ANTIPARALEL DUPLEX/G-QUADRUPLEX DESIGN ON CPG MOTIF MODIFICATION TO STRENGTHEN CYTOKINES INVOLVED IN ALLERGIC REACTIONS

• Main Author: Ayu Safitri, Fika
• Format: Dissertations
• Language: Indonesia
• Subjects: Ilmu hayati ; Biologi
• Online Access: https://digilib.itb.ac.id/gdl/view/75156
• Institution: Institut Teknologi Bandung

Allergies are becoming more common around the world, due to a variety of reasons including heredity and the environment. Allergies are caused by an aberrant reaction or overreaction (hypersensitivity) of the immune system in identifying a potentially hazardous antigen/allergen. Allergies are caused by imbalanced T-cells proliferation, which disrupts the homeostasis of innate and adaptive immune cells. Avoiding allergens, using antihistamines, and oligodeoxynucleotide (ODN) immunotherapy are some of the methods used to suppress or minimize allergy symptoms. ODN is commonly utilized as a treatment agent in allergy sufferers because it can affect cellular activity by attaching to receptor proteins and stimulating a balanced condition in the body. Because it can affect cellular function by attaching to receptor proteins and stimulating a balanced condition in the body, ODN is frequently employed as a therapeutic agent in allergy patients. The activation of Toll-like Receptor-9 (TLR-9) with the introduction of an unmethylated Cytosine Phosphate Guanine (CpG) motif is the most common method for using synthetic ODN, however nuclease enzymes are one of the barriers to using CpGODN for therapy. Using Circular Dichroism analysis, 11 CpGODN designs with an antiparallel topology and a distinctive positive peak at 290 nm and a negative peak at 260 nm were obtained in the first step. By adding one or two CpG motifs to the first, third functional strand, or the duplex region present in the D0G0.0.0 section, the Gquadruplex design can preserve an antiparallel topology. Furthermore, adding a base between the G-tetrad and the CpG motif changes the topological topology. The stimulatory effect of the new design G-quadruplex on the RAW 264 mouse macrophage cell line revealed that the longer the strand in the first functional strand, the higher the cytokine mRNA expression, with the optimum length of the strand being 16 nucleotide bases, which increased mRNA expression by 143. 8, 9. 7, and 3. 8 times, respectively. The 11 antiparallel G-quadruplex designs were characterized in the second step of research utilizing PAGE analysis, serum stability testing, and Melting Temperature (MT) study, followed by in vitro testing using dendritic lini cells and mice primary cells. According to the findings, the eleven antiparallel verified designs feature Gquadruplex molecularity, which has quicker mobility than its linear form. After 24 hours of incubation, four designs with one base linkage between the CpG motif and the G-tetrad (D0Ga2a.0.0; D0Ga2c.0.0; D0Gc2c.0.0; D0Ga2a.0.0) performed well with high serum stability compared to other designs with high serum stability. 26.3 percent, 24. 3 percent, 30. 8 percent, and 17. 9 percent, in that order. The highest order of results was achieved in the study of the antiparalel melting temperature value, namely the original design, D0G0.0.0; D0G1.0.0; D0G2.0.0; D0Ga2a.0.0; D0Gac2ac.0.0; D0Gca2ac.0.0; D0Gca2ac.0.0; D0Gca2ca.0.0; D0Gca2ca.0.0; D0Gca2ca.0.0; D0Ga2c.0.0; and D0Gc2a.0.0. Based on the results of the first stage's test on lini sel RAW 264, it was discovered that D0Ga2c.0.0 and D0Gc2c.0.0 had the highest cytokine gene expression compared to other designs, thus the second stage used DC2.4 mouse dendritic lini sel stimulation and primary macrophage cells from the bone. D0Ga2c.0.0 and D0Gc2c.0.0 were employed in mouse Bone Marrow, with D0G2.0.0 as a positive control and D-PBS as a negatif control. On stimulation of DC2.4 cells and BMMCs, D0Gc2c.0.0 expressed the greatest cytokine gene. However, based on residual ODNs values after 24 hours of incubation in serum, D0Ga2c.0.0 will be employed for in vivo experiments in the future. This study aims to find a design that can reduce the effect of nuclease enzyme degradation. In the first and second stages, the D0Ga2c.0.0 design was discovered to be able to reduce the effect of nuclease enzyme degradation. The revised design from the first and second phases was employed as an in vivo application in the third stage of the research to see how it affected mice who had been treated with ovalbumin for asthma allergies. The three comparison treatments employed were natural control, healthy, unwell, and ovalbumin treatment. mIgE in blood plasma from mice on the day before and after ovalbumin therapy increased and decreased in mice after CpG treatment, according to ELISA analysis. In the mOva-IgE study, the same results were found. This implies that CpG can lower total and ovarian mIgE levels in blood plasma. In plasma and BALF (Bronchoalveolar lavage fluid) samples, it displays a favorable connection with increased IFN-? and decreased IL- 5. Histological investigation revealed that, compared to the control group, treatment of G4CpGODN was able to dramatically lower the number of cells engaged in the allergic mechanism and mucus levels. By exploiting a secondary structure known as G-quadruplex, this discovery offers a new way to decrease the effect of nuclease degradation. This immunotherapy works by recognizing the Cytosine Phosphate Guanine (CpG) motif, which is identified by the TLR9 receptor on immune cells. Because immune cells such as macrophages, B cells, and dendritic cells express this receptor, TLR9 agonists can help alleviate allergic symptoms, particularly asthma.