RIG‐I detects infection with live Listeria by sensing secreted bacterial nucleic acids

Immunity against infection with Listeria monocytogenes is not achieved from innate immune stimulation by contact with killed but requires viable Listeria gaining access to the cytosol of infected cells. It has remained ill‐defined how such immune sensing of live Listeria occurs. Here, we report that efficient cytosolic immune sensing requires access of nucleic acids derived from live Listeria to the cytoplasm of infected cells. We found that Listeria released nucleic acids and that such secreted bacterial RNA/DNA was recognized by the cytosolic sensors RIG‐I, MDA5 and STING thereby triggering interferon β production. Secreted Listeria nucleic acids also caused RIG‐I‐dependent IL‐1β‐production and inflammasome activation. The signalling molecule CARD9 contributed to IL‐1β production in response to secreted nucleic acids. In conclusion, cytosolic recognition of secreted bacterial nucleic acids by RIG‐I provides a mechanistic explanation for efficient induction of immunity by live bacteria.     

Chimeric aptamers in cancer cell-targeted drug delivery

Aptamers are single-stranded structured oligonucleotides (DNA or RNA) that can bind to a wide range of targets (“apatopes”) with high affinity and specificity. These nucleic acid ligands, generated from pools of random-sequence by an in vitro selection process referred to as systematic evolution of ligands by exponential enrichment (SELEX), have now been identified as excellent tools for chemical biology, therapeutic delivery, diagnosis, research, and monitoring therapy in real-time imaging. Today, aptamers represent an interesting class of modern pharmaceuticals which with their low immunogenic potential mimic extend many of the properties of monoclonal antibodies in diagnostics, research, and therapeutics. More recently, chimeric aptamer approach employing many different possible types of chimerization strategies has generated more stable and efficient chimeric aptamers with aptamer–aptamer, aptamer–nonaptamer biomacromolecules (siRNAs, proteins) and aptamer–nanoparticle chimeras. These chimeric aptamers when conjugated with various biomacromolecules like locked nucleic acid (LNA) to potentiate their stability, biodistribution, and targeting efficiency, have facilitated the accurate targeting in preclinical trials. We developed LNA-aptamer (anti-nucleolin and EpCAM) complexes which were loaded in iron-saturated bovine lactofeerin (Fe-blf)-coated dopamine modified surface of superparamagnetic iron oxide (Fe₃O₄) nanoparticles (SPIONs). This complex was used to deliver the specific aptamers in tumor cells in a co-culture model of normal and cancer cells. This review focuses on the chimeric aptamers, currently in development that are likely to find future practical applications in concert with other therapeutic molecules and modalities.     

Proteins involved in binding and cellular uptake of nucleic acids

The study of mechanisms of nucleic acid transport across the cell membrane is valuable both for understanding the biological function of extracellular nucleic acids and the practical use of nucleic acids in gene therapy. It has been clearly demonstrated that cell surface proteins are necessary for transport of nucleic acids into cells. A large amount of data has now been accumulated about the proteins that participate in nucleic acid transport. The methods for revealing and identification of these proteins, possible mechanisms of protein-mediated transport of nucleic acids, and cellular functions of these proteins are described.     

Intracellular delivery of nucleic acid by cell-permeable hPP10 peptide

Although gene therapy offers hope against incurable diseases, nonreplicating transduction vectors remain lacking. We have previously characterized a cell-penetrating peptide hPP10 for the delivery of various cargoes; however, whether hPP10 can mediate nucleic acid delivery is still unknown. Here, examining via different ways, we demonstrate that hPP10 stably complexes with plasmid DNA (pDNA) and safely mediates nucleic acid transfection. hPP10 can mediate GFP-, dsRed-, and luciferase-expressing plasmids into cells with nearly the same efficiency as commercial transfection reagents Turbofectin or Lipofect. Furthermore, hPP10 can mediate Cre fusion protein delivery and pDNA transfection simultaneously in the Cre/loxp system in vitro. In addition, hPP10 fused with an RNA-binding domain can mediate delivery of small interfering RNA into cells to silence the reporter gene expression. Collectively, our results suggest that hPP10 is an option for nucleic acid delivery with efficiencies similar to that of commercial reagents.     

Non‐viral nucleic acid delivery to the central nervous system and brain tumors

Gene therapy is a rapidly emerging remedial route for many serious incurable diseases, such as central nervous system (CNS) diseases. Currently, nucleic acid medicines, including DNAs encoding therapeutic or destructive proteins, small interfering RNAs or microRNAs, have been successfully delivered to the CNS with gene delivery vectors using various routes of administration and have subsequently exhibited remarkable therapeutic efficiency. Among these vectors, non‐viral vectors are favorable for delivering genes into the CNS as a result of their many special characteristics, such as low toxicity and pre‐existing immunogenicity, high gene loading efficiency and easy surface modification. In this review, we highlight the main types of therapeutic genes that have been applied in the therapy of CNS diseases and then outline non‐viral gene delivery vectors.     

Cationic nucleopeptides as novel non-covalent carriers for the delivery of peptide nucleic acid (PNA) and RNA oligomers

Cationic nucleopeptides belong to a family of synthetic oligomers composed by amino acids and nucleobases. Their capability to recognize nucleic acid targets and to cross cellular membranes provided the basis for considering them as novel non-covalent delivery agents for nucleic acid pharmaceuticals. Herein, starting from a 12-mer nucleopeptide model, the number of cationic residues was modulated in order to obtain new nucleopeptides endowed with high solubility in acqueous medium, acceptable bio-stability, low cytotoxicity and good capability to bind nucleic acid. Two candidates were selected to further investigate their potential as nucleic acid carriers, showing higher efficiency to deliver PNA in comparison with RNA. Noteworthy, this study encourages the development of nucleopeptides as new carriers to extend the known strategies for those nucleic acid analogues, especially PNA, that still remain difficult to drive into the cells.     

Peptide nucleic acids: Cellular delivery and recognition of DNA and RNA targets

Summary Peptide nucleic acids (PNAs) can be conveniently delivered into cells in complex with DNA and cationic lipid. This advance enables researchers to test the hypothesis that PNAs offer advantages for recognition of DNA or RNA targets within cells. In this review, I describe the intracellular delivery of PNAs as DNA-PNA-cationic lipid complexes and discuss recognition of three classes of nucleic acid target: duplex DNA, single-stranded mRNA, and the ribonucleoprotein telomerase. These targets differ dramatically in their potential for base-paired structure, offering distinct challenges for hybridization by PNAs. It is apparent that PNAs can exert sequence-specific effects within cells, and their full potential has only begun to be explored.     

Peptide nucleic acids: Cellular delivery and recognition of DNA and RNA targets

Summary Peptide nucleic acids (PNAs) can be conveniently delivered into cells in complex with DNA and cationic lipid. This advance enables researchers to test the hypothesis that PNAs offer advantages for recognition of DNA or RNA targets within cells. In this review, I describe the intracellular delivery of PNAs as DNA-PNA-cationic lipid complexes and discuss recognition of three classes of nucleic acid target: duplex DNA, single-stranded mRNA, and the ribonucleoprotein telomerase. These targets differ dramatically in their potential for base-paired structure, offering distinct challenges for hybridization by PNAs. It is apparent that PNAs can exert sequence-specific effects within cells, and their full potential has only begun to be explored.     

Peptide nucleic acids: Cellular delivery and recognition of DNA and RNA targets

Peptide nucleic acids (PNAs) can be conveniently delivered into cells in complex with DNA and cationic lipid. This advance enables researchers to test the hypothesis that PNAs offer advantages for recognition of DNA or RNA targets within cells. In this review, I describe the intracellular delivery of PNAs as DNA-PNA-cationic lipid complexes and discuss recognition of three classes of nucleic acid target: duplex DNA, single-stranded mRNA, and the ribonucleoprotein telomerase. These targets differ dramatically in their potential for base-paired structure, offering distinct challenges for hybridization by PNAs. It is apparent that PNAs can exert sequence-specific effects within cells, and their full potential has only begun to be explored.     

Circulating nucleic acids (CNAs) and cancer—A survey

It has been known for decades that it is possible to detect small amounts of extracellular nucleic acids in plasma and serum of healthy and diseased human beings. The unequivocal proof that part of these circulating nucleic acids (CNAs) is of tumor origin, initiated a surge of studies which confirmed and extended the original observations. In the past few years many experiments showed that tumor-associated alterations can be detected at the DNA and RNA level. At the DNA level the detection of point mutations, microsatellite alterations, chromosomal alterations, i.e. inversion and deletion, and hypermethylation of promoter sequences were demonstrated. At the RNA level the overexpression of tumor-associated genes was shown. These observations laid the foundation for the development of assays for an early detection of cancer as well as for other clinical means.     

Design,synthesis, and analysis of conformationally constrained nucleic acids

In this review I discuss straightforward and general methods to modify nucleic acid structure with disulfide cross-links. A motivating factor in developing this chemistry was the notion that disulfide bonds would be excellent tools to probe the structure, dynamics, thermodynamics, folding, and function of DNA and RNA, much in the way that cystine cross-links have been used to study proteins. The chemistry described has been used to synthesize disulfide cross-linked hairpins and duplexes, higher order structures like triplexes, nonground-state conformations, and tRNAs. Since the cross-links form quantitatively by mild air oxidation and do not perturb either secondary or tertiary structure, this modification should prove quite useful for the study of nucleic acids. © 1998 John Wiley & Sons, Inc. Biopoly 48: 83–96, 1998     

Propaedeutic study for the delivery of nucleic acid-based molecules from PLGA microparticles and stearic acid nanoparticles

We studied the mechanism governing the delivery of nucleic acid-based drugs (NABD) from microparticles and nanoparticles in zero shear conditions, a situation occurring in applications such as in situ delivery to organ parenchyma. The delivery of a NABD molecule from poly(DL-lactide-co-glycolide) (PLGA) microparticles and stearic acid (SA) nanoparticles was studied using an experimental apparatus comprising a donor chamber separated from the receiver chamber by a synthetic membrane. A possible toxic effect on cell biology, as evaluated by studying cell proliferation, was also conducted forjust PLGA microparticles. A mathematical model based on the hypothesis that NABD release from particles is due to particle erosion was used to interpret experimental release data. Despite zero shear conditions imposed in the donor chamber, particle erosion was the leading mechanism for NABD release from both PLGA microparticles and SA nanoparticles. PLGA microparticle erosion speed is one order of magnitude higher than that of competing SA nanoparticles. Finally, no deleterious effects of PLGA microparticles on cell proliferation were detected. Thus, the data here reported can help optimize the delivery systems aimed at release of NABD from micro- and nanoparticles.     

Novel Rath peptide for intracellular delivery of protein and nucleic acids

In the present study, a novel cell penetrating peptide (CPP) named as Rath, has been identified from the avian infectious bursal disease virus. It has the potential to penetrate and translocate cargo molecules into cells independent of temperature. Additionally, it can deliver oligonucleotide in 30 min and antibodies within an hour intracellular to chicken embryonic fibroblast primary cells. As an ideal delivery vehicle, it has the ability to protect the cargo molecules in the presence of serum, nucleases and has minimal or no cytotoxicity at even higher peptide concentrations studied. The biophysical characterizations showed that Rath has a dominant β structure with a small α helix and has remarkable binding ability with protein and DNA. Thus, the characterization of unique Rath peptide to deliver protein or nucleic acid into the cells with non-covalent interaction could be used as an effective delivery method for various cell based assays.     

Inhibition of bacterial translation and growth by peptide nucleic acids targeted to domain II of 23S rRNA.

The objective of this work was to study the inhibitory effects of antisense peptide nucleic acids (PNAs) targeted to domain II of 23S rRNA on bacterial translation and growth. In this paper, we report that PNA(G1138) or peptide-PNA(G1138) targeted to domain II of 23S rRNA can inhibit both translation in vitro (in a cell-free translation system) and bacterial growth in vivo. The inhibitory concentration (IC50) and the minimum inhibiting concentration (MIC) are 0.15 and 10 microM, respectively. The inhibition effect of PNA(G1138) in vitro is somewhat lower than that of tetracycline (IC50 = 0.12 microM), but the MIC of peptide-PNA(G1138) against Escherichia coli is significantly higher than that of tetracycline (MIC = 4 microM). Further studies based on similar colony-forming unit (CFU) assays showed that peptide-PNA(G1138) at 10 microM is bactericidal, but the bactericidal effect is less effective than that of tetracycline. Nevertheless, the results demonstrated that the peptide-PNA(G1138) treatment is bactericidal in a dose- and sequence-dependent manner and that the G1138 site of 23S rRNA is a possible sequence target for designing novel PNA-based antibiotics.     

Rigid nanoparticle-based delivery of anti-cancer siRNA: Challenges and opportunities

Gene therapy is a promising strategy to treat various genetic and acquired diseases. Small interfering RNA (siRNA) is a revolutionary tool for gene therapy and the analysis of gene function. However, the development of a safe, efficient, and targetable non-viral siRNA delivery system remains a major challenge in gene therapy. An ideal delivery system should be able to encapsulate and protect the siRNA cargo from serum proteins, exhibit target tissue and cell specificity, penetrate the cell membrane, and release its cargo in the desired intracellular compartment. Nanomedicine has the potential to deal with these challenges faced by siRNA delivery. The unique characteristics of rigid nanoparticles mostly inorganic nanoparticles and allotropes of carbon nanomaterials, including high surface area, facile surface modification, controllable size, and excellent magnetic/optical/electrical properties, make them promising candidates for targeted siRNA delivery. In this review, recent progresses on rigid nanoparticle-based siRNA delivery systems will be summarized.     

Inositol phosphates compete with nucleic acids for binding to bovine leukemia virus matrix protein: Implications for deltaretroviral assembly

The matrix (MA) domain of retroviral Gag proteins plays a crucial role in virion assembly. In human immunodeficiency virus type 1 (HIV‐1), a lentivirus, the presence of phosphatidylinositol‐(4,5)‐bisphosphate triggers a conformational change allowing the MA domain to bind the plasma membrane (PM). In this study, the MA protein from bovine leukemia virus (BLV) was used to investigate the mechanism of viral Gag binding to the membrane during replication of a deltaretrovirus. Fluorescence spectroscopy was used to measure the binding affinity of MA for two RNA constructs derived from the BLV genome as well as for single‐stranded DNA (ssDNA). The importance of electrostatic interactions and the ability of inositol hexakisphosphate (IP6) to compete with nucleic acids for binding to MA were also investigated. Our data show that IP6 effectively competes with RNA and DNA for BLV MA binding, while [NaCl] of greater than 100 mM is required to produce any observable effect on DNA‐MA binding. These results suggest that BLV assembly may be highly dependent on the specific interaction of the MA domain with components of the PM, as observed previously with HIV‐1. The mode of MA binding to nucleic acids and the implications for BLV assembly are discussed. Proteins 2013; 81:1377–1385. © 2013 Wiley Periodicals, Inc.