Poster abstracts

Poster number 28 submitted by Oliver Munyaradzi

Helical bPNA targeting U-rich RNA/T-rich DNA

Oliver Munyaradzi (Chemistry and Biochemistry, Ohio State University), Dennis Bong (Chemistry and Biochemistry, Ohio State University)

Abstract:
We present helical bifacial peptide nucleic acids (bPNA) that recognize and bind U-rich RNA or T-rich DNA with high affinity and specificity. bPNA is an α-PNA containing lysine residues (K2M) functionalized with two melamines (M) per lysine. Despite previous work from our group demonstrating the ability of bPNA to bind to 6x6 symmetric internal loops containing U/T through formation of U-M-U or T-M-T base triples, enhanced binding affinity still remains to be explored. We hypothesized that lowering the significant entropic cost of triplex formation by pre-organizing the peptide backbone may improve binding affinity. Initial structured bPNAs included α-helical I: (A3K2M)3 and the PP-II helix II: (PPK2M)3. Triplexes containing pre-organized helix I or II and T6C4T6 ssDNA had greater thermal stability (Tm = 60 oC, 61 oC) than those containing unstructured bPNA – (βAK2M)3 (Tm = 57 oC). Although triplexes containing RNA were less stable, those containing helical bPNA were still more stable (Tm = 30 oC for I and II with U6C4U6 vs. 25 oC for unstructured (βAK2M)3.
To reduce the binding footprint to 4x4 symmetric loops, we sought improved binding properties by optimizing display of melamine along the helix face. We synthesized a series of single-turn stapled helical bPNAs containing two K2M residues with varied spacing between them for optimal placement of melamine along the helix face. Triplexes containing stapled K3Z3 (K2MAZAK2MAZ; Z=azido-Ala) and T4C4T4 ssDNA were more stable (Tm = 36.5 oC) than those containing unstapled K3Z3 (Tm = 35.3 oC). The effect of melamine placement along the helix face on the thermodynamics of binding were evaluated by van’t Hoff analysis of melting curves. Binding studies with RNA are underway, while future work will explore inclusion of other nucleobases to extend this work beyond U-rich RNA or T-rich DNA.

Keywords: RNA-binding peptide, stapled, U-rich