Oligonucleotide Complexes Research Articles

One-pot, degradable, silica nanocarriers with encapsulated oligonucleotides for mitochondrial specific targeting

Mutations in nuclear and mitochondrial genes are responsible for severe chronic disorders such as mitochondrial myopathies. Gene therapy using antisense oligonucleotides is a promising strategy to treat mitochondrial DNA (mtDNA) diseases by blocking the replication of the mutated mtDNA. However, transport vehicles are needed for intracellular, mitochondria-specific transport of oligonucleotides. Nanoparticle (NP) based vectors such as large pore mesoporous silica nanoparticles (LP) often rely on surface complexation of oligonucleotides exposing them to nucleases and limiting mitochondria targeting and controlled release ability. In this work, stable, fluorescent, hollow silica nanoparticles (HSN) that encapsulate and protect oligonucleotides in the hollow core were synthesized by a facile one-pot procedure. Both rhodamine B isothiocyanate and bis[3-(triethoxysilyl)propyl]tetrasulfide were incorporated in the HSN matrix by co-condensation to enable cell tracing, intracellular-specific degradation and controlled oligonucleotide release. We also synthesized LP as a benchmark to compare the oligonucleotide loading and release efficacy of our HSN. Mitochondria targeting was enabled by NP functionalization with cationic, lipophilic Triphenylphosphine (TPP) and, for the first time a fusogenic liposome based carrier, previously reported under the name MITO-Porter. HSN exhibited high oligonucleotide incorporation ratios and release dependent on intracellular degradation. Further, MITO-Porter capping of our NP enabled delayed, glutathione (GSH) responsive oligonucleotide release and mitochondria targeting at the same efficiency as TPP functionalized NP. Overall, our NP are promising vectors for anti-gene therapy of mtDNA disease as well as many other monogenic disorders worldwide.Graphical

Advancing Cyclic Ion Mobility Mass Spectrometry Methods for Studying Biomolecules: Toward the Conformational Dynamics of Mega Dalton Protein Aggregates.

Native mass spectrometry is a powerful tool for the analysis of noncovalent complexes. When coupled with high-resolution ion mobility, this technique can be used to investigate the conformational changes induced in said complexes by different solution or gas-phase conditions. In this study, we describe how a new-generation high-resolution ion mobility instrument equipped with a cyclic ion mobility cell can be utilized for the analysis of large biomolecular systems, including temperature-induced protein aggregates of masses greater than 1.5 MDa, as well as a 63 kDa oligonucleotide complex. The effects of and the interplay between the voltages applied to the different components of the cyclic ion mobility spectrometry system on ion transmission and arrival time distribution were demonstrated using biomolecules covering the m/z range 2000-10,000. These data were used to establish a theoretical framework for achieving the best separation in the cyclic ion mobility system. Finally, the cyclic ion mobility mass spectrometer was coupled with a temperature-controlled electrospray ionization source to investigate high-mass protein aggregation. This analysis showed that it was possible to continuously monitor the change in abundance for several conformations of MDa aggregates with increasing temperature. This work significantly increases the range of biomolecules that can be analyzed by both cyclic ion mobility and temperature-controlled electrospray ionization mass spectrometry, providing new possibilities for high-resolution ion mobility analysis.

Polycation Architecture Affects Complexation and Delivery of Short Antisense Oligonucleotides: Micelleplexes Outperform Polyplexes.

Herein, we examine the complexation and biological delivery of a short single-stranded antisense oligonucleotide (ASO) payload with four polymer derivatives that form two architectural variants (polyplexes and micelleplexes): a homopolymer poly(2-dimethylaminoethyl methacrylate) (D), a diblock polymer poly(ethylene glycol)methylether methacrylate-block-poly(2-dimethylaminoethyl methacrylate) (ObD), and two micelle-forming variants, poly(2-dimethylaminoethyl methacrylate)-block-poly(n-butyl methacrylate) (DB) and poly(ethylene glycol)methylether methacrylate-block-poly(2-dimethylaminoethyl methacrylate)-block-poly(n-butyl methacrylate) (ObDB). Both polyplexes and micelleplexes complexed ASOs, and the incorporation of an Ob brush enhances colloidal stability. Micellplexes are templated by the size and shape of the unloaded micelle and that micelle-ASO complexation is not sensitive to formulation/mixing order, allowing ease, versatility, and reproducibility in packaging short oligonucleotides. The DB micelleplexes promoted the largest gene silencing, internalization, and tolerable toxicity while the ObDB micelleplexes displayed enhanced colloidal stability and highly efficient payload trafficking despite having lower cellular uptake. Overall, this work demonstrates that cationic micelles are superior delivery vehicles for ASOs denoting the importance of vehicle architecture in biological performance.

Molecular Dynamics of Oligonucleotide DNA Complexes with Phosphatidylserine

A formation of DNA-lipid complexes was studied by molec.docking and dynamics methods using the interaction of DNA with phosphatidylserine (PS). We have previously shown that some fatty acids and phosphatidylglycerol can specifically bind to oligonucleotide DNA (dA)20•(dT)20. It is shown that (dA)20•(dT)20 and phosphatidylserine formed a stable complex with a 6.3 kcal/mol binding energy and the PS molecule located in the minor groove of DNA. This complex contains 342 atom groups (interatomic distance ≤3.4 Å). The types of bonds in the PS-DNA(oligonucleotide) complex are suggested as hydrogen bonds, and hydrophobic and dipole-induced dipole interactions. In our previous study, a similar arrangement with close binding energy of 5.8 kcal/mol was shown for phosphatidylglycerol complex with the same oligonucleotide (complex contained 354 groups of atoms). The present study is of interest due to the importance of such complexes for applied biochemistry and biotechnology, genetics and molecular biology, cytology and pharmacology, nanotechnology, and self-assembly smart materials.

Successful Incorporation of Exosome-Capturing Antibody-siRNA Complexes into Multiple Myeloma Cells and Suppression of Targeted mRNA Transcripts.

Simple SummaryAlthough nucleic acid medicines are expected to function as new therapeutic agents, their targeted delivery into cancer cells, particularly hematologic cancer cells, via systemic administration, is limited. Based on our previous finding that tumor cell-derived exosomes are preferentially incorporated into their parental cancer cells, we previously demonstrated that anti-CD63 monoclonal antibody (mAb)-oligonucleotide complexes targeting exosomal microRNAs with linear oligo-D-arginine (Arg) linkers (9mer) were transferred into solid cancer cells and inhibited exosomal miRNA functions. To challenge the delivery of siRNAs into hematologic cancer cells, we developed exosome-capturing anti-CD63 mAb-conjugated small interfering RNAs (siRNA) with branched Arg linkers (9+9mer). Anti-CD63 mAb-conjugated complexes were incorporated into multiple myeloma (MM) cells. Moreover, these exosome-capturing mAb-conjugated siRNAs successfully decreased the mRNA transcript levels of targeted mRNAs in the MM cells. This technology could lead to a breakthrough in drug delivery systems for hematologic cancer therapy.Nucleic acid medicines have been developed as new therapeutic agents against various diseases; however, targeted delivery of these reagents into cancer cells, particularly hematologic cancer cells, via systemic administration is limited by the lack of efficient and cell-specific delivery systems. We previously demonstrated that monoclonal antibody (mAb)-oligonucleotide complexes targeting exosomal microRNAs with linear oligo-D-arginine (Arg) linkers were transferred into solid cancer cells and inhibited exosomal miRNA functions. In this study, we developed exosome-capturing anti-CD63 mAb-conjugated small interfering RNAs (siRNAs) with branched Arg linkers and investigated their effects on multiple myeloma (MM) cells. Anti-CD63 mAb-conjugated siRNAs were successfully incorporated into MM cells. The incorporation of exosomes was inhibited by endocytosis inhibitors. We also conducted a functional analysis of anti-CD63 mAb-conjugated siRNAs. Ab-conjugated luciferase+ (luc+) siRNAs significantly decreased the luminescence intensity in OPM-2-luc+ cells. Moreover, treatment with anti-CD63 mAb-conjugated with MYC and CTNNB1 siRNAs decreased the mRNA transcript levels of MYC and CTNNB1 to 52.5% and 55.3%, respectively, in OPM-2 cells. In conclusion, exosome-capturing Ab-conjugated siRNAs with branched Arg linkers can be effectively delivered into MM cells via uptake of exosomes by parental cells. This technology has the potential to lead to a breakthrough in drug delivery systems for hematologic cancers.

The critical role of mobile phase pH in the performance of oligonucleotide ion-pair liquid chromatography-mass spectrometry methods.

The critical role of mobile phase pH in the performance of oligonucleotide ion-pair liquid chromatography-mass spectrometry methods.

A novel β-glucan–oligonucleotide complex selectively delivers siRNA to APCs via Dectin-1

Delivering therapeutic nucleic acids to targeted cells and organs has been a challenge for decades. A novel technology to deliver oligonucleotide therapeutics to immune cells is here described. In this approach, a macromolecular complex of oligonucleotides and the β-1,3-glucan schizophyllan (SPG) is selectively delivered to cells expressing a lectin receptor, Dectin-1, via SPG–Dectin-1 interaction. Detailed investigation of Dectin-1-expressing cells revealed that Dectin-1 is expressed in all subsets of monocytes as well as dendritic cell (DC) populations, including conventional DCs (cDCs) and plasmacytoid DCs (pDCs), in humans. The expression patterns in mice and humans are comparable, except for the expression in pDCs. The results indicate that Dectin-1 is expressed on cells capable of professional antigen presentation, except for B cells. We chose CD40 as a target gene for small interfering RNA (siRNA) as CD40 expression in antigen-presenting cells (APCs), particularly in DCs, plays critical roles in regulating immune responses. Dose-dependent cellular uptake of siCD40–SPG complexes was confirmed in cells expressing Dectin-1. Gene silencing activity was confirmed in vitro by the reduction of CD40 mRNA and by the site-specific cleavage of CD40 mRNA as determined by the 5’ RNA ligase-mediated rapid amplification of cDNA ends (5’RLM-RACE) technique. In vivo activity of siCD40–SPG complexes was demonstrated as the reduced CD40 protein expression in monocytes and DCs in mice. Furthermore, the in vivo activity of siCD40–SPG targeting human CD40 was confirmed in cynomolgus monkeys by the 5’RLM-RACE technique. In conclusion, we have demonstrated the receptor-ligand binding-mediated delivery of siRNA targeting immune-regulating monocytes and DCs via the interaction of SPG and its receptor, Dectin-1. As monocytes and DCs play central roles in inducing and controlling immune responses, Dectin-1-targeted delivery of nucleic acids should provide a useful tool for developing drugs to treat a wide range of diseases, including autoimmune diseases, allergy, and cancer, as well as transplantation.

Phosphorus and sulfur SAD phasing of the nucleic acid-bound DNA-binding domain of interferon regulatory factor 4.

Pivotal to the regulation of key cellular processes such as the transcription, replication and repair of DNA, DNA-binding proteins play vital roles in all aspects of genetic activity. The determination of high-quality structures of DNA-binding proteins, particularly those in complexes with DNA, provides crucial insights into the understanding of these processes. The presence in such complexes of phosphate-rich oligonucleotides offers the choice of a rapid method for the routine solution of DNA-binding proteins through the use of long-wavelength beamlines such as I23 at Diamond Light Source. This article reports the use of native intrinsic phosphorus and sulfur single-wavelength anomalous dispersion methods to solve the complex of the DNA-binding domain (DBD) of interferon regulatory factor 4 (IRF4) bound to its interferon-stimulated response element (ISRE). The structure unexpectedly shows three molecules of the IRF4 DBD bound to one ISRE. The sole reliance on native intrinsic anomalous scattering elements that belong to DNA-protein complexes renders the method of general applicability to a large number of such protein complexes that cannot be solved by molecular replacement or by other phasing methods.

Polymer-Colloid Complexes Based on Cationic Imidazolium Amphiphile, Polyacrylic Acid and DNA Decamer.

The solution behavior and physicochemical characteristics of polymer–colloid complexes based on cationic imidazolium amphiphile with a dodecyl tail (IA-12) and polyacrylic acid (PAA) or DNA decamer (oligonucleotide) were evaluated using tensiometry, conductometry, dynamic and electrophoretic light scattering and fluorescent spectroscopy and microscopy. It has been established that PAA addition to the surfactant system resulted in a ca. 200-fold decrease in the aggregation threshold of IA-12, with the hydrodynamic diameter of complexes ranging within 100–150 nm. Electrostatic forces are assumed to be the main driving force in the formation of IA-12/PAA complexes. Factors influencing the efficacy of the complexation of IA-12 with oligonucleotide were determined. The nonconventional mode of binding with the involvement of hydrophobic interactions and the intercalation mechanism is probably responsible for the IA-12/oligonucleotide complexation, and a minor contribution of electrostatic forces occurred. The latter was supported by zeta potential measurements and the gel electrophoresis technique, which demonstrated the low degree of charge neutralization of the complexes. Importantly, cellular uptake of the IA-12/oligonucleotide complex was confirmed by fluorescence microscopy and flow cytometry data on the example of M-HeLa cells. While single IA-12 samples exhibit roughly similar cytotoxicity, IA-12–oligonucleotide complexes show a selective effect toward M-HeLa cells (IC50 1.1 µM) compared to Chang liver cells (IC50 23.1 µM).

Lipid nanoparticles loaded with ribonucleoprotein–oligonucleotide complexes synthesized using a microfluidic device exhibit robust genome editing and hepatitis B virus inhibition

The clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) system has considerable therapeutic potential for use in treating a wide range of intractable genetic and infectious diseases including hepatitis B virus (HBV) infections. While non-viral delivery technologies for the CRISPR/Cas system are expected to have clinical applications, difficulties associated with the clinically relevant synthesis of formulations and the poor efficiency of delivery severely hinder therapeutic genome editing. We report herein on the production of a lipid nanoparticle (LNP)-based CRISPR/Cas ribonucleoprotein (RNP) delivery nanoplatform synthesized using a clinically relevant mixer-equipped microfluidic device. DNA cleavage activity and the aggregation of Cas enzymes was completely avoided under the optimized synthetic conditions. The optimized formulation, which was identified through 2 steps of design of experiments, exhibited excellent gene disruption (up to 97%) and base substitution (up to 23%) without any apparent cytotoxicity. The addition of negative charges to the RNPs by complexing single-stranded oligonucleotide (ssON) significantly enhanced the delivery of both Cas9 and Cpf1 RNPs. The optimized formulation significantly suppressed both HBV DNA and covalently closed circular DNA (cccDNA) in HBV-infected human liver cells compared to adeno-associated virus type 2 (AAV2). These findings represent a significant contribution to the development of CRISPR/Cas RNP delivery technology and its practical applications in genome editing therapy.

How Human H1 Histone Recognizes DNA.

Linker H1 histone is one of the five main histone proteins (H1, H2A, H2B, H3, and H4), which are components of chromatin in eukaryotic cells. Here we have analyzed the patterns of DNA recognition by free H1 histone using a stepwise increase of the ligand complexity method; the affinity of H1 histone for various single- and double-stranded oligonucleotides (d(pN)n; n = 1–20) was evaluated using their competition with 12-mer [32P]labeled oligonucleotide and protein–oligonucleotide complex delaying on nitrocellulose membrane filters. It was shown that minimal ligands of H1 histone (like other DNA-dependent proteins and enzymes) are different mononucleotides (dNMPs; Kd = (1.30 ± 0.2) × 10−2 M). An increase in the length of single-stranded (ss) homo- and hetero-oligonucleotides (d(pA)n, d(pT)n, d(pC)n, and d(pN)n with different bases) by one nucleotide link regardless of their bases, leads to a monotonic increase in their affinity by a factor of f = 3.0 ± 0.2. This factor f corresponds to the Kd value = 1/f characterizing the affinity of one nucleotide of different ss d(pN)n for H1 at n = 2–6 (which are covered by this protein globule) is approximately 0.33 ± 0.02 M. The affinity of five out of six DNA nucleotide units is approximately 25 times lower than for one of the links. The affinity of duplexes of complementary homo- and hetero-d(pN)20 is only 1.3–3.3-fold higher in comparison with corresponding ss oligonucleotides. H1 histone forms mainly weak additive contacts with internucleoside phosphate groups of ssDNAs and one chain of double-stranded DNAs, but not with the bases.

Synthesis, chirality-dependent conformational and biological properties of siRNAs containing 5'-(R)- and 5'-(S)-C-methyl-guanosine.

Various chemical modifications have been identified that enhance potency of small interfering RNAs (siRNAs) and that reduce off-target effects, immune stimulation, and toxicities of metabolites of these therapeutic agents. We previously described 5′-C-methyl pyrimidine nucleotides also modified at the 2′ position of the sugar. Here, we describe the synthesis of 2′-position unmodified 5′-(R)- and 5′-(S)-C-methyl guanosine and evaluation of these nucleotides in the context of siRNA. The (R) isomer provided protection from 5′ exonuclease and the (S) isomer provided protection from 3′ exonuclease in the context of a terminally modified oligonucleotide. siRNA potency was maintained when these modifications were incorporated at the tested positions of sense and antisense strands. Moreover, the corresponding 5′ triphosphates were not substrates for mitochondrial DNA polymerase. Models generated based on crystal structures of 5′ and 3′ exonuclease oligonucleotide complexes with 5′-(R)- and 5′-(S)-C-methyl substituents attached to the 5′- and 3′-terminal nucleotides, respectively, provided insight into the origins of the observed protections. Structural properties of 5′-(R)-C-methyl guanosine incorporated into an RNA octamer were analysed by X-ray crystallography, and the structure explains the loss in duplex thermal stability for the (R) isomer compared with the (S) isomer. Finally, the effect of 5′-C-methylation on endoribonuclease activity has been explained.

Development of Antibody-Oligonucleotide Complexes for Targeting Exosomal MicroRNA.

MicroRNAs in exosomes (exosomal miRNAs) are considered as significant targets for cancer therapy. Anti-miR oligonucleotides are often used for the functional inhibition of miRNAs; however, there are no studies regarding the regulation of exosomal miRNA functions. In this study, we attempted to develop a novel drug delivery system using anti-exosome antibody–anti-miR oligonucleotide complexes (ExomiR-Tracker) to hijack exosomes to carry anti-miR oligonucleotides inside exosome-recipient cells. We found that ExomiR-Tracker bound to the exosomes, and then the complexes were introduced into the recipient cells. We also found that anti-miR oligonucleotides introduced into the recipient cells can exhibit inhibitory effects on exosomal miRNA functions in vitro and in vivo. We believe that our strategy would be a promising one for targeting exosomal miRNAs.

Inhibition of post-trabeculectomy fibrosis via topically instilled antisense oligonucleotide complexes co-loaded with fluorouracil

Trabeculectomy is the mainstay of surgical glaucoma treatment, while the success rate was unsatisfying due to postoperative scarring of the filtering blebs. Clinical countermeasures for scar prevention are intraoperative intervention or repeated subconjunctival injections. Herein, we designed a co-delivery system capable of transporting fluorouracil and anti-TGF-β2 oligonucleotide to synergistically inhibit fibroblast proliferation via topical instillation. This co-delivery system was built based on a cationic dendrimer core (PAMAM), which encapsulated fluorouracil within hydrophobic cavity and condensed oligonucleotide with surface amino groups, and was further modified with hyaluronic acid and cell-penetrating peptide penetratin. The co-delivery system was self-assembled into nanoscale complexes with increased cellular uptake and enabled efficient inhibition on proliferation of fibroblast cells. In vivo studies on rabbit trabeculectomy models further confirmed the anti-fibrosis efficiency of the complexes, which prolonged survival time of filtering blebs and maintained their height and extent during wound healing process, exhibiting an equivalent effect on scar prevention compared to intraoperative infiltration with fluorouracil. Qualitative observation by immunohistochemistry staining and quantitative analysis by Western blotting both suggested that TGF-β2 expression was inhibited by the co-delivery complexes. Our study provided a potential approach promising to guarantee success rate of trabeculectomy and prolong survival time of filtering blebs.

Lung-targeted delivery of TGF-β antisense oligonucleotides to treat pulmonary fibrosis

Pulmonary fibrosis is a serious respiratory disease, with limited therapeutic options. Since TGF-β is a critical factor in the fibrotic process, downregulation of this cytokine has been considered a potential approach for disease treatment. Herein, we designed a new lung-targeted delivery technology based on the complexation of polymeric antisense oligonucleotides (pASO) and dimeric human β-defensin 23 (DhBD23). Antisense oligonucleotides targeting TGF-β mRNA were polymerized by rolling circle amplification and complexed with DhBD23. After complexation with DhBD23, pASO showed improved serum stability and enhanced uptake by fibroblasts in vitro and lung-specific accumulation upon intravenous injection in vivo. The pASO/DhBD23 complex delivered into the lung downregulated target mRNA, and subsequently alleviated lung fibrosis in mice, as demonstrated by western blotting, quantitative reverse-transcriptase PCR (qRT-PCR), immunohistochemistry, and immunofluorescence imaging. Moreover, as the complex was prepared only with highly biocompatible materials such as DNA and human-derived peptides, no systemic toxicity was observed in major organs. Therefore, the pASO/DhBD23 complex is a promising gene therapy platform with lung-targeting ability to treat various pulmonary diseases, including pulmonary fibrosis, with low side effects.

Incorporation of Antisense Oligonucleotides into Lipophilic Concatemeric Complexes Provides Their Effective Penetration into Cells

The development of highly effective molecular and biological tools to facilitate the penetration of therapeutic nucleic acids into cells opens a direct way to their successful application as drugs. It has been shown that the incorporation of single-stranded antisense oligonucleotides into concatemeric complexes enhances their binding to the membrane structures of eukaryotic cells, and the use of a cholesterol residue attached to one of the oligonucleotide strands considerably improves the delivery of concatemeric complexes of oligonucleotides to their intracellular target. In the present work, the efficiency of the formation of concatemeric structures from oligonucleotides carrying lipophilic fragments such as lithocholic acid, oleylamide of lithocholic acid, and cholesterol, attached to the 5′-end of the oligonucleotide through oligomethylene linkers of different length has been studied. It has been found that all modified oligonucleotides are capable of effectively “assembling” into concatemeric complexes; however, effective delivery into cells was observed only in the case of concatemeric complexes formed by oligonucleotides carrying a cholesterol residue attached through an aminohexanol linker. It has been shown that antisense oligonucleotides delivered to cells as part of these cholesterol-containing concatemeric complexes effectively inhibit the expression of the target gene.

Poly(1-vinylimidazole) Prospects in Gene Delivery

Polymeric amines are being studied intensively as components of systems for gene delivery in genetic engineering and gene therapy of genetic disorders, including cancer. Despite remarkable achievements in the field, polymeric amines, such as polyethyleneimine, show some disadvantages. Strong interaction between the amine-containing polymer and nucleic acid hampers the release of nucleic acid in the cell cytoplasm. Amine groups can interact with the cell membrane which results in cell death. These limitations of polymeric amines stimulated an investigation of new structures for gene delivery. Imidazole-containing polymers have attracted attention as lesser basic substances, while they are able to interact with polymeric acids. Further development of imidazole-based gene delivery agents requires knowledge about some fundamental aspects of interaction between nucleic acids, and polymeric imidazoles. In this work, we studied the complexation of poly(1-vinylimidazole) and oligomeric DNA. We found that the number of active sites capable of binding with negatively charged phosphate groups is comparable with the number of protonated imidazole units in the case of high molecular weight polymer. The increase in polymer charge by 1-bromopropane quaternizating 1%−5% imidazole units or by decreasing the pH to 6.5−7 considerably increased the ability of poly(1-vinylimidazole) to interact with oligonucleotides. The pH sensitivity of this interaction is interesting for cancer gene therapy because the tumours have a lowered intercellular pH (stable oligonucleotide complex) and a higher extracellular pH which can lead to complex dissociation. Minimal critical length for complexation of quaternized poly(1-vinylimidazole) and DNA is below eight units which corresponds to polymers with amine groups. Fluorescence-tagged poly(1-vinylimidazole) samples were obtained and their potential for monitoring the polymer and polymer-oligonucleotide complex internalization into living cells was demonstrated.

Polyelectrolyte Complexation of Oligonucleotides by Charged Hydrophobic-Neutral Hydrophilic Block Copolymers.

Polyelectrolyte complex micelles (PCMs, core-shell nanoparticles formed by complexation of a polyelectrolyte with a polyelectrolyte-hydrophilic neutral block copolymer) offer a solution to the critical problem of delivering therapeutic nucleic acids, Despite this, few systematic studies have been conducted on how parameters such as polycation charge density, hydrophobicity, and choice of charged group influence PCM properties, despite evidence that these strongly influence the complexation behavior of polyelectrolyte homopolymers. In this article, we report a comparison of oligonucleotide PCMs and polyelectrolyte complexes formed by poly(lysine) and poly((vinylbenzyl) trimethylammonium) (PVBTMA), a styrenic polycation with comparatively higher charge density, increased hydrophobicity, and a permanent positive charge. All of these differences have been individually suggested to provide increased complex stability, but we find that PVBTMA in fact complexes oligonucleotides more weakly than does poly(lysine), as measured by stability versus added salt. Using small angle X-ray scattering and electron microscopy, we find that PCMs formed from both cationic blocks exhibit very similar structure-property relationships, with PCM radius determined by the cationic block size and shape controlled by the hybridization state of the oligonucleotides. These observations narrow the design space for optimizing therapeutic PCMs and provide new insights into the rich polymer physics of polyelectrolyte self-assembly.

Chitosan enhances gene delivery of oligonucleotide complexes with magnetic nanoparticles-cell-penetrating peptide.

Gene-based therapies, including the delivery of oligonucleotides, offer promising methods for the treatment of cancer cells. However, they have various limitations including low efficiency. Herein, cell-penetrating peptides (CPPs)-conjugated chitosan-modified iron oxide magnetic nanoparticles (CPPs-CTS@MNPs) with high biocompatibility as well as high efficiency were tested for the delivery of oligonucleotides such as plasmid pGL3, splice correction oligonucleotides, and small-interfering RNA. A biocompatible nanocomposite, in which CTS@MNPs was incorporated in non-covalent complex with CPPs-oligonucleotide, is developed. Modifying the surface of magnetic nanoparticles with cationic chitosan-modified iron oxide improved the performance of magnetic nanoparticles-CPPs for oligonucleotide delivery. CPPs-CTS@MNPs complexes enhance oligonucleotide transfection compared to CPPs@MNPs or CPPs. The hydrophilic character of CTS@MNPs improves complexation with plasmid pGL3, splice correction oligonucleotides, and small-interfering RNA payload, which consequently resulted in not only strengthening the colloidal stability of the constructed complex but also improving their biocompatibility. Transfection using PF14-splice correction oligonucleotides-CTS@MNPs showed sixfold increase of the transfection compared to splice correction oligonucleotides-PF14 that showed higher transfection than the commercially available lipid-based vector Lipofectamine™ 2000. Nanoscaled CPPs-CTS@MNPs comprise a new family of biomaterials that can circumvent some of the limitations of CPPs or magnetic nanoparticles.

Development of a simple coarse-grained DNA model for analysis of oligonucleotide complex formation

In this paper, we develop a coarse-grained nucleotide model for the purpose of simulating large-scale aptamer-based hydrogel network formation in future research. In the model, each nucleotide is represented by a single interaction site containing sugar, phosphate and base. Discontinuous molecular dynamics (DMD) simulations are performed to simulate formation and denaturation of oligonucleotide duplexes as a function of temperature. The simulated melting temperatures of oligonucleotide duplexes are calculated in simulations of systems with different sequences, lengths and concentrations of oligonucleotides, and compared to data from the OligoAnalyzer tool. The denaturation of oligonucleotide triplexes containing a hybridised structure of three different oligonucleotides is analysed using both simulations and experiments. The nucleotide model is found to be a good predictor of the oligonucleotide’s hybridised state for both duplexes and triplexes. This coarse-grained model has wide ranging applications in the development or optimisation of DNA-based technologies including DNA origami, DNA-enabled hydrogels and DNA-based biosensors.