Extending Recognition by Peptide Nucleic Acids (PNAs):  Binding to Duplex DNA and Inhibition of Transcription by Tail-Clamp PNA−Peptide Conjugates

Kaihatsu, Kunihiro; Shah, Rajiv; Zhao, Xin; Corey, David R.

doi:10.1021/bi035194k

Cited by 73 publications

(64 citation statements)

References 39 publications

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“…In the last years, different approaches have emerged to correct a point mutation at the DNA level without provoking doublestrand breaks (DSB): chimeric RNA-DNA oligonucleotides, which were first designed by Kmiec's group (Yoon et al, 1996); single-stranded oligonucleotides, studied for the first time in 1988 (Moerschell et al, 1988); bifunctional triplehelix-forming oligonucleotides, formed by two domains, a triplex forming oligonucleotide (TFO) domain and a repair domain (Chan et al, 1999;Culver et al, 1999); and several approaches of peptide nucleic acids (PNA) (Nielsen et al, 1991;Egholm et al, 1993), bis-PNA (Rogers et al, 2002;Chin et al, 2008), tc-PNA (Bentin et al, 2003;Kaihatsu et al, 2003), pc-PNA (Lohse et al, 1999) (Demidov et al, 2002;Lonkar et al, 2009), and more recently PNA-single-stranded oligodeoxynucleotides (ssODNs) (Kayali et al, 2010;NikAhd and Bertoni, 2014).…”

Section: Introductionmentioning

confidence: 99%

Repair of Single-Point Mutations by Polypurine Reverse Hoogsteen Hairpins

Solé

Villalobos²,

Ciudad³

et al. 2014

Human Gene Therapy Methods

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Polypurine reverse Hoogsteen hairpins (PPRHs) are formed by two intramolecularly bound antiparallel homopurine domains linked by a five-thymidine loop. One of the homopurine strands binds with antiparallel orientation by Watson-Crick bonds to the polypyrimidine target sequence, forming a triplex. We had previously reported the ability of PPRHs to effectively bind dsDNA displacing the fourth strand away from the newly formed triplex. The main goal of this work was to explore the possibility of repairing a point mutation in mammalian cells using PPRHs as tools. These repair-PPRHs contain different combinations of extended sequences of DNA with the corrected nucleotide to repair the point mutation. As a model we used the dihydrofolate reductase gene. On the one hand, we demonstrate in vitro that PPRHs bind specifically to their polypyrimidine target sequence, opening the two strands of the dsDNA, and allowing the binding of a given repair oligonucleotide to the displaced strand of the DNA. Subsequently, we show at a cellular level (Chinese ovary hamster cells) that repair-PPRHs are able to correct a single-point mutation in a dihydrofolate reductase minigene bearing a nonsense mutation, both in an extrachromosomal location and when the mutated plasmid was stably transfected into the cells. Finally, this methodology was successfully applied to repair a single-point mutation at the endogenous locus, using the DA5 cell line with a deleted nucleotide in exon six of the dhfr gene.

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Section: Introductionmentioning

confidence: 99%

Repair of Single-Point Mutations by Polypurine Reverse Hoogsteen Hairpins

Solé

Villalobos²,

Ciudad³

et al. 2014

Human Gene Therapy Methods

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“…The tail sequence was incorporated in order to destabilize target intramolecular interactions and to allow the clamp portion of the tail-clamp to form the triplex efficiently (Figure 2). Tail sequences have been fused to other types of molecules with different purposes: to increase the strand invasion of double-stranded DNA by bis-PNAs, [36,37] for example, or, with catalytic RNAs, to increase the targeting efficiency of ribozyme M1 RNA (M1GS RNA ribozymes). [38,39] We performed a comparative study on the interaction of the structured sequence 50pyr with tail-clamps, in which the clamp portion was either parallel-or antiparallel-stranded, and including 8-aminopurine nucleotide modifications when possible.…”

Section: Discussionmentioning

confidence: 99%

“Parallel” and “Antiparallel Tail‐Clamps” Increase the Efficiency of Triplex Formation with Structured DNA and RNA Targets

et al. 2005

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Sequence-specific triple-helix structures can be formed by parallel and antiparallel DNA clamps interacting with single-stranded DNA or RNA targets. Single-stranded nucleic acid molecules are known to adopt secondary structures that might interfere with intermolecular interactions. We demonstrate the correlation between a secondary structure involving the target--a stable stem predicted by in silico folding and experimentally confirmed by thermal stability and competition analyses--and an inhibitory effect on triplex formation. We overcame structural impediments by designing a new type of clamp: "tail-clamps". A combination of gel-shift, kinetic analysis, UV thermal melting and thermodynamic techniques was used to demonstrate that tail-clamps efficiently form triple helices with a structured target sequence. The performance of parallel and antiparallel tail-clamps was compared: antiparallel tail-clamps had higher binding efficiencies than parallel tail-clamps both with structured DNA and RNA targets. In addition, the reported triplex-stabilizing property of 8-aminopurine residues was confirmed for tail-clamps. Finally, we discuss the possible use of this improved triplex technology as a new tool for applications in molecular biology.

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“…To determine if the optimized bisPNA could arrest transcription elongation in mammalian cells, as was previously reported for in vitro transcription (14,15), PNA was bound to anchor sites generated in enhanced GFP (EGFP) reporter plasmids and transfected into cultured human U-2 OS cells. Due to the high stability of EGFP protein, we used a destabilized form, d2EGFP, with a half-life of only 2 h (43).…”

Section: Effect Of Bispna Triplex On Destabilized Green Fluorescence mentioning

confidence: 99%

Zorro locked nucleic acid induces sequence‐specific gene silencing

Heinonen

Svahn

et al. 2007

FASEB j.

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Locked nucleic acids (LNAs) are synthetic analogs of nucleic acids that contain a bridging methylene carbon between the 2' and 4' positions of the ribose ring. In this study, we generated a novel sequence-specific antigene molecule "Zorro LNA", which simultaneously binds to both strands, and that induced effective and specific strand invasion into DNA duplexes and potent inhibition of gene transcription, also in a cellular context. By comparing the Zorro LNA with linear LNA as well as an optimized bisPNA (peptide nucleic acid) oligonucleotide directed against the same target sites, respectively, we found that the Zorro LNA construct was unique in its ability to arrest gene transcription in mammalian cells. To our knowledge, this is the first time that in mammalian cells, gene transcription was blocked by a nucleic acid analog in a sequence-specific way using low but saturated binding of a blocking agent. This offers a novel type of antigene drug that is easy to synthesize.

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Extending Recognition by Peptide Nucleic Acids (PNAs): Binding to Duplex DNA and Inhibition of Transcription by Tail-Clamp PNA−Peptide Conjugates

Cited by 73 publications

References 39 publications

Repair of Single-Point Mutations by Polypurine Reverse Hoogsteen Hairpins

Repair of Single-Point Mutations by Polypurine Reverse Hoogsteen Hairpins

“Parallel” and “Antiparallel Tail‐Clamps” Increase the Efficiency of Triplex Formation with Structured DNA and RNA Targets

Zorro locked nucleic acid induces sequence‐specific gene silencing

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