Chemistry and Biology of DNA
Synthesis and evaluation of structure-selective DNA ligands
DNA is often regarded as a passive storehouse of genetic information. However, certain DNAs that fold into stable non-duplex structures may actively regulate gene expression and cellular replication. A three-step approach towards understanding the importance of non-duplex DNA is being pursued. First, novel compounds that selectively bind, report, or modify non-duplex DNA structures are being developed by rational design, chemical synthesis and combinatorial optimization. Molecules exhibiting interesting DNA selectivity are then tested in living cells for their ability to regulate gene expression and inhibit telomerase activity. The structure-activity relationships discovered using this approach are used to guide the future design and refinement of new compounds, thereby enriching our repertoire of molecular tools for exploring non-duplex DNA function.
Kinetics and thermodynamics of non-covalent DNA binding interactions
The equilibrium between duplex and G-quadruplex / i-motif structures observed for GC-rich DNA can be shifted by changes in pH, temperature, and by the selective binding of molecules to G-quadruplex or i-motif structures. We are using a variety of biophysical techniques including surface plasmon resonance, circular dichroism, fluorescence, and calorimetry to probe the association/dissociation kinetics and thermodynamics of small molecules that perturb this equilibrium. By shifting this equilibrium towards G-quadruplex / i-motif structures, these molecules may provide a means to down regulate expression of genes containing GC boxes in their promoter regions, including various oncogenes.
Synthesis of interstand cross links in vitro and in living cells
Preparing well-defined DNA-DNA interstrand cross-links (ICLs) is a challenging synthetic problem, and an important prerequisite for studies aimed at deciphering the mechanisms by which ICLs exert their extreme cytoxicity. Two strategies for generating well-defined ICLs that result from exposure to the anti-cancer agents chloroethyl nitrosourea (CENU) and nitrogen mustard are being developed. We use phosphoramidite chemistry to incorporate stable ICL precursors (as modified bases) into single-stranded DNA. Following purification and hybridization to a complementary DNA stand, the ICL precursors are activated for cross-linking by either photolysis or by enzymatic oxidation. This approach is being used to prepare ICL DNA for biophysical and biological studies aimed at revealing the mechanisms by which ICLs can be repaired by certain tissue types.
- “Lanthanide-mediated phosphoester hydrolysis and phosphate elimination from phosphopeptides” Nathan W. Luedtke, Alanna Schepartz, Chem. Commun., 2005, 5426-5428.
- “The DNA and RNA specificity of eilatin Ru(II) complexes as compared to eilatin and ethidium” Nathan W. Luedtke, Judy S. Hwang, Edith C. Glazer, Dali Gut, Moshe Kol, Yitzhak Tor, Nucleic Acids Res., 2003, 5732-5740.
- “Affinity and specificity of aminoglycoside dimers and acridine conjugates to the HIV-1 Rev response element” Nathan W. Luedtke, Qi Liu, Yitzhak Tor, Biochemistry, 2003, 11391-11403.
- “Fluorescence-based methods for evaluating the RNA affinity and specificity of HIV-1 Rev-RRE inhibitors” Nathan W. Luedtke, Yitzhak Tor, Biopolymers, 2003, 103-119.
- “Guanidinoglycosides: A novel family of RNA ligands” Nathan W. Luedtke, Tracy J. Baker, Murray Goodman, Yitzhak Tor, J. Am. Chem. Soc., 2000, 12035-12036.
- “A novel solid-phase assembly for identifying potent and selective RNA ligands” Nathan W. Luedtke, Yitzhak Tor, Angew. Chem. Intl. Ed., 2000, 1788-1790.
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