Cellular localization and allele-selective inhibition of mutant huntingtin protein by peptide nucleic acid oligomers containing the fluorescent nucleobase [bis-o-(aminoethoxy)phenyl]pyrrolocytosine

Abstract

Peptide nucleic acid (PNA) is a successful DNA/RNA mimic. A major challenge for research is to invent chemically modified PNAs that retain the favorable properties of the parent compound while improving biological recognition. Here, we test modified PNAs containing [bis-o-(aminoethoxy)phenyl]pyrrolocytosine bases designed to engage guanine with an additional hydrogen bond. We observe elevated melting temperatures, localization to cellular compartments, and allele-selective inhibition of mutant huntingtin protein expression.

Figure 1

Structure of [bis-o-(aminoethoxy)phenyl]pyrrolocytosine.

Figure 2

Modified PNAs selectively inhibit mutant (mu) relative to wild-type (wt) HTT expression in fibroblast cell line GM04281. A. Top, western analysis the effects of PNAs I–VI on HTT expression. Bottom, quantitation of inhibition of mutant and wild-type HTT by PNAs I–VI. PNAs were added at 1 μM concentration. B–F. Effects of PNAs II–VI on HTT expression at varied concentrations. Experiments were performed in triplicate. Expression is relative to expression to untreated cells.

Figure 3

Fluorescent microscopy of PNA II in living fibroblasts. PNA was added at 1 μM concentration. A. One day or B. Nine days after PNA transfection. Left, Differential interference contrast microscopy (DIC) image; middle, PNA fluorescent; right, overlay of DIC and fluorescent images. C. PNA was co-localized with endosome marker Transferrin. 1 μM of Htt2 was co-incubated with 25 μg/mL of Transferrin-Alexa Fluor 633 for 15 h in fibroblast cells. Upper left, DIC image; upper right, PNA alone; lower left, transferrin fluorescence; lower right, overlay of PNA and transferrin images.