Peptide nucleic acids (PNA) are artificial structural mimics of nucleic acids capable of sequence specific hybridization to both RNA and DNA. Thus they have obvious potential as gene targeting agents ...for drug discovery approaches. An overview with emphasis on recent progress on RNA "interference" (antisense), targeting of duplex DNA and gene targeted repair and transcription interference using PNA, as well as on PNA delivery and potential PNA anti-infectives is given.
PNA oligomers conjugated to bacteria penetrating peptides (BPPs), such as (KFF)3K, targeting essential bacterial genes, such as acpP, can inhibit bacterial growth at one-digit micromolar ...concentrations. It has been found that the LPS of the outer membrane of Gram-negative bacteria is a barrier for cellular uptake of (KFF)3K-eg1-PNA and that the SbmA transporter protein is involved in the passage through the inner membrane. We now further elucidate the uptake mechanism of (KFF)3K-eg1-PNA by showing that the peptide part of (KFF)3K-eg1-PNA is unstable and is degraded by peptidases in the medium of a bacterial culture (t 1/2 < 5 min) and inside the bacteria. Analysis of peptide–PNA conjugates present in the periplasmic space and the cytoplasm showed the presence of mainly PNA with only the FFK tripeptide and without a peptide, at a concentration 10-fold that added to the medium. Furthermore, the two main degradation products showed no antibacterial effect when added directly to a bacterial culture and the antibacterial effect decreased with peptide length, thereby demonstrating that an intact peptide is indeed crucial for uptake but not for intracellular antisense activity. Most surprisingly, it was found that although the corresponding series of the proteolytically stable D-form (kff)3k-eg1-PNAs exhibited an analogous reduction of activity with peptide length, the activity was dependent on the presence of SbmA for the shorter peptides (which is not the case with the full length peptide). Therefore, our results suggest that the BPP is necessary for crossing both the LPS/outer membrane as well as the inner membrane and that full length (KFF)3K may spontaneously pass the inner membrane. Thus, SbmA dependence of (KFF)3K-eg1-PNA is ascribed to peptide degradation in the bacterial medium and in periplasmic space. Finally, the results show that stability and metabolism (by bacterial proteases/peptidases) should be taken into consideration upon design and activity/uptake analysis of BPPs (and antimicrobial peptides).
Chronic hepatitis B virus (HBV) infection remains a major health problem worldwide. Because current anti-HBV treatments are only virostatic, there is an urgent need for development of alternative ...antiviral approaches. In this context, cell-penetrating peptides (CPPs) and cationic polymers, such as chitosan (CS), appear of particular interest as nonviral vectors due to their capacity to facilitate cellular delivery of bioactive cargoes including peptide nucleic acids (PNAs) or DNA vaccines. We have investigated the ability of a PNA conjugated to different CPPs to inhibit the replication of duck hepatitis B virus (DHBV), a reference model for human HBV infection. The in vivo administration of PNA-CPP conjugates to neonatal ducklings showed that they reached the liver and inhibited DHBV replication. Interestingly, our results indicated also that a modified CPP (CatLip) alone, in the absence of its PNA cargo, was able to drastically inhibit late stages of DHBV replication. In the mouse model, conjugation of HBV DNA vaccine to modified CS (Man-CS-Phe) improved cellular and humoral responses to plasmid-encoded antigen. Moreover, other systems for gene delivery were investigated including CPP-modified CS and cationic nanoparticles. The results showed that these nonviral vectors considerably increased plasmid DNA uptake and expression. Collectively promising results obtained in preclinical studies suggest the usefulness of these safe delivery systems for the development of novel therapeutics against chronic hepatitis B.
DNA and RNA oxidations have been linked to diseases such as cancer, arteriosclerosis, neurodegeneration and diabetes. The prototype base modification studied is the 8-hydroxylation of guanine. DNA ...integrity is maintained by elaborate repair systems and RNA integrity is less studied but relies mainly on degradation.
DNA and RNA oxidations are measured by very similar techniques. The scope of this review is to highlight the preferred methods of measurement of oxidized nucleic acid metabolites, to highlight novel findings particularly in RNA oxidation, and to present the interpretation of the measurements.
Tissue levels are snap-shots of the level in a specific organ or cell system and reflect the balance between formation rate and elimination rate (repair), and must be interpreted as such. Urinary excretion is a global measure of oxidative stress in an organism and is therefore best suited for situations or diseases where large parts or the entire organism is stressed by oxidation. It represents the body average rate by which either RNA or DNA is oxidized and is interpreted as oxidative stress. Oxidations of RNA and DNA precursors have been demonstrated and the quantitative importance is debated.
Careful experimental designs and appropriate choice of methodology are paramount for correct testing of hypotheses related to oxidative stress, and pitfalls are plentiful. There is accumulating evidence that DNA oxidation is associated with disease, particularly cancer, and recent evidence points at an association between RNA oxidation and neurodegenerative diseases and diabetes. This article is part of a Special Issue entitled Current methods to study reactive oxygen species - pros and cons and biophysics of membrane proteins. Guest Editor: Christine Winterbourn.
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•Urinary excretion of oxidized guanine species measures global RNA and DNA oxidations.•The guanine 8-oxo modifications are used as prototype for RNA and DNA oxidations.•HPLC with tandem mass spectrometry is the state of the art method.•DNA oxidation is associated with cancer development.•RNA oxidation is associated with the development of diabetes complications.
Discovery of novel antimicrobial agents against Pseudomonas aeruginosa able to inhibit bacterial growth as well as the resulting inflammatory response is a key goal in cystic fibrosis research. We ...report in this paper that a peptide nucleic acid (PNA3969) targeting the translation initiation region of the essential acpP gene of P. aeruginosa, and previously shown to inhibit bacterial growth, concomitantly also strongly inhibits induced up-regulation of the pro-inflammatory markers IL-8, IL-6, G-CSF, IFN-γ, IP-10, MCP-1 and TNF-α in IB3-1 cystic fibrosis cells infected by P. aeruginosa PAO1. Remarkably, no effect on PAO1 induction of VEGF, GM-CSF and IL-17 was observed. Analogous experiments using a two base mis-match control PNA did not show such inhibition. Furthermore, no significant effects of the PNAs were seen on cell growth, apoptosis or secretome profile in uninfected IB3-1 cells (with the exception of a PNA-mediated up-regulation of PDGF, IL-17 and GM-CSF). Thus, we conclude that in cell culture an antimicrobial PNA against P. aeruginosa can inhibit the expression of pro-inflammatory cytokines otherwise induced by the infection. In particular, the effects of PNA-3969 on IL-8 gene expression are significant considering the key role of this protein in the cystic fibrosis inflammatory process exacerbated by P. aeruginosa infection.
A series of amino- and guanidino-terminating 3- and 4-generation 2,4-diaminobutanoic acid (Dab) dendrons have been robustly synthesized on a solid phase and characterized as cellular delivery agents ...in antisense peptide nucleic acid (PNA) conjugates in the pLuc705 HeLa cell splice switching system. The dendron–PNA conjugates exhibited splice correction activity at one digit micromolar concentrations, and guanidino-terminating dendrons were significantly more effective than analogous amine terminating ones. Furthermore, introduction of lipophilic groups such as phenyl, alkyl, or fatty acids increased efficacy, but also increased cellular toxicity. Fluorescence microscopy analyses supported an endosomal uptake mechanism and furthermore predominantly showed colocalization with late endosomes and lysosomes. The robust solid phase synthesis should make such Dab–dendrons a useful platform for further in vitro as well as in vivo optimization.
The past decade has provided exciting insights into a novel class of central (small) RNA molecules intimately involved in gene regulation. Only a small percentage of our DNA is translated into ...proteins by mRNA, yet 80% or more of the DNA is transcribed into RNA, and this RNA has been found to encompass various classes of novel regulatory RNAs, including, e.g., microRNAs. It is well known that DNA is constantly oxidized and repaired by complex genome maintenance mechanisms. Analogously, RNA also undergoes significant oxidation, and there are now convincing data suggesting that oxidation, and the consequent loss of integrity of RNA, is a mechanism for disease development. Oxidized RNA is found in a large variety of diseases, and interest has been especially devoted to degenerative brain diseases such as Alzheimer disease, in which up to 50–70% of specific mRNA molecules are reported oxidized, whereas other RNA molecules show virtually no oxidation. The iron-storage disease hemochromatosis exhibits the most prominent general increase in RNA oxidation ever observed. Oxidation of RNA primarily leads to strand breaks and to oxidative base modifications. Oxidized mRNA is recognized by the ribosomes, but the oxidation results in ribosomal stalling and dysfunction, followed by decreased levels of functional protein as well as the production of truncated proteins that do not undergo proper folding and may result in protein aggregation within the cell. Ribosomal dysfunction may also signal apoptosis by p53-independent pathways. There are very few reports on interventions that reduce RNA oxidation, one interesting observation being a reduction in RNA oxidation by ingestion of raw olive oil. High urinary excretion of 8-oxo-guanosine, a biomarker for RNA oxidation, is highly predictive of death in newly diagnosed type 2 diabetics; this demonstrates the clinical relevance of RNA oxidation. Taken collectively the available data suggest that RNA oxidation is a contributing factor in several diseases such as diabetes, hemochromatosis, heart failure, and β-cell destruction. The mechanism involves free iron and hydrogen peroxide from mitochondrial dysfunction that together lead to RNA oxidation that in turn gives rise to truncated proteins that may cause aggregation. Thus RNA oxidation may well be an important novel contributing mechanism for several diseases.
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► RNA oxidation is more abundant than DNA oxidation. ► High levels of oxidized RNA are found in degenerative brain diseases. ► General RNA oxidation is measured by 8-oxoGuo excretion into urine. ► General RNA oxidation predicts death in type 2 diabetes. ► RNA may be a novel and important mechanism of diseases such as diabetes, degenerative brain diseases, and hemochromatosis.
Antisense peptide nucleic acid (PNA) oligomers constitute a novel class of potential antibiotics that inhibit bacterial growth via specific knockdown of essential gene expression. However, discovery ...of efficient, nontoxic delivery vehicles for such PNA oligomers has remained a challenge. In the present study we show that antimicrobial peptides (AMPs) with an intracellular mode of action can be efficient vehicles for bacterial delivery of an antibacterial PNA targeting the essential acpP gene. The results demonstrate that buforin 2-A (BF2-A), drosocin, oncocin 10, Pep-1-K, KLW-9,13-a, (P59→W59)-Tat48–60, BF-2A-RXR, and drosocin-RXR are capable of transporting PNA effectively into E. coli (MICs of 1–4 μM). Importantly, presence of the inner-membrane peptide transporter SbmA was not required for antibacterial activity of PNA–AMP conjugates containing Pep-1-K, KLW-9,13-a, or drosocin-RXR (MICs of 2–4 μM).
Peptide Nucleic Acid (PNA)-peptide conjugates targeting essential bacterial genes are showing promise as antisense antimicrobials in drug discovery. Optimization has focused on selection of target ...genes and exact localization around the ribosome binding site, but surprisingly a length optimum around 10-12 nucleobases has been found. Addressing this observation, we have investigated the relationship between PNA-length, PNA-RNA duplex stability and antimicrobial activity in
in more detail. For PNAs of identical length of ten nucleobases the expected reverse correlation between the thermal stability (Tm) of the PNA-RNA duplex and the MIC for single mismatched PNAs was found. Also the expected direct correlation between the length of the PNA and the PNA-RNA duplex stability was found. Nonetheless, 10-mer PNAs in a 6-18 mer extension series of (KFF)
K- and (RXR)
conjugates were the most active as antisense antimicrobials in both wild type
MG1655 and AS19, suggesting that the size constraint is related to the bacterial uptake of PNA-peptide conjugates. This conclusion was supported by flow cytometry data showing higher bacterial uptake of shorter PNA fluorophore labeled conjugates. Interestingly, the size-limited uptake seems independent on outer membrane integrity (AS19), and thus the results suggest that the inner membrane limits the molecular size for peptide-PNA passage.
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