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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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Related in: MedlinePlus

(A) PAGE (8%) of cis-ligated sequences with DB921 not in the gel. Electrophoresis was done as in Figure 2. For each duplex, 0 indicates DNA that is not incubated with compound prior to electrophoresis, and the ratios (compound to binding site) indicate DNA is incubated with compound prior to electrophoresis. The colored arrows indicate 189 bp for each duplex. The red arrow of M2 indicates 100 bp, while the red arrow of M1 indicates 105 bp of the random sequence marker. (B) PAGE (8%) was done for 2 h 20 min at 200 V and compound was included in the gel. Experiments were done with the same cis sequences as in (A) and with trans-ligated DNA sequences of AAAAA (tA5) and ATATA (tAT). The colored arrows indicate 189 bp and the double intensity band of M2 is 100 bp.
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Figure 3: (A) PAGE (8%) of cis-ligated sequences with DB921 not in the gel. Electrophoresis was done as in Figure 2. For each duplex, 0 indicates DNA that is not incubated with compound prior to electrophoresis, and the ratios (compound to binding site) indicate DNA is incubated with compound prior to electrophoresis. The colored arrows indicate 189 bp for each duplex. The red arrow of M2 indicates 100 bp, while the red arrow of M1 indicates 105 bp of the random sequence marker. (B) PAGE (8%) was done for 2 h 20 min at 200 V and compound was included in the gel. Experiments were done with the same cis sequences as in (A) and with trans-ligated DNA sequences of AAAAA (tA5) and ATATA (tAT). The colored arrows indicate 189 bp and the double intensity band of M2 is 100 bp.

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)

(A) PAGE (8%) of cis-ligated sequences with DB921 not in the gel. Electrophoresis was done as in Figure 2. For each duplex, 0 indicates DNA that is not incubated with compound prior to electrophoresis, and the ratios (compound to binding site) indicate DNA is incubated with compound prior to electrophoresis. The colored arrows indicate 189 bp for each duplex. The red arrow of M2 indicates 100 bp, while the red arrow of M1 indicates 105 bp of the random sequence marker. (B) PAGE (8%) was done for 2 h 20 min at 200 V and compound was included in the gel. Experiments were done with the same cis sequences as in (A) and with trans-ligated DNA sequences of AAAAA (tA5) and ATATA (tAT). The colored arrows indicate 189 bp and the double intensity band of M2 is 100 bp.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 3: (A) PAGE (8%) of cis-ligated sequences with DB921 not in the gel. Electrophoresis was done as in Figure 2. For each duplex, 0 indicates DNA that is not incubated with compound prior to electrophoresis, and the ratios (compound to binding site) indicate DNA is incubated with compound prior to electrophoresis. The colored arrows indicate 189 bp for each duplex. The red arrow of M2 indicates 100 bp, while the red arrow of M1 indicates 105 bp of the random sequence marker. (B) PAGE (8%) was done for 2 h 20 min at 200 V and compound was included in the gel. Experiments were done with the same cis sequences as in (A) and with trans-ligated DNA sequences of AAAAA (tA5) and ATATA (tAT). The colored arrows indicate 189 bp and the double intensity band of M2 is 100 bp.
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