Recognition of Chromosomal DNA by PNAs

Kaihatsu, Kunihiro; Janowski, Bethany A.; Corey, David R.

doi:10.1016/j.chembiol.2003.09.014

Cited by 81 publications

(76 citation statements)

References 70 publications

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“…They are true DNA mimics, because they form Watson-Crick bonds with DNA and RNA, but are of higher thermal stability than natural duplexes due to the lack of electrostatic repulsion (11). They are also resistant to proteases and nucleases, and thus afford a significantly greater biological stability in culture and in vivo (12).…”

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confidence: 99%

“…A unique aspect of PNAs is that amino acids can be covalently added to the peptide backbone at either end of the sequence of bases. Of particular relevance for the present studies, the PNA/DNA interaction may occur through single-strand invasion (11,13). In this case the PNA, which can be of mixed sequence design, hybridizes with one strand of DNA through Watson-Crick base pairing and simply replaces the other strand of the double helix.…”

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confidence: 99%

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Design of embedded chimeric peptide nucleic acids that efficiently enter and accurately reactivate gene expression in vivo

Chen

Peterson²,

Iancu‐Rubin³

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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Pharmacological treatments designed to reactivate fetal γ-globin can lead to an effective and successful clinical outcome in patients with hemoglobinopathies. However, new approaches remain highly desired because such treatments are not equally effective for all patients, and toxicity issues remain. We have taken a systematic approach to develop an embedded chimeric peptide nucleic acid (PNA) that effectively enters the cell and the nucleus, binds to its target site at the human fetal γ-globin promoter, and reactivates this transcript in adult transgenic mouse bone marrow and human primary peripheral blood cells. In vitro and in vivo DNAbinding assays in conjunction with live-cell imaging have been used to establish and optimize chimeric PNA design parameters that lead to successful gene activation. Our final molecule contains a specific γ-promoter-binding PNA sequence embedded within two amino acid motifs: one leads to efficient cell/nuclear entry, and the other generates transcriptional reactivation of the target. These embedded PNAs overcome previous limitations and are generally applicable to the design of in vivo transcriptional activation reagents that can be directed to any promoter region of interest and are of direct relevance to clinical applications that would benefit from such a need.γ-globin gene reactivation | primary human CD34+ cells

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confidence: 99%

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confidence: 99%

Design of embedded chimeric peptide nucleic acids that efficiently enter and accurately reactivate gene expression in vivo

Chen

Peterson²,

Iancu‐Rubin³

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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“…PNAs hybridize to complementary sequences by Watson-Crick base-pairing and have an outstanding ability to invade double-stranded DNA (1)(2)(3)(4)(5). Reports have appeared suggesting that PNAs can also target duplex DNA inside cells (6)(7)(8).…”

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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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“…Examples of synthetic targeting agents include pyrrole-imidazole polyamides and peptide nucleic acids (19,20). Despite the elegance of these strategies, however, their widespread application has been limited by the need for specialized chemical synthesis methods and by constraints associated with their genomic targeting (i.e., locus specificity) (19,21).…”

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Genomic targeting of epigenetic probes using a chemically tailored Cas9 system

Liszczak

Brown

Kim

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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Recent advances in the field of programmable DNA-binding proteins have led to the development of facile methods for genomic localization of genetically encodable entities. Despite the extensive utility of these tools, locus-specific delivery of synthetic molecules remains limited by a lack of adequate technologies. Here we combine the flexibility of chemical synthesis with the specificity of a programmable DNA-binding protein by using protein trans-splicing to ligate synthetic elements to a nuclease-deficient Cas9 (dCas9) in vitro and subsequently deliver the dCas9 cargo to live cells. The versatility of this technology is demonstrated by delivering dCas9 fusions that include either the small-molecule bromodomain and extra-terminal family bromodomain inhibitor JQ1 or a peptide-based PRC1 chromodomain ligand, which are capable of recruiting endogenous copies of their cognate binding partners to targeted genomic binding sites. We expect that this technology will allow for the genomic localization of a wide array of small molecules and modified proteinaceous materials.

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Recognition of Chromosomal DNA by PNAs

Cited by 81 publications

References 70 publications

Design of embedded chimeric peptide nucleic acids that efficiently enter and accurately reactivate gene expression in vivo

Design of embedded chimeric peptide nucleic acids that efficiently enter and accurately reactivate gene expression in vivo

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

Genomic targeting of epigenetic probes using a chemically tailored Cas9 system

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