Probing Specific Sequences on Single DNA Molecules with Bioconjugated Fluorescent Nanoparticles

TLDR

Nanometer‑sized fluorescent particles covalently linked to DNA‑binding proteins provide brighter, more photostable probes for specific sequences on stretched single DNA molecules than single organic fluorophores. The study demonstrates conjugating the restriction enzyme EcoRI to 20‑nm fluorescent nanoparticles to create nanoconjugates that retain DNA‑binding and cleavage activity. The nanoconjugates were prepared by covalently attaching EcoRI to 20‑nm fluorescent nanoparticles, and single DNA molecules stretched and immobilized on a solid surface were visualized by multicolor fluorescence microscopy to detect nanoparticle binding at specific sites. EcoRI–nanoparticle conjugates bind specific DNA sequences without Mg²⁺ but do not cleave, and their direct visualization on single DNA molecules enables new optical gene‑mapping approaches and fundamental studies of DNA–protein interactions.

Abstract

Nanometer-sized fluorescent particles (latex nanobeads) have been covalently linked to DNA binding proteins to probe specific sequences on stretched single DNA molecules. In comparison with single organic fluorophores, these nanoparticle probes are brighter, are more stable against photobleaching, and do not suffer from intermittent on/off light emission (blinking). Specifically, we demonstrate that the site-specific restriction enzyme EcoRI can be conjugated to 20-nm fluorescent nanoparticles and that the resulting nanoconjugates display DNA binding and cleavage activities of the native enzyme. In the absence of cofactor magnesium ions, the EcoRI conjugates bind to specific sequences on double-stranded DNA but do not initiate enzymatic cutting. For single DNA molecules that are stretched and immobilized on a solid surface, nanoparticles bound at specific sites can be directly visualized by multicolor fluorescence microscopy. Direct observation of site-specific probes on single DNA molecules opens new possibilities in optical gene mapping and in the fundamental study of DNA−protein interactions.