Oligonucleotide drugs have the characteristics of targeting, modifiability and high biosafety. Recent studies have shown that oligonucleotide can be used to make biosensors, vaccine adjuvants, and has the functions of inhibiting alveolar bone resorption, promoting jaw and alveolar bone regeneration, anti-tumor, destroying plaque biofilm, and precise control of drug release. Therefore, it has a broad application prospect in the field of stomatology. This article reviews the classification, action mechanism and research status of oligonucleotide in stomatology. The aim is to provide ideas for further research and application of oligonucleotide.
Humans ; Alveolar Bone Loss ; Biofilms ; Bone Regeneration ; Oligonucleotides ; Oral Medicine

Objective To explore the aptamer specific binding blood group antigen-binding adhesin (BabA) of Helicobacter pylori (H.pylori) for blocking of H.pylori adhering host cell. Methods H.pylori strain was cultured and its genome was extracted as templates to amplify the BabA gene by PCR with designed primers. The BabA gene obtained was cloned and constructed into prokaryotic expression plasmid, which was induced by isopropyl beta-D-galactoside (IPTG) and purified as target. The single stranded DNA (ssDNA) aptamers that specifically bind to BabA were screened by SELEX. Enzyme-linked oligonucleotide assay (ELONA) was used to detect and evaluate the characteristics of candidate aptamers. The blocking effect of ssDNA aptamers on H.pylori adhesion was subsequently verified by flow cytometry and colony counting at the cell level in vitro and in mouse model of infection, respectively. Meanwhile, the levels of cytokines, interleukin 6 (IL-6), IL-8, tumor necrosis factor α (TNF-α), IL-10 and IL-4 in the homogenate of mouse gastric mucosa cells were detected by ELISA. Results The genome of H.pylori ATCC 43504 strains was extracted and the recombinant plasmid pET32a-BabA was constructed. After induction and purification, the relative molecular mass (M_r) of the recombinant BabA protein was about 39 000. The amino acid sequence of recombinent protein was consistent with BabA protein by peptide mass fingerprint (PMF). Five candidate aptamers were selected to bind to the above recombinent BabA protein by SELEX. The aptamers A10, A30 and A42 identified the same site, while A3, A16 and the above three aptamers identified different sites respectively. The aptamer significantly blocked the adhesion of H.pylori in vitro. Animal model experiments showed that the aptamers can block the colonization of H.pylori in gastric mucosa by intragastric injection and reduce the inflammatory response. The levels of IL-4, IL-6, IL-8 and TNF-α in gastric mucosal homogenates in the model group with aptamer treatment were lower than that of model group without treatment. Conclusion Aptamers can reduce the colonization of H.pylori in gastric mucosa via binding BabA to block the adhesion between H.pylori and gastric mucosal epithelial cells.
Animals ; Mice ; Helicobacter pylori/genetics* ; Interleukin-4 ; Interleukin-6 ; Interleukin-8 ; Tumor Necrosis Factor-alpha ; Stomach ; Oligonucleotides ; Adhesins, Bacterial/genetics* ; Blood Group Antigens

RNA therapeutics inhibit the expression of specific proteins/RNAs by targeting complementary sequences of corresponding genes or encode proteins for the synthesis desired genes to treat genetic diseases. RNA-based therapeutics are categorized as oligonucleotide drugs (antisense oligonucleotides, small interfering RNA, RNA aptamers), and mRNA drugs. The antisense oligonucleotides and small interfering RNA for treatment of genetic diseases have been approved by the FDA in the United States, while RNA aptamers and mRNA drugs are still in clinical trials. Chemical modifications can be applied to RNA drugs, such as pseudouridine modification of mRNA, to reduce immunogenicity and improve the efficacy. The secure and effective delivery systems such as lipid-based nanoparticles, extracellular vesicles, and virus-like particles are under development to address stability, specificity, and safety issues of RNA drugs. This article provides an overview of the specific molecular mechanisms of eleven RNA drugs currently used for treating genetic diseases, and discusses the research progress of chemical modifications and delivery systems of RNA drugs.
Aptamers, Nucleotide ; RNA, Small Interfering/therapeutic use* ; RNA, Messenger ; Oligonucleotides, Antisense/therapeutic use*

OBJECTIVE: To construct a mouse model of Glanzmann's thrombasthenia (GT) with ITGA2B c.2659 C>T (p.Q887X) nonsense mutation by CRISPR/Cas9 technology, and then further explore the expression and function of glycoprotein αIIbβ3 on the surface of platelet membrane. METHODS: The donor oligonucleotide and gRNA vector were designed and synthesized according to the ITGA2B gene sequence. The gRNA and Cas9 mRNA were injected into fertilized eggs with donor oligonucleotide and then sent back to the oviduct of surrogate mouse. Positive F0 mice were confirmed by PCR genotyping and sequence analysis after birth. The F1 generation of heterozygous GT mice were obtained by PCR and sequencing from F0 bred with WT mice, and then homozygous GT mice and WT mice were obtained by mating with each other. The phenotype of the model was then further verified by detecting tail hemorrhage time, saphenous vein bleeding time, platelet aggregation, expression and function of αIIbβ3 on the surface of platelet. RESULTS: The bleeding time of GT mice was significantly longer than that of WT mice (P<0.01). Induced by collagen, thrombin, and adenosine diphosphate (ADP), platelet aggregation in GT mice was significantly inhibited (P<0.01, P<0.01, P<0.05). Flow cytometry analysis showed that the expression of αIIbβ3 on the platelet surface of GT mice decreased significantly compared with WT mice (P<0.01), and binding amounts of activated platelets to fibrinogen were significantly reduced after thrombin stimulation (P<0.01). The spreading area of platelet on fibrinogen in GT mice was significantly smaller than that in WT mice (P<0.05). CONCLUSION A GT mouse model with ITGA2B c.2659 C>T (p.Q887X) nonsense mutation has been established successfully by CRISPR/Cas9 technology. The aggregation function of platelet in this model is defective, which is consistent with GT performance.
Animals ; CRISPR-Cas Systems ; Codon, Nonsense ; Disease Models, Animal ; Fibrinogen/genetics* ; Humans ; Integrin alpha2/genetics* ; Mice ; Oligonucleotides ; Platelet Glycoprotein GPIIb-IIIa Complex/genetics* ; RNA, Guide ; Thrombasthenia/genetics* ; Thrombin/genetics*

Bar-code (tag) microarrays of yeast gene-deletion collections facilitate the systematic identification of genes required for growth in any condition of interest. Anti-sense strands of amplified bar-codes hybridize with ~10,000 (5,000 each for up- and down-tags) different kinds of sense-strand probes on an array. In this study, we optimized the hybridization processes of an array for fission yeast. Compared to the first version of the array (11 µm, 100K) consisting of three sectors with probe pairs (perfect match and mismatch), the second version (11 µm, 48K) could represent ~10,000 up-/down-tags in quadruplicate along with 1,508 negative controls in quadruplicate and a single set of 1,000 unique negative controls at random dispersed positions without mismatch pairs. For PCR, the optimal annealing temperature (maximizing yield and minimizing extra bands) was 58℃ for both tags. Intriguingly, up-tags required 3× higher amounts of blocking oligonucleotides than down-tags. A 1:1 mix ratio between up- and down-tags was satisfactory. A lower temperature (25℃) was optimal for cultivation instead of a normal temperature (30℃) because of extra temperature-sensitive mutants in a subset of the deletion library. Activation of frozen pooled cells for >1 day showed better resolution of intensity than no activation. A tag intensity analysis showed that tag(s) of 4,316 of the 4,526 strains tested were represented at least once; 3,706 strains were represented by both tags, 4,072 strains by up-tags only, and 3,950 strains by down-tags only. The results indicate that this microarray will be a powerful analytical platform for elucidating currently unknown gene functions.
Oligonucleotides ; Polymerase Chain Reaction ; Schizosaccharomyces ; Yeasts

Breast cancer is among the most common malignancies affecting women and reproductively intact female dogs, resulting in death from metastatic disease if not treated effectively. To better manage the disease progression, canine mammary tumor (CMT) cells derived from malignant canine mammary cancers were fused to autologous dendritic cells (DCs) to produce living hybrid-cell fusion vaccines for canine patients diagnosed with spontaneous mammary carcinoma. The high-speed sorting of rare autologous canine patient DCs from the peripheral blood provides the autologous component of fusion vaccines, and fusion to major histocompatibility complex-unmatched CMT cells were produced at high rates. The vaccinations were delivered to each patient following a surgical resection 3 times at 3-week intervals in combination with immuno-stimulatory oligonucleotides and Gemcitabine adjunct therapy. The immunized patient animals survived 3.3-times longer (median survival 611 days) than the control patients (median survival 184 days) and also appeared to exhibit an enhanced quality of life. A comparison of vaccinated patients diagnosed with inflammatory mammary carcinoma resulted in a very short median survival (42 days), suggesting no effect of vaccination. The data showed that the development of autologous living DC-based vaccine strategies in patient animals designed to improve the management of canine mammary carcinoma can be successful and may allow an identification of the antigens that can be translatable to promote effective immunity in canine and human patients.
Animals ; Breast Neoplasms ; Breast ; Dendritic Cells ; Disease Progression ; Dogs ; Female ; Histocompatibility ; Humans ; Hybrid Cells ; Oligonucleotides ; Quality of Life ; Vaccination ; Vaccines

OBJECTIVE: MicroRNA-155 (miR-155) is significantly highly expressed in breast cancer, lung cancer, liver cancer and other malignant tumors. This study was to design and construct a radiolabeled probe targeting miR-155 for in vivo imaging in breast cancer. METHODS: Anti-miR-155 oligonucleotide (AMO-155) was chemically synthesized with 2' OMe modification. Its 5' end was linked with acetyl amine group. After chelated with a bifunctional chelator NHS-MAG3, AMO-155 was radiolabeled with ^99mTc using stannous chloride. The serum stability was evaluated at cellular level. In vivo imaging was performed in MCF-7 tumor bearing mice after the administration of ^99mTc radiolabeled AMO-155 and scramble control probes, respectively. Furthermore, the blocked imaging of tumor bearing mice was obtained after the injection of unlabeled AMO-155 2 hours ahead. MCF-7 and MDA-MB-231 tumor bearing mice with different expression level of miR-155 were imaged, respectively. Quantitative real-time PCR (qRT-PCR) was used to identify the expression level of miR-155 in the bearing tumors. RESULTS: ^99mTc-AMO-155 was prepared with high radiolabeled efficiency (97%), radiochemical purity (greater than 98%), and radioactive specific activity (3.75 GBq/μg). ^99mTc-AMO-155 was stable in fresh human serum for 12 hours. After the administration via tail vein, ^99mTc-AMO-155 displayed significant accumulation in MCF-7 bearing tumors with high expression level of miR-155, whereas ^99mTc-control showed little accumulation. After blocked with unlabeled AMO-155, the tumor could not be visualized clearly after the administration of ^99mTc-AMO-155. Furthermore, ^99mTc-AMO-155 could show the differential expression of miR-155 in vivo. MCF-7 tumor was shown with significantly higher radioactive accumulation than MDA-MB-231, based on its higher expression level of miR-155, which was verified by qRT-PCR. CONCLUSION ^99mTc-labeled AMO-155 with chemical modification showed good serum stability and in vivo tumor targeting ability. This study provides a potential probe for in vivo imaging of breast cancer.
Animals ; Breast Neoplasms/diagnostic imaging* ; Cell Line, Tumor ; Female ; Humans ; Mice ; MicroRNAs/analysis* ; Oligonucleotides, Antisense ; Oligopeptides ; Radiopharmaceuticals ; Succinimides ; Technetium ; Tissue Distribution

Echinostoma cinetorchis is an oriental intestinal fluke causing significant pathological damage to the small intestine. The aim of this study was to determine a full-length cDNA sequence of E. cinetorchis endoribonuclease (RNase H; EcRNH) and to elucidate its molecular biological characters. EcRNH consisted of 308 amino acids and showed low similarity to endoribonucleases of other parasites (<40%). EcRNH had an active site centered on a putative DDEED motif instead of DEDD conserved in other species. A recombinant EcRNH produced as a soluble form in Escherichia coli showed enzymatic activity to cleave the 3′-O-P bond of RNA in a DNA-RNA duplex, producing 3′-hydroxyl and 5′-phosphate. These findings may contribute to develop antisense oligonucleotides which could damage echinostomes and other flukes.
Amino Acids ; Catalytic Domain ; DNA, Complementary ; Echinostoma* ; Endoribonucleases ; Escherichia coli ; Intestine, Small ; Oligonucleotides, Antisense ; Parasites ; Ribonuclease H* ; Ribonucleases* ; RNA ; Trematoda

After transcription, RNAs are always associated with RNA binding proteins (RBPs) to perform biological activities. RBPs can interact with target RNAs in sequence- and structure-dependent manner through their unique RNA binding domains. In development and progression of carcinogenesis, RBPs are aberrantly dysregulated in many human cancers with various mechanisms, such as genetic alteration, epigenetic change, noncoding RNA-mediated regulation, and post-translational modifications. Upon deregulation in cancers, RBPs influence every step in the development and progression of cancer, including sustained cell proliferation, evasion of apoptosis, avoiding immune surveillance, inducing angiogenesis, and activating metastasis. To develop therapeutic strategies targeting RBPs, RNA interference-based oligonucleotides or small molecule inhibitors have been screened based on reduced RBP-RNA interaction and changed level of target RNAs. Identification of binding RNAs with high-throughput techniques and integral analysis of multiple datasets will help us develop new therapeutic drugs or prognostic biomarkers for human cancers.
Apoptosis ; Biomarkers ; Carcinogenesis ; Cell Proliferation ; Chemoprevention ; Dataset ; Epigenomics ; Humans ; Neoplasm Metastasis ; Oligonucleotides ; Protein Processing, Post-Translational ; RNA* ; RNA-Binding Proteins*

STUDY DESIGN: In vitro cell culture model. PURPOSE: To investigate the effect of small interfering RNA (siRNA) on Fas expression, apoptosis, and proliferation in serum-deprived rat disc cells. OVERVIEW OF LITERATURE: Synthetic siRNA can trigger an RNA interference (RNAi) response in mammalian cells and precipitate the inhibition of specific gene expression. However, the potential utility of siRNA technology in downregulation of specific genes associated with disc cell apoptosis remains unclear. METHODS: Rat disc cells were isolated and cultured in the presence of either 10% fetal bovine serum (FBS) (normal control) or 0% FBS (serum deprivation to induce apoptosis) for 48 hours. Fas expression, apoptosis, and proliferation were determined. Additionally, siRNA oligonucleotides against Fas (Fas siRNA) were transfected into rat disc cells to suppress Fas expression. Changes in Fas expression were assessed by reverse transcription-polymerase chain reaction and semiquantitatively analyzed using densitometry. The effect of Fas siRNA on apoptosis and proliferation of rat disc cells were also determined. Negative siRNA and transfection agent alone (Mock) were used as controls. RESULTS: Serum deprivation increased apoptosis by 40.3% (p<0.001), decreased proliferation by 45.3% (p<0.001), and upregulated Fas expression. Additionally, Fas siRNA suppressed Fas expression in serum-deprived cultures, with 68.5% reduction at the mRNA level compared to the control cultures (p<0.001). Finally, Fas siRNA–mediated suppression of Fas expression significantly inhibited apoptosis by 9.3% and increased proliferation by 21% in serum-deprived cultures (p<0.05 for both). CONCLUSIONS: The observed dual positive effect of Fas siRNA might be a powerful therapeutic approach for disc degeneration by suppression of harmful gene expression.
Animals ; Apoptosis* ; Cell Culture Techniques ; Cell Proliferation ; Densitometry ; Down-Regulation ; Gene Expression ; In Vitro Techniques ; Intervertebral Disc Degeneration ; Intervertebral Disc* ; Oligonucleotides ; Rats* ; RNA Interference ; RNA, Messenger ; RNA, Small Interfering ; Transfection