Intracellular Uptake and Inhibition of Gene Expression by PNAs and PNA−Peptide Conjugates

Kaihatsu, Kunihiro; Huffman, Kenneth E.; Corey, David R.

doi:10.1021/bi048519l

Cited by 71 publications

(57 citation statements)

References 33 publications

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“…Moreover, PNAs are resistant to both nucleases and proteases, and their neutral backbone increases their hybridization affinities to complementary RNA and DNA strands [65]. PNAs have been used successfully to target chromosomal DNA at transcription start sites to inhibit gene expression in cells [66][67][68]. In LNA molecules, the deoxyribose moiety is modified by introducing a methylene bridge between the 2'-O, 4'-O, and 4'-C.…”

Section: Chemical Modifications Of Tfosmentioning

confidence: 99%

DNA triple helices: Biological consequences and therapeutic potential

Jain

Wang²,

Vásquez³

2008

Biochimie

252

199

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DNA structure is a critical element in determining its function. The DNA molecule is capable of adopting a variety of non-canonical structures, including three-stranded (i.e. triplex) structures, which will be the focus of this review. The ability to selectively modulate the activity of genes is a longstanding goal in molecular medicine. DNA triplex structures, either intermolecular triplexes formed by binding of an exogenously applied oligonucleotide to a target duplex sequence, or naturally occurring intramolecular triplexes (H-DNA) formed at endogenous mirror repeat sequences, present exploitable features that permit site-specific alteration of the genome. These structures can induce transcriptional repression and site-specific mutagenesis or recombination. Triplex-forming oligonucleotides (TFOs) can bind to duplex DNA in a sequence specific fashion with high affinity, and can be used to direct DNA-modifying agents to selected sequences. H-DNA plays important roles in vivo and is inherently mutagenic and recombinogenic, such that elements of the H-DNA structure may be pharmacologically exploitable. In this review we discuss the biological consequences and therapeutic potential of triple helical DNA structures. We anticipate that the information provided will stimulate further investigations aimed toward improving DNA triplexrelated gene targeting strategies for biotechnological and potential clinical applications.

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Section: Chemical Modifications Of Tfosmentioning

confidence: 99%

DNA triple helices: Biological consequences and therapeutic potential

Jain

Wang²,

Vásquez³

2008

Biochimie

252

199

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“…Whether such endosomes then release the PNA into the cytosol, because of a ''proton sponge'' effect of the Lys residues, in order to travel to another cytosolic compartment, or whether there is a fusion of PNAcontaining endosomes with the microRNA-containing cytosolic compartments must be the subject of further experimentation. MicroRNA may be a more susceptible cytosolic target than mRNA, since a PNA complementary to human caveolin-1 mRNA conjugated to a Lys-rich signal peptide was unable to reduce gene expression after 56 h incubation in HeLa cells or primary endothelial cells (Kaihatsu et al 2004), and additional Lys residues were shown also not to be sufficient to obtain nuclear activity for PNA (Turner et al 2005b;Abes et al 2007). …”

Section: Discussionmentioning

confidence: 99%

miR-122 targeting with LNA/2′-O-methyl oligonucleotide mixmers, peptide nucleic acids (PNA), and PNA–peptide conjugates

Fabani¹,

Gait²

2007

RNA

241

236

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MicroRNAs are small noncoding RNAs that regulate many cellular processes in a post-transcriptional mode. MicroRNA knockdown by antisense oligonucleotides is a useful strategy to explore microRNA functionality and as potential therapeutics. MicroRNA-122 (miR-122) is a liver-specific microRNA, the main function of which has been linked with lipid metabolism and liver homeostasis. Here, we show that lipofection of an antisense oligonucleotide based on a Locked Nucleic Acids (LNA)/29-Omethyl mixmer or electroporation of a Peptide Nucleic Acid (PNA) oligomer is effective at blocking miR-122 activity in human and rat liver cells. These oligonucleotide analogs, evaluated for the first time in microRNA inhibition, are more effective than standard 29-O-methyl oligonucleotides in binding and inhibiting microRNA action. We also show that microRNA inhibition can be achieved without the need for transfection or electroporation of the human or rat cell lines, by conjugation of an antisense PNA to the cell-penetrating peptide R 6 -Penetratin, or merely by linkage to just four Lys residues, highlighting the potential of PNA for future therapeutic applications as well as for studying microRNA function.

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“…This is evident from microscopy of live cells that show a punctate distribution of fluorescently labeled PNA and studies that show co-localization of PNAs with endosomal markers (13,(20)(21)(22)(23)(24). One solution, therefore, is to increase the amount of PNA released from endosomes.…”

Section: Improving the Potency Of Agpnasmentioning

confidence: 99%

Inhibiting Gene Expression with Peptide Nucleic Acid (PNA)−Peptide Conjugates That Target Chromosomal DNA

Corey

2007

Biochemistry

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Peptide nucleic acids (PNAs) are a nonionic DNA/RNA mimic that can recognize complementary sequences by Watson-Crick base-pairing. The neutral PNA backbone facilitates recognition of duplex DNA by strand invasion, suggesting that antigene PNAs (agPNAs) can be important tools for exploring the structure and function of chromosomal DNA inside cells. However, before agPNAs can enter wide use it will be necessary to develop straightforward strategies for introducing them into cells. Here we demonstrate that agPNA-peptide conjugates can target promoter DNA and block progesterone receptor (PR) gene expression inside cells. Thirty-six agPNA-peptide conjugates were synthesized and tested. We observed inhibition of gene expression using cationic peptides containing either arginine or lysine residues, with eight or more cationic amino acids being preferred. Both thirteen and nineteen base agPNA-peptide conjugates were inhibitory. Inhibition was observed in human cancer cell lines expressing either high or low levels of progesterone receptor. Modification of agPNA-peptide conjugates with hydrophobic amino acids or small molecule hydrophobic moities yielded improved potency. Inhibition by agPNAs did not require cationic lipid or any other additive, but adding agents to cell growth media that promote endosomal release caused modest increases in agPNA potency. These data demonstrate that chromosomal DNA is accessible to agPNA-peptide conjugates and that chemical modifications can improve potency.Peptide nucleic acids 1 (PNAs) are a class of DNA/RNA mimic with an uncharged amide backbone (1). PNAs hybridize to complementary sequences by Watson-Crick base-pairing and have an outstanding ability to invade double-stranded DNA (1-5). Reports have appeared suggesting that PNAs can also target duplex DNA inside cells (6)(7)(8). Recently, we reported that antigene PNAs (agPNAs) that target chromosomal DNA at transcription start sites inhibit gene expression (9). These data suggest that PNAs may be valuable tools for exploring promoter function and for controlling gene expression at the level of the chromosome.For our initial experiments with agPNAs, we delivered them into cultured human cells in complex with complementary DNA oligonucleotides and cationic lipid (9,10). This method is a variation of standard protocols for lipid-mediated transfection. The DNA binds to the PNA, the lipid binds to the DNA, and the PNA is transported into cells as cargo by the DNA/lipid complex.This method has worked well and can lead to potent inhibition of gene expression in the presence of nanomolar concentrations of agPNA (9,10). However, the combination of steps (annealing DNA and PNA, lipid transfection) is likely to discourage full exploitation of the substantial potential of agPNAs as tools for probing chromosomal DNA in cell culture. Animal * To whom correspondence should be addressed. Email: david.corey@utsouthwestern MATERIALS AND METHODS Synthesis of PNA-Peptide ConjugatesPNA-peptide conjugates were synthesized as previously describe...

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Intracellular Uptake and Inhibition of Gene Expression by PNAs and PNA−Peptide Conjugates

Cited by 71 publications

References 33 publications

DNA triple helices: Biological consequences and therapeutic potential

DNA triple helices: Biological consequences and therapeutic potential

miR-122 targeting with LNA/2′-O-methyl oligonucleotide mixmers, peptide nucleic acids (PNA), and PNA–peptide conjugates

Inhibiting Gene Expression with Peptide Nucleic Acid (PNA)−Peptide Conjugates That Target Chromosomal DNA

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