Sequence-Specific Minor Groove Binders in Labeling and Single-Molecule Analysis of DNA

The ability to address specific sequences within DNA is of tremendous interest in biotechnology and biomedicine. Various technologies have been established over the past few decades, such as nicking enzymes and methyltransferase-directed sequence-specific labeling, transcription activator-like effector nucleases (TALENs), the CRISPR-Cas9 system, and polyamides of heterocycles as sequence-specific DNA minor groove binders. Pyrrole-imidazole polyamides have been reported to recognize predetermined DNA sequences, and some successful attempts have demonstrated their potential in regulating gene expression. However, few studies on single-molecule labeling and analysis of DNA have been explored, particularly at single-targeting sites. In this study, we rationally designed and synthesized a set of functional minor groove binders, varying in structures, sequence information addressed, and methods of dye introduction. Their potential for sequence-specific labeling and single-molecule DNA analysis was evaluated through chromatographic and on-surface optical assays. First results indicated that, while they yielded excellent imaging output, the labeling specificity of the hairpin polyamides for single-molecule use was hindered by single-mismatch sites. To address this issue, and in an unprecedented approach, we devised a competitive binding strategy that utilizes ethidium bromide as a nonspecific binder to competitively block the mismatches, significantly enhancing the labeling specificity. These findings provide valuable insights into the use of hairpin polyamides as sequence-programmable and enzyme-free DNA labelers in the field of sequence-specific DNA recognition and modification.