Reversible and Selective Bimolecular Interactions Using yPNA

2019-10-29T20:06:19Z (GMT) by Taylor D. Canady
On-demand selective regulation of gene expression in living cells is a central goal of chemical biology and antisense therapeutic development. While significant advances
have allowed regulatory modulation through inserted genetic elements, on-demand control of the expression/translation state of a given native gene by complementary sequence interactions remains a technical challenge. Toward this objective, in the second chapter, we demonstrate the reversible suppression of a luciferase gene in cell-free translation using Watson-Crick base pairing between the mRNA and a complementary gamma-modified peptide nucleic acid (γPNA) sequence with a non-complementary toehold. Exploiting the favorable thermodynamics
of γPNA–γPNA interactions, the antisense sequence can be removed by hybridization of a second, fully complementary γPNA, through a strand displacement reaction, allowing
translation to proceed. Additionally, we characterize the displacement reaction via surface plasmon resonance (SPR). The third chapter continues the theme of reversible translation control and SPR measurements of strand displacement. However, we do so through the orthogonal
recognition capability of chiral γPNA. In addition to reversible translation control, we characterize the chimeric probe displacement reaction via surface plasmon resonance
(SPR) and demonstrate specific chiral recognition of the chimeric γPNA probes. The fourth chapter explores increasing the selectivity of complementary γPNA by
incorporating intramolecular stem-loop structure. Using SPR, we investigated the selectivity of a structured γPNA against several mutations types and at different target
positions. To this end, we identify several mutations which are kinetically discriminated against compared to an unstructured γPNA control. Additionally, we replicated the
enhanced selectivity findings in an antisense knockdown assay.