RUI: Exploring the Utility of Antiparallel Triplex-forming CNA Dimers as Sequence-specific DNA Major Groove Binding Agents

With this award, the Chemistry of Life Processes Program in the Chemistry Division is funding Dr. Thomas Minehan from California State University, Northridge to design and synthesize hybrid molecules that can recognize DNA sequences. Nucleic acid-like bases are attached to a protein-like (carbamate) polymer to create carbamate nucleic acid (CNA) hybrid molecules that can recognize the sequences of double-stranded DNA. Large dye molecules are added to one end of the CNAs to distinguish DNA from RNA. Finally, an organic molecule is added to the opposite end to create CNA-dye dimers that can recognize DNA sequences that are important in controlling the expression of genes that impact human health, such as cancer. This project trains undergraduate and masters-level graduate students at the host institution in organic synthesis as well as in the biophysical and biochemical techniques to assess the ability of synthetic molecules to affect gene expression. In addition, an outreach program introduces local high school students to summer research in STEM areas. This project explores the development of DNA major-groove binding compounds that can directly compete with transcription factors, activators, and repressors for their binding sites to regulate gene expression by small-molecule therapeutics. The objective of this project is to synthesize hybrid molecules containing carbamate nucleic acids (molecules capable of sequence-specific nucleic acid binding via triplex formation and reverse-Hoogsteen hydrogen bonding) coupled with triphenylmethane dyes (molecules capable of selective binding of the DNA major groove over the RNA major groove) and dimerization moieties. These synthetic monomers and dimers are quantitatively and qualitatively characterized for binding to duplex DNA and RNA using ultraviolet-visible absorption and circular dichroism spectroscopy, isothermal titration calorimetry (ITC), and electrophoretic mobility shift assay (EMSA) analysis. The DNA sequence selectivity and DNA versus RNA binding selectivity of the synthetic compounds are characterized through determination of their dissociation constants (Kd) for binding and enthalpy and entropy of binding, which further guides the redesign of the CNA base for ligand optimization. In addition, the ability of the CNA-dye conjugate dimers to selectively and tightly bind transcription factor consensus sequences and inhibit the binding of c-Myc/Max, AP1, and STAT6 to those sequences in vitro and in vivo are evaluated through ITC, EMSA, and luciferase reporter experiments, shedding light on the therapeutic and biotechnological potential of this class of compounds. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.