Fluorescence of free and bound to DNA of compounds 1–3.

<p>Comparison of fluorescence intensity of compounds <b>1</b> (circles)<b>, 2</b> (squares) and <b>3 (</b>triangles) in the presence (A) or absence (B) of magnesium cations. Open symbols, fluorescence of free (unbound) <b>1–</b>3; filled symbols, fluorescence of <b>1–3</b> in complexes with pUC19 DNA (10 μM; bp). Excitation: 440 nm, fluorescence detection: 480 nm.</p

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<p><b>Dependence of DNA binding affinity of compounds 1 (A) and 3 (B) on the ionic strength of solution.</b> DNA binding constants of <b>1</b> and <b>3</b> obtained in solutions that contained 5mM MgCl₂ (filled circles) and no MgCl₂ (open circles). Linear approximations shown by solid lines with the slope SK = ∂logK/∂log[KCl].</p

Salt dependent thermodynamic parameters of binding of 1 and 3 to DNA calculated on the basis of fluorescence measurements.

<p>Salt dependent thermodynamic parameters of binding of 1 and 3 to DNA calculated on the basis of fluorescence measurements.</p

Binding of compounds 1–3 to the pUC19 plasmid DNA monitored by electrophoretic mobility in 1% agarose gel.

<p>The plasmid was incubated with the compounds at indicated concentrations (μM) in BB-Mg buffer containing 5 mM MgCl₂ (A) or BB buffer (same buffer with no MgCl₂) (B). Migration of the free compound is shown at the highest concentration (25 μM) for each drug (arrows). Bottom panels in A and B: electrophoresis with EtBr in the gel and in the running buffer.</p

Changes of CD spectra of compound 3 upon binding to DNA.

<p>CD spectra of unbound compound <b>3</b> in the absence of DNA (open circles) and in complexes with DNA (filled circles) in BB-Mg (A) or BB (B). DNA concentration increased in the range 5–50 μM (bp), the concentration of <b>3</b> was 20 μM.</p

Table_1_Deep Sequencing Revealed a CpG Methylation Pattern Associated With ALDH1L1 Suppression in Breast Cancer.DOCX

<p>Hypermethylation of promoter CpG islands is generally recognized epigenetic mechanism responsible for gene silencing in cancer. However, molecular details on how this epigenetic mark triggers the process of gene downregulation are still elusive. Here, we used deep bisulfite sequencing and qPCR analysis to investigate the pattern of CpG methylation of ALDH1L1 promoter region and its association with the gene expression level in 16 paired breast cancer (BC) samples of different clinical stages. Expression of ALDH1L1 gene was suppressed in all examined BC samples up to 200-fold, and average hypermethylation level of the promoter region correlated positively with ALDH1L1 downregulation. We determined the role of every individual CpG site within the ALDH1L1 promoter, including upstream untranscribed region, first untranslated exon, and the start of the first intron, in aberrant gene expression by correlation analysis. The search revealed CpG sites which methylation has the highest impact on intensity of gene transcription. The majority of such CpG sites are located in a compact region in the first intron of the ALDH1L1 gene. These results assist in unraveling of dynamic nature of CpG promoter hypermethylation as well as demonstrate the efficiency of deep bisulfite sequencing in search for novel epigenetic markers in cancer.</p