Conjugation of fluorescent proteins with DNA oligonucleotides.

This work describes the synthesis of covalent ssDNA conjugates of six fluorescent proteins, ECFP, EGFP, E(2)GFP, mDsRed, Dronpa, and mCherry, which were cloned with an accessible C-terminal cystein residue to enable site-selective coupling using a heterobispecific cross-linker. The resulting conjugates revealed similar fluorescence emission intensity to the unconjugated proteins, and the functionality of the tethered oligonucleotide was proven by specific Watson-Crick base pairing to cDNA-modified gold nanoparticles. Fluorescence spectroscopy analysis indicated that the fluorescence of the FP is quenched by the gold particle, and the extent of quenching varied with the intrinsic spectroscopic properties of FP as well as with the configuration of surface attachment. Since this study demonstrates that biological fluorophores can be selectively incorporated into and optically coupled with nanoparticle-based devices, applications in DNA-based nanofabrication can be foreseen.

Single-molecule investigations of a photoswitchable nanodevice.

Due to the specificity of Watson-Crick base pairing, DNA is an excellent molecule for the fabrication of nanostructures. It has been shown that DNA can be used as a scaffold for positioning proteins and synthetic molecules with nanometer accuracy. As the next step in adding complexity and functionality to these nanodevices, optical addressability is incorporated. The fluorescent protein Dronpa, which can be optically switched between a fluorescent state and a dark state, is mounted on a DNA scaffold in the proximity of a synthetic fluorophore. Hence, the system can be optically switched between the dark state and an optically active state that undergoes Förster resonance energy transfer. As nanodevices operate as individual units, the functionality of the device is analyzed using single-molecule microscopy. The physical characteristics of nanodevices make them well suited as probes for investigating cellular processes or as shuttles for gene therapy. Hence, the functionality of the nanodevice is verified in the context of cellular measurements.

DNA-directed assembly of supramolecular fluorescent protein energy transfer systems.

Fluorescent proteins with a wide variety of physicochemical properties have evolved in the past few years. The use of these proteins for applications in biomolecular nanosciences requires their precise positioning at the nanometer length scale. To address this challenge, we report here on the self-organization of DNA-tagged fluorescent probes to construct a set of photofunctional supramolecular complexes which include the enhanced yellow fluorescent protein (EYFP). The optical functionality is based on the strongly distance dependent fluorescence resonance energy transfer (FRET), occurring between the donor (EYFP) and an acceptor fluorophore, i.e., the fluorescent dye Atto647. The photophysical properties of four bimolecular FRET complexes, each possessing a well-defined donor-acceptor distance defined by the length of the interconnecting DNA backbone, are investigated by two-dimensional photoluminescence excitation spectroscopy (2D-PLE).

A single-molecule Förster resonance energy transfer analysis of fluorescent DNA-protein conjugates for nanobiotechnology.

The development of nanobiotechnological devices requires the ability to build various components with nanometer accuracy. DNA is a well-established nanoscale building block that self assembles due to specific interactions that are encoded in its sequence. Recently, it has become possible to couple proteins to DNA, thereby expanding the capabilities of DNA for use with molecular photonics and bioelectronics. Here, we present the design and characterization of a supramolecular Förster resonance energy transfer (FRET) system by using a fluorescent protein bound to single-stranded DNA (ssDNA), a fluorophore attached to a second ssDNA molecule, and a complementary strand for hybridizing the two fluorophores together. The FRET efficiency was studied by using both ensemble and single-pair FRET measurements. The distance between the two fluorophores was determined from the single-pair FRET efficiency and could be described by a simple cylindrical model for the DNA. Hence, DNA can be used as a scaffold for positioning fluorescent proteins, as well as traditional fluorophores, with nanometer accuracy and shows great potential for use in the future of nanobiotechnology.

Synthesis of fluorescent oligonucleotide--EYFP conjugate: towards supramolecular construction of semisynthetic biomolecular antennae.

A novel species of DNA--protein conjugate was synthesized by chemically linking DNA oligonucleotides to Aequorea victoria green fluorescent protein mutant EYFP. An additional cysteine was added to the C-terminus of the EYFP by genetic engineering and used to covalently attach amino-modified oligonucleotide with the aid of the heterobifunctional crosslinker sSMCC. EYFP maintained its fluorescence upon conjugation. The oligonucleotide provides an additional binding site to the fluorescent protein, and hence, the EYFP conjugate could be specifically hybridized with both complementary DNA-protein conjugates in-solution as well as immobilized at capture oligonucleotides attached to a solid substrate. These studies are paving the way for future applications in the self-assembly of photoactive supramolecular complexes, such as artificial light-harvesting systems.

Covalent coupling of DNA oligonucleotides and streptavidin.

Semisynthetic DNA-protein conjugates are synthesized by covalent coupling of thiol-modified DNA oligonucleotides and streptavidin. The resulting conjugates have a binding capacity for four equivalents of biotin and one nucleic acid of complementary sequence. The conjugates are purified to homogeneity by ultrafiltration and chromatography and characterized by photometry and gel electrophoresis. Subsequently, the conjugates are applied as molecular linkers in the DNA-directed immobilization of a biotinylated enzyme on a microplate, containing complementary capture oligonucleotides.