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Induced topological changes in DNA complexes: influence of DNA sequences and small molecule structures.

Hunt RA, Munde M, Kumar A, Ismail MA, Farahat AA, Arafa RK, Say M, Batista-Parra A, Tevis D, Boykin DW, Wilson WD - Nucleic Acids Res. (2011)

Bottom Line: The changes caused by binding of the compounds are sequence dependent, but generally the topological effects on AAAAA and AAATT are similar as are the effects on TTTAA and ATATA.A total of 13 compounds with a variety of structural differences were evaluated for topological changes to DNA.Similar, but generally smaller, effects are seen with TTTAA.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, Georgia State University, Atlanta, GA 30302, USA.

ABSTRACT
Heterocyclic diamidines are compounds with antiparasitic properties that target the minor groove of kinetoplast DNA. The mechanism of action of these compounds is unknown, but topological changes to DNA structures are likely to be involved. In this study, we have developed a polyacrylamide gel electrophoresis-based screening method to determine topological effects of heterocyclic diamidines on four minor groove target sequences: AAAAA, TTTAA, AAATT and ATATA. The AAAAA and AAATT sequences have the largest intrinsic bend, whereas the TTTAA and ATATA sequences are relatively straight. The changes caused by binding of the compounds are sequence dependent, but generally the topological effects on AAAAA and AAATT are similar as are the effects on TTTAA and ATATA. A total of 13 compounds with a variety of structural differences were evaluated for topological changes to DNA. All compounds decrease the mobility of the ATATA sequence that is consistent with decreased minor groove width and bending of the relatively straight DNA into the minor groove. Similar, but generally smaller, effects are seen with TTTAA. The intrinsically bent AAAAA and AAATT sequences, which have more narrow minor grooves, have smaller mobility changes on binding that are consistent with increased or decreased bending depending on compound structure.

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PAGE (8%) of the DNA sequences (Figure 2) with multiple DB compounds. (A) The duplexes AAAAA and ATATA with five selected compounds, and (B) TTTAA and AAATT with the same five compounds. For each duplex, DNAs are incubated with compound prior to electrophoresis at a ratio of 2:1 (compound to binding site). The colored arrows indicate 189 bp for each duplex. The red arrow M1 indicates 105 bp of the random sequence marker. Electrophoresis was done as in Figure 2 and compound was not included in the gel.
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Figure 4: PAGE (8%) of the DNA sequences (Figure 2) with multiple DB compounds. (A) The duplexes AAAAA and ATATA with five selected compounds, and (B) TTTAA and AAATT with the same five compounds. For each duplex, DNAs are incubated with compound prior to electrophoresis at a ratio of 2:1 (compound to binding site). The colored arrows indicate 189 bp for each duplex. The red arrow M1 indicates 105 bp of the random sequence marker. Electrophoresis was done as in Figure 2 and compound was not included in the gel.

Mentions: Ligation ladders were separated using 8% native polyacrylamide (1.5 mm thick, 20 cm long) prepared from a 40% acrylamide stock solution (29:1 bis-acrylamide:acyrlamide; EMD, Gibbstwon, NJ, USA) in 1× TBE buffer (0.089 M Tris, 0.089 M boric acid, 2.0 mM EDTA, pH 8.3). Each sample had a final volume of 20 µl, which contained the following: 10 µl of 2 µM DNA, 4 µl of 6× load dye (Promega, Madison, WI, USA), the volume of compound needed for the desired ratio of compound to binding site and double distilled water to bring to the final volume, if needed. For the 1:1 and 2:1 ratios 3 and 6 µl of compound (13.3 µM) were added, respectively. Electrophoresis was done at 200 V (10 V/cm) for 140 min at 25°C ± 0.1°C in 1× TBE buffer. The quite different observed mobilities with compounds in adjacent lanes indicate that when using the 140-min time period, there is no significant interference from inter-lane diffusion when different compounds are used in the gel lanes. The lack of interference is also supported by the observation that compounds have the same mobilities when studied alone (Figures 2 and 3) or on a gel with other compounds (Figure 4).Figure 3.


Induced topological changes in DNA complexes: influence of DNA sequences and small molecule structures.

Hunt RA, Munde M, Kumar A, Ismail MA, Farahat AA, Arafa RK, Say M, Batista-Parra A, Tevis D, Boykin DW, Wilson WD - Nucleic Acids Res. (2011)

PAGE (8%) of the DNA sequences (Figure 2) with multiple DB compounds. (A) The duplexes AAAAA and ATATA with five selected compounds, and (B) TTTAA and AAATT with the same five compounds. For each duplex, DNAs are incubated with compound prior to electrophoresis at a ratio of 2:1 (compound to binding site). The colored arrows indicate 189 bp for each duplex. The red arrow M1 indicates 105 bp of the random sequence marker. Electrophoresis was done as in Figure 2 and compound was not included in the gel.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3105405&req=5

Figure 4: PAGE (8%) of the DNA sequences (Figure 2) with multiple DB compounds. (A) The duplexes AAAAA and ATATA with five selected compounds, and (B) TTTAA and AAATT with the same five compounds. For each duplex, DNAs are incubated with compound prior to electrophoresis at a ratio of 2:1 (compound to binding site). The colored arrows indicate 189 bp for each duplex. The red arrow M1 indicates 105 bp of the random sequence marker. Electrophoresis was done as in Figure 2 and compound was not included in the gel.
Mentions: Ligation ladders were separated using 8% native polyacrylamide (1.5 mm thick, 20 cm long) prepared from a 40% acrylamide stock solution (29:1 bis-acrylamide:acyrlamide; EMD, Gibbstwon, NJ, USA) in 1× TBE buffer (0.089 M Tris, 0.089 M boric acid, 2.0 mM EDTA, pH 8.3). Each sample had a final volume of 20 µl, which contained the following: 10 µl of 2 µM DNA, 4 µl of 6× load dye (Promega, Madison, WI, USA), the volume of compound needed for the desired ratio of compound to binding site and double distilled water to bring to the final volume, if needed. For the 1:1 and 2:1 ratios 3 and 6 µl of compound (13.3 µM) were added, respectively. Electrophoresis was done at 200 V (10 V/cm) for 140 min at 25°C ± 0.1°C in 1× TBE buffer. The quite different observed mobilities with compounds in adjacent lanes indicate that when using the 140-min time period, there is no significant interference from inter-lane diffusion when different compounds are used in the gel lanes. The lack of interference is also supported by the observation that compounds have the same mobilities when studied alone (Figures 2 and 3) or on a gel with other compounds (Figure 4).Figure 3.

Bottom Line: The changes caused by binding of the compounds are sequence dependent, but generally the topological effects on AAAAA and AAATT are similar as are the effects on TTTAA and ATATA.A total of 13 compounds with a variety of structural differences were evaluated for topological changes to DNA.Similar, but generally smaller, effects are seen with TTTAA.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, Georgia State University, Atlanta, GA 30302, USA.

ABSTRACT
Heterocyclic diamidines are compounds with antiparasitic properties that target the minor groove of kinetoplast DNA. The mechanism of action of these compounds is unknown, but topological changes to DNA structures are likely to be involved. In this study, we have developed a polyacrylamide gel electrophoresis-based screening method to determine topological effects of heterocyclic diamidines on four minor groove target sequences: AAAAA, TTTAA, AAATT and ATATA. The AAAAA and AAATT sequences have the largest intrinsic bend, whereas the TTTAA and ATATA sequences are relatively straight. The changes caused by binding of the compounds are sequence dependent, but generally the topological effects on AAAAA and AAATT are similar as are the effects on TTTAA and ATATA. A total of 13 compounds with a variety of structural differences were evaluated for topological changes to DNA. All compounds decrease the mobility of the ATATA sequence that is consistent with decreased minor groove width and bending of the relatively straight DNA into the minor groove. Similar, but generally smaller, effects are seen with TTTAA. The intrinsically bent AAAAA and AAATT sequences, which have more narrow minor grooves, have smaller mobility changes on binding that are consistent with increased or decreased bending depending on compound structure.

Show MeSH
Related in: MedlinePlus