| 1. |
- Bergquist, Helen, et al.
(författare)
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Disruption of Higher Order DNA Structures in Friedreich's Ataxia (GAA)(n) Repeats by PNA or LNA Targeting
- 2016
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Ingår i: PLOS ONE. - : PLOS. - 1932-6203. ; 11:11
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Tidskriftsartikel (refereegranskat)abstract
- Expansion of (GAA)(n) repeats in the first intron of the Frataxin gene is associated with reduced mRNA and protein levels and the development of Friedreich's ataxia. (GAA)(n) expansions form non-canonical structures, including intramolecular triplex (H-DNA), and Rloops and are associated with epigenetic modifications. With the aim of interfering with higher order H-DNA (like) DNA structures within pathological (GAA)(n) expansions, we examined sequence-specific interaction of peptide nucleic acid (PNA) with (GAA)(n) repeats of different lengths (short: n= 9, medium: n= 75 or long: n= 115) by chemical probing of triple helical and single stranded regions. We found that a triplex structure (H-DNA) forms at GAA repeats of different lengths; however, single stranded regions were not detected within the medium size pathological repeat, suggesting the presence of a more complex structure. Furthermore, (GAA) 4-PNA binding of the repeat abolished all detectable triplex DNA structures, whereas (CTT) 5-PNA did not. We present evidence that (GAA) 4-PNA can invade the DNA at the repeat region by binding the DNA CTT strand, thereby preventing non-canonical-DNA formation, and that triplex invasion complexes by (CTT) 5-PNA form at the GAA repeats. Locked nucleic acid (LNA) oligonucleotides also inhibited triplex formation at GAA repeat expansions, and atomic force microscopy analysis showed significant relaxation of plasmid morphology in the presence of GAA-LNA. Thus, by inhibiting disease related higher order DNA structures in the Frataxin gene, such PNA and LNA oligomers may have potential for discovery of drugs aiming at recovering Frataxin expression.
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| 4. |
- Bergquist, Helen, 1979-, et al.
(författare)
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Structure-Specific Recognition of Friedreich’s Ataxia (GAA)n Repeats by Benzoquinoquinoxaline Derivatives
- 2009
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Ingår i: ChemBioChem. - : Wiley. - 1439-4227 .- 1439-7633. ; 10:16, s. 2629-2637
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Tidskriftsartikel (refereegranskat)abstract
- Expansion of GAA triplet repeats in intron 1 of the FXN gene reduces frataxin expression and causes Friedreich's ataxia. (GAA)nrepeats form non-B-DNA structures, including triple helix H-DNA and higher-order structures (sticky DNA). In the proposed mechanisms of frataxin gene silencing, central unanswered questions involve the characterization of non-B-DNA structure(s) that are strongly suggested to play a role in frataxin expression. Here we examined (GAA)nbinding by triplex-stabilizing benzoquinoquinoxaline (BQQ) and the corresponding triplex-DNA-cleaving BQQ-1,10-phenanthroline (BQQ-OP) compounds. We also examined the ability of these compounds to act as structural probes for H-DNA formation within higher-order structures at pathological frataxin sequences in plasmids. DNA-complex-formation analyses with a gel-mobility-shift assay and sequence-specific probing of H-DNA-forming (GAA)nsequences by single-strand oligonucleotides and triplex-directed cleavage demonstrated that a parallel pyrimidine (rather than purine) triplex is the more stable motif formed at (GAA)nrepeats under physiologically relevant conditions.
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| 5. |
- Dryselius, Rikard, et al.
(författare)
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Variable coordination of cotranscribed genes in Escherichia coli following antisense repression
- 2006
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Ingår i: BMC Microbiology. - : Springer Science and Business Media LLC. - 1471-2180. ; 6, s. 97-
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Tidskriftsartikel (refereegranskat)abstract
- Background: A majority of bacterial genes belong to tight clusters and operons, which complicates gene functional studies using conventional knock-out methods. Antisense agents can down-regulate the expression of genes without disrupting the genome because they bind mRNA and block its expression. However, it is unclear how antisense inhibition affects expression from genes that are cotranscribed with the target. Results: To examine the effects of antisense inhibition on cotranscribed genes, we constructed a plasmid expressing the two reporter genes gfp and DsRed as one transcriptional unit. Incubation with antisense peptide nucleic acid (PNA) targeted to the mRNA start codon region of either the upstream gfp or the downstream DsRed gene resulted in a complete expression discoordination from this artificial construct. The same approach was applied to the three cotranscribed genes in the endogenously expressed lac-operon (lacZ, Y and A) and partial downstream expression coordination was seen when the lacZ start codon was targeted with antisense PNA. Targeting the lacY mRNA start codon region showed no effect on the upstream lacZ gene expression whereas expression from the downstream lacA gene was affected as strongly as the lacY gene. Determination of lacZ and lacY mRNA levels revealed a pattern of reduction that was similar to the Lac-proteins, indicating a relation between translation inhibition and mRNA degradation as a response to antisense PNA treatment. Conclusion: The results show that antisense mediated repression of genes within operons affect cotranscribed genes to a variable degree. Target transcript stability appears to be closely related to inhibition of translation and presumably depends on translating ribosomes protecting the mRNA from intrinsic decay mechanisms. Therefore, for genes within operons and clusters it is likely that the nature of the target transcript will determine the inhibitory effects on cotranscribed genes. Consequently, no simple and specific methods for expression control of a single gene within polycistronic operons are available, and a thorough understanding of mRNA regulation and stability is required to understand the results from both knock-down and knock-out methods used in bacteria.
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| 7. |
- Nikravesh, Abbas, et al.
(författare)
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Antisense PNA Accumulates in Escheria coli and mediates a Long Post-antibiotic Effect
- 2008
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Ingår i: Molecular Therapy. - : Elsevier BV. - 1525-0016 .- 1525-0024. ; 15:8, s. 1537-1542
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Tidskriftsartikel (refereegranskat)abstract
- Antisense agents that target growth-essential genes display surprisingly potent bactericidal properties. In particular, peptide nucleic acid (PNA) and phosphorodiamidate morpholino oligomers linked to cationic carrier peptides are effective in time kill assays and as inhibitors of bacterial peritonitis in mice. It is unclear how these relatively large antimicrobials overcome stringent bacterial barriers and mediate killing. Here we determined the transit kinetics of peptide–PNAs and observed an accumulation of cell-associated PNA in Escherichia coli and slow efflux. An inhibitor of drug efflux pumps did not alter peptide–PNA potency, indicating a lack of active efflux from cells. Consistent with cell retention, the post-antibiotic effect (PAE) of the anti-acyl carrier protein (acpP) peptide–PNA was greater than 11 hours. Bacterial cell accumulation and a long PAE are properties of significant interest for antimicrobial development.
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