Spiropyran as a potential molecular diagnostic tool for double-stranded RNA detection

Abstract Background Long double-stranded RNAs (dsRNAs) are duplex RNAs that can induce immune response when present in mammalian cells. These RNAs are historically associated with viral replication, but recent evidence suggests that human cells naturally encode endogenous dsRNAs that can regulate antiviral machineries in cellular contexts beyond immune response. Results In this study, we use photochromic organic compound spiropyran to profile and quantitate dsRNA expression. We show that the open form of spiropyran, merocyanine, can intercalate between RNA base pairs, which leads to protonation and alteration in the spectral property of the compound. By quantifying the spectral change, we can detect and quantify dsRNA expression level, both synthetic and cellular. We further demonstrate that spiropyrans can be used as a molecular diagnostic tool to profile endogenously expressed dsRNAs. Particularly, we show that spiropyrans can robustly detect elevated dsRNA levels when colorectal cancer cells are treated with 5-aza-2′-deoxycytidine, an FDA-approved DNA-demethylating agent used for chemotherapy, thus demonstrating the use of spiropyran for predicting responsiveness to the drug treatment. Conclusion As dsRNAs are signature of virus and accumulation of dsRNAs is implicated in various degenerative disease, our work establishes potential application of spiropyrans as a simple spectral tool to diagnose human disease based on dsRNA expression

Mitochondrial double-stranded RNAs govern the stress response in chondrocytes to promote osteoarthritis development

© 2022 The Author(s)Protein kinase R (PKR) is an immune response protein that becomes activated by double-stranded RNAs (dsRNAs). PKR overactivation is associated with degenerative diseases with inflammation, including osteoarthritis (OA), but the dsRNA activator remains largely unknown. Here, we find that mitochondrial dsRNA (mt-dsRNA) expression and its cytosolic efflux are facilitated in chondrocytes under OA-eliciting conditions, leading to innate immune activation. Moreover, mt-dsRNAs are released to the extracellular space and activate Toll-like receptor 3 at the plasma membrane. Elevated levels of mt-dsRNAs in the synovial fluids and damaged cartilage of OA patients and in the cartilage of surgery-induced OA mice further support our data. Importantly, autophagy prevents PKR activation and protects chondrocytes from mitochondrial stress partly by removing cytosolic mtRNAs. Our study provides a comprehensive understanding of innate immune activation by mt-dsRNAs during stress responses that underlie the development of OA and suggests mt-dsRNAs as a potential target for chondroprotective intervention.Y

Mitochondrial double-stranded RNAs govern the stress response in chondrocytes to promote osteoarthritis development

© 2022 The Author(s)Protein kinase R (PKR) is an immune response protein that becomes activated by double-stranded RNAs (dsRNAs). PKR overactivation is associated with degenerative diseases with inflammation, including osteoarthritis (OA), but the dsRNA activator remains largely unknown. Here, we find that mitochondrial dsRNA (mt-dsRNA) expression and its cytosolic efflux are facilitated in chondrocytes under OA-eliciting conditions, leading to innate immune activation. Moreover, mt-dsRNAs are released to the extracellular space and activate Toll-like receptor 3 at the plasma membrane. Elevated levels of mt-dsRNAs in the synovial fluids and damaged cartilage of OA patients and in the cartilage of surgery-induced OA mice further support our data. Importantly, autophagy prevents PKR activation and protects chondrocytes from mitochondrial stress partly by removing cytosolic mtRNAs. Our study provides a comprehensive understanding of innate immune activation by mt-dsRNAs during stress responses that underlie the development of OA and suggests mt-dsRNAs as a potential target for chondroprotective intervention.11Nsciescopu

Double-stranded RNA induction asa potential dynamic biomarkerfor DNA-demethylating agents

© 2022 The Author(s)Hypomethylating agents (HMAs), such as azacitidine and decitabine, induce cancer cell death by demethylating DNAs to promote the expression of tumor-suppressor genes. HMAs also reactivate the transcription of endogenous double-stranded RNAs (dsRNAs) that trigger the innate immune response and subsequent apoptosis via viral mimicry. However, the expression patterns of endogenous dsRNAs and their relevance in the efficacy of HMAs remain largely uninvestigated. Here, we employ amidine-conjugated spiropyran (Am-SP) to examine the dynamic expression pattern of total dsRNAs regulated by HMAs. By analyzing the bone-marrow aspirates of myelodysplastic syndrome or acute myeloid leukemia patients who received the HMAs, we find a dramatic increase in total dsRNA levels upon treatment only in patients who later benefited from the therapy. We further apply our approach in solid tumor cell lines and show that the degree of dsRNA induction correlates with the effectiveness of decitabine in most cases. Notably, when dsRNA induction is accompanied by increased expression of nc886 RNA, decitabine becomes ineffective. Collectively, our study establishes the potential application of monitoring the total dsRNA levels by a small molecule as an analytical method and a dynamic marker to predict the clinical outcome of the HMA therapy.N