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

Ali et al. BMC Biomedical Engineering (2019) 1:6 https://doi.org/10.1186/s42490-019-0008-x BMC Biomedical Engineering RESEARCH ARTICLE Open Access Spiropyran as a potential molecular diagnostic tool for double-stranded RNA detection Ahsan Ausaf Ali, Minjeong Kang, Raisa Kharbash* and Yoosik Kim* 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 FDAapproved 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. Keywords: Spiropyran, Double-stranded RNA, Biosensor, UV-vis spectroscopy, DNA-demethylating agent, Drug responsiveness, Innate immune response Background Long double-stranded RNAs (dsRNAs) are analogous to DNAs in that they both exist in duplex helical structures. Historically, these dsRNAs are associated with virus as they are believed to be byproducts of viral replication of positive sense RNA viruses [1]. Consequently, when expressed in mammalian cells, long dsRNAs are recognized by innate immune response proteins which induce interferons, suppress translation, and initiate apoptosis programs [2]. Although dsRNAs are strongly associated with immune response to viral infection, increasing evidences suggest that human cells naturally express endogenous dsRNAs that can regulate antiviral machineries * Correspondence: ; Department of Chemical and Biomolecular Engineering and KI for Health Science and Technology (KIHST), Korea Advanced Institute of Science and Technology (KAIST), Daejeon 34141, South Korea in various cellular contexts such as during the cell cycle and response to stressors [3, 4]. Recently, two research groups have independently shown that mitochondrial RNAs can exist as intermolecular dsRNAs and are recognized by immune response proteins to regulate antiviral signaling [5, 6]. Moreover, accumulation of endogenously encoded dsRNAs is related to the onset of autoimmune [7] and age-related macular degeneration [8]. dsRNAs also play a key role during cellular response to chemotherapy where treatment of the DNA-demethylating agent leads to cell death by inducing the transcription of endogenous dsRNA genes, which subsequently activate antiviral machineries [9, 10]. One interesting aspect of the dsRNA-mediated antiviral signaling is that immune response proteins recognize specific structural signatures of the RNA such as the double-stranded secondary structure rather than © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Ali et al. BMC Biomedical Engineering (2019) 1:6 specific sequences of the RNA [11]. Therefore, to investigate the potential of endogenous dsRNAs as novel signaling molecules and their function during the onset of human disease, we need to develop a quantitative approach to measure the collective expression level of all types of dsRNAs with minimal sequence specificity. Photochromic spiropyrans undergo drastic structural changes between closed spiropyran (SP) and open merocyanine (MC) isoforms. Due to their photo-switching properties, spiropyran derivatives have a wide range of applications, from design of smart nanomaterials to optical regulation of biomacromolecules [12]. Specifically, spiropyrans have been used in solution, blended in nanomaterials and polymers to develop sensors for various target molecules [13–21]. These sensors rely on the colorimetric and spectral variations in the spiropyrans occurring due to protonation by and complex formation with the target material. Recently, numerous studies have investigated and characterized photo-switchable interactions between spiropyran derivatives and biological molecules such as DNA [22–26]. They found that only the open form can interact with DNA, which can be monitored using characteristic changes in the UV-Vis absorbance spectrum of the compound [22–26]. These studies revealed that considerable changes occur in the absorbance spectrum due to intercalation with DNA base pairs and that the degree of the change is correlated with the amount and the sequences of the DNA present. In contrast to the spiropyran-DNA interaction, which has been under extensive investigation in recent years, little has been done to examine possible interaction between spiropyran derivatives and RNAs. Previous studies with spiropyrans have primarily focused on tertiary structured short hairpin RNAs and their qualitative interactions as aptamers via surface plasmon resonance [27] and nanopores [28]. More recently, studies utilized spectral properties of gold nanoparticles and quantum dots to detect and quantify expression of specific RNA targets such as mRNAs [29, 30], single-stranded RNAs (ssRNAs) [31, 32], and short double-stranded RNAs (dsRNAs) [33]. However, they all used RNA probes that are complementary to the target RNAs and thus, their approach is limited to detecting just one or few RNA transcripts [34]. In this study, we investigate the potential of using spiropyrans as a tool to detect and profile the overall expression of dsRNAs. Our goal is three folds: 1) To establish spiropyran as a molecule that can interact with dsRNAs, 2) To characterize the interactions between dsRNA and spiropyran, and 3) To apply spiropy (...truncated)