Journal: bioRxiv
Article Title: Template-Directed RIG-I Agonist Assembly for Targeted Cancer Immunotherapy
doi: 10.1101/2022.12.08.519592
Figure Lengend Snippet: a) Design of the mir-21-template directed RIG-I agonist, ss-ppp-miRNA-21. The antisense ss-miRNA-21 oligonucleotide, designated as ss-ppp-miRNA-21, was designed to be fully complementary to the target miRNA-21 and to incorporate a 5’ triphosphate modification; b) Biogenesis and function of miRNA (black solid arrow). Starting in the nucleus, imperfectly or perfectly complementary miRNA:miRNA* duplexes are produced in the cytoplasm, where they are incorporated into the Argonaute-containing miRNA induced silencing complex (miRISC), unwound, and the mature miRNA strand retained in miRISC, while the complementary strand is released and degraded. miRISC is guided by the mature miRNA, which directs it to complementary sites in target mRNAs, resulting in translational repression and/or mRNA degradation; c) Activation of RIG-I signaling pathway (red dashed arrow). Upon entry into the cell, ss-ppp-miRNA-21 competes effectively with endogenous mRNA targets (mostly only partially complementary) to bind to the miRNA in the miRISC complex, resulting in release from the RISC and formation of a 5’-ppp blunt ended double stranded RNA. The blunt-ended 5’ppp-dsRNA can be readily captured by RIG-I and trigger its activation. RIG-I signaling in the tumor cells leads to type I IFN-driven immune response and preferential activation of programmed tumor cell death, releasing various immunological factors into the TME; d) Activation of cell-mediated immunity (dark red solid/dashed arrow). RIG-I signaling promotes rapid rise of type I IFNs and direct cancer cell death, releasing IFNs and pro-inflammatory cytokines as well as tumor antigens (TAs). Both innate immune cells and the adaptive immune system are energized in the remodeled TME. Leukocytes such as NK cells and macrophages boost their cytolytic activity in response to an IFN-rich environment. Importantly, an increase in IFNs and TAs triggers an adaptive immune response, resulting in the maturation and activation of macrophages and DCs, and enhanced antigen presentation to T-lymphocytes in tumor draining lymph nodes. Naïve T cells are then activated, propagated, differentiated, and transported to the TME, where they can perform an effective anti-tumor response by direct or indirect cytolytic activity mediated by perforin and granzymes, and the secretion of cytokines like IFNγ and TNFα. Following cancer cell death, the majority of tumor-specific effector CD8+ T cells die via apoptosis, but some survive to mature into long-lived protective memory CD8+ T cells. RIG-I, retinoic acid induced gene 1; IFN, interferon; NK, natural killer; DC, dendritic cell; TME, tumor microenvironment; TA, tumor antigen; APC, antigen-presenting cell; PRR, pattern-recognition receptor; TNFα, tumor necrosis factor alpha; Treg, T-regulatory cells. (Parts of the figure were drawn by using pictures from Servier Medical Art, provided by Servier, licensed under a Creative Commons Attribution 3.0 unported license).
Article Snippet: Briefly, B16-F10 cells (10,000 or 20,000 cells/well) were seeded in 96-well plates (Corning, Tewksbury, MA) and treated with ds-ppp-RNA positive control (1 μg/mL, (Catalog Code: tlrl-3prnac, InvivoGen), ss-ppp-miRNA-21 (2, 4 or 8 μg/ml) or ss-miRNA-21 (2, 4 or 8 μg/ml) for 48 h. Plates were allowed to equilibrate to room temperature.
Techniques: Modification, Produced, Activation Assay, Activity Assay
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