Short Double-stranded Oligonucleotides Research Articles

Amphiphilic pH-sensitive polypeptides for siRNA delivery

The effective delivery of nucleic acid therapeutics into tumor cells is of great importance in terms of cancer gene therapy. Successful application of siRNAs is restricted by the requirement to overcome several biological barriers, i.e. cell membrane penetration, endosomal escape, enzymatic degradation in cytosol, etc. Cationic polymers are promising delivery systems due to their biodegradability and effective binding with siRNA. A key characteristic of the particle trafficking into the cell is a noticeable change in pH during this process. This change can be used as a natural stimulus for pH-sensitive polymer particles. Such polymers are able to buffer the environment of a late endosome that leads to an abnormal flow of ions and water followed by endosomal swelling and disrupt and quick escape of the carriers into a cytoplasm. In present work, we synthesized a series of amphiphilic positively charged biocompatible pH-sensitive polypeptides forming polyplexes with nucleic acids. Four amino acids were used to compose the delivery systems, namely l-lysine, l-glutamic acid, l-phenylalanine and l-histidine. The number and the ratio of amino acids were varied in order to establish the optimal composition. The characteristics of polypeptides were studied by 1H NMR spectroscopy, high performance liquid chromatography and static light scattering. All synthesized polypeptides tended to a self-assembly in aqueous media and formed nanoparticles with hydrodynamic diameters ranging from 140 to 360 nm. The formation of polyplexes with short double-stranded oligonucleotides as a physicochemical siRNA model as well as the release of cargo in buffer media mimicking lysosome (pH∼5) and cytosol or bloodstream (pH 7.4) were studied. Cellular uptake of the particles was efficient and their cytotoxicity was negligible. Successful RNAi-mediated down-regulation of GFP gene expression was revealed in MDA-MB-231/GFP breast cancer cells. The amphiphilic polypeptides obtained can be considered as promising non-viral candidates for siRNA delivery.

1052. Characterisation of the DNA binding properties of ridinilazole, a selective antibiotic currently in phase III trials for the treatment of Clostridioides difficile

Background Clostridioides difficile infection (CDI) is recognised by the CDC as an “urgent threat” in the USA, responsible for nearly 13,000 deaths, and carries an economic burden ranging from &5.4 to &6.3 billion per year. In a phase II study, ridinilazole was shown to be effective at treating CDI and decreasing subsequent recurrence compared to vancomycin. However, the precise mechanism of action of ridinilazole has yet to be fully elucidated. We now present data that reveals ridinilazole clearly co-localises with DNA in C. difficile and binds with high affinity to the minor groove of DNA. These interactions are predicted to have consequences on cellular functions within C. difficile.MethodsHigh resolution confocal microscopy was used to track the intracellular localisation of ridinilazole in C. difficile. Fluorescence intensity was used to characterise the DNA binding properties of ridinilazole; sequence specificity was demonstrated with AT- or GC-rich DNA polymers, and tight binding was shown using short double-stranded oligonucleotides. Hanging drop vapour diffusion enabled co-crystallisation and subsequent structural determination of DNA-bound ridinilazole. ResultsConfocal microscopy revealed clear co-localisation of ridinilazole to the DNA within C. difficile. Ridinilazole demonstrated a dose-dependent increase in fluorescence in response to increasing concentration of target DNA. Fluorescence binding studies revealed that ridinilazole shows a preference towards AT-rich DNA sequences. Tight binding characteristics were demonstrated by ridinilazole in complex with short double-stranded oligonucleotides, returning dissociation constants (Kd) of 20 – 50 nM. Crystallisation enabled co-structures of ridinilazole bound to the minor groove of double-stranded DNA oligonucleotides to be solved.ConclusionRidinilazole demonstrates tight binding with sequence specificity within the minor groove of DNA and co-localises with DNA in C. difficle. Further analysis is ongoing to fully understand this novel mechanism of action, the downstream consequences of these interactions and how they contribute to the bactericidal activity of ridinilazole.Disclosures Clive Mason, PhD, Summit Therapeutics (Employee, Shareholder) Tim Avis, n/a, Summit therapeutics (Shareholder) Chris Coward, PhD, Summit Therapeutics (Employee, Scientific Research Study Investigator, Shareholder) David Powell, PhD, Summit Therapeutics (Employee) Kevin W. Garey, Pharm.D., M.S., FASHP, Summit Therapeutics (Research Grant or Support)

Versatile CRISPR/Cas9 Systems for Genome Editing in Ustilago maydis.

The phytopathogenic smut fungus Ustilago maydis is a versatile model organism to study plant pathology, fungal genetics, and molecular cell biology. Here, we report several strategies to manipulate the genome of U. maydis by the CRISPR/Cas9 technology. These include targeted gene deletion via homologous recombination of short double-stranded oligonucleotides, introduction of point mutations, heterologous complementation at the genomic locus, and endogenous N-terminal tagging with the fluorescent protein mCherry. All applications are independent of a permanent selectable marker and only require transient expression of the endonuclease Cas9hf and sgRNA. The techniques presented here are likely to accelerate research in the U. maydis community but can also act as a template for genome editing in other important fungi.

Development and Mechanism of Small Activating RNA Targeting CEBPA, a Novel Therapeutic in Clinical Trials for Liver Cancer

Small activating RNAs (saRNAs) are short double-stranded oligonucleotides that selectively increase gene transcription. Here, we describe the development of an saRNA that upregulates the transcription factor CCATT/enhancer binding protein alpha (CEBPA), investigate its mode of action, and describe its development into a clinical candidate. A bioinformatically directed nucleotide walk around the CEBPA gene identified an saRNA sequence that upregulates CEBPA mRNA 2.5-fold in human hepatocellular carcinoma cells. A nuclear run-on assay confirmed that this upregulation is a transcriptionally driven process. Mechanistic experiments demonstrate that Argonaute-2 (Ago2) is required for saRNA activity, with the guide strand of the saRNA shown to be associated with Ago2 and localized at the CEBPA genomic locus using RNA chromatin immunoprecipitation (ChIP) assays. The data support a sequence-specific on-target saRNA activity that leads to enhanced CEBPA mRNA transcription. Chemical modifications were introduced in the saRNA duplex to prevent activation of the innate immunity. This modified saRNA retains activation of CEBPA mRNA and downstream targets and inhibits growth of liver cancer cell lines in vitro. This novel drug has been encapsulated in a liposomal formulation for liver delivery, is currently in a phase I clinical trial for patients with liver cancer, and represents the first human study of an saRNA therapeutic.

Microfluidic self-assembly of folate-targeted monomolecular siRNA-lipid nanoparticles.

Non-viral delivery of nucleic acids for therapies based on RNA interference requires a rational design and optimal self-assembly strategies. Nucleic acid particles need to be small, stable and functional in terms of selective cell uptake and controlled release of encapsulated nucleic acids. Here we report on small (∼38 nm) monomolecular nucleic acid/lipid particles (mNALPs) that contain single molecules of short double-stranded oligonucleotides covered by a tight, highly curved lipid bilayer. The particles consist of DOPE, DOTAP, DOPC and DSPE-PEG(2000) and are assembled with 21 bp double-stranded DNA or small interfering RNA by solvent exchange on a hydrodynamic-focusing microfluidic chip. In comparison to vortex mixing by hand this method increases the encapsulation efficiency by 20%, and yields particles with a narrower size distribution, negligible aggregate formation and high stability in blood plasma and serum. Modification of mNALPs with folate-conjugated PEG-lipids results in specific binding and uptake by epithelial carcinoma KB cells overexpressing folate receptors. Binding is significantly reduced by competitive inhibition using free folate and is not observed with non-targeted mNALPs, revealing high specificity. The functionalized mNALPs show gene silencing in the presence of chloroquine, an endosome-destabilizing agent. Together, the robust self-assembly of small-sized mNALPs with their high stability and receptor-specific cell uptake demonstrate that the tight, PEG-grafted lipid-bilayer encapsulation may offer a promising approach towards the delivery of short double-stranded oligonucleotides.

Self-Assembly of Stable Monomolecular Nucleic Acid Lipid Particles with a Size of 30 nm

The design of efficient nucleic acid complexes is key to progress in genetic research and therapies based on RNA interference. For optimal transport within tissue and across extracellular barriers, nucleic acid carriers need to be small and stable. In this Article, we prepare and characterize mono-nucleic acid lipid particles (mono-NALPs). The particles consist of single short double-stranded oligonucleotides or single siRNA molecules each encapsulated within a closed shell of a cationic-zwitterionic lipid bilayer, furnished with an outer polyethylene glycol (PEG) shield. The particles self-assemble by solvent exchange from a solution containing nucleic acid mixed with the four lipid components DOTAP, DOPE, DOPC, and DSPE-PEG(2000). Using fluorescence correlation spectroscopy, we monitor the formation of mono-NALPs from short double-stranded oligonucleotides or siRNA and lipids into monodisperse particles of approximately 30 nm in diameter. Small angle neutron and X-ray scattering and transmission electron microscopy experiments substantiate a micelle-like core-shell structure of the particles. The PEGylated lipid shell protects the nucleic acid core against degradation by nucleases, sterically stabilizes the mono-NALPs against disassembly in collagen networks, and prevents nonspecific binding to cells. Hence, PEG-lipid shielded mono-NALPs are the smallest stable siRNA lipid system possible and may provide a structural design to be built upon for the development of novel nucleic acid delivery systems with enhanced biodistribution in vivo.

Phosphorothioation of Oligonucleotides Strongly Influences the Inhibition of Bacterial (M.HhaI) and Human (Dnmt1) DNA Methyltransferases

Methyltransferase inhibitors: Short double-stranded oligonucleotides that have a hemimethylated target sequence and 5-fluoro-2'-deoxycytidine as a suicide inhibitor as well as their phosphorothioated analogues were tested for their ability to inhibit the bacterial methyltransferase M.HhaI and the human Dnmt1 in vitro.The cytidine analogue 5-fluoro-2'-deoxycytidine (dC(F)) is a mechanism-based inhibitor of DNA methyltransferases. We report the synthesis of short 18-mer dsDNA oligomers containing a triple-hemimethylated CpG motive as a recognition sequence for the human methyltransferase Dnmt1. The DNA strands carry within these CpG islands dC(F) building blocks that function as mechanism-based inhibitors of the analyzed methyltransferases. In addition, we replaced the phosphodiester backbones at defined positions by phosphorothioates. These hypermodified DNA strands were investigated as inhibitors of the DNA methyltransferases M.HhaI and Dnmt1 in vitro. We could show that both methylases behave substantially differently in respect to the amount of DNA backbone modification.

Responding to DNA

The body has multiple mechanisms to mount a response to infection, including pathways activated by lipids, proteins, and nucleic acids. However, the mechanism by which DNA viruses trigger an innate immune response that is independent from pathways that involve the Toll-like receptors (TLRs) has been unclear. Muruve et al . found that adenovirus-infected cultured cells stimulated the processing and release of the proinflammatory mediator interleukin-1β (IL-1β) and that this response required the DNA from the virus (delivered by cationic liposomes), but not the capsid, and was independent of TLR signaling. Transfected DNA from bacteria, synthetic DNA, plasmid DNA, or mammalian DNA also triggered IL-1β secretion, although short double-stranded oligonucleotides did not. A minimum size of about 250 base pairs was required to stimulate IL-1β release. IL-1β processing involves the inflammasome, a multiprotein complex that includes a NALP protein (a Nod-like cytosolic receptor), the adaptor protein ASC, and caspase-1. Macrophages from NALP3- or ASC-deficient mice failed to stimulate IL-1β processing and secretion in response to adenovirus infection. However, if the DNA was introduced by cationic liposome delivery, then NALP3 was not required, but ASC and caspase-1 were required, suggesting that DNA delivered by that mechanism may use an inflammasome complex with a different sensor protein. K + efflux is known to be involved in inflammasome activation, and blocking K + efflux pharmacologically prevented IL-1β release by adenovirus infection or DNA transfection. Mice deficient in NALP3, ASC, or caspase-1 exhibited a reduced innate immune response to infection with adenovirus. The identification of this new DNA sensor pathway for innate immunity has implications not only for understanding and potentially manipulating the response to pathogen infection but also in autoimmune disease, which may also involve aberrant activity of the inflammasome. D. A. Muruve, V. Pétrilli, A. K. Zaiss, L. R. White, S. A. Clark, P. J. Ross, R. J. Parks, J. Tschopp, The inflammasome recognizes cytosolic microbial and host DNA and triggers an innate immune response. Nature 452 , 103-107 (2008). [PubMed]

Nuclear-Cytoplasmic Shuttling of the Oncogenic Mouse UNP/USP4 Deubiquitylating Enzyme

The oncogenic deubiquitylating enzyme (DUB) Unp/Usp4, which binds to the retinoblastoma family of tumor suppressor proteins, was originally described as a nuclear protein. However, more recent studies have shown it to be cytoplasmic. In addition, analysis of its subcellular localization has been complicated by the existence of the paralog Usp15. In this study, we resolved this controversy by investigating the localization of exogenously expressed Usp4 (using red fluorescent protein-Usp4) and of endogenous Usp4 (using highly specific antibodies that can distinguish Usp4 from Usp15). We found that by inhibiting nuclear export with leptomycin B, both exogenous and endogenous Usp4 accumulate in the nucleus. Further, using a Rev-green fluorescent protein-based export assay, we confirmed the existence of a nuclear export signal ((133)VEVYLLELKL(142)) in Usp4. In addition, a functional nuclear import signal ((766)QPQKKKK(772)) was also identified, which was specifically recognized by importin alpha/beta. Finally, we show that the equilibrium of Usp4 subcellular localization varies between different cell types. Usp4 is thus the first DUB reported to have nucleocytoplasmic shuttling properties. The implications of this shuttling for its function as a DUB are discussed.

Bacteriophage T4 Strand Transfer Protein UvsX Tolerates Symmetric and Asymmetric Heterologies in Short Double-stranded Oligonucleotides

The UvsX protein of bacteriophage T4 catalyzes strand transfer from double-stranded DNA to homologous single-stranded DNA to generate both paranemic and plectonemic joints. We demonstrate here that UvsX mediates strand transfer efficiently from synthetic double-stranded donor oligonucleotides of 30 to 117 bp in length to circular single-stranded recipient M13mp19 DNA. Recovery of a diagnostic BamHI-restriction site, activated in the recipient after strand transfer, demonstrates that recipient and donated strands are perfectly base-paired after the exchange reaction has taken place. The transfer reaction progresses with greatest efficiency using donor DNA with a 3′ overhang. Use of donor DNA having recessed 3′ ends or blunt ends reduces the transfer efficiency by half. Single-stranded heterologies, centrally located in either strand of the donor DNA and forming either heteroduplex loops or a bulge in the donor are transferred with 80 to 100% efficiency. Also, a centrally located C/C-mismatch in the donor does not affect the transfer efficiency. Double-stranded heterologies are tolerated by the UvsX-catalyzed reaction but have different effects on the transfer efficiencies, depending on length and location in the molecule. A heterology of 24 bp located at the proximal end (start of transfer), the distal end (termination of transfer) and at each end of the donor molecule results in transfer efficiencies of 100%, 50% and 50 to 60%, respectively. Strand transfer efficiency is markedly reduced to about 15% if the 24 bp heterology is at a central location. However, insertion of a 4 bp heterology at this position yields a transfer efficiency of about 30%. Also, large double-stranded heterologies of 187 bp at the proximal end or 590 bp at the distal end of control-donor DNAs derived from plasmid digests did not impair the transfer activity of UvsX. This result differs from published results obtained with strand transfer reactions with large distally located heterologies catalyzed by RecA of Escherichia coli in vitro.

Bridging the gap. Joining of nonhomologous ends by DNA polymerases.

DNA double strand breaks with noncomplementary ends can be joined by mechanisms of nonhomologous recombination. In some systems a DNA end with a 3'-protruding single strand (PSS), which does not have a recessed 3'-hydroxyl that can allow for fill-in DNA synthesis, is joined to a blunt end with preservation of the 3'-PSS. It has been proposed that this process occurs via single strand ligation or is facilitated by an alignment protein. We were interested in testing the hypothesis that a DNA polymerase could function as this putative alignment protein. To characterize polymerase activities in this type of reaction, we incubated short double-stranded oligonucleotides that had an excess of one of the strands with an exonuclease-free Klenow fragment of Escherichia coli polymerase I, Taq DNA polymerase from Thermus aquaticus, or an exonuclease-free Stoffel fragment of Taq DNA polymerase. Products were analyzed by using biotinylated oligonucleotides separated by denaturing polyacrylamide gel electrophoresis. To further assess the effect of DNA polymerases on the joining of 3'-PSS ends to blunt ends, we incubated linear plasmid DNA with the polymerases and subjected the DNA to Southern blot and sequence analysis. We determined that these DNA polymerases can use a 3'-PSS end as a template after priming off the 3'-hydroxyl of a blunt end. This implies that the joining of noncomplementary ends in eukaryotic cells could proceed by a similar mechanism.

Preferential hydroxylation by the chemical nuclease meso-tetrakis-(4-N-methylpyridiniumyl)porphyrinatomanganeseIII pentaacetate/KHSO5 at the 5' carbon of deoxyriboses on both 3' sides of three contiguous A.T base pairs in short double-stranded oligonucleotides.

Selected double-stranded oligodeoxyribonucleotides have been used to probe, at the molecular level, DNA chain breakages induced by the chemical nuclease mesotetrakis(4-N- methylpyridiniumyl)porphyrinatomanganeseIII pentaacetate/KHSO5. The results show that cleavage selectively occurs on the two 3' sides of three contiguous A.T base pairs (an A.T triplet). Hydroxylation at 5' carbon of the deoxyribose targets represents the initial damage on the sugar-phosphodiester backbone and leaves a 3' phosphate and a 5' aldehyde at the ends. The fragments were separated by HPLC and unambiguously identified through chemical and biochemical reactions and/or sequencing after enzymatic conversion to mononucleosides. Also studied was the degradation of a 22-nucleotide DNA molecule containing two A.T triplets. Gel electrophoresis analyses on the corresponding 5'-32P-end-labeled substrate supported the above cleavage specificity and mechanism.