Sequence-Selective Single-Molecule Alkylation with a Pyrrole–Imidazole Polyamide Visualized in a DNA Nanoscaffold

We demonstrate a novel strategy for visualizing sequence-selective alkylation of target double-stranded DNA (dsDNA) using a synthetic pyrrole–imidazole (PI) polyamide in a designed DNA origami scaffold. Doubly functionalized PI polyamide was designed by introduction of an alkylating agent 1-(chloromethyl)-5-hydroxy-1,2-dihydro-3<i>H</i>-benz­[<i>e</i>]­indole (<i>seco</i>-CBI) and biotin for sequence-selective alkylation at the target sequence and subsequent streptavidin labeling, respectively. Selective alkylation of the target site in the substrate DNA was observed by analysis using sequencing gel electrophoresis. For the single-molecule observation of the alkylation by functionalized PI polyamide using atomic force microscopy (AFM), the target position in the dsDNA (∼200 base pairs) was alkylated and then visualized by labeling with streptavidin. Newly designed DNA origami scaffold named “five-well DNA frame” carrying five different dsDNA sequences in its cavities was used for the detailed analysis of the sequence-selectivity and alkylation. The 64-mer dsDNAs were introduced to five individual wells, in which target sequence AGTXCCA/TGGYACT (XY = AT, TA, GC, CG) was employed as fully matched (X = G) and one-base mismatched (X = A, T, C) sequences. The fully matched sequence was alkylated with 88% selectivity over other mismatched sequences. In addition, the PI polyamide failed to attach to the target sequence lacking the alkylation site after washing and streptavidin treatment. Therefore, the PI polyamide discriminated the one mismatched nucleotide at the single-molecule level, and alkylation anchored the PI polyamide to the target dsDNA

Synthesis and Biological Properties of Highly Sequence-Specific-Alkylating <i>N</i>-Methylpyrrole–<i>N</i>-Methylimidazole Polyamide Conjugates

Four new alkylating <i>N</i>-methylpyrrole-<i>N</i>-methylimidazole (PI) polyamide conjugates (<b>1</b>–<b>4</b>) with seven-base-pair (bp) recognition ability were synthesized. Evaluation of their DNA-alkylating activity clearly showed accurate alkylation at match site­(s). The cytotoxicities of conjugates <b>1</b>–<b>4</b> were determined against six human cancer cell lines, and the effect of these conjugates on the expression levels of the whole human genome in A549 cells were also investigated. A few genes among the top 20 genes were commonly downregulated by each conjugate, which reflects their sequence specificity. Conversely, many of the top 10 genes were commonly upregulated, which may have been caused by alkylation damage to DNA. Moreover, the antitumor activities of the PI polyamide conjugates <b>2</b> and <b>3</b> were investigated using nude mice transplanted with DU145 or A549. The intravenous administration of each liposomal conjugate in water yielded tumor-suppressing effects specifically toward DU145 cells and not A549 cells, which was pertinent to cytotoxicity