Peptide Nucleic Acid (PNA) Hybridization to Nucleic Acid Targets




Nulf, Christopher J.

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Peptide nucleic acid (PNA) is a DNA/RNA mimic that offers many advantages for hybridization to nucleic acid targets. The simple premise of Watson-Crick base-pairing presents PNAs with a number of diverse applications ranging fromnanotechnology to antisense therapeutics. I studied the synthesis and characterization of novel tethered PNA molecules (bisPNAs) designed to assemble two individual DNA molecules through Watson-Crick base pairing. The spacer regions linking the PNAs were varied in length and contained amino acids with different electrostatic properties. I observed that bisPNAs effectively assembled oligonucleotides that were either the exact length of the PNA or that contained overhanging regions that projected outwards. In contrast, DNA assembly was much less efficient if the oligonucleotides contained overhanging regions that projected inwards. Surprisingly, the length of the spacer region between the PNA sequences did not greatly affect the efficiency of DNA assembly. Reasons for inefficient assembly of inward projecting DNA oligonucleotides include non-sequence-specific intramolecular interactions between the overhanging region of the bisPNA and steric conflicts that complicate binding of two inward projecting strands. These results suggested that bisPNA molecules can be used for self-assembling DNA nanostructures. The Hepatitis C Virus (HCV) RNA genome contains a conserved tertiary structure known as the internal ribosomal entry site (IRES) necessary for cap-independent translation. I tested the hypothesis that antisense peptide nucleic acid (PNA) and locked nucleic acid (LNA) oligomers can bind IRES sequences and block translation. Using a lipid-mediated approach to introduce antisense PNAs and LNAs into cells, my data suggested that PNAs and LNAs could inhibit HCV IRES-dependent translation. PNA or LNA oligomers targeting different regions of the HCV IRES demonstrated a sequencespecific dose-response inhibition of translation with EC50 values of 50-150 nM. IRESdirected inhibition of gene expression widens the range of mechanisms for antisense inhibition by PNAs and LNAs and may provide further therapeutic lead compounds for the treatment of HCV. It is important to compare and contrast the biological activities of PNAs against other nucleotide analogs. Presented herein are collaborations involving comparisons of my PNAs against siRNA, 2'-O-methoxyethyl RNA, and morpholinos. Antisense PNAs were demonstrated to cause isoform-specific inhibition of protein expression of Caveolin. imilarly, PNAs demonstrated the ability to re-direct splicing activity of Insulin Receptor a-Subunit pre-mRNA. Also, antisense PNAs targeting the Chordino gene demonstrated "knock-down" morphologies similar to "knock-out" mutants in developing Zebrafish embryos. Collectively, these results suggest that PNAs are comparable in function to other oligonucleotide analogs and across many experimental platforms.

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