Probing DNA bulges with designed helical spirocyclic molecules

Abstract

Because bulged structures (unpaired bases) in nucleic acids are of general biological significance, it has been of interest to design small molecules as specific probes of bulge function. On the basis of our earlier work with the specific DNA bulge-binding metabolite obtained from the enediyne antitumor antibiotic neocarzinostatin chromophore (NCS-chrom), we have prepared three small helical spirocyclic molecules that most closely mimic the natural product. These wedge-shaped molecules resemble the natural product in having the sugar residue attached to the same five-membered ring system. In one instance, the sugar is aminoglucose in beta-glycosidic linkage, and in the other, two enantiomers have the natural sugar N-methylfucosamine in alpha-glycosidic linkage. All three analogues were found to interfere with bulge-specific cleavage by NCS-chrom and the ability of bulged DNA to serve as a template for DNA polymerase 1 in accordance with their binding affinities for DNA containing a two-base bulge. Comparable results were obtained with the analogues for the less efficiently cleaved three-base bulge DNA structures. In each situation, the enantiomers possessing the natural sugar in alpha-glycosidic linkage are the most potent inhibitors of the cleavage reaction. In the DNA polymerase reactions, again, the closest natural product mimics were the most effective in selectively impeding nucleotide extension at the bulge site, presumably by complex formation. These results demonstrate the potential usefulness of bulge-binding compounds in modifying DNA structure and function and support efforts to design and prepare reactive species of these molecules that can covalently modify bulged DNA.

Scheme 1

Proposed mechanism for base-catalyzed NCS-chrom activation and DNA damage.

Scheme 2

Illustration of strand scission by NCS-chrom at a 2-base bulge in a DNA duplex. Arrow points to the target T. Cleavage results in two fragments: (a) having ³²P label at its 5’ end and phosphate at the 3’ end and (b) the unlabeled product, has a nucleoside 5’ aldehyde.

Figure 1

Structures NCS-chrom metabolite 1 (spirolactone 1 in Scheme 1) and synthetic analogues of 1. 1 has N-methylfucosamine in α- glycosidic linkage; 2−4 are synthetic analogues of 1. Analogue 2 has aminoglucose in β- glycosidic linkage; 3 and 4 have N-methylfucosamine in α− glycosidic linkage.

Figure 2

Effect of compound 4 on strand scission at a 2-base bulge in a DNA duplex by NCS-chrom. In a standard cleavage reaction (40 min) containing the bulge duplex 1 of Table 1 (5.5 μM) having 5’-³²P-end label on the bulge strand was treated with NCS-chrom in the absence and presence of varying amounts of 4 as described in Materials and Methods. A gel analysis profile of the products is shown. Lane 1, control duplex DNA without any treatment; lanes 2 and 3, duplicates of DNA treated with only NCS-chrom (15 μM)) ; lanes 4−8, with NCS-chrom in the presence of 4 at concentrations of 10, 20, 40, 80, and 166 μM, respectively. Lanes T+C and G+A show Maxam-Gilbert markers made from the 5’-³²P-end-labeled bulge strand. Arrow points to the target site T8.

Figure 3

Time course of inhibition of NCS-chrom-induced strand scission at the bulge by 4. In experiments similar to those in Figure 2, bulge duplex 2 of Table 1(10 μM) having 5’-³²P-end label on the 10-mer strand was treated with NCS-chrom (21 μM). When present, 4 was at a concentration of 150 μM. At times indicated, aliquots were withdrawn to stop the reaction. After gel separation of the products, the gel band intensities were quantitated.

Figure 4

Dose response for analogues 2, 3, and 4 in the inhibition of bulge-specific cleavage by NCS-chrom (20 μM): Strand cleavage reactions similar to those in Figure 3 were performed using 5’-³²P-end-labeled bulge duplex in the absence and presence of varying amounts of the indicated analogues. After separation of the products on a sequencing gel, the band intensities were quantitated.

Figure 5

Effect of 4 on strand scission at 3-base bulges in DNA. Duplexes 3 and 4 (5 μM each) of Table 1 having ³²P label on the 5’ end of the bulge strand were treated for 30 min with NCS-chrom (45 μM) in the absence and presence of compound 4 (67 μM). A gel profile of the reaction products is shown. Lanes 1−7 represent DNA duplex 3. Lane 3, control DNA without any addition; lanes 4 and 5, duplicates of DNA treated with NCS-chrom; lanes 6 and 7, duplicates of DNA treated with NCS-chrom in the presence of compound 4. Lanes 8−14 represent DNA duplex 4. Lane 8, control DNA without any addition; lanes 9 and 10, DNA treated with NCS-chrom; lanes 11 and 12, DNA treated with NCS-chrom in the presence of compound 4. Lanes T+C and G+A show Maxam-Gilbert markers made from the 5’-³²P-end labeled bulge strands. Arrows indicate the bulge site of cleavage.

Figure 6

Putative folding pattern of: A. 31-mer (Table 1, entry 9) and B. 29-mer (Table 1, entry 10). Arrow and the bold letters indicate the 2-base bulge. C is the 10-mer primer that was annealed to the 3’-end of the 31-mer and the 29-mer in polymerase reactions.

Figure 7

Effect of 4 on DNA polymerase-dependent primer extension (time course) on a template containing a putative 2-base bulge. 5’-³²P-end-labeled 10-mer primer was annealed to the 31-mer (Figure 6A). Its extension by DNA polymerase at various times was followed, in the absence and presence of 4 (100 μM) as described in Materials and Methods. Samples were analyzed on a sequencing gel. Lanes 3, 4, 5 and 9, 10, 11 are duplicate controls lacking the test compound and lanes 6, 7 and 8 represent reactions containing the test compound at 15, 45 and 90 min of incubation, respectively. Arrows indicate the length of the synthesis products at the bulge region of the template.

Figure 8

Effect of 4 on primer extension on a DNA template containing or lacking a bulge. In experiments similar to those in Figure 7, 5’-³²P-end-labeled 10-mer was extended on the 31-mer template (lanes 1−4) and on the 29-mer derived from it by deletion of its 2-base bulge (lanes 5−8). Lanes 1 and 2 are control reactions for 1 h and 2 h respectively; lanes 3 and 4, reactions in the presence of test compound 4 for 1 h and 2 h respectively. Similarly, lanes 5 and 6 have control reactions for 1 h and 2 h, and lanes 7 and 8 represent those with the test compound for 1 h and 2 h respectively. Compound 4 was present at a level of 100 μM. Arrows indicate the bulge region containing products of length 21−23 nucleotides.