Mechanism of Pyrene Derivatives with Human Tumor-Related DNA

    doi: 10.3390/molecules171214159

    Figure Lengend Snippet: CD and ICD spectra of DNA solutions (5 μmol/L) with the addition of 1-PBO (0–20 μmol/L). ( A ) CDspectra of p53 DNA; ( B ) CDspectra of C-myc DNA; ( C ) ICDspectra of p53 DNA and ( D ) ICDspectra of C-myc DNA.

    Article Snippet: The average lifetime of 1-PBO with DNA interaction is shorter than that of unbound 1-PBO, suggesting that 1-PBO does affect the DNA double helix structure, similar with some groove binding and intercalation agents in previous studies [ , ].

    Techniques:

    Quenching extents of pyrene derivatives solutions (1 μmol/L) with the addition of KI (0–20 μmol/L) in the absence and presence of C-myc DNA (10 μmol/L). ( A ) 1-OHP and ( B ) 1-PBO.

    Journal: Molecules

    Article Title: Studies on the Interaction Mechanism of Pyrene Derivatives with Human Tumor-Related DNA

    doi: 10.3390/molecules171214159

    Figure Lengend Snippet: Quenching extents of pyrene derivatives solutions (1 μmol/L) with the addition of KI (0–20 μmol/L) in the absence and presence of C-myc DNA (10 μmol/L). ( A ) 1-OHP and ( B ) 1-PBO.

    Article Snippet: The average lifetime of 1-PBO with DNA interaction is shorter than that of unbound 1-PBO, suggesting that 1-PBO does affect the DNA double helix structure, similar with some groove binding and intercalation agents in previous studies [ , ].

    Techniques:

    Fluorescence spectra of the pyrenederivatives solution (1 μmol/L) with the addition of p53 DNA (0-20 μmol/L). ( A ) 1-OHP and ( B ) 1-PBO. The arrows show the fluorescence changes at peak wavelength with the DNA concentration increased.

    Journal: Molecules

    Article Title: Studies on the Interaction Mechanism of Pyrene Derivatives with Human Tumor-Related DNA

    doi: 10.3390/molecules171214159

    Figure Lengend Snippet: Fluorescence spectra of the pyrenederivatives solution (1 μmol/L) with the addition of p53 DNA (0-20 μmol/L). ( A ) 1-OHP and ( B ) 1-PBO. The arrows show the fluorescence changes at peak wavelength with the DNA concentration increased.

    Article Snippet: The average lifetime of 1-PBO with DNA interaction is shorter than that of unbound 1-PBO, suggesting that 1-PBO does affect the DNA double helix structure, similar with some groove binding and intercalation agents in previous studies [ , ].

    Techniques: Fluorescence, Concentration Assay

    LSS-11 interacts with DNA and increases its stability ( A ) UV-Vis spectra and ( B ) fluorescent emission spectra of LSS-11 (50 μM) with increasing concentrations of CT DNA (0 to 200 μM). ( C ) Scatchard plot of the fluorescent intensity of LSS-11 at 570 nm with increasing concentrations of CT DNA [DNA], F stands for fluorescent intensity and F0 refers to fluorescent intensity without CT DNA. ( D ) Increased Tm of a 63 bp DNA fragment in the presence of LSS-11 at indicated concentrations.

    Journal: Oncotarget

    Article Title: A novel triazolonaphthalimide induces apoptosis and inhibits tumor growth by targeting DNA and DNA-associated processes

    doi: 10.18632/oncotarget.16962

    Figure Lengend Snippet: LSS-11 interacts with DNA and increases its stability ( A ) UV-Vis spectra and ( B ) fluorescent emission spectra of LSS-11 (50 μM) with increasing concentrations of CT DNA (0 to 200 μM). ( C ) Scatchard plot of the fluorescent intensity of LSS-11 at 570 nm with increasing concentrations of CT DNA [DNA], F stands for fluorescent intensity and F0 refers to fluorescent intensity without CT DNA. ( D ) Increased Tm of a 63 bp DNA fragment in the presence of LSS-11 at indicated concentrations.

    Article Snippet: On the other hand, the binding of LSS-11 to RNA suggests the interaction depends on neither the B-form DNA double helix nor intercalation, since RNA only partially forms double helix in A-form geometry.

    Techniques:

    LSS-11 binds DNA in vitro and in cell by minor groove binding ( A ) Fluorescent microscopic photography showing cells stained by Hoechst33258 (blue), LSS-11 (green) or both. Please note the mutual exclusive distribution of Hoechst33258 and LSS-11 fluorescence in nucleus. ( B ) and ( C ) Competitive fluorescent spectra titration of LSS-11 to Hoechst33258 (B) or EB (C). ( D ) Agarose electrophoresis of pUC-19 plasmid incubated with indicated concentrations of LSS-11 and stained with EB, pUC-19 digested by endonuclease was employed as a positive control.

    Journal: Oncotarget

    Article Title: A novel triazolonaphthalimide induces apoptosis and inhibits tumor growth by targeting DNA and DNA-associated processes

    doi: 10.18632/oncotarget.16962

    Figure Lengend Snippet: LSS-11 binds DNA in vitro and in cell by minor groove binding ( A ) Fluorescent microscopic photography showing cells stained by Hoechst33258 (blue), LSS-11 (green) or both. Please note the mutual exclusive distribution of Hoechst33258 and LSS-11 fluorescence in nucleus. ( B ) and ( C ) Competitive fluorescent spectra titration of LSS-11 to Hoechst33258 (B) or EB (C). ( D ) Agarose electrophoresis of pUC-19 plasmid incubated with indicated concentrations of LSS-11 and stained with EB, pUC-19 digested by endonuclease was employed as a positive control.

    Article Snippet: On the other hand, the binding of LSS-11 to RNA suggests the interaction depends on neither the B-form DNA double helix nor intercalation, since RNA only partially forms double helix in A-form geometry.

    Techniques: In Vitro, Binding Assay, Staining, Fluorescence, Titration, Electrophoresis, Plasmid Preparation, Incubation, Positive Control

    LSS-11 inhibits DNA polymerase, topoisomerase II and minimal TA promoter-drove luciferase reporter expression ( A ) The impact of indicated concentrations of LSS-11 on decatenation of kDNA by Topo II. “D” refers to decatenated DNA, “L” means linear DNA, kDNA without any treatment was included as a blank control. ( B ) The impact of LSS-11 on the thermal stabilities of Topo IIα and Topo I determined by CETSA as described in Materials and methods. Quantification of band intensity was performed by ImageJ and shown as bar graph in the lower panel. ( C ) The impact of LSS-11 on the amplification efficiency of Taq DNA polymerase in real time fluorescent quantitative PCR. 63 bp and 216 bp represent the sizes of amplicons. ( D ) The impact of LSS-11 on the efficiency of isothermal helicase-dependent amplifications. Lane M, marker; lane P, positive control. ( E ) Relative luciferase activities of SW480 cells transfected with pGL-6 TA luciferase reporter, the luciferase activity (arbitrary unit) was normalized by the luciferase activity of cells treated with vehicle only. *p

    Journal: Oncotarget

    Article Title: A novel triazolonaphthalimide induces apoptosis and inhibits tumor growth by targeting DNA and DNA-associated processes

    doi: 10.18632/oncotarget.16962

    Figure Lengend Snippet: LSS-11 inhibits DNA polymerase, topoisomerase II and minimal TA promoter-drove luciferase reporter expression ( A ) The impact of indicated concentrations of LSS-11 on decatenation of kDNA by Topo II. “D” refers to decatenated DNA, “L” means linear DNA, kDNA without any treatment was included as a blank control. ( B ) The impact of LSS-11 on the thermal stabilities of Topo IIα and Topo I determined by CETSA as described in Materials and methods. Quantification of band intensity was performed by ImageJ and shown as bar graph in the lower panel. ( C ) The impact of LSS-11 on the amplification efficiency of Taq DNA polymerase in real time fluorescent quantitative PCR. 63 bp and 216 bp represent the sizes of amplicons. ( D ) The impact of LSS-11 on the efficiency of isothermal helicase-dependent amplifications. Lane M, marker; lane P, positive control. ( E ) Relative luciferase activities of SW480 cells transfected with pGL-6 TA luciferase reporter, the luciferase activity (arbitrary unit) was normalized by the luciferase activity of cells treated with vehicle only. *p

    Article Snippet: On the other hand, the binding of LSS-11 to RNA suggests the interaction depends on neither the B-form DNA double helix nor intercalation, since RNA only partially forms double helix in A-form geometry.

    Techniques: Luciferase, Expressing, Amplification, Real-time Polymerase Chain Reaction, Marker, Positive Control, Transfection, Activity Assay

    LSS-11 induces DNA damage and DNA damage response ( A ) Cells were treated with indicated concentrations of LSS-11 then DNA fragmentation was visualized by single cell electrophoresis as described in Materials and methods. ( B ) SW480 and LoVo cells were treated with indicated concentrations of LSS-11 for 72 h or 0.1 μM LSS11 for specified time, then harvested and blotted by antibodies against indicated proteins, GAPDH was blotted as an internal control to assure equal loading.

    Journal: Oncotarget

    Article Title: A novel triazolonaphthalimide induces apoptosis and inhibits tumor growth by targeting DNA and DNA-associated processes

    doi: 10.18632/oncotarget.16962

    Figure Lengend Snippet: LSS-11 induces DNA damage and DNA damage response ( A ) Cells were treated with indicated concentrations of LSS-11 then DNA fragmentation was visualized by single cell electrophoresis as described in Materials and methods. ( B ) SW480 and LoVo cells were treated with indicated concentrations of LSS-11 for 72 h or 0.1 μM LSS11 for specified time, then harvested and blotted by antibodies against indicated proteins, GAPDH was blotted as an internal control to assure equal loading.

    Article Snippet: On the other hand, the binding of LSS-11 to RNA suggests the interaction depends on neither the B-form DNA double helix nor intercalation, since RNA only partially forms double helix in A-form geometry.

    Techniques: Electrophoresis

    Top view and all structure view of the molecular docking top-ranked poses of the AO derivatives intercalated in the d(ACGTACGT) 2 sequence. ( a ) 125 I-C 3 ; ( b ) 125 I-C 5 ; ( c ) 125 I-C 8 ; ( d ) 99m Tc-C 3 ; ( e ) 99m Tc-C 5 and ( f ) 99m Tc-C 8 . Distance between the 125 I or 99m Tc atom relative to the DNA helical axis is displayed in Å.

    Journal: Scientific Reports

    Article Title: Evaluation of Acridine Orange Derivatives as DNA-Targeted Radiopharmaceuticals for Auger Therapy: Influence of the Radionuclide and Distance to DNA

    doi: 10.1038/srep42544

    Figure Lengend Snippet: Top view and all structure view of the molecular docking top-ranked poses of the AO derivatives intercalated in the d(ACGTACGT) 2 sequence. ( a ) 125 I-C 3 ; ( b ) 125 I-C 5 ; ( c ) 125 I-C 8 ; ( d ) 99m Tc-C 3 ; ( e ) 99m Tc-C 5 and ( f ) 99m Tc-C 8 . Distance between the 125 I or 99m Tc atom relative to the DNA helical axis is displayed in Å.

    Article Snippet: These results provide the first evidence that 99m Tc can induce DNA damage with similar efficiency to that of 125 I, when both are positioned at comparable distances to the double helix.

    Techniques: Sequencing

    Cleavage of supercoiled ϕX174 DNA by 99m Tc-C 3 , 99m Tc -C 5 and 99m Tc -C 8 . Incubation was performed for 24 hours at 4 °C in Tris-HCl buffer (pH 7.4) with different activities of the complexes (in μCi), in the presence or absence of DMSO (0.2 M). SC, OC and Lin are supercoiled, open circular and linear forms of DNA, respectively. “DNA 0 h” is the DNA control without any incubation.

    Journal: Scientific Reports

    Article Title: Evaluation of Acridine Orange Derivatives as DNA-Targeted Radiopharmaceuticals for Auger Therapy: Influence of the Radionuclide and Distance to DNA

    doi: 10.1038/srep42544

    Figure Lengend Snippet: Cleavage of supercoiled ϕX174 DNA by 99m Tc-C 3 , 99m Tc -C 5 and 99m Tc -C 8 . Incubation was performed for 24 hours at 4 °C in Tris-HCl buffer (pH 7.4) with different activities of the complexes (in μCi), in the presence or absence of DMSO (0.2 M). SC, OC and Lin are supercoiled, open circular and linear forms of DNA, respectively. “DNA 0 h” is the DNA control without any incubation.

    Article Snippet: These results provide the first evidence that 99m Tc can induce DNA damage with similar efficiency to that of 125 I, when both are positioned at comparable distances to the double helix.

    Techniques: Incubation

    Induction of DNA damage by 99m Tc-C 3 and 125 I-C 5 in PC3 cells: ( a ) Fluorescence images of untreated PC3 cells (control) or cells exposed to 99m Tc-C 3 and 125 I-C 5 for 24 h. Cells were immunostained for γ-H2AX. DAPI was used to visualize the nuclei; ( b ) and ( c ) Average number of foci per cell in PC3 cells incubated during 2 and 24 hours with 99m Tc-C 3 and 125 I-C 5 , respectively.

    Journal: Scientific Reports

    Article Title: Evaluation of Acridine Orange Derivatives as DNA-Targeted Radiopharmaceuticals for Auger Therapy: Influence of the Radionuclide and Distance to DNA

    doi: 10.1038/srep42544

    Figure Lengend Snippet: Induction of DNA damage by 99m Tc-C 3 and 125 I-C 5 in PC3 cells: ( a ) Fluorescence images of untreated PC3 cells (control) or cells exposed to 99m Tc-C 3 and 125 I-C 5 for 24 h. Cells were immunostained for γ-H2AX. DAPI was used to visualize the nuclei; ( b ) and ( c ) Average number of foci per cell in PC3 cells incubated during 2 and 24 hours with 99m Tc-C 3 and 125 I-C 5 , respectively.

    Article Snippet: These results provide the first evidence that 99m Tc can induce DNA damage with similar efficiency to that of 125 I, when both are positioned at comparable distances to the double helix.

    Techniques: Fluorescence, Incubation

    Deposited energies in the DNA segment with the decay sources ( 125 I and 99m Tc) at different distances from the DNA center: ( a ) deposited energies per Auger electron emitted; ( b ) deposited energies normalized per decay.

    Journal: Scientific Reports

    Article Title: Evaluation of Acridine Orange Derivatives as DNA-Targeted Radiopharmaceuticals for Auger Therapy: Influence of the Radionuclide and Distance to DNA

    doi: 10.1038/srep42544

    Figure Lengend Snippet: Deposited energies in the DNA segment with the decay sources ( 125 I and 99m Tc) at different distances from the DNA center: ( a ) deposited energies per Auger electron emitted; ( b ) deposited energies normalized per decay.

    Article Snippet: These results provide the first evidence that 99m Tc can induce DNA damage with similar efficiency to that of 125 I, when both are positioned at comparable distances to the double helix.

    Techniques: