title: DNA visualization in single molecule studies carried out with optical tweezers: Covalent versus non-covalent attachment of fluorophores.
abstract: 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.
authors.affiliation: Institute of Fundamental Sciences, Massey University, Palmerston North 4442, New Zealand; MacDiarmid Institute for Advanced Materials and Nanotechnology, Wellington 6140, New Zealand.
authors.forename: Sandy
authors.initials: S
authors.lastname: Suei
authors.affiliation: Institute of Fundamental Sciences, Massey University, Palmerston North 4442, New Zealand.
authors.forename: Allan
authors.initials: A
authors.lastname: Raudsepp
authors.affiliation: Institute of Fundamental Sciences, Massey University, Palmerston North 4442, New Zealand; Riddet Institute, Massey University, Palmerston North 4442, New Zealand.
authors.forename: Lisa M
authors.initials: LM
authors.lastname: Kent
authors.affiliation: Institute of Fundamental Sciences, Massey University, Palmerston North 4442, New Zealand; MacDiarmid Institute for Advanced Materials and Nanotechnology, Wellington 6140, New Zealand.
authors.forename: Stephen A J
authors.initials: SA
authors.lastname: Keen
authors.affiliation: Institute of Fundamental Sciences, Massey University, Palmerston North 4442, New Zealand.
authors.forename: Vyacheslav V
authors.initials: VV
authors.lastname: Filichev
authors.affiliation: Institute of Fundamental Sciences, Massey University, Palmerston North 4442, New Zealand; MacDiarmid Institute for Advanced Materials and Nanotechnology, Wellington 6140, New Zealand; Riddet Institute, Massey University, Palmerston North 4442, New Zealand. Electronic address: m.williams@massey.ac.nz.
authors.forename: Martin A K
authors.initials: MA
authors.lastname: Williams
mesh_terms: D004247:DNA / Q000032:analysis*; D005456:Fluorescent Dyes / Q000737:chemistry*; D052898:Optical Tweezers; D016133:Polymerase Chain Reaction
chemical_list: D005456:Fluorescent Dyes; D004247:DNA
keywords: DNA; Fluorescent visualization; Mechanical properties; Optical tweezers; Single molecules