Sequence-Specific Minor\nGroove Binders in Labeling\nand Single-Molecule Analysis of DNA

The ability to address specific sequences\nwithin DNA\nis of tremendous\ninterest in biotechnology and biomedicine. Various technologies have\nbeen established over the past few decades, such as nicking enzymes\nand methyltransferase-directed sequence-specific labeling, transcription\nactivator-like effector nucleases (TALENs), the CRISPR-Cas9 system,\nand polyamides of heterocycles as sequence-specific DNA minor groove\nbinders. Pyrrole-imidazole polyamides have been reported to recognize\npredetermined DNA sequences, and some successful attempts have demonstrated\ntheir potential in regulating gene expression. However, few studies\non single-molecule labeling and analysis of DNA have been explored,\nparticularly at single-targeting sites. In this study, we rationally\ndesigned and synthesized a set of functional minor groove binders,\nvarying in structures, sequence information addressed, and methods\nof dye introduction. Their potential for sequence-specific labeling\nand single-molecule DNA analysis was evaluated through chromatographic\nand on-surface optical assays. First results indicated that, while\nthey yielded excellent imaging output, the labeling specificity of\nthe hairpin polyamides for single-molecule use was hindered by single-mismatch\nsites. To address this issue, and in an unprecedented approach, we\ndevised a competitive binding strategy that utilizes ethidium bromide\nas a nonspecific binder to competitively block the mismatches, significantly\nenhancing the labeling specificity. These findings provide valuable\ninsights into the use of hairpin polyamides as sequence-programmable\nand enzyme-free DNA labelers in the field of sequence-specific DNA\nrecognition and modification.