Nucleic Acid Core Research Articles

A Bioswitchable MiRNA Delivery System: Tetrahedral Framework DNA-Based miRNA Delivery System for Applications in Wound Healing.

The human body's primary line of defense, the skin, is especially prone to harm. Although microRNA (miRNA)-based therapies have attracted increasing attention for skin wound healing, their applications remain limited owing to a range of issues. Tetrahedral framework DNA (tFNA), a nanomaterial possessing nucleic acid characteristics, exhibits an excellent biocompatibility, in addition to anti-inflammatory and transdermal delivery capabilities, and can accelerate skin wound healing. Due to its potential to exert synergistic action with therapeutic miRNA, tFNA has been considered an ideal vehicle for miRNA therapy. The design and synthesis of a bioswitchable miRNA delivery system (BiRDS) is reported, which contains three miRNAs as well as a nucleic acid core to maximize the loading capacity while preserving the characteristics of tFNA. A high stability, excellent permeability of cells as well as tissues and good biological compatibility are demonstrated. By selectively inhibiting heparin-binding epidermal growth factor (HB-EGF), the BiRDS can inhibit the NF-κB pathway while simultaneously controlling the PTEN/Akt pathway. As a result, the BiRDS helps wound healing go through the inflammation to the proliferative phase. This study demonstrates the advantages of the BiRDS in miRNA-based therapy and provides new research ideas for the treatment of skin-related diseases.

A Tetrahedral Framework DNA-Based Bioswitchable miRNA Inhibitor Delivery System: Application to Skin Anti-Aging.

MicroRNA (miR)-based therapy shows strong potential; however, structural limitations pose a challenge in fully exploiting its biomedical functionality. Tetrahedral framework DNA (tFNA) has proven to be an ideal vehicle for miR therapy. Inspired by the ancient Chinese myth "Sun and Immortal Birds," a novel bioswitchable miR inhibitor delivery system (BiRDS) is designed with three miR inhibitors (the three immortal birds) and a nucleic acid core (the central sun). The BiRDS fuses miR inhibitors within the framework, maximizing their loading capacity, while allowing the system to retain the characteristics of small-sized tFNA and avoiding uncertainty associated with RNA exposure in traditional loading protocols. The RNase H-responsive sequence at the tail of each "immortal bird" enables the BiRDS to transform from a 3D to a 2D structure upon entering cells, promoting the delivery of miR inhibitors. To confirm the application potential, the BiRDS is used to deliver the miR-31 inhibitor, with antiaging effects on hair follicle stem cells, into a skin aging model. Superior skin penetration ability and RNA delivery are observed with significant anti-aging effects. These findings demonstrate the capability and editability of the BiRDS to improve the stability and delivery efficacy of miRs for future innovations.

DDX1 vesicles control calcium-dependent mitochondrial activity in mouse embryos

The DEAD box protein DDX1, previously associated with 3’-end RNA processing and DNA repair, forms large aggregates in the cytoplasm of early mouse embryos. Ddx1 knockout causes stalling of embryos at the 2-4 cell stages. Here, we identify a DDX1-containing membrane-bound calcium-containing organelle with a nucleic acid core. We show that aggregates of these organelles form ring-like structures in early-stage embryos which we have named Membrane Associated RNA-containing Vesicles. We present evidence that DDX1 is required for the formation of Membrane Associated RNA-containing Vesicles which in turn regulate the spatial distribution of calcium in embryos. We find that Ddx1 knockout in early embryos disrupts calcium distribution, and increases mitochondria membrane potential, mitochondrial activity, and reactive oxygen species. Sequencing analysis of embryos from Ddx1 heterozygote crosses reveals downregulation of a subset of RNAs involved in developmental and mitochondrial processes in the embryos with low Ddx1 RNA. We propose a role for Membrane Associated RNA-containing Vesicles in calcium-controlled mitochondrial functions that are essential for embryonic development.

Front Cover: Chemically Tuned Intracellular Gene Delivery by Core‐Shell Nanoparticles: Effects of Proton Buffering, Acid Degradability, and Membrane Disruption (ChemMedChem 7/2022)

The Front Cover shows the release of plasmid DNA to the cytoplasm from a core-shell nanoparticle (CSNP) consisting of a condensed nucleic acid core with protamine sulfate and a polymeric shell that is chemically tuned for facilitated escape from the mildly acidic endosome due to its varying protonation, acid-degradability, and membrane disruption capabilities. The transnuclear transport of the plasmid DNA is aided by protamine sulfate enabling efficient expression of the genetic payload. Cover design by Tae Won Yoon. More information can be found in the Research Article by Soo Kyung Cho, Young Jik Kwon et al.

Surface-Engineered Mucus Penetrating Nucleic Acid Delivery Systems with Cell Penetrating Peptides for the Lungs.

Nucleic acids, both DNA and small RNAs, have emerged as potential therapeutics for the treatment of various lung disorders. However, delivery of nucleic acids to the lungs is challenging due to the barrier property imposed by mucus, which is further reinforced in disease conditions such as chronic obstructive pulmonary disease and asthma. The presence of negatively charged mucins imparts the electrostatic barrier property, and the mesh network structure of mucus provides steric hindrance to the delivery system. To overcome this, the delivery system either needs to be muco-inert with a low positive charge such that the interactions with mucus are minimized or should have the ability to transiently dismantle the mucus structure for effective penetration. We have developed a mucus penetrating system for the delivery of both small RNA and plasmid DNA independently. The nucleic acid core consists of a nucleic acid (pDNA/siRNA) and a cationic/amphipathic cell penetrating peptide. The mucus penetrating coating consists of the hydrophilic biopolymer chondroitin sulfate A (CS-A) conjugated with a mucolytic agent, mannitol. We hypothesize that the hydrophilic coating of CS-A would reduce the surface charge and decrease the interaction with negatively charged mucins, while the conjugated mannitol residues would disrupt the mucin-mucin interaction or decrease the viscosity of mucus by increasing the influx of water into the mucus. Our results indicate that CS-A-mannitol-coated nanocomplexes possess reduced surface charge, reduced viscosity of artificial mucus, and increased diffusion in mucin suspension as well as increased penetration through the artificial mucus layer as compared to the non-coated ones. Further, the coated nanocomplexes showed low cytotoxicity as well as higher transfection in A-549 and BEAS-2B cells as compared to the non-coated ones.

Chemically Tuned Intracellular Gene Delivery by Core-Shell Nanoparticles: Effects of Proton Buffering, Acid Degradability, and Membrane Disruption.

Nanoparticles consisting of a condensed nucleic acid core surrounded by protective layers which aid to overcome extracellular and intracellular hurdles to gene delivery (i. e., core-shell nanoparticles, CSNPs) synthetically mimic viruses. The outer shells shield the core and are particularly designed to enable facilitated release of the gene payload into the cytoplasm, the major limiting step in intracellular gene delivery. The hypothetical proton sponge effect and degradability in response to a stimulus (i. e., mildly acidic pH in the endosome) are two prevailing, although contested, principles in designing effective carriers for intracellular gene delivery via endosomal escape. Utilizing the highly flexible chemical-tuning of the polymeric shell via surface-initiated photo-polymerization of the various monomers at different molecular ratios, the effects of proton buffering capacity, acid-degradability, and endosomal membrane-lysis property on intracellular delivery of plasmid DNA by CSNPs were investigated. This study demonstrated the equivalently critical roles of proton buffering and acid-degradability in achieving efficient intracellular gene delivery, independent of cellular uptake. Extended proton buffering resulted in further improved transfection as long as the core structure was not compromised. The results of the study present a promising synthetic strategy to the development of an efficient, chemically-tunable gene delivery carrier.

Virus Assembly Pathways Inside a Host Cell.

Simple RNA viruses self-assemble spontaneously and encapsulate their genome into a shell called the capsid. This process is mainly driven by the attractive electrostatics interaction between the positive charges on capsid proteins and the negative charges on the genome. Despite its importance and many decades of intense research, how the virus selects and packages its native RNA inside the crowded environment of a host cell cytoplasm in the presence of an abundance of nonviral RNA and other anionic polymers has remained a mystery. In this paper, we perform a series of simulations to monitor the growth of viral shells and find the mechanism by which cargo-coat protein interactions can impact the structure and stability of the viral shells. We show that coat protein subunits can assemble around a globular nucleic acid core by forming nonicosahedral cages, which have been recently observed in assembly experiments involving small pieces of RNA. We find that the resulting cages are strained and can easily be split into fragments along stress lines. This suggests that such metastable nonicosahedral intermediates could be easily reassembled into the stable native icosahedral shells if the larger wild-type genome becomes available, despite the presence of a myriad of nonviral RNAs.

Packaging of DNA origami in viral capsids: towards synthetic viruses.

We report a new type of nanoparticle, consisting of a nucleic acid core (>7500 nt) folded into a 35 nm DNA origami sphere, encapsulated by a capsid composed of all three SV40 virus capsid proteins. Compared to the prototype reported previously, whose capsid consists of VP1 only, the new nanoparticle closely adopts the unique intracellular pathway of the native SV40, suggesting that the proteins of the synthetic capsid retain their native viral functionality. Some of the challenges in the design of such near-future composite drugs destined for gene delivery are discussed.

A Synthetic Carrier of Nucleic Acids Structured as a Neutral Phospholipid Envelope Tightly Assembled on Polyplex Surface.

Synthetic carriers of nucleic acids remain inefficient for practical applications due to their insufficient functions as compared with viral vectors developed by evolution. Here, a synthetic carrier is designed to structurally mimic lentivirus, a widely-used viral vector in therapeutic developments, for its neutral phospholipid membrane tightly anchored on the surface of a packed nucleic acid core. Unlike the reported lipopolyplexes of which the surface membrane around the nucleic acid core is formed from charged lipids, the stable attachment of the neutral lipids to each polyplex core in the present system is achieved through preadsorbed micelles of multicarboxyl amphiphilic molecules as lipid bilayer anchors. The adsorbed micelles are under a tension of deformation due to the electrostatic attraction of the head groups to the cationic surface and their "thermodynamic responsibility" to cover the hydrophobic tails in water. When liposomes of neutral phospholipids approach, the hydrophobic tail groups of the adsorbed micelles may insert into the lipid bilayer matrix to induce them to fuse around polyplex and relieve the thermodynamic tension. The formed neutral phospholipid membrane may encapsulate the polyplex core stably, prevent siRNA from prephagocytic leaking and degrading, and immobilize functional agents with increased capacity.

Spherical Nucleic Acid Architecture Can Improve the Efficacy of Polycation-Mediated siRNA Delivery

Clinical translation of small interfering RNA (siRNA) nanocarriers is hindered by limited knowledge regarding the parameters that regulate interactions between nanocarriers and biological systems. To address this, we investigated the influence of polycation-based nanocarrier architecture on intracellular siRNA delivery. We compared the cellular interactions of two polycation-based siRNA carriers that have similar size and surface charge but different siRNA orientation: (1) polyethylenimine-coated spherical nucleic acids (PEI-SNAs), in which polyethylenimine is wrapped around a spherical nucleic acid core containing radially oriented siRNA and (2) randomly assembled polyethylenimine-siRNA polyplexes that lack controlled architecture. We found that PEI-SNAs undergo enhanced and more rapid cellular uptake than polyplexes, suggesting a prominent role for architecture in cellular uptake. Confocal microscopy studies demonstrated that while PEI-SNAs and polyplexes exhibit similar intracellular stability, PEI-SNAs undergo decreased accumulation within lysosomes, identifying another advantage conferred by their architecture. Indeed, these advantageous cellular interactions enhanced the gene silencing potency of PEI-SNAs by 10-fold relative to polyplexes. Finally, cytocompatibility studies showed that PEI-SNAs exhibit decreased toxicity per PEI content relative to polyplexes, allowing the use of more polycation. Our studies provide critical insight into design considerations for engineering siRNA carriers and warrant future investigation of how nanocarrier architecture influences cellular-, organ-, and organism-level interactions.

Functionalizing polyplex surface through self-assembly of a rationally designed tri-block copolymer membrane around the nucleic acid core for systemic delivery

Functionalizing polyplex surface through self-assembly of a rationally designed tri-block copolymer membrane around the nucleic acid core for systemic delivery

Molecular Characterization of Salmonella Bacteriophages Isolated from Natural Environment and its Potential Role in Phage Therapy

Bacteriophages or phages are the viruses that infect bacteria. Phages like all viruses are made up of a nucleic acid core which is surrounded by a protein coat. The majority of phages are known to contain DNA but some contain RNA also. In this study, four salmonella phages were isolated and were subjected to mollecular characterization. All the four Salmonella phages were found to contain DNA as their genetic material. Comparison of protein profiles of phages by SDS-PAGE revealed near identical protein profiles for phages SaP2 and SaP4. Study of storage stability of phage indicated that the titer of the Salmonella phage SaP1 almost remains unaltered during the 14 days storage at different temperatures ranging from - 20oC, 0oC and 4oC. This study shows that the isolated phage have good storage stability. The isolated phages also show narrow host range.Bangladesh J Microbiol, Volume 29, Number 1, June 2012, pp 33-36

Computational virology: From the inside out

Viruses typically pack their genetic material within a protein capsid. Enveloped viruses also have an outer membrane made up of a lipid bilayer and membrane-spanning glycoproteins. X-ray diffraction and cryoelectron microscopy provide high resolution static views of viral structure. Molecular dynamics (MD) simulations may be used to provide dynamic insights into the structures of viruses and their components. There have been a number of simulations of viral capsids and (in some cases) of the inner core of RNA or DNA packaged within them. These simulations have generally focussed on the structural integrity and stability of the capsid and/or on the influence of the nucleic acid core on capsid stability. More recently there have been a number of simulation studies of enveloped viruses, including HIV-1, influenza A, and dengue virus. These have addressed the dynamic behaviour of the capsid, the matrix, and/or of the outer envelope. Analysis of the dynamics of the lipid bilayer components of the envelopes of influenza A and of dengue virus reveals a degree of biophysical robustness, which may contribute to the stability of virus particles in different environments. Significant computational challenges need to be addressed to aid simulation of complex viruses and their membranes, including the need to integrate structural data from a range of sources to enable us to move towards simulations of intact virions.This article is part of a Special Issue entitled: Membrane Proteins edited by J.C. Gumbart and Sergei Noskov.

Polywraplex, Functionalized Polyplexes by Post‐Polyplexing Assembly of a Rationally Designed Triblock Copolymer Membrane

A core–shell structured synthetic carrier, polywraplex, is reported to overcome the hurdles along the inter‐ and intracellular pathways of systemic delivery of siRNA, yet remain structurally simple and easy‐to‐formulate. The core is a cationic polyplex formed of siRNA with polyethylene imine (PEI) and polyspermine‐imidazole‐4,5‐imine (PSI), respectively, and the shell is a self‐assembled unilamella membrane of PEG45‐PCL20‐mototriose‐COO−, a triblock copolymer possessing multicarboxyl saccharide block to guide adsorption to each polyplex surface, a hydrophobic central block to form a protecting layer around the nucleic acid core, and a PEG block functioning as a steric stabilization out‐layer to extend in vivo circulation. The hydrophobic layer limits the anionic charges of the guiding block within a 2D surface to prevent them from penetrating into the polyplex, a common cause for prephagocytic siRNA leaking by polyelectrolytes in vivo. Cell targeting agents may be conjugated to the distal end of the PEG block and assembled on polyplex surface in optimal population. Chemical characterizations comprising consequent fluorescent imaging, dynamic laser scattering, zeta potential, as well as electrophoresis confirm polywraplex formation and its protection to siRNA against leaking and degradation in serum. Cellular and in vivo (mice) assays of biotin‐conjugated polywraplexes suggest prolonged circulation and tumor tissue targeting.

Isolation and characterization of the DNA and protein binding activities of adenovirus core protein V.

The structure of adenovirus outer capsid was revealed recently at 3- to 4-Å resolution (V. Reddy, S. Natchiar, P. Stewart, and G. Nemerow, Science 329:1071-1075, 2010, http://dx.doi.org/10.1126/science.1187292); however, precise details on the function and biochemical and structural features for the inner core still are lacking. Protein V is one the most important components of the adenovirus core, as it links the outer capsid via association with protein VI with the inner DNA core. Protein V is a highly basic protein that strongly binds to DNA in a nonspecific manner. We report the expression of a soluble protein V that exists in monomer-dimer equilibrium. Using reversible cross-linking affinity purification in combination with mass spectrometry, we found that protein V contains multiple DNA binding sites. The binding sites from protein V mediate heat-stable nucleic acid associations, with some of the binding sites possibly masked in the virus by other core proteins. We also demonstrate direct interaction between soluble proteins V and VI, thereby revealing the bridging of the inner DNA core with the outer capsid proteins. These findings are consistent with a model of nucleosome-like structures proposed for the adenovirus core and encapsidated DNA. They also suggest an additional role for protein V in linking the inner nucleic acid core with protein VI on the inner capsid shell. Scant knowledge exists of how the inner core of adenovirus containing its double-stranded DNA (dsDNA) genome and associated proteins is organized. Here, we report a purification scheme for a recombinant form of protein V that allowed analysis of its interactions with the nucleic acid core region. We demonstrate that protein V exhibits stable associations with dsDNA due to the presence of multiple nucleic acid binding sites identified both in the isolated recombinant protein and in virus particles. As protein V also binds to the membrane lytic protein VI molecules, this core protein may serve as a bridge from the inner dsDNA core to the inner capsid shell.

E6AP/UBE3A Ubiquitin Ligase Harbors Two E2∼ubiquitin Binding Sites

By exploiting (125)I-polyubiquitin chain formation as a functional readout of enzyme activity, we have quantitatively examined the mechanism of human E6AP/UBE3A for the first time. Initial rate studies identify UbcH7 as the cognate E2 carrier protein for E6AP, although related Ubc5 isoforms and the ISG15-specific UbcH8 paralog also support E6AP with reduced efficacy due to impaired binding and catalytic competence. Initial rates of polyubiquitin chain formation displayed hyperbolic kinetics with respect to UbcH7 concentration (K(m) = 57.6 ± 5.7 nM and kcat = 0.032 ± 0.001 s(-1)) and substrate inhibition above 2 μM. Competitive inhibition by an isosteric UbcH7C86S-ubiquitin oxyester substrate analog (K(i) = 64 ± 18 nM) demonstrates that Km reflects intrinsic substrate affinity. In contrast, noncompetitive inhibition by a UbcH7C86A product analog (K(i) = 7 ± 0.7 μM) and substrate inhibition at high concentrations require two functionally distinct E2∼ubiquitin substrate binding sites. The kinetics of polyubiquitin chain formation reflect binding at a cryptic Site 1 not previously recognized that catalyzes E6AP∼ubiquitin thioester formation. Subsequent binding of E2∼ubiquitin at the canonical Site 2 present in the extant crystal structure is responsible for polyubiquitin chain elongation. Other rate studies show that the conserved -4 Phe(849) residue is required for polyubiquitin chain formation rather than target protein conjugation as originally suggested. The present studies unambiguously preclude earlier models for the mechanism of Hect domain-catalyzed conjugation through the canonical binding site suggested by the crystal structure and define a novel two-step mechanism for formation of the polyubiquitin degradation signal.

Light Chain of Factor VIII Is Sufficient for Accelerating Cleavage of von Willebrand Factor by ADAMTS13 Metalloprotease

We previously demonstrated that coagulation factor VIII (FVIII) accelerates proteolytic cleavage of von Willebrand factor (VWF) by A disintegrin and metalloprotease with thrombospondin type 1 repeats (ADAMTS13) under fluid shear stress. In this study, the structural elements of FVIII required for the rate-enhancing effect and the biological relevance of this cofactor activity are determined using a murine model. An isolated light chain of human FVIII (hFVIII-LC) increases proteolytic cleavage of VWF by ADAMTS13 under shear in a concentration-dependent manner. The maximal rate-enhancing effect of hFVIII-LC is ∼8-fold, which is comparable with human full-length FVIII and B-domain deleted FVIII (hFVIII-BDD). The heavy chain (hFVIII-HC) and the light chain lacking the acidic (a3) region (hFVIII-LCΔa3) have no effect in accelerating VWF proteolysis by ADAMTS13 under the same conditions. Although recombinant hFVIII-HC and hFVIII-LCΔa3 do not detectably bind immobilized VWF, recombinant hFVIII-LC binds VWF with high affinity (K(D), ∼15 nM). Moreover, ultra-large VWF multimers accumulate in the plasma of fVIII(-/-) mice after hydrodynamic challenge but not in those reconstituted with either hFVIII-BDD or hFVIII-LC. These results suggest that the light chain of FVIII, which is not biologically active for clot formation, is sufficient for accelerating proteolytic cleavage of VWF by ADAMTS13 under fluid shear stress and (patho) physiological conditions. Our findings provide novel insight into the molecular mechanism of how FVIII regulates VWF homeostasis.

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.

Automation Highlights from the Literature

Automation Highlights from the Literature

Hydration forces as a tool for the optimization of core–shell nanoparticle vectors for cancer gene therapy

The high cationic charge density of the polymers used in synthetic gene therapy vectors makes these systems toxic and induces non-specific interactions with blood components. Covalent modification of the positively charged terminal amines with hydroxyl, acetyl, poly(ethylene glycol) or methyl groups reduces not only the toxicity but also the condensation capacity, leading to lower transfection efficiency and changes in nanoparticle biodistribution. The colloidal properties of such nanoparticulate systems can be optimised based on the hydration forces active on their surface. Specifically, a core–shell strategy was used to optimise hydration forces and stabilise the vector system by separating vector functions into those provided by the core, where polypropylenimine dendrimers (DAB-Am16) mediate complexation and transfection functionality, and those linked to the shell, where a hyaluronic acid (HA) coat affords colloidal stability and systemic targeting. This core–shell vector allows a 6-fold reduction in the amount of cationic polymer used to form the nucleic acid core complex, compared to conventional nanoparticles. The HA shell was optimised in terms of thickness and density by maximising short-range hydration forces under physiological conditions. The resulting vector particles also allow specific targeting and transfection of CD44 positive cancer cells. The HA coated particles show reduced toxicity (2.5-fold) and increased transfection (8-fold) compared to the conventional dendriplex. In conclusion, we demonstrate a strategy for independent optimisation of vector core and shell functionality which allows specific targeting and increases the therapeutic index of the non-viral vector system 20-fold.