Inhibiting Gene Expression with Locked Nucleic Acids (LNAs) That Target Chromosomal DNA

Beane, Randall L.; Ram, Rosalyn; Gabillet, Sylvie; Arar, Khalil; Monia, Brett P.; Corey, David R.

doi:10.1021/bi700227g

Cited by 36 publications

(28 citation statements)

References 44 publications

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“…We observed inhibition of PR expression by conjugates 4, 5, and 7 containing eight, ten, or twelve lysines respectively ( Inhibitory agPNA-peptide conjugates 4, 5, and 7 blocked expression of both PR-B and PR-A, a result that had been observed previously with agPNAs delivered into cells in complex with DNA and lipid (9) as well as with siRNAs (duplex RNAs that are complementary to mRNA) or antigene RNAs (agRNAs, duplex RNAs that are complementary to promoter DNA) (29,30). Inhibition of both PR-B and PR-A was also observed by antigene locked nucleic acid (LNA) oligomers (31). These data reveal a linkage between reduced expression of PR-B and PR-A regardless of the chemical properties of the oligomers used (PNA, duplex RNA, LNA), target sequence (mRNA or promoter DNA), and the method of cellular delivery (cationic lipid or attached cationic peptide).…”

Section: Antigene Inhibition By Pna-lysine Conjugatessupporting

confidence: 60%

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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Section: Antigene Inhibition By Pna-lysine Conjugatessupporting

confidence: 60%

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

Corey

2007

Biochemistry

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“…LNA modifications within a TFO can increase the binding affinity to the target duplex DNA, and can increase resistance to digestion by nucleases [73]. Beane et al, have demonstrated that LNAs can recognize chromosomal target sequences and efficiently block endogenous expression of the progesterone and androgen receptors [74]. An LNA analogue ENA, containing a 2'-O, 4'-Cethylene bridge has also been reported to form stable triplex at physiological pH [75].…”

Section: Chemical Modifications Of Tfosmentioning

confidence: 99%

DNA triple helices: Biological consequences and therapeutic potential

Jain

Wang²,

Vásquez³

2008

Biochimie

259

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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“…LNA bases contain a methylene bridge that connects the 2 0 -oxygen of the ribose with the 4 0 -carbon, which makes it more stable than a regular oligonucleotide and suitable for therapeutic application (Beane et al 2007, Mook et al 2007). LNA-ASO binds to RNA, prevents transcription, and activates RNAse H resulting in cleavage of the target RNA molecule (Petersen & Wengel 2003, Vester & Wengel 2004.…”

Section: Apoptosis Of Neuroblastoma Cell Lines After Birc5 Knock Downmentioning

confidence: 99%

Knockdown of survivin (BIRC5) causes apoptosis in neuroblastoma via mitotic catastrophe

Lamers¹,

Ploeg²,

Schild³

et al. 2011

Endocrine-Related Cancer

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BIRC5 (survivin) is one of the genes located on chromosome arm 17q in the region that is often gained in neuroblastoma. BIRC5 is a protein in the intrinsic apoptotic pathway that interacts with XIAP and DIABLO leading to caspase-3 and caspase-9 inactivation. BIRC5 is also involved in stabilizing the microtubule-kinetochore dynamics. Based on the Affymetrix mRNA expression data, we here show that BIRC5 expression is strongly upregulated in neuroblastoma compared with normal tissues, adult malignancies, and non-malignant fetal adrenal neuroblasts. The overexpression of BIRC5 correlates with an unfavorable prognosis independent of the presence of 17q gain. Silencing of BIRC5 in neuroblastoma cell lines by various antisense molecules resulted in massive apoptosis as measured by PARP cleavage and FACS analysis. As both the intrinsic apoptotic pathway and the chromosomal passenger complex can be therapeutically targeted, we investigated in which of them BIRC5 exerted its essential anti-apoptotic role. Immunofluorescence analysis of neuroblastoma cells after BIRC5 silencing showed formation of multinucleated cells indicating mitotic catastrophe, which leads to apoptosis via P53 and CASP2. We show that BIRC5 silencing indeed resulted in activation of P53 and we could rescue apoptosis by CASP2 inhibition. We conclude that BIRC5 stabilizes the microtubules in the chromosomal passenger complex in neuroblastoma and that the apoptotic response results from mitotic catastrophe, which makes BIRC5 an interesting target for therapy.

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Inhibiting Gene Expression with Locked Nucleic Acids (LNAs) That Target Chromosomal DNA

Cited by 36 publications

References 44 publications

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

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

DNA triple helices: Biological consequences and therapeutic potential

Knockdown of survivin (BIRC5) causes apoptosis in neuroblastoma via mitotic catastrophe

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