Molecular engineering of Cyanine–Hoechst hybrid materials for nanoscale DNA and chromatin imaging

Abstract Chromatin is a hierarchically organized soft material whose nanoscale structure and heterogeneity regulate essential genomic functions. Resolving this organization requires molecular imaging materials that combine selective DNA binding with photophysical properties compatible with nanoscale localization and energy transfer under biologically relevant conditions. Conventional bisbenzimide (Hoechst) DNA stains provide robust targeting of nuclear DNA but limited applicability for super-resolution imaging. Here, we investigate the modular molecular engineering strategy in which cyanine chromophores (Cyanine3, Cyanine5, or Cyanine7) are covalently integrated with a bisbenzimide DNA-binding motif through an aliphatic spacer to form hybrid fluorescent materials. This design decouples DNA recognition from optical functionality, allowing independent optimization of binding affinity and photophysical performance. In the resulting conjugates, the cyanine units retain their intrinsic brightness and spectral properties, while the bisbenzimide ligand preserves high-affinity minor-groove binding to nuclear DNA. The Cyanine3–Hoechst and Cyanine5–Hoechst hybrids enable high-contrast imaging of nuclear DNA in fixed and permeabilized cells using long-wavelength excitation (>500 nm), with strongly suppressed extranuclear background. The substantial spectral overlap between Cyanine3 emission and Cyanine5 absorption further enables efficient Förster resonance energy transfer within the nuclear environment, providing a route to probe nanoscale proximity and organization in chromatin. Owing to their favorable photophysical stability and brightness, these hybrid materials also support single-molecule localization microscopy, revealing nanostructured features within the nucleus that are not resolved in diffraction-limited images. This approach provides a versatile platform for developing next-generation imaging materials tailored to the study of chromatin as a dynamic soft matter system.