Introduction of sugar-modified nucleotides into CpG-containing antisense oligonucleotides inhibits TLR9 activation.

Antisense oligonucleotides (ASOs) are synthetic single-stranded oligonucleotides that bind to RNAs through Watson-Crick base pairings. They are actively being developed as therapeutics for various human diseases. ASOs containing unmethylated deoxycytidylyl-deoxyguanosine dinucleotide (CpG) motifs are known to trigger innate immune responses via interaction with toll-like receptor 9 (TLR9). However, the TLR9-stimulatory properties of ASOs, specifically those with lengths equal to or less than 20 nucleotides, phosphorothioate linkages, and the presence and arrangement of sugar-modified nucleotides-crucial elements for ASO therapeutics under development-have not been thoroughly investigated. In this study, we first established SY-ODN18, an 18-nucleotide phosphorothioate oligodeoxynucleotide with sufficient TLR9-stimulatory activity. We demonstrated that an unmethylated CpG motif near its 5'-end was indispensable for TLR9 activation. Moreover, by utilizing various sugar-modified nucleotides, we systematically generated model ASOs, including gapmer, mixmer, and fully modified designs, in accordance with the structures of ASO therapeutics. Our results illustrated that introducing sugar-modified nucleotides in such designs significantly reduces TLR9-stimulatory activity, even without methylation of CpG motifs. These findings would be useful for drug designs on several types of ASOs.

Targeting c-Myc with decoy oligodeoxynucleotide-loaded polycationic nanoparticles inhibits cell growth and induces apoptosis in cancer stem-like cells (NTERA-2).

BACKGROUND: An increase in cancer stem cell (CSC) populations and their resistance to common treatments could be a result of c-Myc dysregulations in certain cancer cells. In the current study, we investigated anticancer effects of c-Myc decoy ODNs loaded-poly (methacrylic acid-co-diallyl dimethyl ammonium chloride) (PMA-DDA)-coated silica nanoparticles as carriers on cancer-like stem cells (NTERA-2). METHODS AND RESULTS: The physicochemical characteristics of the synthesized nanocomposites (SiO2@PMA-DDA-DEC) were analyzed using FT-IR, DLS, and SEM techniques. UV-Vis spectrophotometer was applied to analyze the release pattern of decoy ODNs from the nanocomposite. Furthermore, uptake, cell viability, apoptosis, and cell cycle assays were used to investigate the anticancer effects of nanocomposites loaded with c-Myc decoy ODNs on NTERA-2 cancer cells. The results of physicochemical analytics demonstrated that SiO2@PMA-DDA-DEC nanocomposites were successfully synthesized. The prepared nanocomposites were taken up by NTERA-2 cells with high efficiency, and could effectively inhibit cell growth and increase apoptosis rate in the treated cells compared to the control group. Moreover, SiO2@PMA-DDA nanocomposites loaded with c-Myc decoy ODNs induced cell cycle arrest at the G0/G1 phase in the treated cells. CONCLUSIONS: The conclusion drawn from this study is that c-Myc decoy ODN-loaded SiO2@PMA-DDA nanocomposites can effectively inhibit cell growth and induce apoptosis in NTERA-2 cancer cells. Moreover, given that a metal core is incorporated into this synthetic nanocomposite, it could potentially be used in conjunction with irradiation as part of a decoy-radiotherapy combinational therapy in future investigations.

Programming peptide-oligonucleotide nano-assembly for engineering of neoantigen vaccine with potent immunogenicity.

Background: Neoantigen nanovaccine has been recognized as a promising treatment modality for personalized cancer immunotherapy. However, most current nanovaccines are carrier-dependent and the manufacturing process is complicated, resulting in potential safety concerns and suboptimal codelivery of neoantigens and adjuvants to antigen-presenting cells (APCs). Methods: Here we report a facile and general methodology for nanoassembly of peptide and oligonucleotide by programming neoantigen peptide with a short cationic module at N-terminus to prepare nanovaccine. The programmed peptide can co-assemble with CpG oligonucleotide (TLR9 agonist) into monodispersed nanostructures without the introduction of artificial carrier. Results: We demonstrate that the engineered nanovaccine promoted the codelivery of neoantigen peptides and adjuvants to lymph node-residing APCs and instigated potent neoantigen-specific T-cell responses, eliciting neoantigen-specific antitumor immune responses with negligible systemic toxicity. Furthermore, the antitumor T-cell immunity is profoundly potentiated when combined with anti-PD-1 therapy, leading to significant inhibition or even complete regression of established melanoma and MC-38 colon tumors. Conclusions: Collectively, this work demonstrates the feasibility and effectiveness of personalized cancer nanovaccine preparation with high immunogenicity and good biosafety by programming neoantigen peptide for nanoassembly with oligonucleotides without the aid of artificial carrier.

Tuning CpG motif position in nanostructured DNA for efficient immune stimulation.

It was previously demonstrated that polypod-like nanostructured DNA (polypodna) comprising three or more oligodeoxynucleotides (ODNs) were useful for the delivery of ODNs containing cytosine-phosphate-guanine (CpG) motifs, or CpG ODNs, to immune cells. Although the immunostimulatory activity of single-stranded CpG ODNs is highly dependent on CpG motif sequence and position, little is known about how the position of the motif affects the immunostimulatory activity of CpG motif-containing nanostructured DNAs. In the present study, four series of polypodna were designed, each comprising a CpG ODN with one potent CpG motif at varying positions and 2-5 CpG-free ODNs, and investigated their immunostimulatory activity using Toll-like receptor-9 (TLR9)-positive murine macrophage-like RAW264.7 cells. Polypodnas with the CpG motif in the 5'-overhang induced more tumor necrosis factor-α release than those with the motif in the double-stranded region, even though their cellular uptake were similar. Importantly, the rank order of the immunostimulatory activity of single-stranded CpG ODNs changed after their incorporation into polypodna. These results indicate that the CpG ODN sequence as well as the motif location in nanostructured DNAs should be considered for designing the CpG motif-containing nanostructured DNAs for immune stimulation.

Surface charge-dependent cytokine production using near-infrared emitting silicon quantum dots.

Toll-like receptor 9 (TLR-9) is a protein that helps our immune system identify specific DNA types. Upon detection, CpG oligodeoxynucleotides signal the immune system to generate cytokines, essential proteins that contribute to the body's defence against infectious diseases. Native phosphodiester type B CpG ODNs induce only Interleukin-6 with no effect on interferon-α. We prepared silicon quantum dots containing different surface charges, such as positive, negative, and neutral, using amine, acrylate-modified Plouronic F-127, and Plouronic F-127. Then, class B CpG ODNs are loaded on the surface of the prepared SiQDs. The uptake of ODNs varies based on the surface charge; positively charged SiQDs demonstrate higher adsorption compared to SiQDs with negative and neutral surface charges. The level of cytokine production in peripheral blood mononuclear cells was found to be associated with the surface charge of SiQDs prior to the binding of the CpG ODNs. Significantly higher levels of IL-6 and IFN-α induction were observed compared to neutral and negatively charged SiQDs loaded with CpG ODNs. This observation strongly supports the notion that the surface charge of SiQDs effectively regulates cytokine induction.

The orientation of CpG conjugation on aluminum oxyhydroxide nanoparticles determines the immunostimulatory effects of combination adjuvants.

In subunit vaccines, aluminum salts (Alum) are commonly used as adjuvants, but with limited cellular immune responses. To overcome this limitation, CpG oligodeoxynucleotides (ODNs) have been used in combination with Alum. However, current combined usage of Alum and CpG is limited to linear mixtures, and the underlying interaction mechanism between CpG and Alum is not well understood. Thus, we propose to chemically conjugate Alum nanoparticles and CpG (with 5' or 3' end exposed) to design combination adjuvants. Our study demonstrates that compared to the 3'-end exposure, the 5'-end exposure of CpG in combination adjuvants (Al-CpG-5') enhances the activation of bone-marrow derived dendritic cells (BMDCs) and promotes Th1 and Th2 cytokine secretion. We used the SARS-CoV-2 receptor binding domain (RBD) and hepatitis B surface antigen (HBsAg) as model antigens to demonstrate that Al-CpG-5' enhanced antigen-specific antibody production and upregulated cytotoxic T lymphocyte markers. Additionally, Al-CpG-5' allows for coordinated adaptive immune responses even at lower doses of both CpG ODNs and HBsAg antigens, and enhances lymph node transport of antigens and activation of dendritic cells, promoting Tfh cell differentiation and B cell activation. Our novel Alum-CPG strategy points the way towards broadening the use of nanoadjuvants for both prophylactic and therapeutic vaccines.

Layer-by-layer nanoparticle encapsulating all-trans retinoic acid and CpG as a mucosal adjuvant targeting colorectal cancer.

Colorectal cancer (CRC) ranks among the most prevalent cancers globally, demanding innovative therapeutic strategies. Immunotherapy, a promising avenue, employs cancer vaccines to activate the immune system against tumors. However, conventional approaches fall short of eliciting robust responses within the gastrointestinal (GI) tract, where CRC originates. Harnessing the potential of all-trans retinoic acid (ATRA) and cytosine-phosphorothioate-guanine (CpG), we developed layered nanoparticles using a layer-by-layer assembly method to co-deliver these agents. ATRA, crucial for gut immunity, was efficiently encapsulated alongside CpG within these nanoparticles. Administering these ATRA@CpG-NPs, combined with ovalbumin peptide (OVA), effectively inhibited orthotopic CRC growth in mice. Our approach leveraged the inherent benefits of ATRA and CpG, demonstrating superior efficacy in activating dendritic cells, imprinting T cells with gut-homing receptors, and inhibiting tumor growth. This mucosal adjuvant presents a promising strategy for CRC immunotherapy, showcasing the potential for targeting gut-associated immune responses in combating colorectal malignancies.

Emerging nanoparticle platforms for CpG oligonucleotide delivery.

Unmethylated cytosine-phosphate-guanine (CpG) oligodeoxynucleotides (ODNs), which were therapeutic DNA with high immunostimulatory activity, have been applied in widespread applications from basic research to clinics as therapeutic agents for cancer immunotherapy, viral infection, allergic diseases and asthma since their discovery in 1995. The major factors to consider for clinical translation using CpG motifs are the protection of CpG ODNs from DNase degradation and the delivery of CpG ODNs to the Toll-like receptor-9 expressed human B-cells and plasmacytoid dendritic cells. Therefore, great efforts have been devoted to the advances of efficient delivery systems for CpG ODNs. In this review, we outline new horizons and recent developments in this field, providing a comprehensive summary of the nanoparticle-based CpG delivery systems developed to improve the efficacy of CpG-mediated immune responses, including DNA nanostructures, inorganic nanoparticles, polymer nanoparticles, metal-organic-frameworks, lipid-based nanosystems, proteins and peptides, as well as exosomes and cell membrane nanoparticles. Moreover, future challenges in the establishment of CpG delivery systems for immunotherapeutic applications are discussed. We expect that the continuously growing interest in the development of CpG-based immunotherapy will certainly fuel the excitement and stimulation in medicine research.

Identification of Low-Density Lipoprotein Receptor-Related Protein 1 as a CXCL14 Receptor Using Chemically Synthesized Tetrafunctional Probes.

CXCL14 is a primordial CXC-type chemokine that transports CpG oligodeoxynucleotides (ODN) into endosomes and lysosomes in dendritic cells, thereby leading to the activation of the Toll-like receptor 9 (TLR9)-mediated innate immune system. However, the underlying molecular mechanism by which the CXCL14-CpG ODN complex enters cells remains elusive. Herein, we describe the chemical synthesis of CXCL14-derived photoaffinity probes and their application to the identification of target receptors for CXCL14 using quantitative proteomics. By utilizing native chemical ligation and maleimide-thiol coupling chemistry, we synthesized site-specifically modified CXCL14-based photoaffinity probes that contain photoreactive 2-aryl-5-carboxytetrazole (ACT) and a hydrazine-labile cleavable linker. CXCL14-based probes were found to be capable of binding CpG ODN to immune cells, whose bioactivities were comparable to native CXCL14. Application of CXCL14-derived probes to quantitative proteomic experiments enabled the identification of dozens of target receptor candidates for CXCL14 in mouse macrophage-derived RAW264.7 cells, and we discovered that low-density lipoprotein receptor-related protein 1 (LRP1) is a novel receptor for CXCL14 by competitive proteome profiling. We further showed that disruption of LRP1 affected the incorporation of the CXCL14-CpG ODN complex in the cells. Overall, this report highlights the power of synthetic CXCL14-derived photoaffinity probes combined with chemical proteomics to discover previously unidentified receptors for CXCL14, which could promote an understanding of the molecular functions of CXCL14 and the elaborate machinery of innate immune systems.

Synthesis of Nucleotides Bearing the 2'-O-Trifluoromethyl Group and Their Application in RNA Analogs Preparation.

The integration of fluorine atoms into biologically active organic compounds has proved to be a vital technique in small molecule drugs. This technique can substantially enhance crucial properties, including metabolic stability, lipophilicity, and bioavailability, often with a mere addition of a single fluorine atom or a trifluoromethyl group. Over the past few decades, this concept has also been applied in nucleic acid chemistry. A commonly employed 2'-OH substitution is the introduction of a 2'-deoxy-2'-fluoro (2'-F) group. The strong electronegativity of fluorine prompts the modified siRNA to readily adopt a C3'-endo conformation, resulting in significant advantages in terms of binding affinity. To enrich the toolbox of chemical modification of oligonucleotides, the replacement of the 2'-OH with the 2'-O-trifluoromethyl group has been developed in RNA analog synthesis. Oligodeoxynucleotides containing the 2'-O-trifluoromethyl group can greatly increase the thermal stability of DNA/RNA duplexes depending on the position and amount of the modification. Moreover, 2'-O-trifluoromethylated oligodeoxynucleotide also exhibited a slightly higher resistance to snake venom phosphodiesterase than the unmodified oligodeoxynucleotide. The 2'-O-trifluoromethylated oligonucleotides can emerge as a label to study RNA structure and function as well, or to develop DNA/RNA-based diagnostics. Hence, it is necessary to report an effective method for the synthesis, deprotection, purification, and characterization of oligonucleotides bearing a 2'-O-trifluoromethyl group. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Preparation of 6-N-benzoyl-5'-O-dimethoxytrityl-2'-O-trifluoromethyl adenosine 3'-(2-cyanoethyl N,N-diisopropyl)phosphoramidite Basic Protocol 2: Preparation of 4-N-acetyl-5'-O-dimethoxytrityl-2'-O-trifluoromethyl cytidine 3'-(2-cyanoethyl N,N-diisopropyl)phosphoramidite Basic Protocol 3: Preparation of 2-N-isobutyryl-5'-O-dimethoxytrityl-2'-O-trifluoromethyl guanine 3'-(2-cyanoethyl N,N-diisopropyl)phosphoramidite Basic Protocol 4: Preparation of 5'-O-dimethoxytrityl-2'-O-2-trifluoromethyl uridine 3'-(2-cyanoethyl N,N-diisopropyl) phosphoramidite Basic Protocol 5: Solid-phase synthesis of 2'-O-trifluoromethylated RNA analogs Basic Protocol 6: Deprotection and purification of 2'-O-trifluoromethyl-RNAs.

Glycogen for lysosome-targeted CpG ODNs delivery and enhanced cancer immunotherapy.

CpG oligodeoxynucleotides (ODNs) strongly activate the immune system after binding to toll-like receptor 9 (TLR9) in lysosome, which demonstrated significant potential in cancer immunotherapy. However, their therapeutic efficacy is limited by drawbacks such as rapid degradation and poor cellular uptake. Although encouraging progress have been made on developing various delivery systems for CpG ODNs, safety risks of the synthetic nanocarriers as well as the deficient CpG ODNs release within lysosome remain big obstacles. Herein, we developed a novel nanovector for lysosome-targeted CpG ODNs delivery and enhanced cancer immunotherapy. Natural glycogen was simply aminated (NH2-Gly) through grafting with diethylenetriamine (DETA), which was spherical in shape with diameter of approximately 40 nm. NH2-Gly possessed good biocompatibility. Cationic NH2-Gly complexed CpG ODNs well and protected them from nuclease digestion. NH2-Gly significantly enhanced the cellular uptake of CpG ODNs. Efficient CpG ODNs release was observed in the presence of α-glucosidase that mimicking the environment of lysosome. Consequently, NH2-Gly/CpG complexes triggered potent antitumor immunity and effectively inhibit the tumor growth without causing any toxic effect or tissue damages. This work highlights the promise of glycogen for lysosome-targeted on-command delivery of CpG ODNs, which brings new hope for precision cancer immunotherapy.

Influence of the Charge Ratio of Guanine-Quadruplex Structure-Based CpG Oligodeoxynucleotides and Cationic DOTAP Liposomes on Cytokine Induction Profiles.

Cationic liposomes, specifically 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) liposomes, serve as successful carriers for guanine-quadruplex (G4) structure-based cytosine-guanine oligodeoxynucleotides (CpG ODNs). The combined benefits of CpG ODNs forming a G4 structure and a non-viral vector carrier endow the ensuing complex with promising adjuvant properties. Although G4-CpG ODN-DOTAP complexes show a higher immunostimulatory effect than naked G4-CpG ODNs, the effects of the complex composition, especially charge ratios, on the production of the pro-inflammatory cytokines interleukin (IL)-6 and interferon (IFN)-α remain unclear. Here, we examined whether charge ratios drive the bifurcation of cytokine inductions in human peripheral blood mononuclear cells. Linear CpG ODN-DOTAP liposome complexes formed micrometer-sized positively charged agglomerates; G4-CpG ODN-DOTAP liposome complexes with low charge ratios (0.5 and 1.5) formed ~250 nm-sized negatively charged complexes. Notably, low-charge-ratio (0.5 and 1.5) complexes induced significantly higher IL-6 and IFN-α levels simultaneously than high-charge-ratio (2 and 2.5) complexes. Moreover, confocal microscopy indicated a positive correlation between the cellular uptake of the complex and amount of cytokine induced. The observed effects of charge ratios on complex size, surface charge, and affinity for factors that modify cellular-uptake, intracellular-activity, and cytokine-production efficiency highlight the importance of a rational complex design for delivering and controlling G4-CpG ODN activity.

Mass Spectrometric Analysis of the Active Site Tryptic Peptide of Recombinant O6-Methylguanine-DNA Methyltransferase Following Incubation with Human Colorectal DNA Reveals the Presence of an O6-Alkylguanine Adductome.

Human exposure to DNA alkylating agents is poorly characterized, partly because only a limited range of specific alkyl DNA adducts have been quantified. The human DNA repair protein, O6-methylguanine O6-methyltransferase (MGMT), irreversibly transfers the alkyl group from DNA O6-alkylguanines (O6-alkGs) to an acceptor cysteine, allowing the simultaneous detection of multiple O6-alkG modifications in DNA by mass spectrometric analysis of the MGMT active site peptide (ASP). Recombinant MGMT was incubated with oligodeoxyribonucleotides (ODNs) containing different O6-alkGs, Temozolomide-methylated calf thymus DNA (Me-CT-DNA), or human colorectal DNA of known O6-MethylG (O6-MeG) levels. It was digested with trypsin, and ASPs were detected and quantified by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry. ASPs containing S-methyl, S-ethyl, S-propyl, S-hydroxyethyl, S-carboxymethyl, S-benzyl, and S-pyridyloxobutyl cysteine groups were detected by incubating MGMT with ODNs containing the corresponding O6-alkGs. The LOQ of ASPs containing S-methylcysteine detected after MGMT incubation with Me-CT-DNA was <0.05 pmol O6-MeG per mg CT-DNA. Incubation of MGMT with human colorectal DNA produced ASPs containing S-methylcysteine at levels that correlated with those of O6-MeG determined previously by HPLC-radioimmunoassay (r2 = 0.74; p = 0.014). O6-CMG, a putative O6-hydroxyethylG adduct, and other potential unidentified MGMT substrates were also detected in human DNA samples. This novel approach to the identification and quantitation of O6-alkGs in human DNA has revealed the existence of a human DNA alkyl adductome that remains to be fully characterized. The methodology establishes a platform for characterizing the human DNA O6-alkG adductome and, given the mutagenic potential of O6-alkGs, can provide mechanistic information about cancer pathogenesis.

Lipid Nanoparticle Delivery Alters the Adjuvanticity of the TLR9 Agonist CpG by Innate Immune Activation in Lymphoid Tissue.

Pharmacological strategies to activate innate immune cells are of great relevance in the context of vaccine design and anticancer immune therapy, to mount broad immune responses able to clear infection and malignant cells. Synthetic CpG oligodeoxynucleotides (CpG-ODNs) are short single-stranded DNA molecules containing unmethylated CpG dinucleotides and a phosphorothioate backbone. Class B CpG ODNs activate robust innate immune responses through a TLR9-dependent NF-κB signaling pathway. This feature is attractive to exploit in the context of vaccine design and cancer immunotherapy. Soluble CpG-ODNs cause hepatic toxicity, which reduces its therapeutic applicability. The formulation of class B CpG ODN1826 in lipid nanoparticles (LNPs) containing an ionizable cationic lipid that complexes CpG through electrostatic interaction is reported. Upon local administration, LNP-formulated CpG drains to lymph nodes and triggers robust innate immune activation. Unformulated, soluble, CpG, by contrast, is unable to induce robust innate activation in draining lymph nodes and is distributed systemically. In a vaccination setting, LNP-formulated CpG, admixed with a protein antigen, induces higher antigen-specific antibody titers and T cell responses than antigen admixed with unformulated soluble CpG.

Solid-phase synthesis of oligodeoxynucleotides using nucleobase N-unprotected oxazaphospholidine derivatives bearing a long alkyl chain.

In this study, we developed a new approach for the solid-phase synthesis of oligodeoxynucleotides (ODNs) using nucleobase-unprotected oxazaphospholidine derivatives. We tackled the problem of the difficult purification of N-unprotected monomers due to their high affinity to silica gel by introducing a tetrahydrogeranyl group into the oxazaphospholidine monomers, thereby enhancing the lipophilicity and facilitating the isolation. In addition, the cyclic structure of oxazaphospholidine enabled a hydroxy-group-selective condensation with sufficient efficiency. Unmodified and boranophosphate/phosphate chimeric ODNs were successfully synthesized using this strategy. This synthetic method can be expected to afford ODNs containing base-labile functional groups.

Post-Synthetic Nucleobase Modification of Oligodeoxynucleotides by Sonogashira Coupling and Influence of Alkynyl Modifications on the Duplex-Forming Ability.

Recently, it was reported that the alkynyl modification of nucleobases mitigates the toxicity of antisense oligonucleotides (ASO) while maintaining the efficacy. However, the general effect of alkynyl modifications on the duplex-forming ability of oligonucleotides (ONs) is unclear. In this study, post-synthetic nucleobase modification by Sonogashira coupling in aqueous medium was carried out to efficiently evaluate the physiological properties of various ONs with alkynyl-modified nucleobases. Although several undesired reactions, including nucleobase cyclization, were observed, various types of alkynyl-modified ONs were successfully obtained via Sonogashira coupling of ONs containing iodinated nucleobases. Evaluation of the stability of the duplex formed by the synthesized alkynyl-modified ONs showed that the alkynyl modification of pyrimidine was less tolerated than that of purine, although both the modifications occurred in the major groove of the duplex. These results can be attributed to the bond angle of the alkyne on the pyrimidine and the close proximity of the alkynyl substituents to the phosphodiester backbone. The synthetic method developed in this study may contribute to the screening of the optimal chemical modification of ASO because various alkynyl-modified ONs that are effective in reducing the toxicity of ASO can be easily synthesized by this method.

Integration of Protein Nanocage with CpG Motifs: A Virus-Mimicked Core-Shell Nanostructure to Ignite Antitumor Immunity.

The tumor microenvironment typically possesses immunosuppressive properties that hinder the effectiveness of antitumor immune responses, even in the context of immunotherapies. However, it is observed that pathogenic microorganisms can trigger strong immune responses during infection, offering a potential means to counteract the immunosuppressive environment of tumors. In this study, a protein nanocage called CpG@HBc nanocages (NCs) is developed, which mimics the structure of the hepatitis B virus and combines with an immunostimulatory component known as cytosine phosphoguanosine oligonucleotide (CpG). By delivering these immunostimulatory agents, CpG@HBc NCs are able to effectively reverse the suppressive tumor microenvironment, resulting in the inhibition of poorly immunogenic tumors in mice. Through high-dimensional mass cytometry (CyTOF) analysis, remarkable alterations in immune responses is observed induced by CpG@HBc. Treatment with immunogenic CpG@HBc NCs, along with co-injection of an OX40 agonist, sensitized colorectal cancer tumors to T cell immune responses, resulting in significant impairment of tumor growth and robust immune activation. Furthermore, CpG@HBc NCs induced long-term antitumor immunological memory, protecting tumor-cured mice from tumor rechallenge. Overall, these findings highlight the potential of a virus-inspired protein nanocage to mimic anti-viral immunity and offer a unique therapeutic approach for cancer immunotherapy.

Solvolysis of 2'-deoxyribonucleosides and oligo-2'-deoxyribonucleotides in aqueous pyrrolidine or ammonia solutions.

To assess the feasibility of high-temperature aminolysis of deoxyribooligonucleotides containing rare bases as a method to determine their base sequence, the 2'-ß-D-deoxyribosides of 5-bromouracil, 2-aminopurine, uracil, adenine, cytosine, 5-methylcytosine, hypoxanthine, N6-methyladenine, N4-ethylcytosine, and guanine were compared as to their rate of degradation in 0.5 M aqueous pyrrolidine at 110 °C, conditions used earlier in the analysis of oligonucleotides containing only the canonical bases. The reaction mixtures were analyzed by chromatography on Zorbax XDB-CN and UV absorption spectroscopy. The first-order rate constants for the nucleoside degradations decreased in the above order, spanning a wide range of reactivities. Some of these nucleosides were also tested in 0.5 M aqueous ammonia at 110 °C, giving similar first-order rate constants, except for 2'-deoxyguanosine, which is much more reactive with ammonia, due to the lower basicity of this reagent, leaving a larger proportion of the nucleoside in the non-ionized form, susceptible to nucleophilic attack at the base. Short oligothymidylates containing a single 2-aminopurine, adenine, guanine, or cytosine unit in central position were tested in pyrrolidinolysis, to determine the cleavage rates at these sites and the dependence of these cleavage rates on oligonucleotide length. A model decadeoxyribonucleotide containing all four canonical bases was also pyrrolidinolyzed, followed by ion-exchange chromatography, to deduce the nucleotide sequence from the resulting chromatographic profile.

Molecular Bottlebrushes for Immunostimulatory CpG ODN Delivery: Relationship among Cation Density, Complex Formation Ability, and Cytotoxicity.

Artificially designed short single-stranded DNA sequences containing unmethylated CG (CpG ODNs) are agonists for toll-like receptor 9 (TLR9); thus, they have great potential as vaccine adjuvants for cancer immunotherapy and preventing infectious diseases. To deliver effectively CpG ODNs into cells bearing TLR9, nanoparticle polyion complexes of cationic polymers that are able to ingest multiple CpG ODN molecules have been developed; however, their structures and synthesized polycations are hard to control and bioincompatible, respectively. To solve these issues, we designed cationic molecular bottlebrushes (CMBs) with branches that are made from copolymers of 2-methacryloyloxyethyl phosphorylcholine and 2-methacryloyloxyethyl trimethylammonium chloride. Several instrumental methods were carried out to determine the structure of a CMB and its complex with CpG ODNs. The complexation did not change the overall shape of the original CMB, and the bound CpG ODNs were captured by the outer layer of the CMB. The moderation of cations was important to reduce toxicity and improve secretion of inflammatory cytokines.

Development of a new synthetic method for oligodeoxynucleotides using 3'-H-phosphonamidate derivatives.

In this study, we developed a new approach for the solution-phase synthesis of oligodeoxynucleotides (ODNs) using nucleoside 3'-H-phosphonamidate derivatives as monomers. The H-phosphonamidate monomers having a heterocyclic amino group as the leaving group reacted with an alcohol to form an internucleotidic H-phosphonate diester under mild basic conditions without using additives. The resultant internucleotidic linkage was converted into a more stable linkage, such as an S-cyanoethyl phosphorothioate diester. Moreover, under the conditions for detritylation, the unreacted H-phosphonamidate monomer was converted into a water-soluble compound, which was easily removed by extraction. Thus, only simple extractions were required to purify intermediates, and the solution-phase synthesis of trithymidine diphosphorothioate from the monomer was achieved with only one silica gel column chromatography purification. This method was applied to deoxyadenosine, deoxycytidine, and deoxyguanosine derivatives. This strategy enables us to reduce the number of reagents and simplify the purification process.