Synthesis and Applications of Covalent Protein-DNA Conjugates.

Using maltose-binding protein (MBP) and green fluorescent protein mutant EYFP as a model systems, we demonstrate the feasibility of this approach, as well as the integrity and functionality of the DNA–protein conjugates synthesized.

Synthesis of covalent DNA–protein conjugates by …

Synthesis and Applications of Covalent Protein-DNA Conjugates

Synthesis and Applications of Covalent Protein ..

Protein-oligonucleotide conjugates have been used in a variety of biological and biotechnological applications., Oligodeoxynucleotide (ODN) tags have been attached to proteins in order to encode multiplexed protein libraries, to immobilize proteins to surfaces in the fabrication of protein arrays, , and to identify targets in protein- and immuno-diagnostics via PCR or mass spectroscopy. Conjugating antisense ODNs to proteins has been shown to enhance cellular delivery of the nucleic acid component. In addition, ODNs attached to proteins have been used as architectural tools, as a means of spatially organizing multiple proteins into multimeric assemblies. Different strategies have been used to connect proteins to ODNs, using both noncovalent and covalent chemistries. Synthesizing discrete protein-ODN conjugates via noncovalent (e.g., biotin-streptavidin and nickel-histidine ) interactions is convenient, but the resulting conjugates can reversibly dissociate during purification and subsequent handling. Covalent protein-ODN conjugates have also been synthesized by connecting appropriately functionalized ODNs and proteins, using a wide variety of chemistries. Some of these chemistries involve direct attachment of the ODN to an amino acid residue on the protein surface—usually a lysine amine or a cysteine thiol—while other, more complex schemes require post-translational modification of the protein,, or the involvement of a catalytic or reactive protein domain.

Synthesis and applications of covalent protein-DNA conjugates 2009 ..

Synthetic protein-DNA conjugates are valuable tools with applications in fields including nanobiotechnology, bioanalytical chemistry and molecular diagnostics, and various synthetic methods for their production have been developed during the last three decades. The present article reviews current methodologies for the synthesis of covalent protein-DNA conjugates with particular focus on the regiospecifity and stoichiometry of these reactions.

the design of covalent and non-covalent DNA-protein conjugates, ..
Semisynthetic DNA-protein conjugates are synthesized by either covalent coupling chemistries, or by means of reversible biomolecular recognition systems.

of covalent DNA–protein conjugates ..

In summary, we have demonstrated the utility of SPAAC reaction for site-specific attachment of DNA to two model proteins. The efficiency of the SPAAC reaction was found to be comparable to that of the Cu-catalyzed click reaction. In addition, the SPAAC bioconjugation method described herein proceeds without Cu catalyst and does not affect the activity of the protein and DNA involved. Moreover, in comparison to other fusion protein methodologies, the method described here requires a shorter recognition tag (C-terminal CVIA). We envision that our method can be an excellent alternate route to Cu-catalyzed click reaction for synthesizing myriads of protein-DNA conjugates with structural and functional importance.

16/03/2005 · Semisynthetic DNA–protein conjugates are versatile tools for many applications in bioanalytics and nanobiotechnology

applications, Bioorganic Chemistry ..

Synthetic protein-DNA conjugates are valuable tools with applications in fields including nanobiotechnology, bioanalytical chemistry and molecular diagnostics, and various synthetic methods for their production have been developed during the last three decades. The present article reviews current methodologies for the synthesis of covalent protein-DNA conjugates with particular focus on the regiospecifity and stoichiometry of these reactions.

Schaeffer, P. M. & Dixon, N. E. (2009). Synthesis and applications of covalent protein-DNA conjugates. Australian Journal of Chemistry, 62 (10), 1328-1332.

The developments of semisynthetic DNA-protein conjugates.

Characterization. The DNA-dye conjugates synthesized in the Carell lab will be hybridized to unmodified complementary DNA strands and characterized by optical spectroscopy. First, UV melting experiments will be performed on the duplexes. We expect to find that the melting temperatures of the dye-functionalized duplexes will be higher than that of the corresponding unmodified duplex due to intercalation of the attached dye into the helix. The melting temperature of the unmodified duplex will also be measured in the presence of free thiazole orange. These experiments will help to evaluate which of the different linkers and attachment sites are optimal for permitting intercalation into the DNA helix in preparation for synthesis of the poly-functionalized DNA strands needed to make the covalent nanotags. Fluorescence spectra will also be measured for the DNA-dye conjugates in both single- and double-stranded forms. If the covalently attached TO dye successfully intercalates into the DNA, a large fluorescence enhancement should be observed. Again, comparing the different TO analogues will help to determine the best dye for synthesis of the covalent nanotags. Alternative Nanostructures. In addition to the synthetic efforts described above, experiments were performed to assess alternative nanostructures that would be much brighter than the assemblies tested previously. These included nanogel structures prepared from crosslinked DNA units [3] as well as a tetrahedron assembled from four partially complementary strands [4]. While we found it difficult to control the size of the gels in the first approach, the fluorescent tetrahedra were well behaved. These assemblies exhibited the efficient energy transfer previously reported for the two-dimensional nanotags and also showed significantly enhanced stability to nuclease enzymes. This latter property will be important for applications requiring imaging in complex biological media or intracellular environments. A manuscript is in preparation describing these results. Plans. Our goal for the next year is to characterize the monofunctionalized DNA strands recently synthesized by the Carell group and determine the best dye to use for moving forward. Then, three or four partially complementary strands will be prepared, each having 5 covalently attached dyes. The resulting self-assembled nanostructures will be characterized by gel electrophoresis and fluorescence spectroscopy and then used for labeling polystyrene beads and mammalian cells as done previously. References 1. Benvin, A. L.; et al, J. Am. Chem. Soc. 2007, 129, 2025-2034. 2. Gierlich, J.; et al, Org. Lett. 2006, 8, 3639-3642. 3. Matsuura, K.; et al, Chem. Commun. 2003, 376-377. 4. Goodman, R. P.; et al, Science 2005, 310, 1661-1665.