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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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Sequence of DNA duplexes used (left) and 8% PAGE gel of DB75 with the duplexes (right). The colored arrows indicate the 189-bp duplex for each ladder. The red arrow of M2 indicates 100 bp, while the red arrow of M1 indicates 189 bp of the random sequence marker. Electrophoresis was done for 2 h 20 min at 200 V and compound was not included in the gel. For each duplex, 0 indicates DNA that is not incubated with compound prior to electrophoresis, and the ratio 2:1 (compound to binding site) indicates DNA incubated with compound prior to electrophoresis. The brightest bands near the top of the lanes are circular products.
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Figure 2: Sequence of DNA duplexes used (left) and 8% PAGE gel of DB75 with the duplexes (right). The colored arrows indicate the 189-bp duplex for each ladder. The red arrow of M2 indicates 100 bp, while the red arrow of M1 indicates 189 bp of the random sequence marker. Electrophoresis was done for 2 h 20 min at 200 V and compound was not included in the gel. For each duplex, 0 indicates DNA that is not incubated with compound prior to electrophoresis, and the ratio 2:1 (compound to binding site) indicates DNA incubated with compound prior to electrophoresis. The brightest bands near the top of the lanes are circular products.

Mentions: Two 21-mer duplexes (AAAAA and ATATA in Figure 2) from Tevis et al. (18) and two new duplexes (TTTAA and AAATT in Figure 2) were used in this study. The marker, M1, is also a 21 mer with the same base composition as the test duplexes, however; there is no AT binding site (18). Marker, M2, is a commercially available 20 bp ladder (Bayou Biolabs, Harahan, LA, USA) with a double intensity band at 100 bp. High-performance liquid chromatography (HPLC) purified and lyophilized DNA oligomers were purchased from Integrated DNA Technologies, Inc. (IDT, Coraville, IA, USA). The concentration of single-strand DNA was brought to ∼1 mM using water. The exact concentration was then determined using the extinction coefficient calculated by the nearest neighbor approximation. Then the complementary strands (100 µM) were combined in a 1:1 ratio based on the determined concentrations and annealed in 1× ligation buffer (New England BioLabs, Ipswich, MA, USA) containing 50 mM Tris–HCl, 10 mM MgCl2, 10 mM dithiothreitol, 1 mM ATP and 25 mg/ml bovine serum albumin.Figure 2.


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)

Sequence of DNA duplexes used (left) and 8% PAGE gel of DB75 with the duplexes (right). The colored arrows indicate the 189-bp duplex for each ladder. The red arrow of M2 indicates 100 bp, while the red arrow of M1 indicates 189 bp of the random sequence marker. Electrophoresis was done for 2 h 20 min at 200 V and compound was not included in the gel. For each duplex, 0 indicates DNA that is not incubated with compound prior to electrophoresis, and the ratio 2:1 (compound to binding site) indicates DNA incubated with compound prior to electrophoresis. The brightest bands near the top of the lanes are circular products.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 2: Sequence of DNA duplexes used (left) and 8% PAGE gel of DB75 with the duplexes (right). The colored arrows indicate the 189-bp duplex for each ladder. The red arrow of M2 indicates 100 bp, while the red arrow of M1 indicates 189 bp of the random sequence marker. Electrophoresis was done for 2 h 20 min at 200 V and compound was not included in the gel. For each duplex, 0 indicates DNA that is not incubated with compound prior to electrophoresis, and the ratio 2:1 (compound to binding site) indicates DNA incubated with compound prior to electrophoresis. The brightest bands near the top of the lanes are circular products.
Mentions: Two 21-mer duplexes (AAAAA and ATATA in Figure 2) from Tevis et al. (18) and two new duplexes (TTTAA and AAATT in Figure 2) were used in this study. The marker, M1, is also a 21 mer with the same base composition as the test duplexes, however; there is no AT binding site (18). Marker, M2, is a commercially available 20 bp ladder (Bayou Biolabs, Harahan, LA, USA) with a double intensity band at 100 bp. High-performance liquid chromatography (HPLC) purified and lyophilized DNA oligomers were purchased from Integrated DNA Technologies, Inc. (IDT, Coraville, IA, USA). The concentration of single-strand DNA was brought to ∼1 mM using water. The exact concentration was then determined using the extinction coefficient calculated by the nearest neighbor approximation. Then the complementary strands (100 µM) were combined in a 1:1 ratio based on the determined concentrations and annealed in 1× ligation buffer (New England BioLabs, Ipswich, MA, USA) containing 50 mM Tris–HCl, 10 mM MgCl2, 10 mM dithiothreitol, 1 mM ATP and 25 mg/ml bovine serum albumin.Figure 2.

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