DNA visualization in single molecule studies carried out with optical tweezers: Covalent versus non-covalent attachment of fluorophores

• A new methodology that enables the visualization of single chains of DNA using covalent attachment.
• Covalently bound fluorophores do not significantly modify the mechanical properties of the DNA.
• SYBR gold is a significantly brighter fluorescent probe when bound to DNA.
• The interaction of the groove binder SYBR gold has a large effect on the mechanical properties of DNA.

In this study, we investigated the use of the covalent attachment of fluorescent dyes to double-stranded DNA (dsDNA) stretched between particles using optical tweezers (OT) and compared the mechanical properties of the covalently-functionalized chain to that of unmodified DNA and to DNA bound to a previously uncharacterized groove-binder, SYBR-gold. Modified DNA species were obtained by covalently linking azide-functionalized organic fluorophores onto the backbone of DNA chains via the alkyne moieties of modified bases that were incorporated during PCR. These DNA molecules were then constructed into dumbbells by attaching polystyrene particles to the respective chain ends via biotin or digoxigenin handles that had been pre-attached to the PCR primers which formed the ends of the synthesized molecule. Using the optical tweezers, the DNA was stretched by separating the two optically trapped polystyrene particles. Displacements of the particles were measured in 3D using an interpolation-based normalized cross-correlation method and force-extension curves were calculated and fitted to the worm-like chain model to parameterize the mechanical properties of the DNA. Results showed that both the contour and persistence length of the covalently-modified dsDNAs were indistinguishable from that of the unmodified dsDNA, whereas SYBR–gold binding perturbed the contour length of the chain in a force-dependent manner.