Oligonucleotide Research Articles

Effect of oligonucleotide MT01 delivered by N-isopropylacrylamide modified polyethyleneimine for bone regeneration.

Objective: This study aimed to investigate the regulatory effect of N-isopropylacrylamide-modified polyethyleneimine (PEN)-delivered oligodeoxynucleotide (ODN) MT01 on bone regeneration in vitro and in vivo. Methods: A polyethylenimine (PEI) derivative, PEN, was constructed through Michael addition and employed as a carrier for ODN MT01 transfection. PEN/MT01 nanocomposites were characterized using agarose gel retardation assay, size distribution, zeta potential and transmission electron microscopy. The Cell Counting Kit-8 (CCK-8) assay was used to detect the effect of PEN on cell viability. Alkaline phosphatase (ALP) staining was used to detect the osteogenic differentiation ability of PEN/MT01 nanocomposite. Real-time quantitative PCR (q RT-PCR) and enzyme-linked immunosorbent assay (ELISA) were used to detect the regulatory effects of PEN/MT01 nanocomposite on osteogenic differentiation gene expression. Rat model was observed using the skull defect method and verified using micro-computed tomography (CT), serum biochemical indices, hematoxylin and eosin (H&E) staining and Immunohistochemistry (IHC). Results: PEN had good biological properties and could deliver MT01 well to achieve efficient transmission of MT01. PEN/MT01 nanocomposites were effectively transfected into MC3T3-E1 cells at a ratio of 6.0. CCK-8 assay displayed that PEN had no cytotoxicity to MC3T3-E1 cells. Additionally, PEN/MT01 nanocomposites could promote the expression of osteogenic genes. In vivo results revealed that PEN/MT01 nanocomposites could promote bone regeneration more effectively than the other groups. Conclusion: PEN has good biocompatibility and low toxicity, which is a good carrier for ODN MT01. PEN-delivered MT01 can be potentially employed as a useful approach to achieving bone regeneration.

CpG ODN 2102 promotes antibacterial immune responses and enhances vaccine-induced protection in golden pompano (Trachinotusovatus).

CpG oligodeoxynucleotides (ODNs) are oligodeoxynucleotides containing CpG motifs and can be recognized by toll-like receptor 9 (TLR9), activating the host's immune responses. In this study, ten different CpG ODNs were designed and synthesized to study the antibacterial immune responses of CpG ODNs in golden pompano (Trachinotus ovatus). Results showed that CpG ODN 2102 significantly improved the immunity of golden pompano against bacteria. Besides, CpG ODN 2102 promoted the proliferation of head kidney lymphocytes and activated the head kidney macrophages. When TLR9-specific small interfering RNA (siRNA) was used to interfere with TLR9 expression, the immune responses were decreased. Moreover, the expression levels of myeloid differentiation primary response 88 (Myd88), p65, tumor necrosis factor receptor-associated factor 6 (TRAF6), and tumor necrosis factor-alpha (TNF-α) in the TLR9-knockdown golden pompano kidney (GPK) cells were significantly reduced. The nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) promoter activity of the TLR9-knockdown GPK cells was also significantly reduced. In vivo, the antibacterial immune effects induced by CpG ODN 2102 in golden pompano were mostly abolished when TLR9 expression was knocked down. These results suggested that TLR9 was involved in the immune responses induced by CpG ODN 2102. CpG ODN 2102 also enhanced the protective effect of the Vibrio harveyi vaccine pCTssJ, where the survival rate of golden pompano was significantly improved by 20%. In addition, CpG ODN 2102 enhanced the messenger RNA (mRNA) expression levels of TLR9, Myxovirus resistance (Mx), interferon γ (IFN-γ), TNF-α, interleukin (IL)-1β, IL-8, major histocompatibility complex class (MHC) Iα, MHC IIα, Immunoglobulin D (IgD), and IgM. Therefore, TLR9 was involved in the antibacterial immune responses induced by CpG ODN 2102 and CpG ODN 2102 possessed adjuvant immune effects. These results enlarged our knowledge of the antibacterial immunity of fish TLRs signaling pathway and had important implications for exploring natural antibacterial molecules in fish and developing new vaccine adjuvants.

Nano-particulate Toll-like Receptor 9 Agonist Potentiates the Antitumor Activity of Anti-Glypican-1 Antibody.

Monoclonal antibodies (mAbs) that target tumor antigens have recently been developed. Their antitumor activity is mainly achieved through antibody-dependent cellular cytotoxicity (ADCC) via effector cells such as tumor-infiltrated macrophages and natural killer (NK) cells. CpG oligodeoxynucleotides (ODNs) have potent antitumor activity and are considered to increase the tumor infiltration of macrophages and NK cells; however, a completely solubilized novel CpG-schizophyllan (SPG) complex, K3-SPG, displays more potent antitumor activity. We recently reported the significant antitumor activity of anti-glypican-1 (GPC1) mAb against GPC1-positive esophageal squamous cell carcinoma (ESCC) via ADCC. The aim of this study was to evaluate the potential synergistic antitumor activity of anti-GPC1 mAb and K3-SPG and elucidate the underlying mechanisms using a xenograft model of GPC1-positive human ESCC cells. The established human esophageal cancer cell line TE14 was subcutaneously injected into SCID mice. Xenograft mice were treated with anti-GPC1 mAb, K3-SPG, or their combination. Antitumor activity was evaluated by measuring the tumor volume. For FACS analysis, agents were administrated, and tumors were resected 1 day after the final treatment. Anti-GPC1 mAb or K3-SPG monotherapy showed dose-dependent antitumor activity, and combination therapy with anti-GPC1 mAb and K3-SPG showed antitumor activity (p=0.0859). Flow cytometry revealed significantly increased numbers of macrophages (p=0.0133) and of the ratio of activated NK cells/total NK cells (p=0.0058) following K3-SPG or combination therapy. Combination therapy with K3-SPG and anti-GPC1 mAb or another antitumor mAb may represent a new cancer treatment option acting via ADCC.

Enhancement of the thermal stability of G-quadruplex structures by urea

The solute urea has been used extensively as a denaturant in protein folding studies; double-stranded nucleic acid structures are also destabilized by urea, but comparatively less than proteins. In previous research, the solute has been shown to strongly destabilize folded G-quadruplex DNA structures. This contribution demonstrates the stabilizing effect of urea on the G-quadruplex formed by the oligodeoxyribonucleotide (ODN), G3T (d[5′-GGGTGGGTGGGTGGG-3′]), and related sequences in the presence of sodium or potassium cations. Stabilization is observed up to 7 M urea, which was the highest concentration we investigated. The folded structure of G3T has three G-tetrads and three loops that consist of single thymine residues. ODNs related to G3T, in which the thymine residues in the loop are substituted by adenosine residues, also exhibit enhanced stability in the presence of molar concentrations of urea. The circular dichroism (CD) spectra of these ODNs in the presence of urea are consistent with that of a G-quadruplex. As the urea concentration increases, the spectral intensities of the peaks and troughs change, while their positions change very little. The heat-induced transition from the folded to unfolded state, Tm, was measured by monitoring the change in the UV absorption as a function of temperature. G-quadruplex structures with loops containing single bases exhibited large increases in Tm with increasing urea concentrations. These data imply that the loop region play a significant role in the thermal stability of tetra-helical DNA structures in the presence of the solute urea.

Piezo2 Channel Upregulation is Involved in Mechanical Allodynia in CYP-Induced Cystitis Rats

Mechanical sensing Piezo2 channel in primary sensory neurons has been shown contribute to mechanical allodynia in somatic chronic pain conditions. Interstitial cystitis (IC)-associated pain is often triggered by bladder filling, a presentation that mimics the mechanical allodynia. In the present study, we aimed to examine the involvement of sensory Piezo2 channel in IC-associated mechanical allodynia using a commonly employed cyclophosphamide (CYP)-induced IC model rat. Piezo2 channels in dorsal root ganglia (DRGs) was knocked down by intrathecal injections of Piezo2 anti-sense oligodeoxynucleotides (ODNs) in CYP-induced cystitis rats, and mechanical stimulation–evoked referred bladder pain was measured in the lower abdomen overlying the bladder using von Frey filaments. Piezo2 expression at the mRNA, protein, and functional levels in DRG neurons innervating the bladder was detected by RNA-fluorescence in situ hybridization, western blotting, immunofluorescence, and Ca2+ imaging, respectively. We found that Piezo2 channels were expressed on most (> 90%) of the bladder primary afferents, including afferents that express CGRP, TRPV1 and stained with isolectin B4. CYP-induced cystitis was associated with Piezo2 upregulation in bladder afferent neurons at the mRNA, protein, and functional levels. Knockdown of Piezo2 expression in DRG neurons significantly suppressed mechanical stimulation–evoked referred bladder pain as well as bladder hyperactivity in CYP rats compared to CYP rats treated with mismatched ODNs. Our results suggest upregulation of Piezo2 channels is involved in the development of bladder mechanical allodynia and bladder hyperactivity in CYP-induced cystitis. Targeting Piezo2 might be an attractive therapeutic approach for IC-related bladder pain.

Diverse pathogen-associated molecular patterns affect transcription of genes in the toll-like receptor signaling pathway in goat blood

Pathogen-associated molecular patterns (PAMPs) such as lipopolysaccharide (LPS), peptidoglycan (PGN), Polyinosinic-polycytidylic acid (poly I:C), and CpG Oligodeoxynucleotides (ODN) are recognized by Toll-like receptors (TLR). This study aimed to investigate the effect of diverse PAMPs on the transcription of TLR signaling pathway genes in goat blood. Whole blood was collected from 3 female BoerXSpanish goats and treated with the following PAMPs: 10 µg/ml LPS, PGN, CpG ODN (2216), CpG ODN (2006), and 12.5 µg/ml Poly I:C. Blood-treated PBS served as a control. The expression of 84 genes in the human TLR signaling pathway RT2 PCR Array (Qiagen) was evaluated using real-time PCR. Treatment with PBS affected the expression of 74 genes, Poly I:C affected the expression of 40 genes, t ODN 2006 affected the expression of 50 genes, ODN 2216 affected the expression of 52 genes, LPS affected the expression of 49 genes, while PGN affected the expression of 49 genes. Our results show that PAMPs modulated and increased the expression of genes in the TLR signaling pathway. These results highlight important insights into how the host responds to different pathogens and may help design adjuvants for therapeutics and vaccines that target different.

Polybutylene terephthalate-based stationary phase for ion-pair-free reversed-phase liquid chromatography of small interfering RNA. Part 2: Use for selective comprehensive two-dimensional liquid chromatography

With the increasing numbers of nucleic acid-based pharmaceuticals like antisense oligonucleotides (ASO), small interfering ribonucleic acid (siRNA) entering the market, research facilities, pharmaceutical industries and also regulatory authorities have been looking for efficient analytical methods for these synthetic oligonucleotides (ON). Besides of conventional one-dimensional (1D) reversed-phase liquid chromatography with or without ion-pairing (IP-RP-LC, RP-LC), hydrophilic liquid chromatography (HILIC) and mixed-mode chromatography (MMC), two-dimensional (2D) approaches combining two orthogonal chromatographic techniques also become more relevant due to the high structural complexity of oligonucleotides. Recently, we tested a polybutylene terephthalate(PBT)-based stationary phase under ion-pairing free RP mode for the liquid chromatography electrospray ionization mass spectrometry (LC-ESI-MS) analysis of siRNA (Patisiran). In this study, retention profile and chromatographic orthogonality, respectively, were compared to other LC-modes like HILIC, IP-RPLC, another ion-pair free cholesterol-bonded RPLC and MMC considering their normalized retention times. Finally, because of higher orthogonality, the ion-pairing free PBT-bonded RPLC as first dimension (1D) was hyphenated with HILIC in the second dimension (2D) in a selective comprehensive 2D-LC setup leading to an enhanced resolution for peak purity evaluation of the main ON entities.

Nucleic Acid Probes in Bio-Imaging and Diagnostics: Recent Advances in ODN-Based Fluorescent and Surface-Enhanced Raman Scattering Nanoparticle and Nanostructured Systems.

Raman nanoparticle probes are a potent class of optical labels for the interrogation of pathological and physiological processes in cells, bioassays, and tissues. Herein, we review the recent advancements in fluorescent and Raman imaging using oligodeoxyribonucleotide (ODN)-based nanoparticles and nanostructures, which show promise as effective tools for live-cell analysis. These nanodevices can be used to investigate a vast number of biological processes occurring at various levels, starting from those involving organelles, cells, tissues, and whole living organisms. ODN-based fluorescent and Raman probes have contributed to the achievement of significant advancements in the comprehension of the role played by specific analytes in pathological processes and have inaugurated new possibilities for diagnosing health conditions. The technological implications that have emerged from the studies herein described could open new avenues for innovative diagnostics aimed at identifying socially relevant diseases like cancer through the utilization of intracellular markers and/or guide surgical procedures based on fluorescent or Raman imaging. Particularly complex probe structures have been developed within the past five years, creating a versatile toolbox for live-cell analysis, with each tool possessing its own strengths and limitations for specific studies. Analyzing the literature reports in the field, we predict that the development of ODN-based fluorescent and Raman probes will continue in the near future, disclosing novel ideas on their application in therapeutic and diagnostic strategies.

A novel Cas9 fusion protein promotes targeted genome editing with reduced mutational burden in primary human cells.

Precise genome editing requires the resolution of nuclease-induced DNA double strand breaks (DSBs) via the homology-directed repair (HDR) pathway. In mammals, this is typically outcompeted by non-homologous end-joining (NHEJ) that can generate potentially genotoxic insertion/deletion mutations at DSB sites. Because of higher efficacy, clinical genome editing has been restricted to imperfect but efficient NHEJ-based approaches. Hence, strategies that promote DSB resolution via HDR are essential to facilitate clinical transition of HDR-based editing strategies and increase safety. Here we describe a novel platform that consists of a Cas9 fused to DNA repair factors to synergistically inhibit NHEJ and favor HDR for precise repairing of Cas-induced DSBs. Compared to canonical CRISPR/Cas9, the increase in error-free editing ranges from 1.5-fold to 7-fold in multiple cell lines and in primary human cells. This novel CRISPR/Cas9 platform accepts clinically relevant repair templates, such as oligodeoxynucleotides (ODNs) and adeno-associated virus (AAV)-based vectors, and has a lower propensity to induce chromosomal translocations as compared to benchmark CRISPR/Cas9. The observed reduced mutational burden, resulting from diminished indel formation at on- and off-target sites, provides a remarkable gain in safety and advocates this novel CRISPR system as an attractive tool for therapeutic applications depending on precision genome editing.

Abstract 5164: Liposomal Delivery of an Immunostimulatory CpG Induces Robust Antitumor Immunity and Long-Term Immune Memory by Reprogramming Tumor-Associated Macrophages

Abstract Background: The efficacy of current T cell-based immunotherapy is known to be limited by various factors that induce immune suppression in the tumor microenvironment (TME). Tumor-associated macrophages (TAMs) are representative TME-comprising immune suppressive cells and influence tumor growth and progression. TAMs are considered as an attractive target for cancer immunotherapy. However, current TAMs-targeting strategies are not sufficient to induce continuous antitumor responses. Here, we suggest a new strategy that can reprogram TAMs and further bridge innate-to-adaptive immunity by utilizing immunostimulatory CpG oligodeoxynucleotides (ODNs). In this study, we investigated the new role of TLR9 agonist CpG ODN as an immunotherapeutic agent and tested whether liposomal delivery of CpG could modulate the function of TAMs and lead to remodeling of the TME. Methods: A liposome-based nanomaterial encapsulating CpG ODN was constructed. A small lipid nanoparticle (SLNP) used as the CpG delivery platform in this study was made of a cationic cholesterol derivative and two biocompatible phospholipids. The CpG-encapsulating SLNP (SLNP@CpG) was prepared by a thin-film formation and rehydration method. In vitro studies were conducted to assess whether SLNP@CpG could reprogram macrophages. To assess antitumor therapeutic efficacy of the SLNP@CpG, two subcutaneous tumor models were established in mice and tumor growth and survival rates of mice were monitored. For tumor rechallenge experiment, mice showing complete tumor regression were rechallenged with same cancer cells and their splenocytes were analyzed to confirm the development of immune memory. To elucidate the immunological mechanism of SLNP@CpG-mediated antitumor efficacy, changes of immune cell subpopulations in treated tumors were analyzed by flow cytometry. Results: SLNP@CpG enhanced macrophage-mediated phagocytosis of cancer cells and tumor antigen cross-presentation, and skewed the polarization state of macrophages in vitro. Intratumorally injected SLNP@CpG exerted its therapeutic efficacy in an established E.G7-OVA tumor via uptake by TAMs. SLNP@CpG treatment significantly suppressed the E.G7 tumor growth and also considerably prolonged the survival of mice, with 83.3% of mice becoming tumor-free. Local administration of SLNP@CpG resisted E.G7 tumor rechallenge by inducing immunological memory and long-term antitumor immunity. Local administration of SLNP@CpG even exerted its antitumor efficacy in an aggressive B16-F10 melanoma by remodeling TME towards immune stimulation and tumor elimination. Conclusion: Liposomal delivery of CpG via local treatment reprogrammed TAMs by enhancing phagocytic activity and repolarizing M2 to M1 phenotype and also reshaped immunosuppressive TME, leading to antitumor immunity and long-term memory responses. Citation Format: Yujin Kim, Sangyong Jon. Liposomal Delivery of an Immunostimulatory CpG Induces Robust Antitumor Immunity and Long-Term Immune Memory by Reprogramming Tumor-Associated Macrophages. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5164.

STAT3/NF‑κB decoy oligodeoxynucleotides inhibit atherosclerosis through regulation of the STAT/NF‑κB signaling pathway in a mouse model of atherosclerosis.

Atherosclerosis is a progressive chronic inflammatory condition that is the cause of most cardiovascular and cerebrovascular diseases. The transcription factor nuclear factor‑κB (NF‑κB) regulates a number of genes involved in the inflammatory responses of cells that are critical to atherogenesis, and signal transducer and activator of transcription (STAT)3 is a key transcription factor in immunity and inflammation. Decoy oligodeoxynucleotides (ODNs) bind to sequence‑specific transcription factors and limit gene expression by interfering with transcription invitro and invivo. The present study aimed to investigate the beneficial functions of STAT3/NF‑κB decoy ODNs in liposaccharide (LPS)‑induced atherosclerosis in mice. Atherosclerotic injuries of mice were induced via intraperitoneal injection of LPS and the mice were fed an atherogenic diet. Ring‑type STAT3/NF‑κB decoy ODNs were designed and administered via an injection into the tail vein of the mice. To investigate the effect of STAT3/NF‑κB decoy ODNs, electrophoretic mobility shift assay, western blot analysis, histological analysis with hematoxylin and eosin staining, Verhoeff‑Van Gieson and Masson's trichrome staining were performed. The results revealed that STAT3/NF‑κB decoy ODNs were able to suppress the development of atherosclerosis by attenuating morphological changes and inflammation in atherosclerotic mice aortae, and by reducing pro‑inflammatory cytokine secretion through inhibition of the STAT3/NF‑κB pathway. In conclusion, the present study provided novel insights into the antiatherogenic molecular mechanism of STAT3/NF‑κB decoy ODNs, which may serve as an additional therapeutic intervention to combat atherosclerosis.

Effective, Rapid, and Small-Scale Bioconjugation and Purification of "Clicked" Small-Molecule DNA Oligonucleotide for Nucleic Acid Nanoparticle Functionalization.

Nucleic acid-based therapeutics involves the conjugation of small molecule drugs to nucleic acid oligomers to surmount the challenge of solubility, and the inefficient delivery of these drug molecules into cells. "Click" chemistry has become popular conjugation approach due to its simplicity and high conjugation efficiency. However, the major drawback of the conjugation of oligonucleotides is the purification of the products, as traditionally used chromatography techniques are usually time-consuming and laborious, requiring copious quantities of materials. Herein, we introduce a simple and rapid purification methodology to separate the excess of unconjugated small molecules and toxic catalysts using a molecular weight cut-off (MWCO) centrifugation approach. As proof of concept, we deployed "click" chemistry to conjugate a Cy3-alkyne moiety to an azide-functionalized oligodeo-xynucleotide (ODN), as well as a coumarin azide to an alkyne-functionalized ODN. The calculated yields of the conjugated products were found to be 90.3 ± 0.4% and 86.0 ± 1.3% for the ODN-Cy3 and ODN-coumarin, respectively. Analysis of purified products by fluorescence spectroscopy and gel shift assays demonstrated a drastic amplitude of fluorescent intensity by multiple folds of the reporter molecules within DNA nanoparticles. This work is intended to demonstrate a small-scale, cost-effective, and robust approach to purifying ODN conjugates for nucleic acid nanotechnology applications.

Systematic Investigation of Tether Length and Phosphorus Configuration in Backbone Constrained Macrocyclic Nucleic Acids to Modulate Binding Kinetics for RNA.

We recently described a chemical strategy to pre-organize a trinucleotide subunit in a conformation suitable for Watson-Crick base pairing for modulating the binding kinetics of single-stranded oligonucleotides (ONs) using bis-phosphonate esters bridging hydrocarbon tethers to provide 11- and 15-membered macrocyclic analogues. In this manuscript, we describe the synthesis of all eight P-stereoisomers of macrocyclic 12-, 13-, 14-, and 16-membered hydrocarbon-bridged nucleotide trimers, their incorporation into ONs, and biophysical characterization of the modified ONs. The size of the macrocyclic tether and configuration at phosphorus had profound effects on hybridization kinetics. ONs containing 12- and 13-membered rings exhibited faster on-rates (up to 5-fold) and off-rates (up to 161-fold). In contrast, ONs using the larger ring size macrocycles generally exhibited smaller changes in binding kinetics relative to unmodified DNA. Interestingly, several of the analogues retained significant binding affinity for RNA based on their dissociation constants, despite being modestly destabilizing in the thermal denaturation experiments, highlighting the potential utility of measuring dissociation constants versus duplex thermal stability when evaluating novel nucleic acid analogues. Overall, our results provide additional insights into the ability of backbone-constrained macrocyclic nucleic acid analogues to modulate hybridization kinetics of modified ONs with RNA.

Immunogenicity of adjuvanted plant-produced SARS-CoV-2 Beta spike VLP vaccine in New Zealand white rabbits.

The outbreak of the SARS-CoV-2 global pandemic heightened the pace of vaccine development with various vaccines being approved for human use in a span of 24months. The SARS-CoV-2 trimeric spike (S) surface glycoprotein, which mediates viral entry by binding to ACE2, is a key target for vaccines and therapeutic antibodies. Plant biopharming is recognized for its scalability, speed, versatility, and low production costs and is an increasingly promising molecular pharming vaccine platform for human health. We developed Nicotiana benthamiana-produced SARS-CoV-2 virus-like particle (VLP) vaccine candidates displaying the S-protein of the Beta (B.1.351) variant of concern (VOC), which triggered cross-reactive neutralising antibodies against Delta (B.1.617.2) and Omicron (B.1.1.529) VOCs. In this study, immunogenicity of the VLPs (5µg per dose) adjuvanted with three independent adjuvants i.e. oil-in-water based adjuvants SEPIVAC SWETM (Seppic, France) and "AS IS" (Afrigen, South Africa) as well as a slow-release synthetic oligodeoxynucleotide (ODN) adjuvant designated NADA (Disease Control Africa, South Africa) were evaluated in New Zealand white rabbits and resulted in robust neutralising antibody responses after booster vaccination, ranging from 1:5341 to as high as 1:18204. Serum neutralising antibodies elicited by the Beta variant VLP vaccine also showed cross-neutralisation against the Delta and Omicron variants with neutralising titres ranging from 1:1702 and 1:971, respectively. Collectively, these data provide support for the development of a plant-produced VLP based candidate vaccine against SARS-CoV-2 based on circulating variants of concern.

Using Microphysiological System for the Development of Treatments for Joint Inflammation and Associated Cartilage Loss-A Pilot Study.

Osteoarthritis (OA) is a painful and disabling joint disease affecting millions worldwide. The lack of clinically relevant models limits our ability to predict therapeutic outcomes prior to clinical trials, where most drugs fail. Therefore, there is a need for a model that accurately recapitulates the whole-joint disease nature of OA in humans. Emerging microphysiological systems provide a new opportunity. We recently established a miniature knee joint system, known as the miniJoint, in which human bone-marrow-derived mesenchymal stem cells (hBMSCs) were used to create an osteochondral complex, synovial-like fibrous tissue, and adipose tissue analogs. In this study, we explored the potential of the miniJoint in developing novel treatments for OA by testing the hypothesis that co-treatment with anti-inflammation and chondroinducing agents can suppress joint inflammation and associated cartilage degradation. Specifically, we created a "synovitis"-relevant OA model in the miniJoint by treating synovial-like tissues with interleukin-1β (IL-1β), and then a combined treatment of oligodeoxynucleotides (ODNs) suppressing the nuclear factor kappa beta (NF-κB) genetic pathway and bone morphogenic protein-7 (BMP-7) was introduced. The combined treatment with BMP-7 and ODNs reduced inflammation in the synovial-like fibrous tissue and showed an increase in glycosaminoglycan formation in the cartilage portion of the osteochondral complex. For the first time, this study demonstrated the potential of the miniJoint in developing disease-modifying OA drugs. The therapeutic efficacy of co-treatment with NF-κB ODNs and BMP-7 can be further validated in future clinical studies.

Synthesis of 6-Alkynylated Purine-Containing DNA via On-Column Sonogashira Coupling and Investigation of Their Base-Pairing Properties

Synthetic unnatural base pairs have been proven to be attractive tools for the development of DNA-based biotechnology. Our group has very recently reported on alkynylated purine-pyridazine pairs, which exhibit selective and stable base-pairing via hydrogen bond formation between pseudo-nucleobases in the major groove of duplex DNA. In this study, we attempted to develop an on-column synthesis methodology of oligodeoxynucleotides (ODNs) containing alkynylated purine derivatives to systematically explore the relationship between the structure and the corresponding base-pairing ability. Through Sonogashira coupling of the ethynyl pseudo-nucleobases and CPG-bound ODNs containing 6-iodopurine, we have demonstrated the synthesis of the ODNs containing three NPu derivatives (NPu1, NPu2, NPu3) as well as three OPu derivatives (OPu1, OPu2, OPu3). The base-pairing properties of each alkynylated purine derivative revealed that the structures of pseudo-nucleobases influence the base pair stability and selectivity. Notably, we found that OPu1 bearing 2-pyrimidinone exhibits higher stability to the complementary NPz than the original OPu, thereby demonstrating the potential of the on-column strategy for convenient screening of the alkynylated purine derivatives with superior pairing ability.

Surfactant like peptides for targeted gene delivery to cancer cells

Surfactant like peptides (SLPs) are a class of amphiphilic peptides widely used for drug delivery and tissue engineering. However, there are very few reports on their application for gene delivery. The current study was aimed at development of two new SLPs, named (IA)4K and (IG)4K, for selective delivery of antisense oligodeoxynucleotides (ODNs) and small interfering RNA (siRNA) to cancer cells. The peptides were synthesized by Fmoc solid phase synthesis. Their complexation with nucleic acids was studied by gel electrophoresis and DLS. The transfection efficiency of the peptides was assessed in HCT 116 colorectal cancer cells and human dermal fibroblasts (HDFs) using high content microscopy. The cytotoxicity of the peptides was assessed by standard MTT test. The interaction of the peptides with model membranes was studied using CD spectroscopy. Both SLPs delivered siRNA and ODNs to HCT 116 colorectal cancer cells with high transfection efficiency which was comparable to the commercial lipid-based transfection reagents, but with higher selectivity for HCT 116 compared to HDFs. Moreover, both peptides exhibited very low cytotoxicity even at high concentrations and long exposure time. The current study provides more insights into the structural features of SLPs required for nucleic acid complexation and delivery and can therefore serve as a guide for the rational design of new SLPs for selective gene delivery to cancer cells to minimize the adverse effects in healthy tissues.

NF-κB Decoy ODN-Loaded Poly(Lactic-co-glycolic Acid) Nanospheres Inhibit Alveolar Ridge Resorption.

Residual ridge resorption combined with dimensional loss resulting from tooth extraction has a prolonged correlation with early excessive inflammation. Nuclear factor-kappa B (NF-κB) decoy oligodeoxynucleotides (ODNs) are double-stranded DNA sequences capable of downregulating the expression of downstream genes of the NF-κB pathway, which is recognized for regulating prototypical proinflammatory signals, physiological bone metabolism, pathologic bone destruction, and bone regeneration. The aim of this study was to investigate the therapeutic effect of NF-κB decoy ODNs on the extraction sockets of Wistar/ST rats when delivered by poly(lactic-co-glycolic acid) (PLGA) nanospheres. Microcomputed tomography and trabecular bone analysis following treatment with NF-κB decoy ODN-loaded PLGA nanospheres (PLGA-NfDs) demonstrated inhibition of vertical alveolar bone loss with increased bone volume, smoother trabecular bone surface, thicker trabecular bone, larger trabecular number and separation, and fewer bone porosities. Histomorphometric and reverse transcription-quantitative polymerase chain reaction analysis revealed reduced tartrate-resistant acid phosphatase-expressing osteoclasts, interleukin-1β, tumor necrosis factor-α, receptor activator of NF-κB ligand, turnover rate, and increased transforming growth factor-β1 immunopositive reactions and relative gene expression. These data demonstrate that local NF-κB decoy ODN transfection via PLGA-NfD can be used to effectively suppress inflammation in a tooth-extraction socket during the healing process, with the potential to accelerate new bone formation.

Advances in the mechanisms and applications of inhibitory oligodeoxynucleotides against immune-mediated inflammatory diseases.

Inhibitory oligodeoxynucleotides (ODNs) are short single-stranded DNA, which capable of folding into complex structures, enabling them to bind to a large variety of targets. With appropriate modifications, the inhibitory oligodeoxynucleotides exhibited many features of long half-life time, simple production, low toxicity and immunogenicity. In recent years, inhibitory oligodeoxynucleotides have received considerable attention for their potential therapeutic applications in immune-mediated inflammatory diseases (IMIDs). Inhibitory oligodeoxynucleotides could be divided into three categories according to its mechanisms and targets, including antisense ODNs (AS-ODNs), DNA aptamers and immunosuppressive ODNs (iSup ODNs). As a synthetic tool with immunomodulatory activity, it can target RNAs or proteins in a specific way, resulting in the reduction, increase or recovery of protein expression, and then regulate the state of immune activation. More importantly, inhibitory oligodeoxynucleotides have been used to treat immune-mediated inflammatory diseases, including inflammatory disorders and autoimmune diseases. Several inhibitory oligodeoxynucleotide drugs have been developed and approved on the market already. These drugs vary in their chemical structures, action mechanisms and cellular targets, but all of them could be capable of inhibiting excessive inflammatory responses. This review summarized their chemical modifications, action mechanisms and applications of the three kinds of inhibitory oligodeoxynucleotidesin the precise treatment of immune-mediated inflammatory diseases.

Oligoribonucleotide-Mediated Blockade of DNA Extension by Taq DNA Polymerases Increases Specificity and Sensitivity for Detecting Single-Nucleotide Differences.

Blocking PCR is a method that inhibits amplification of DNA possessing a nucleotide sequence complementary to that of a blocker; the method can be used to suppress amplification of target wild-type DNA while amplifying mutated DNA. Previously, we demonstrated that an oligoribonucleotide (ORN) functions as a cost-effective and sequence-specific blocker. This blocking PCR system, named ORN interference-PCR (ORNi-PCR), is compatible with DNA polymerases lacking 5'-3' exonuclease activity but not with those possessing the activity (e.g., Taq DNA polymerase), which can remove a hybridized ORN during DNA extension. Here, we demonstrate that under specific experimental conditions, an intact or phosphorothioated ORN strongly suppresses extension of target DNA by Taq DNA polymerases. This method was applied successfully to real-time ORNi-PCR and one-step real-time reverse transcription-ORNi-PCR using a dual-labeled fluorescent probe to detect a single-nucleotide mutation in DNA and RNA in a sequence-specific manner. The results reaffirm the utility of blocking PCR and provide technical hints for its improvement.