Inhibition of Gene Expression Inside Cells by Peptide Nucleic Acids:  Effect of mRNA Target Sequence, Mismatched Bases, and PNA Length

Abstract

Genome sequencing has revealed thousands of novel genes, placing renewed emphasis on chemical approaches for controlling gene expression. Antisense oligomers designed directly from the information generated by sequencing are one option for achieving this control. Here we explore the rules governing the inhibition of gene expression by peptide nucleic acids (PNAs) inside cells. PNAs are a DNA/RNA mimic in which the phosphate deoxyribose backbone has been replaced by uncharged linkages. Binding to complementary sequences is not hindered by electrostatic repulsion and is characterized by high rates of association and elevated affinities. Here we test the hypothesis that the favorable properties of PNAs offer advantages for recognition of mRNA and antisense inhibition of gene expression in vivo. We have targeted 27 PNAs to 18 different sites throughout the 5'-untranslated region (5'-UTR), start site, and coding regions of luciferase mRNA. PNAs were introduced into living cells in culture as PNA-DNA-lipid complexes, providing a convenient high throughput method for cellular delivery. We find that PNAs targeted to the terminus of the 5'-UTR are potent and sequence-specific antisense agents. PNAs fifteen to eighteen bases in length were optimal inhibitors. The introduction of one or two mismatches abolished inhibition, and complementary PNAs targeted to the sense strand were also inactive. In striking contrast to effective inhibition by PNAs directed to the terminal region, PNAs complementary to other sites within the 5'-UTR do not inhibit gene expression. We also observe no inhibition by PNAs complementary to the start site or rest of the coding region, nor do we detect inhibition by PNAs that are highly C/G rich and possess extremely high affinities for their target sequences. Our results suggest that PNAs can block binding of the translation machinery but are less able to block the progress of the ribosome along mRNA. The high specificity of antisense inhibition by PNAs emphasizes both the promise and the challenges for PNAs as antisense agents and provides general guidelines for using PNAs to probe the molecular recognition of biological targets inside cells.

The peptide nucleic acids (PNAs), powerful tools for molecular genetics and cytogenetics

“…It was also reported that intron -exon splice junctions were very sensitive targets for PNA antisense probes because correct mRNA splicing can be altered by PNA binding. 15 The good stability of PNA oligomers, their strong binding efficiency and their lack of toxicity at even relatively high concentrations suggests that PNAs could constitute highly efficient compounds for effective antisense/antigene application. However, despite the initial 18 Another strategy adapted to improve the in vivo delivery of PNA can be their incorporation into liposomes.…”

Preparation and Evaluation of 99mTc-Epidermal Growth Factor Receptor (EGFR)-Peptide Nucleic Acid for Visualization of EGFR Messenger RNA Expression in Malignant Tumors

“…PNA has been reported to resist nuclease and protease degradation (29) and can enhance acid specificity and inhibit mRNA expression (30,31). Furthermore, these modifications can increase the flexibility and binding affinities of PNA for its complementary single-stranded PNA, exceeding that of comparable DNAs (28).…”

Molecular Imaging of bcl-2 Expression in Small Lymphocytic Lymphoma Using 111In-Labeled PNA–Peptide Conjugates

“…Relative to DNA and RNA oligonucleotides, these modifications increase the flexibility and binding affinity of PNA to complementary DNA or mRNA. PNAs have been shown to enhance nucleic acid specificity and inhibit protein expression (22,23). However, the major disadvantage of PNA is its poor cellular uptake.…”