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Given that our knowledge of DNA repair is limited because of the complexity of the DNA system, a technique called UVA micro-irradiation has been developed that can be used to visualize the recruitment of DNA repair proteins at double-strand break (DSB) sites. Interestingly, Hoechst 33258 was used under micro-irradiation to sensitize 5-bromouracil (BrU)-labelled DNA, causing efficient DSBs. However, the molecular basis of DSB formation under UVA micro-irradiation remains unknown. Herein, we investigated the mechanism of DSB formation under UVA micro-irradiation conditions. Our results suggest that the generation of a uracil-5-yl radical through electron transfer from Hoechst 33258 to BrU caused DNA cleavage preferentially at self-complementary 5′-AABrUBrU-3′ sequences to induce DSB. We also investigated the DNA cleavage in the context of the nucleosome to gain a better understanding of UVA micro-irradiation in a cell-like model. We found that DNA cleavage occurred in both core and linker DNA regions although its efficiency reduced in core DNA.

Introducing novel building blocks to solid-phase peptide synthesis, we readily synthesized long-chain hairpin pyrrole-imidazole (PI) polyamide-chlorambucil conjugates 3 and 4 via the introduction of an amino group into a GABA (γ-turn) contained in 3, to target CAG/CTG repeat sequences, which are associated with various hereditary disorders. A high-resolution denaturing polyacrylamide sequencing gel revealed sequence-specific alkylation both strands at the N3 of adenines or guanines in CAG/CTG repeats by conjugates 3 and 4, with 11bp recognition. In vitro transcription assays using conjugate 4 revealed that specific alkylation inhibited the progression of RNA polymerase at the alkylating sites. Chiral substitution of the γ-turn with an amino group resulted in higher binding affinity observed in SPR assays. These assays suggest that conjugates 4 with 11bp recognition has the potential to cause specific DNA damage and transcriptional inhibition at the alkylating sites.

5-Bromouracil ( Br U) was incorporated into three types of synthetic RNA and the products of the photoirradiated Br U-containing RNAs were investigated using HPLC and MS analysis. The photoirradiation of r(GCA Br UGC) 2 and r(CGAA Br UUGC)/r(GCAAUUCG) in A-form RNA produced the corresponding 2′-keto adenosine ( keto A) product at the 5′-neighboring nucleotide, such as r(GC keto AUGC) and r(CGA keto AUUGC), respectively. The photoirradiation of r(CGCG Br UGCG)/r(C m GCAC m GCG) in Z-form RNA produced the 2′-keto guanosine ( keto G) product r(CGC keto GUGCG), whereas almost no products were observed from the photoirradiation of r(CGCG Br UGCG)/r(C m GCAC m GCG) in A-form RNA. The present results indicate clearly that hydrogen (H) abstraction by the photochemically generated uracil-5-yl radical selectively occurs at the C2′ position to provide a 2′-keto RNA product.