Non-Watson-Crick interactions between PNA and DNA inhibit the ATPase activity of bacteriophage T4 Dda helicase

Tackett, Alan J.; Corey, David R.; Raney, Kevin D.

doi:10.1093/nar/30.4.950

Cited by 47 publications

(48 citation statements)

References 34 publications

(54 reference statements)

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“…Although it is possible to hybridize at initially very high temperatures [15], we prefer to hybridize plasmids at 37 • C in order to avoid increased nicking. Under these conditions we have found that a substantial fraction of PNA interacts with the plasmid in an unspecific way ( Figure 6) and it has also been shown by others that PNA can stack to unrelated DNA in a non-specific manner [19]. This unspecific binding can easily be removed by purifying hybridized plasmids on standard DNA purification spin columns.…”

Section: Visualizing Pna-nucleic Acid Interactionssupporting

confidence: 67%

Adding functional entities to plasmids

Svahn

Lundin

et al. 2004

J. Gene Med.

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Non-viral gene therapy constitutes an alternative to the more common use of viral-mediated gene transfer. Most gene transfer methods using naked DNA are based upon non-sequence-specific interactions between the nucleic acid and cationic lipids (lipoplex) or polymers (polyplex). We have developed a technology in which functional entities hybridize in a sequence-specific manner to the nucleic acid (bioplex). This technology is still in its infancy, but has the potential to become a useful tool, since it allows the construction of highly defined complexes containing a variety of functional entities. In its present form the bioplex technology is based upon the use of peptide/nucleic acids (PNA) as anchors. Single, or multiple, functional entities are directly coupled to the anchors. By designing plasmids, or oligonucleotides, with the corresponding anchor target sequence, complexes with desired composition can easily be generated. The long-term aim is to combine functional entities in order to achieve optimal, synergistic interactions allowing enhanced gene transfer in vivo.

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Section: Visualizing Pna-nucleic Acid Interactionssupporting

confidence: 67%

Adding functional entities to plasmids

Svahn

Lundin

et al. 2004

J. Gene Med.

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“…We cannot exclude that is in part associated with nonspecific cytotoxicity of PNA molecules, as already suggested by other groups. 10,21 Notwithstanding, these results emphasize the essential A peptide carrier for PNA-mediated targeting of cell cycle progression MC Morris et al role of Pep-2 in improving the bioavailability of antisense HypNA-pPNA and PNA molecules and demonstrate the selectivity and specificity of the antisense sequence used in this study.…”

Section: Resultsmentioning

confidence: 51%

“…18 The solubility of PNAs and their tendency to self-aggregate are important factors to be considered for the biological use of PNAs. [21][22][23] PNA solubility is often sequence-dependent, which usually leads to several restrictions in PNA probe design and can be improved by incorporation of Lys residues or ethylene glycol linkers (eg1 ¼ 8 amino-2-6 dioxaoctanoic acid). 18,[21][22][23][24] Efimov et al 25,26 have developed a novel oligonucleotide mimic, a dimeric oligomer that consists of a phosphonate analog of PNA (pPNA) and a PNA-like monomer based on a trans-4-hydroxyl-L-proline (HypNA).…”

Section: Introductionmentioning

confidence: 99%

Combination of a new generation of PNAs with a peptide-based carrier enables efficient targeting of cell cycle progression

et al. 2004

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The design of potent systems for the delivery of charged and noncharged molecules that target genes of interest remains a challenge. We describe a novel technology that combines a new generation of peptide nucleic acids (PNAs), or HypNApPNAs, with a new noncovalent peptide-based delivery system, Pep-2, which promotes efficient delivery of PNAs into several cell lines. We have validated the potential of this technology by showing that Pep2-mediated delivery of an antisense HypNApPNA chimera directed specifically against cyclin B1 induces rapid and robust downregulation of its protein levels and efficiently blocks cell cycle progression of several cell lines, as well as proliferation of cells derived from a breast cancer. Pep-2-based delivery system was shown to be 100-fold more efficient in delivering HypNA-pPNAs than classical cationic lipidbased methods. Whereas Pep-2 is essential for improving the bioavailability of PNAs and HypNA-pPNAs, the latter contribute significantly to the efficiency and specificity of the biological response. We have found that Pep-2/HypNA-pPNA strategy promotes potent antisense effects, which are approximately 25-fold greater than with classical antisense oligonucleotide directed specifically against the same cyclin B1 target. Taken together, these data demonstrate that peptide-mediated delivery of HypNA-pPNAs constitutes a very promising technology for therapeutic applications.

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“…The PNA 14-mer combined excellent specificity and the fastest association rate among the inhibitors tested (Table 1). Its relatively high K i value (12.5 nM) ( Table 1) can be explained by a certain propensity of hydrophobic PNA to self-aggregate (29), which likely reduced the effective concentration of PNA single strands, although we tried to minimize this problem by preparing, handling, and quantifying PNA solutions at 80°C. At explicitly smaller oligomer sizes, however, LNA may become advantageous over PNA.…”

Section: Discussionmentioning

confidence: 99%

Antisense Inhibition of RNase P

Gruegelsiepe

Brandt

Hartmann

2006

Journal of Biological Chemistry

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We explored bacterial RNase P as a drug target using antisense oligomers against the P15 loop region of Escherichia coli RNase P RNA. An RNA 14-mer, or locked nucleic acid (LNA) and peptide nucleic acid (PNA) versions thereof, disrupted local secondary structure in the catalytic core, forming hybrid duplexes over their entire length. Binding of the PNA and LNA 14-mers to RNase P RNA in vitro was essentially irreversible and even resisted denaturing PAGE. Association rates for the RNA, LNA, and PNA 14-mers were ϳ10 M ؊1 s؊1 with a rate advantage for PNA and were thus rather fast despite the need to disrupt local structure. Conjugates in which the PNA 14-mer was coupled to an invasive peptide via a novel monoglycine linker showed RNase P RNA-specific growth inhibition of E. coli cells. Cell growth could be rescued when expressing a second bacterial RNase P RNA with an unrelated sequence in the target region. We report here for the first time specific and growthinhibitory drug targeting of RNase P in live bacteria. This is also the first example of a duplex-forming oligomer that invades a structured catalytic RNA and inactivates the RNA by (i) trapping it in a state in which the catalytic core is partially unfolded, (ii) sterically interfering with substrate binding, and (iii) perturbing the coordination of catalytically relevant Mg 2؉ ions.Antisense-based inhibition of bacterial cell growth as a strategy to combat prokaryotic pathogens has been little-explored mainly because of the low cellular uptake efficiency of antisense agents. We report here on antisense-based inhibition of the catalytic RNA subunit of Ribonuclease P (RNase P) 2 from Escherichia coli. Bacterial RNase P is an essential ribonucleoprotein enzyme responsible for the 5Ј-end maturation of tRNAs (1) whose architecture largely differs from that of eukaryotic RNase P enzymes, a first prerequisite for a favorable drug target.Also, its cellular abundance is low compared with ribosomal RNA or tRNA; for example, E. coli cells contain a 60 -100-fold molar excess of ribosomes over RNase P RNA (2). Thus, compared with ribosomes, lower intracellular drug concentrations may be required to deplete cellular RNase P activity below a threshold essential for cell growth and survival. Because the enzyme contains a stable catalytic RNA subunit expected to turnover slowly relative to most mRNAs, de novo transcription rates of its RNA subunit are predicted to be relatively low, suggesting that its inactivation will result in a rather persistent phenotype.Previously, the so-called P15 loop region of E. coli-type RNase P RNAs, known to interact with the 3Ј-CCA portion of precursor tRNA (ptRNA) substrates (3), was demonstrated to be a very effective target site for antisense-like inhibition strategies (4, 5). In a related but conceptually different approach termed oligonucleotide-directed misfolding of RNA, E. coli RNase P RNA was screened with consecutive DNA 12-mers for inhibition of ptRNA processing in a reaction mixture in which RNase P RNA was newly transcribed in t...

show abstract

Non-Watson-Crick interactions between PNA and DNA inhibit the ATPase activity of bacteriophage T4 Dda helicase

Cited by 47 publications

References 34 publications

Adding functional entities to plasmids

Adding functional entities to plasmids

Combination of a new generation of PNAs with a peptide-based carrier enables efficient targeting of cell cycle progression

Antisense Inhibition of RNase P

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