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.
The identification of binding sites for small molecules in genomic DNA is important in various applications. Previously, we demonstrated rapid transcriptional activation by our small molecule SAHA-PIP. However, it was not clear whether the strong biological effects exerted by SAHA-PIP were attributable to its binding specificity. Here, we used high-throughput sequencing (Bind-n-seq) to determine the binding specificity of SAHA-PIPs. Sequence specificity bias was determined for SAHA-PIPs (3 and 4), and this showed enhanced 6 bp sequence-specific binding compared with hairpin PIPs (1 and 2). This finding allowed us to investigate the role of the β-alanine that links SAHA to PIP, and led in turn to the design of ββ-PIPs (5 and 6), which showed enhanced binding specificity. Overall, we demonstrated the importance of β-moieties for the binding specificity of PIPs and the use of cost-effective high-throughput screening of these small molecules for binding to the DNA minor groove.
Ultraviolet (UV) radiation causes cellular DNA damage, among which cyclobutane pyrimidine dimers (CPDs) are responsible for a variety of genetic mutations. Although several approaches have been developed for detection of CPDs, conventional methods require time-consuming steps. Aquaphotomics, a new approach based on near-infrared spectroscopy (NIRS) and multivariate analysis that determines interactions between water and other components of the solution, has become an effective method for qualitative and quantitative parameters measurement in the solutions. NIR spectral patterns of UVC-irradiated and nonirradiated DNA solutions were evaluated using aquaphotomics for detection of UV-induced CPDs. Groups of UV-irradiated and nonirradiated DNA samples were classified (87.5% accuracy) by soft independent modeling of class analogy (SIMCA). A precise regression model calculated from NIR water spectral patterns based on UVC doses (r Val = 0.9457) and the concentration of cis-syn cyclobutane thymine dimers (cis-syn T<>Ts; r Val = 0.9993) was developed using partial least squares regression (PLSR), while taking advantage of water spectral patterns, particularly around 1400–1500 nm. Our results suggested that, in contrast to DNA, the formation of cis-syn T<>Ts increased the strongly hydrogen bonded water. Additionally, NIRS could qualitatively and quantitatively detect cis-syn T<>Ts in isolated DNA aqueous solutions upon UVC exposure.
Tet family proteins have the ability to convert 5-methylcytosine (mC) to 5-hydroxymethylcytosine, and further to 5-formylcytosine and 5-carboxycytosine. We found that CGmCGCG can be the substrate of Tet protein, and observed iterative oxidation of mC by HPLC analysis. We also demonstrated that Tet protein favours single-stranded DNA over double-stranded DNA.
In a previous study, we found that 2-deoxyribonolactone is effectively generated in the specific 5-bromouracil (BrU)-substituted sequence 5′-(G/C)[A]n
= 1,2BrUBrU-3′ and proposed that a formed uracil-5-yl radical mainly abstracts the C1′ hydrogen from the 5′-side of BrUBrU under 302-nm irradiation condition. In the present work, we performed photoirradiation of BrU-substituted DNA in the presence of a hydrogen donor, tetrahydrofuran, to quench the uracil-5-yl radical to uracil and then subjected the sample to uracil DNA glycosylase digestion. Slab gel sequence analysis indicated that uracil residues were formed at the hot-spot sequence of 5′-(G/C)[A]n
= 1,2BrUBrU-3′ in 302-nm irradiation of BrU-substituted DNA. Furthermore, we found that the uracil residue was also formed at the reverse sequence 5′-BrUBrU[A]n
= 1,2(G/C)-3′, which suggests that both 5′-(G/C)[A]n
= 1,2BrUBrU-3′ and 5′-BrUBrU[A]n
= 1,2(G/C)-3′ are hot-spot sequences for the formation of the uracil-5-yl radical.
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