Antisense and Antigene Applications

Originally conceived as agents for double-stranded DNA binding, the unique properties of PNAs as a DNA mimics were first exploited for gene therapy drug design. PNAs can inhibit transcription (antigene) and transcription (antisense) of genes by tight binding to DNA or mRNA.

Peptide nucleic acid-mediated inhibition of gene transcription is mainly due to the formation of invaded strand complexes or strand displacement in a DNA target. Several in vitro studies have shown that the binding of PNA or bis-PNA to dsDNA can efficiently block tran-scriptional elongation and inhibit the binding of tran-scriptional factors and helicases.[3] PNA targeted against the promoter region of a gene can form stable PNA-DNA complexes that restrict the DNA access of the polymerase, whereas PNA complexes located far from the promoter can block the polymerase progression and lead to the production of truncated RNA transcripts. Nielsen et al.[4] have demonstrated that even an 8-mer PNA can efficiently block transcriptional elongation by (PNA)2-DNA triplex formation.

Peptide nucleic acids are able to interact with mRNA independently of the RNA secondary structure. However, unlike other antisense agents, PNA-RNA duplexes are not recognized by RNase-H. Studies on the mechanisms of antisense activity have demonstrated that PNA inhibits expression differently than antisense oligonucleotides acting through RNase-H-mediated degradation of the mRNA-oligonucleotide hybrid. Because PNAs are not substrates for RNAse, their antisense effect acts through steric interference of either RNA processing, transport into cytoplasm, or translation, caused by binding to the mRNA.

Application of PNAs as antisense reagents was first demonstrated in 1992. The nuclear microinjection of a 15-mer PNA targeting the translation start region of SV40 large T antigen mRNA inhibited transcription in cell extracts.[5] This inhibition was both sequence specific and dose dependent. More recently, Mologni et al.[6] reported the effect of three different types of antisense PNAs on the in vitro expression of PML/RARalpha gene. It was also reported that intron-exon splice junctions are very sensitive targets for PNA antisense probes because correct mRNA splicing can be altered by PNA binding.[7]

Although these in vitro results strongly emphasized the potential of PNAs for antigene and antisense applications, the limiting factors for in vivo use of PNAs are the weak uptake of PNAs by living cells. Several modifications of PNAs have led to significant improvements. Cellular uptake can be speeded up by coupling PNA to DNA oligomers, to receptor ligands, or more efficiently, to peptides such as cell-penetrating peptides that are rapidly internalized by mammalian cells. Reports have demonstrated that PNAs conjugated to such peptides are efficiently taken up by eukaryotic cells.[8,9] Another strategy adapted to improve the in vivo delivery of PNA can be their incorporation into liposomes.

Getting Started With Dumbbells

Getting Started With Dumbbells

The use of dumbbells gives you a much more comprehensive strengthening effect because the workout engages your stabilizer muscles, in addition to the muscle you may be pin-pointing. Without all of the belts and artificial stabilizers of a machine, you also engage your core muscles, which are your body's natural stabilizers.

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