The expression of host miRNAs is affected by virus invasion, which also affects virus replication. Increasing evidence has demonstrated that miRNA influences RNA virus multiplication by binding directly to the RNA virus genome. Here, the knowledge relating to miRNAs’ relationships between host miRNAs and RNA viruses are discussed. Caenorhabditis elegans in the 1990s, in which lin-4, a small non-coding RNA (ncRNA), was discovered (C. elegans were able to bind to the 3' untranslated region (3’UTR) of post-transcriptional products, thus regulating gene expression in order to control the development of C. elegans (http://www.mirbase.org/) had been established, it became possible to search the database for miRNA sequences from different species.The discovery of microRNA (miRNA) began with scovered . Later,  elegans . Since tvia virus invasion and other mechanisms. Furthermore, the expression of miRNAs in the organism changes following viral infection, suggesting that they may be involved in the virus infection process itself or in the innate immunity of the organism as modulators of cellular gene post-transcriptional expression  is an ancient, dominating, and strong antiviral defense mechanism, whereas RNAi plays an antiviral role only in undifferentiated embryonic stem cells in vertebrates . The antAs for the virus, we know that many experimental results have shown that a virus infection can alter the miRNA expression profiles of host cells . Due to via polymerase II and partly by polymerase III, which includes thousands of nucleotides and contains at least a hairpin structure  helps the RISC complex target transcripts through the principle of base complementation . But, inAccumulating evidence demonstrates that host miRNAs participate in the replication and pathogenesis of the viruses by binding directly to the RNA of many RNA viruses. During RNA virus infection, miRNA can then target RNA virus genes in a manner similar to the way they target host genes . In geneMany relevant examples have proved that miRNA inhibits viral proliferation and replication by targeting viral RNA and gene silencing. For example, the infectious bursal disease virus (IBDV), a member of the family Birnaviridae, is targeted by gga-miR-21 that downregulates the expression of IBDV VP1 at the translational level rather than the mRNA level, which therefore represses IBDV replication . Similarin vivo. Another example is the porcine epidemic diarrhea virus (PEDV), which belongs to the genus Alphacoronavirus of the Coronavirus family, a single-stranded, positive-sense RNA virus. It was confirmed that miR-221-5p acted as a dose-dependent inhibitor for PEDV, by directly targeting the 3’UTR of the PEDV genomic RNA, and stimulating NF-κB signaling via p65 nuclear translocation, to upregulate IFN-β and related ISGs  . The gented ISGs . Due to in vivo than miRNA-binding sites that have positive effects on viral replication.In the course of a previously published study, it was found that mutations in miRNA targets within some RNA viruses could render the functions of miRNAs targeting RNA viruses ineffective. These phenomena probably occur because RNA polymerases lack the same collation activity as DNA polymerases and miRNA sequences are highly conserved, thus allowing RNA viruses to mutate and fleetly escape from the host’s miRNA inhibition. For instance, as a member of the Orthomyxoviridae family, the influenza A virus (IAV) is a negative single-stranded RNA virus and has multiple subtype strains. A study verified that hsa-miR- 1307-3p, as a novel potent suppressor, could bind directly to the NS1 RNA of IAV to suppress NS1 expression and influenza virus replication, but a new mutation has been identified in the NS1 gene, which is targeted by hsa-miR- 1307-3p, of more than 100 strain types represented by A(H1N1)pdm09 (a subtype strain of IAV), which invalidated the inhibition of miRNA . Analogoin vivo. Besides this, after human immunodeficiency virus type 1 (HIV-1)- infected resting primary CD4+ T cells, the significantly enriched miRNAs including, miR-28, miR-150, miR-223, and miR-382 potently inhibited HIV-1 production by targeting the 3’UTR of the HIV-1 RNA and related host proteins  mosquitoes. Ae. aegypti miR-2944b-5p can bind to the 3’UTR of CHIKV and reduce CHIKV replication, meanwhile regulating the cellular target, vps-13 (vacuolar protein sorting)  . CHIKV msorting) . This miin vitro, endogenous miR-548g-3p can bind to the conserved stem-loop promoter (SLA) (a promoter for DENV RNA transcription) in the 5’NTR of the DENV genome to suppress DENV intracellular replication , a single-stranded RNA virus with the Flaviviridae family, only has an ORF and requires liver-specific miR-122 interactions with the sequences from the HCV RNA 5’UTR to maintain a high viral RNA abundance in the liver . The 5’Ulication . Based olication . Besideslication . AGO2 anThese examples indicate the importance of the miRNA binding site location for targeting the virus RNA. If the binding site is located in the folding of the secondary structure of RNA, SL structure, or the binding site of some proteins, the corresponding miRNA may have an important influence on the expression or function of this genome . InteresEukaryotic miRNAs participate in the regulation of most physiological activities within cells, including RNA virus infection. RNA viruses use RNA as a gene carrier, and miRNAs bind to RNA in a base complementary way, which allows some miRNAs to directly target the RNA within RNA viruses. Most of this binding occurs in the ORF or 3’UTR of the RNA virus RNA, whereas miRNA works with RNA-binding proteins to inhibit RNA viral replication. In addition, some cellular miRNAs are able to bind to RNA viral RNA to avoid the cleavage of the related enzymes or to stabilize the spatial structure of the RNA to positively regulate RNA virus replication. However, as there is a lack of any proofreading function, RNA viruses can escape from miRNA through gene mutation in the face of the miRNA’s direct targeted inhibition. From the above-cited publications, which include many examples, on the one hand, RNA viruses utilize some specific cellular miRNAs to create an intracellular environment conducive to the transmission of viral infection. Yet, on the other hand, it is commonplace that most miRNAs that directly or indirectly strongly inhibit RNA virus replication are manipulated by the RNA virus to reduce expression, or are shunned by RNA viruses with genetic mutations. Moreover, miRNAs in eukaryotes are highly conserved and are not easily mutated, which makes it difficult for miRNAs to be dominant antiviral factors against RNA viruses in nature.via deletion , Sichuan Veterinary Medicine and Drug Innovation Group of China Agricultural Research System (CARS-SVDIP), and China Agricultural Research System (CARS-42-17).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


N6-methyladenosine (m6A) RNA, seemingly valuable biomarkers in CRCs. For ncRNAs, there exists a “molecular sponge effect” between long non-coding RNAs (lncRNAs), circular RNAs (circRNAs), and microRNAs (miRNAs). The detection of exosomes is a novel method in CRC monitoring, especially for predicting metastasis. There is a close relationship between exosomes and EMT in CRCs. This review summarizes the close relationship between epigenetic changes and EMT in CRCs and emphasizes the crucial function of exosomes in regulating the EMT process.Worldwide, colorectal cancer (CRC) ranks as the third most common malignancy, and the second most deadly with nearly one million attributable deaths in 2020. Metastatic disease is present in nearly 25% of newly diagnosed CRC, and despite advances in chemotherapy, less than 20% will remain alive at 5 years. Epigenetic change plays a key role in the epithelial-to-mesenchymal transition (EMT), which is a crucial phenotype for metastasis and mainly includes DNA methylation, non-coding RNAs (ncRNAs), and  Furthermore, it is the most common cause of concern and anxiety for patients with CRCs. Based on previous studies, the mortality of metastatic CRCs is much higher than that of primary CRCs, especially for liver metastasis. Metastatic cancers severely influence the 5-year survival rate and quality of life of patients  have become significant public health problems worldwide, and their incidence has increased to twice in the top 10 cancers . Monitorpatients . TherefoSNAI1, SNAI2, Zeb1, Zeb2, and Twist, called EMT-TFs, and some non-coding RNAs (ncRNAs) can influence the EMT process to regulate the migration and infiltration of tumor cells  is a vital phenotype in its metastasis. EMT is a reversible process that promotes tumor cells exfoliating into circulation and damaging the intercellular skeletal structure. These exfoliated tumor cells could reach distal cites and form metastatic loci. The transcription factors (TFs) or cells , 6. EMT The liquid biopsy (LB) technology is a novel method used to detect the genesis, progress of recurrence, and metastasis in CRC patients. Circulating tumor cells (CTCs), circulating tumor DNAs (ctDNAs), and cell-free RNAs are detectable biomarkers in serum . In addiExosomes are tiny vesicles derived from tumor cells that contain DNA, ncRNAs, epigenetic modulation, and metabolites. Especially in comparison to obtaining tissue biopsies, liquid biopsies, including the relatively noninvasive venipuncture, or sampling other bodily fluids is often safer and more convenient. According to previous studies, exosomes play a pivotal role in predicting cancer metastasis. Comparably, exosome detection is safer and more convenient in malignant cancers, especially for CRCs. LB has shown considerable accuracy in CRC testing. Its sensitivity and specificity can reach up to 60% and 90%, respectively . TherefoThe reverse EMT process is characterized by the downregulation of E-cadherin, desmoplakin, claudins, and β-catenin and the upregulation of N-cadherin, vimentin, fibronectin, and Snail1/Snail2 mediate the migration and invasive metastasis of cancer cells –13. Whetvia the detection of epigenetic biomarkers, such as DNA methylation, overexpression of ncRNAs, and N6-methyladenosine (m6A) modification. These epigenetic changes are regulated by genes and can be inherited by the next generation, but they cannot be transmitted into proteins to form phenotypes. Their influence is so strong that they have gained wide attention , microsatellite instability (MSI), and the CpG island (CGI) methylator phenotype (CIMP) pathways are three principle pathways involved in the genesis and development of CRCs. CIMP is related to the hypermethylation of promoters or the upstream regulator regions that usually contain CGIs, reported to be related to the detection of CRCs , 15. Thettention , 20. Sevttention –23.6A modification for RNA are two epigenetic modifications affecting the transcription and expression of critical genes. In addition, ncRNA is another group that cannot be transcribed, but has high regulatory ability for coding genes. These two mechanisms can undoubtedly regulate the EMT process in CRC metastatic events.Regarding epigenetic changes, 5-methylcytosine (5mC) methylation for DNA and mDNA methylation occurs when a methyl group is transferred to the carbon-5′ position of the cytosine base (5-methylcytosine) concentrated in CGIs under the catalytic action of DNA methyltransferase enzymes . The hypSDC2 methylation is a carcinogenic biomarker in CTCs tested in patients with CRC  and helicase-like transcription factor (HLTF), has prognostic value for therapy and metastatic location in CRC patients (SEPT9 and SDC2 (SEPT9 and OSMR (77.0%)  , SEPT9 aand OSMR , SEPT9 aYCD2/VHL , APC/MGMF2A/WIF1 , SYNE1 and FOXE1 , WIF1/NPNPY/PENK , THBD an C9orf50 , and the/TMEM90B  and MGMT1A/SEPT9  were rep markers , 58, wit markers , 59.Methylated DNA analysis in exosomes has been reported for gastric cancer, prostate cancer, lymphoma, breast cancer, and gingivitis . AlthougNcRNAs are a class of small RNA fragments or circulars in the non-coding regions. Although an association between DNA methylation in vesicles and CRCs exists, no study on their relationship has been conducted. The ncRNAs in CTCs originate from three main pathways, namely, membrane-bound vesicles, apoptotic bodies, and RNA-binding proteins (RBPs), which have been elucidated in detail in a previous review . The ncRMiRNAs were the first discovered ncRNAs that can regulate the functions of genes and proteins. The sequence of miRNAs is short and can form complementary pairs with mRNAs. Most miRNAs are inhibitory and can lead to the degradation of mRNAs, which decreases the expressions of target proteins. Many cytoplasmic miRNAs have been discovered that can regulate expression of N-cadherin, Vimentin, MMPs, E-cadherin, Claudin, and EpCAM, thereby influencing the direction of EMT through molecular regulation in targeted pathways –69. Go eSOX4 , which is produced by cancer-associated fibroblasts (CAFs) and released into serum, is associated with the metastasis of CRCs. The upregulation of UCA1 can change the expressions of the genes in EMT (BANCR is an oncogenic molecule that enhanced the aggressiveness of CRC metastasis (LNCRNA-ATB) can suppress the expression of E-cadherin in EMT to promote CRC metastasis  of m6A modification under the catalytic function of methyltransferase 3 (METTL3)  . The earand ZEB2 , 92. Theand ZEB2 . Most ci6A modification could alter its local RNA structure at the terminal loop region of primary miRNAs (pri-miRNAs) to promote their processing through nuclear transcripts and alternative splicing by modulating RALY binding  and the m6A/METTL-associated complex (MACOM). MAC consists of METTL3 and METTL14, which can form stable heterodimers  could increase export into the cytoplasm in the circNSUN2/IGF2BP2/HMGA2 ternary complex (6A modification enhances the metastasis risk of CRC cells (In total, 11 readers, 7 writers, and 2 erasers have already been found. The term “writers” often denotes methyltransferase as the beginning of mity. The methyltrarodimers . BesidesmiR-1246 . Contrar miR-375 –149. Fur miR-375 , 142. Th complex . In addiRC cells .6A modulation sites, such as YTHDF1, IGF2BP1, IGF2BP3, and EIF3B (FTO (GAS5 can reversely regulate YAP1 via m6A (6A can positively increase with the increased concentrations of circRNAs (RP11 by m6A methylation can accelerate the spread of CRC cells through the upregulation of ZEB1 (6A circRNAs are different from m6A mRNA sites. This indicates that disturbance in the m6A modification of mRNA is not equivalent to that of m6A circRNAs. Therefore, to abolish the effects of m6A modification, then the m6A of circRNAs must be considered as well (6A modification, then the m6A of circRNAs must be considered as well. NcRNAs can regulate the critical enzymes as the downstream target molecules or combined partners. Feedback of the essential enzymes affects the m6A modulation of ncRNAs, and this process can change the cancer-related phenotype and lead to metastasis (Beyond the methyltransferases, the “readers” that can recognize mnd EIF3B , are alsF3B (FTO . Moreove via m6A . SurpriscircRNAs . The upr of ZEB1 . Interes as well , 154. Thtastasis .via the addition of anti-miRNAs and exogenous exosomes containing miRNAs confirmed that ncRNAs negatively regulated the downstream target proteins or mRNA molecules to promote tumor metastasis via EMT (via increasing the permeability of vascular epithelial layers, and miR-27b-3p can regulate this process through the STAT3 pathway (What is an exosome? Exosomes are microvesicles with diameters from 30 to 150 nm. They are derived from normal intestinal cells, CRC cells, and other cancer-related stromal cells . Exosome via EMT –162. In  pathway . There i pathway . The eff pathway .6A methylation of RNA in CRCs could enhance the invasive and migration ability to promote metastasis. The mechanisms of the mutual effect between methylation and ncRNAs may provide a novel direction for future studies.Cancer metastasis has always been a hot topic for scientists and clinicians, and discovering the risk factors and suitable therapeutic targets is a critical part of preventing metastasis. Epigenetic change is a popular context associated with the progression and metastasis of CRCs due to its variability and vulnerability. NcRNAs plays a crucial role in regulating epithelial tissues and the mesenchymal components. The dynamic process determines the tumor metastasis tendency. In this regulation, the sponge effect between lncRNAs/circRNAs/miRNAs/mRNA is critical. In addition, epigenetic modification is a crucial link. The 5mC methylation of DNA and the mThe EMT is a vital regulator in metastasis, while the mechanism and influence factor of EMT is still unclear. As mentioned before, these epigenetic changes are not independent but interacting. The methylation of promoters can regulate the process of alternative splicing and production of ncRNAs. The review interprets a crosstalk interaction in epigenetic changes from DNA methylation, ncRNAs, and m6A methylation. The function of epigenetic changes is powerful and complex in regulating mechanisms in CRC metastasis. At the same time, EMT is a cross point that the DNA methylation, m6A methylation, and ncRNAs can all affect CRC metastasis. They can upregulate or downregulate the biomarkers in EMT such as N-cadherin, Vimentin, MMPs, E-cadherin, Claudin, and EpCAM to control the progress of the epithelial transition.Another novelty of the review is that the exosomes can regulate EMT as a carrier for transporting ncRNAs. The function of exosomes has gained widespread attention in cancers. The exosomes have been proved to be related to cancers detection. This review summarizes the effect of exosomes in CRC metastasis related to EMT. That indicates that exosomes are a vital factor in the tumor micro-environment. The tumor cells or other stromal cells can secret exosomes to regulate EMT. And this review indicates that DNA methylation, ncRNAs, m6A methylation, exosomes, and EMT can all act as potential therapeutic targets in CRC metastasis. The associated relationship and mutual interaction among them are elucidated thoroughly in this review. While prior studies have been published elucidating the mechanisms of exosomes and EMT, the field remains in its relative infancy, with the potential to discover important new therapeutic targets for CRC.The detection of exosomes in LB is a novel method to predict metastasis in CRCs. The variety of stromal cells can produce exosomes to participate in the progression, proliferation, migration, and EMT in CRCs. Epigenetic changes, ncRNAs, and exosomes are not independent of each other; on the contrary, there exists an intrinsic interrelation between them. Different from previous studies, this review elaborated on the potential links between epigenetic changes, exosomes, and EMT in CRCs regarding metastasis. It provided novel sights for the study of probable mechanisms of metastasis in CRCs. There have already been several studies on the mechanisms of exosomes and EMT, but more studies are needed to discover new therapeutic targets for CRCs.MY and HZ designed the study design and wrote the main manuscript. MS contributed to the final proof. All authors reviewed the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


However, although useful to study the contribution of single RNA sub-modules to RNA/protein interactions, these exogenous practices might fail in revealing biologically meaningful contacts occurring in vivo and risk to generate non-physiological artifacts. Therefore, endogenous methods must be preferred, especially for the initial identification of partners specifically interacting with elected RNAs. Here, we apply an endogenous RNA pull-down to lncMN2-203, a neuron-specific lncRNA contributing to the robustness of motor neurons specification, through the interaction with miRNA-466i-5p. We show that both the yield of lncMN2-203 recovery and the specificity of its interaction with the miRNA dramatically increase in the presence of Dextran Sulfate Sodium (DSS) salt. This new set-up may represent a powerful means for improving the study of RNA-RNA interactions of biological significance, especially for those lncRNAs whose role as microRNA (miRNA) sponges or regulators of mRNA stability was demonstrated.Detecting RNA/RNA interactions in the context of a given cellular system is crucial to gain insights into the molecular mechanisms that stand beneath each specific RNA molecule. When it comes to non-protein coding RNA (ncRNAs), and especially to long noncoding RNAs (lncRNAs), the reliability of the RNA purification is dramatically dependent on their abundance. Exogenous methods, in which lncRNAs are  Since the first characterization of RNA, DNA and protein chemical composition, it has becoming increasingly clear the need to carve the interactions that these macromolecules establish in living cells to execute their functions . On thisAmong the RNA-centric approaches, the methodologies allowing to detect RNA/RNA contacts train great attention, also in light of the ever-increasing importance of base-complementarity between transcripts, both in a mechanistic and in a regulative perspective . AlthougLncRNAs represent an heterogenous class of non-protein coding molecules arbitrarily defined as transcripts longer than 200 nucleotides, which regulate gene expression through transcriptional as well as post-transcriptional mechanisms . Many ofin silico tools were developed for the prediction of lncRNA functions based on lncRNA-RNA interactions , with the innovative use of the Dextran Sulphate Sodium (DSS) salt as a hygroscopic chemical additive. By testing the procedure to the motoneuron-expressed lncRNA lncMN2-203 , we founA number of 15 oligonucleotide probes, 90-nucleotides long and carrying a 5’-biotin modification, were designed as in Tris-HCl pH 7.5 50 mM, NaCl 150 mM, MgCl2 3 mM, NP40 0.5%, EDTA 2 mM. Add fresh DTT 1 mM, 1× PIC, and RNase Inhibitors (0.2 U/μl).Tris-HCl pH 7.5 100 mM, NaCl 300 mM, MgCl2 1 mM, SDS 0.2%, Formamide 15%, NP40 0.5%, EDTA 10 mM. Add fresh DTT 1 mM, 1× PIC, and RNase inhibitors (0.2 U/μl).1) Streptavidin Magnesphere paramagnetic beads—Promega.2) Dextran Sulfate Sodium (DSS) salt—SigmaAldrich3) cOmplete™, Mini, EDTA-free Protease Inhibitor Cocktail (PIC)—ROCHE.4) RNase Inhibitor: RiboLock—ThermoFisher Scientific.5) Tris, NaCl, MgCl2, SDS, Formamide, NP40, EDTA and Dithiothreitol (DTT)—SigmaAldrich.6) TRI-Reagent—Zymo Research.7) Direct-zol RNA Miniprep Kit and DNase—Zymo Research.8) Superscript VILO™ cDNA Synthesis Kit—ThermoFisher Scientific.9) SYBR Green Power-UP - ThermoFisher Scientific10) miScript II RT Kit—QIAGEN11) miScript SYBR Green—QIAGEN.1) Gently resuspend cell pellets from EB6 in lysis buffer supplemented with proteases (1X PIC- ROCHE) and RNase inhibitors (0.2 U/μl—Thermo Fisher Scientific). Live, freshly isolated, not frozen cells should be preferred.2) Incubate the cell suspension first on ice (10 min) and then on a rotating wheel for 10 min at 4°C. Centrifuge the cell suspension at 15,000 × g at 4°C for 15 min and collect the supernatant, which represents the total cellular extract, into new tubes. Quantify the extract by protein determination and dilute 1:2 in Hybridization Buffer (HB). From the diluted extracts, collect 1 mg of material for each PD condition and 0.1 mg for the Input . Typically, 10 millions of freshly lysed EB6 yield up to 1 mg of total extract. CRITICAL STEP: since DSS can interfere with enzymatic reactions, collect Input before DSS addition to the EB6 total extract or use specific RNA purification methods to remove possible salt contaminants. A final volume of 0.9 ml in a 1.5 ml vial tube is the ideal condition for hybridization.3) Add DSS salt (SigmaAldrich) to each PD sample at a final concentration of 1% or 2.5%. CRITICAL STEP: DSS can be very viscous when undiluted, be careful to pipette the required volume. Cutting the tip of the pipette could help.4) Dilute 100 pmol of 5′-biotinylated (90-mer long) antisense DNA oligonucleotide probes specifically targeting lnc-MN2 or U1snRNA to a final volume of 50 μL with HB. Heat-denature the probes for 3 min at 80°C and directly add to each sample .5) Mix the specific probes with the cellular extract and incubate at 4°C for, at least, 4 h. Incubate Input and PD samples in parallel. Before concluding the incubation, gently wash 0.1 ml of Streptavidin paramagnetic beads (Promega)/sample with HB on a magnetic rack (Millipore). Repeat this step. CRITICAL STEP: prior to pipette the needed amount of beads, gently flick the bottom of the tube until the particles are completely dispersed.6) Resuspend the Streptavidin paramagnetic beads in 0.1 ml of fresh HB and then add to each PD sample.7) Incubate beads/extract/probe for 1 h at RT (20–25°C). On a magnetic rack, remove the supernatant from the beads and discard as it contains the unbound material. Keep the beads for steps 8–9!8) Carefully wash the beads 4 times (3 min each) on a rotating wheel with HB at RT (20–25°C). After each wash, recover the beads on a magnetic rack.9) For RNA extraction, add Trizol (TRI Reagent- Zymo Research) directly on the washed beads and vortex thoroughly for 2 min to detach the probes. Place back the samples on the magnetic rack and collect the supernatant in a new 1.5 ml tube. CRITICAL STEP: the supernatant contains RNA. Keep it for RNA extraction!10) Purify the RNA with Direct-zol RNA Miniprep Kit (Zymo Research), according to manufacturer’s instructions and treat with DNase at RT (20–25°C). Elute RNA in 30 μL of Elution Buffer (Zymo Research) or DNase/RNase-Free Water. *OK to store RNA at −20°C at this point.11) For long RNA transcript quantification, perform reverse transcription with SuperScript™ VILO™ cDNA Synthesis Kit (ThermoFisher Scientific), according to manufacturer’s instructions and qRT-PCR with SYBR Green Power-UP (ThermoFisher Scientific). For miRNA quantification, perform reverse transcription with miScript II RT Kit (QIAGEN) and qRT-PCR with miScript SYBR Green (QIAGEN). 250–500 μg of RNA for each reverse transcription reaction is the ideal amount.mESCs carrying the Hb9:GFP transgene  are diffThe abovementioned protocol is thought to optimize the performance of the classical endogenous RNA PD procedures, with the aim to increase the recovery of the targeted lncRNA and, consequently, the specific enrichment of its co-precipitated RNA interactors.The efficacy of the procedure was tested on lncMN2-203 , a motorBuilding upon these results, we wondered whether we could improve the cost-effectiveness of the procedure without reducing the performances. The evidence that lncRNA expression is often restricted to specific cell sub-populations causes not negligible concentration constrains . This isWith the aim to reduce unnecessary costs, lncMN2-203 PD was repeated by halving the Input used in our original set-up  to 1 mg In Situ Hybridization (RNA-FISH) experiments for the visualization of both mRNA RNA sequencing technologies showing that the expression of given RNAs can be extremely cell type specific, thus underrated in total extract, especially for those whose expression is further restricted to specific time and space windows .We have tested the feasibility and the outcomes of a new RNA precipitation protocol, by narrowing our attention to lncMN2-203, a lncRNA recently demonstrated to be functionally relevant through a mechanism of lncRNA/miRNA interaction. In principle, we cannot predict any technical restriction that may impede, in the future, downstream transcriptomics for a deeper and unbiased identification of the lncRNA-RNA interactome. Moreover, we believe that the improvements to the endogenous RNA PD protocol herein presented could also be applied to future studies on other RNA partners (e.g. proteins) or other RNA substrates. For instance, it may serve as a beneficial tool for the analysis of difficult-to-precipitate ncRNAs, such as circular RNAs (circRNAs), covalently closed lncRNAs generated by back-splicing of canonical pre-mRNAs . The mec


One of these elements is a three-stemmed pseudoknot, although alternative folds of the frameshift site might have functional roles as well. Here, by complementing ensemble and single-molecule structural analysis of SARS-CoV-2 frameshift RNA variants with functional data, we reveal a conformational interplay of the 5′ and 3′ immediate regions with the FSE and show that the extended FSE exists in multiple conformations. Furthermore, limiting the base pairing of the FSE with neighboring nucleotides can favor or impair the formation of the alternative folds, including the pseudoknot. Our results demonstrate that co-existing RNA structures can function together to fine-tune SARS-CoV-2 gene expression, which will aid efforts to design specific inhibitors of viral frameshifting.The RNA genome of SARS-CoV-2 contains a frameshift stimulatory element (FSE) that allows access to an alternative reading frame through −1 programmed ribosomal frameshifting (PRF). −1PRF in the  Many viruses, including Coronaviruses, employ several recoding strategies that allow access to an overlapping and functional reading frame, thereby increasing the regulatory potential and the coding repertoire of their genomes . One sucin vitro and in infected cells indicate that the frameshift element can also be found in alternative conformations  forming with the 3′ and 5′ immediate ends of the PK and an alternative PK where the SL1 loop base pairs with nucleotides upstream of the FSE , X-ray crystallography, cryo-electron microscopy (Cryo-EM), optical tweezers and chemical probing techniques . These srmations ,16,21,22 the FSE ,24. In s the FSE . In infe the FSE ,22. Desp the FSE ,22. Recearticles . Lastly,articles ,20. Collarticles ,21.in vitro and a in cell-based reporter assay. Taken together, our dynamic- and steady-state analysis of the frameshift RNA suggest that there are alternative conformations of the FSE mediated by cis-acting short-range RNA interactions that fine-tune gene expression of SARS-CoV-2, which would provide important clues for future structure-based drug design studies.To gain a comprehensive view of the conformational heterogeneity of the SARS-CoV-2 FSE, we used an integrative approach with single-molecule optical tweezers (OT) and chemical probing by dimethyl sulfate mutational profiling (DMS-MaP) Figure  and C. TTo generate dual-fluorescence reporter constructs, frameshift sites of SARS-CoV-2 and corresponding mutant/truncated variants were placed between the coding sequences of EGFP and mCherry  by siteOT constructs were based on the wild-type SARS-CoV-2 frameshift site (nucleotides 13475–13541) cloned into the plasmid pMZ_lambda_OT, which encodes for the handle sequences of lambda DNA (2 kb each) flanking the RNA structure of interest ,33. Consin vitro transcription of the RNA samples were generated by PCR using the pMZ_lambda_OT vector. The 3′ handle was labeled during the PCR using a 5′ digoxigenin-labeled reverse primer. The 5′ handle was labeled with biotin-16-dUTP at the 3′ end following PCR using T4 DNA polymerase. The RNA was in vitro transcribed using in-house purified T7 RNA polymerase. Next, DNA handles (5′ and 3′) and in vitro transcribed RNA were annealed in a mass ratio 1:1:1 (5 μg each) by incubation at 95°C for 10 min, 62°C for 2 h, 52°C for 2 h and slow cooling to 4°C in annealing buffer  to yield the optical tweezer suitable construct. Following the annealing, samples were concentrated by ethanol precipitation, pellets were resuspended in 50 μl RNase-free water, and 4 μl aliquots were stored at −80°C until use.5′ and 3′ DNA handles, and the template for 2, 5 mM DTT and 0.05% Tween 20) and 1 μl of RNase inhibitor (Molox). The mixture was incubated for 20 min at room temperature in a final volume of 19 μl and subsequently diluted by the addition of 0.5 ml assay buffer. Separately, 0.8 μl of streptavidin-coated polystyrene beads  were mixed with 1 ml of assay buffer. The flow cell was washed with the assay buffer, and suspensions of streptavidin beads and the complex of OT construct with anti-digoxigenin beads were introduced into the flow cell. During the experiment, single AD and SA beads were trapped in individual traps and brought into proximity to allow the formation of a tether. The beads were moved apart (unfolding) and back together (refolding) at a constant speed (0.05 μm/s) to yield the force-distance (FD) curves. The stiffness was maintained at 0.31 and 0.24 pN/nm for trap 1 (AD bead) and trap 2 (SA bead), respectively. FD data were recorded at a rate of 78125 Hz.Optical tweezers measurements were performed using a commercial dual-trap platform coupled with a microfluidics system  as described before ,33,34. Fhttps://github.com/REMI-HIRI/POTATO . In briI/POTATO . First, 2. Transfections were performed using linear 25 kDa PEI (Polysciences) according to manufacturer's instructions using a 1:12 DNA:PEI ratio. For co-transfections, plasmids and antisense oligonucleotides were mixed in a 1:40 molar ratio and electroporations were performed in OptiMEM (Gibco) using a Nepa21 Super Electroporator (NEPAGENE) following the manufacturer's instructions. 24 h post-transfection, the cells were analyzed by flow cytometry. Antisense oligonucleotides (IDT) were added to the cells in a final concentration of 100 nM. To increase stability of the oligonucleotides, the phosphate backbone was substituted by phosphothioate and three bases at the 5′ and 3′ end were locked nucleic acids (LNAs).HEK293 cells  were maintained in DMEM (Gibco) supplemented with 10% FBS (Gibco) and 100 μg/ml streptomycin and 100 U/ml penicillin at 37°C with 5% COHEK293 cells were transiently transfected with either the control construct or the −1PRF construct encoding for the dual-fluorescence EGFP-mCherry translation reporter as outlined in Figure  control . Data reIn vitro translations in rabbit reticulocyte lysate (RRL) were performed as described before . Typical reactions were 0.05 μM template mRNA, 75% v/v RRL, and 20 μM amino acids. Antisense oligonucleotides (IDT) were added to the reactions in a concentration range from 0–0.5 μM. Reactions were incubated for 1 h at 30°C at which point samples were mixed with three volumes of 1× BOLT™ LDS Sample Buffer (Invitrogen), denatured for 10 min at 70°C, and resolved on NuPAGE™ 4–12% Bis-Tris polyacrylamide gels (Invitrogen). Translation products were detected by western blot. Briefly, after transfer using Trans-Blot (Bio-Rad), nitrocellulose membranes were developed with anti-DYKDDDK-tag antibody  and visualized by incubation with a secondary antibody ). Bands were detected using an Odyssey Clx infrared imager system (LI-COR) and quantified densitometrically using FIJI software  in GraphPad Prism (version 9.2.0) software.2 and 0.3 U/μl of RNasin) and slow cooling (<1°C/min) to 23°C. DMS was diluted in ethanol to a working concentration of 0.85 M. 1/10 volume of DMS working stock was added to the samples to make a final concentration of 85 mM in a total volume of 33 μl. Samples were incubated at 37°C for 6 min and then quenched with 33 μl of beta-mercaptoethanol (14.2 M stock). For the untreated control, ethanol was used instead of DMS. RNA was then purified by Trizol LS (Sigma) according to the manufacturer's instructions.A mix of 37 nM of RNA, 37 nM of blocking primer 5′ [GTAGCTGTCGAGCTCCTGCGAAG] and 37 nM of blocking primer 3′ [GGCGAAGAGCAGGTTGCAGGAT] was first heat denatured at 90°C for 2 min, then annealed by slow cooling (<1°C/min) to 50°C. RNA was then folded by adding the folding buffer  long pseudoknot structure within the FSE, yet analyses of the SARS-CoV-2 whole genome structure suggest that the region including the FSE can adopt multiple conformations in cells ,21. In oWe first asked whether upstream and downstream regions of the FSE can alter frameshifting. For that we used a cell-based dual-fluorescence frameshift reporter, in which the SARS-CoV-2 frameshift site is placed between the EGFP gene in the 0-frame and the mCherry gene in the –1-frame Figure  29). Wi. Wi29). In order to test whether the effect on frameshifting is related to altered conformation of the FSE variants, we next employed single-molecule optical tweezers Figure . Here, FIn the FSE RNA, which in addition to the PK contains the slippery sequence and the spacer, we mostly observed a single unfolding step in 72.2% of the FD trajectories Figure . Most ofPrevious studies also predicted that the 3′ end of the PK can be involved in alternative folds, with unknown functions ,16,20–22Next, we employed the FSE-V2 RNA variant containing the putative attenuator hairpin at the 5′ of the FSE, which was previously reported to decrease frameshifting in SARS-CoV-1 and SARS-CoV-2 ,26 Figu. The un. HoweverWe then evaluated the effect of the alternative stem (AS)  formed by base pairing between the 5′ upstream region of the attenuator hairpin and parts of the FSE including the stem of SL1 ,22. For Overall, based on our single-molecule analysis we conclude that the PK is the major conformation for the FSE, yet the FSE can adopt pseudoknot structures with varying stabilities. Interestingly, addition of 5′ and 3′ proximal nucleotides to the canonical FSE resulted in the loss of the highly stable RNA structures, suggestive of alternative no-PK folds present together with a low stability PK.To investigate the conformational heterogeneity of the FSE and its variants, we next generated structural profiles of the RNAs based on reactivities of RNA to the chemical DMS ,51 Figu. In the In the FSE-V1 and FSE-V2 samples we saw negligible differences compared to the FSE sample Figure . In the et al.  algorithm . DREEM cet al. ,24. Inteet al.  , based on the FSE-V1 mRNA. These mutants were computationally predicted to either disrupt the PK fold or alter equilibrium between the alternative folds identified above  Figure .FSE-M1 was designed to interfere with PK formation by disrupting SL2 whilst leaving SL1 and SL3 of the FSE intact Figure  24). Mo. Mo24). The FSE-M2, contains two point mutations at the 3′ end of SL1 Figure . This muThe FSE-M3 RNA variant has two mutations in the 3′ strand of SL2 Figure . These mAnother FSE mutant contained two substitutions in SL2 (FSE-M4), which was predicted to form an alternative PK structure  24). Ho. Ho24). Thus, we conclude that the correct folding of the pseudoknot is crucial for frameshifting and that alternative folds, such as the 3WJ, can compete with PK formation to decrease frameshifting efficiency.Informed by our analysis of the FSE structural folds, we next tested three antisense oligonucleotides (ASO) designed to alter the balance between PK and alternative folds Figure .The first ASO (FSE-A1) hybridizes to the 3′ strand of SL2 (position 145–158 relative to the FSE-V4) and thus impairs folding of the canonical PK Figures  29). In. In29). The second ASO (FSE-A2) binds to the 5′ end of the PK including the SS and the spacer region (position 69–86), which is a few nucleotides downstream of the attenuator stem Figure . This ASLastly, we tested FSE-A3, which binds 10 nucleotides downstream of the FSE (position 166–182), a region that was not predicted to be part of any of the major conformers Figure . AccordiOverall, these data show that a preclusion of the base pairing of 5′ spacer region and the 3′ strand of the SL2 hampers the formation of the alternative folds, further suggesting that FSE can undergo structural rearrangements during translation, when the slippery nucleotides and the spacer region are occluded by the ribosome.in vitro translation reporter assays , a 3WJ (FSE-V1), or a closed SL1 loop that may preclude PK formation (FSE-V2). Strikingly, in the longer FSE-V3 and FSE-V4 variants DREEM analysis detected a single non-PK conformation containing AS1, yet these variants supported higher levels of frameshifting when compared to FSE-V1 and FSE-V2. One possible explanation is that during translation, RNA structures would be unfolded by the helicase activity of the ribosomes allowing the RNA to resample into alternative conformations. Specifically, as the ribosome moves over the 5′ portion of the AS1 stem, it would liberate 3′ part of the stem allowing the folding of SL1, and eventually the formation of the canonical PK fold Figure . In contWe also suggest that the presence of less-stable structures can be a strategy employed by RNA viruses to avoid logjams during the replication of their genomes ,60. In cin vitro and in cells. Nevertheless, the conformational landscape that we identified has implications for therapeutic interventions. So far, efforts to target FSE have mainly focused on the isolated PK element. However, if it is formed transiently during translation or at different stages of the infection cycle, the PK may not be the most abundant structure in the cell. This would impact the efficiency of small molecule targeting. In addition, (de)stabilization of the alternative conformers may lead to a shift in the possible RNA folds, which may ultimately impact frameshifting and reduce viral replication. Knowledge of functionally relevant alternative conformers of viral frameshift elements could therefore be targeted to increase the efficiency of viral RNA targeting. Although our work provides mechanistic insights on structural transitions of FSE variants under defined conditions, future investigations will also be needed to understand the potential role of RNA-binding proteins and longer range cis-interactions on the structure and function of frameshift elements in the context of viral infection in cells.Finally, we show that ASO targeting of the SL2 prevents the formation of the canonical PK, thus decreasing frameshifting both Data supporting this study is available at 10.5281/zenodo.6626934.https://github.com/REMI-HIRI/POTATO.git)  .gkac1184_Supplemental_FileClick here for additional data file.


Science2000, 289, 448–452) investigated such conversion from one ribozyme to a new ribozyme with distinctly different catalytic functions. Within a neutral network that linked these two prototype ribozymes, a single RNA chain could be identified that exhibited both enzymatic functions. As commented by Schultes and Bartel, this system possessing one sequence with two enzymatic functions serves as a paradigm for an evolutionary system that allows neutral drifts by stepwise mutation from one ribozyme into a different ribozyme without loss of intermittent function. Here, we investigated this complex functional diversification of ancestral ribozymes by analyzing several RNA sequences within this neutral network between two ribozymes with class III ligase activity and with self‐cleavage reactivity. We utilized rapid RNA sample preparation for NMR spectroscopic studies together with SHAPE analysis and in‐line probing to characterize secondary structure changes within the neutral network. Our investigations allowed delineation of the secondary structure space and by comparison with the previously determined catalytic function allowed correlation of the structure‐function relation of ribozyme function in this neutral network.During evolution of an RNA world, the development of enzymatic function was essential. Such enzymatic function was linked to RNA sequences capable of adopting specific RNA folds that possess catalytic pockets to promote catalysis. Within this primordial RNA world, initially evolved self‐replicating ribozymes presumably mutated to ribozymes with new functions. Schultes and Bartel ( The diversity of secondary structures of different ribozymes connected through several mutational steps were investigated. We mapped the mutational landscape in order to understand changes in secondary structure and their link to particular functions. This work further highlights the necessity for the utilization of different complementary techniques for the analysis of RNA secondary structures. Interestingly, a single intersection sequence was found that possessed both functions and was called two‐function ribozyme  and for HDV1 (5.9×10−3) increases 120 times . In SHAPE experiments, each detectable band indicates a flexible and therefore unpaired nucleotide. As for some NMR spectra, SHAPE patterns of closely related ribozymes feature similar characteristics. The sequences of LIG1, INT and HDV1 look similar except for the 3’‐end region of HDV1, containing a mutation as well as an overall decrease in intensity for INT. Figure SHAPE20, 211H,1H‐NOESY spectra of the flanking prototype sequences LIGP and HDVP as well as the spectrum of INT are illustrated. The spectra of LIGP and HDVP furthermore cover spectra of their individual secondary structure elements for comparison. For additional 2D spectra and assignment procedures see Supporting Information . In order to implement the divide‐and‐conquer approach, we designed hairpin models of the RNA sequences LIGP and HDVP. NMR spectra of these hairpin models can be assigned with high confidence, allowing transfer of these initial assignment spectra to the full‐length sequences. This procedure exploits the observation that chemical shifts observed in smaller structural elements remain largely unchanged in larger constructs. We thus investigated unlabeled model hairpins LIGP HP (5’‐CACCCGUU UUCGGGUG‐3’) and HDVP HP (5’‐CAUCCGAAGGUU UUCCUUCGGAUG‐3’). Figure 1H NMR spectra are merged into one new spectrum . The comparison of LIGP and LIG36 lead to new information about previously unassigned base pairs. Thus, we assigned the stem between G51*U73 and U54*G70. Using these data as restraints for secondary structure prediction by online tools  mostly showed higher signal‐to‐noise for imino proton signals at higher temperatures, with few exceptions, e. g. G48 for HDVP and G24 for HP. Especially hairpin imino protons appear to dominate the signal pattern with strong and sharp signals whereas others are less intense and decrease fast at low temperatures. This observation as well as the assignment indicate a highly stable hairpin element in the HDVP secondary structure and a less stable remaining structure.The secondary structure of HDVP was likewise analyzed by NMR experiments at room temperature. Additional data recorded at 278–283 K are shown in the Supporting Information . In addition, we analyzed the structural hairpin HP C43‐G66 as part of the full‐length HDVP. The NMR chemical shift assignment of this hairpin structure helped for the assignment of the HDVP spectra and led to the finding that most signals from hetero‐ and homonuclear experiments of the full‐length construct belong to the C34‐G66 hairpin  and HDVP HP (C34‐G66 in full length RNA) and then used the data to assign the NMR spectra of full length RNAs. The assignment of full length HDVP spectra featured only its HP signals; other signals could not be assigned to the remaining structural elements with certainty. LIGP on the other hand presented signals within the NMR experiments, which covered LIGP HP (C19‐G34) and another hairpin element, G56‐C69. We made use of these data and found a new and slightly different secondary structure for LIGP. This finding was confirmed through the analysis of LIG20. The respective data revealed two hairpin structures, C19‐G34 and A56‐U70, indicating that these structural elements are conserved despite the mutations in sequence. Our analysis indicated that not only the destabilization of HDV structure by mutation (A4) increases ligase activity through increased fold population but also the hairpin structures are stabilized by the following mutations  causing a strong activity increase, too.Our study applies novel methodology to study even large RNA sequence space in considerable detail. Application of complementary technology sensitive to presence (NMR) or absence (in‐line probing) of base‐pairing at nucleotide resolution is essential to reach a structure‐function relationship. The thus established structure‐function relationship shows that enzymatic function of the ribozymes has to be linked to lowly‐populated conformations since the revised secondary structures do not adopt structural characteristics required for catalytic activity;14, 28Documentation: For analytical images see Figures S1‐5, S14 and S38 in the Supporting Information.RNA sample preparation: RNAs were prepared by in‐vitro transcription and solid phase synthesis. The latter ones were purchased from Dharmacon™. The RNAs were deprotected, as described on their website. For in‐vitro transcription[2HPO4/KH2PO4; pH 6.3) by using centrifugal concentrators with 5k MWCO from Sartorius, if RNA was used for NMR experiments. The RNAs were then folded at 95 °C and 10 % D2O and 100 μM DSS were added. We used Shigemi NMR tubes (Shigemi Inc.). For further information about NMR sample preparation see Schnieders et al.SHAPE experiments: RNAs for SHAPE experiments were purified by use of magnetic beads. RNAs were chemically modified with 1M7 at room temperature for 15 min and quenched with Na‐MES (pH 6.0). A reaction mixture of 20 μL contained 10 mM MgCl2, 0.06 μM RNA, 50 mM Na‐HEPES (pH 8) and 5 μL 1M7 mixture . The modified RNA was reverse transcribed with Superscript III reverse transcriptase from Invitrogen. For detection of cDNA fragments an ABI 3100 capillary sequencer was used. The data analysis was performed with the HiTRACECD spectroscopic measurements: Concentrations of RNA samples were adjusted to 100 μM RNA with 25 mM potassium phosphate buffer (NMR samples were used). The measured temperature range was 5 °C–95 °C. CD intensities were measured with a JASCO J‐810 spectropolarimeter. The data was visualized via SigmaPlot™ 12.5 and is shown in the Supporting Information, Figures S12, S22 and S29 in the Supporting Information.In‐line probing: RNAs were in‐vitro transcribed. Leftover free rNTPs were removed by a NAP‐5 column from Merck. The RNAs were precipitated from EtOH with NaOAc and the 5’ ends were dephosphorylated with Shrimp Alkaline Phosphatase (rSAP) from New England Biolabs® (NEB) for 4 h at 37 °C. The 5’ ends were phosphorylated with 32P‐ATP by using T4 Polynucleotide Kinase from NEB® for 40 min at 37 °C. The RNAs were then purified via preparative PAGE. Probing was performed with probing buffer  at room temperature for 15 h. The result was analyzed by denaturing polyacrylamide gels. For imaging phosphor imaging plates and a Typhoon™ Scanner 9400 from GE Healthcare were used.NMR spectroscopy experiments: NMR experiments were performed on Bruker spectrometers with fields ranging from 600 MHz to 950 MHz and from 278 K to 298 K. We applied jump‐return echo experiments from Sklenar and Bax1H and 2D 1H,1H‐NOESY NMR spectra. For heteronuclear experiments we used BEST‐TROSY[15N‐labeled samples, decoupling was obtained through a method by Shaka et al.The authors declare no conflict of interest.1As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.Supporting InformationClick here for additional data file.


Crocus sativus that are rich in secondary metabolites, polysaccharides, and other interfering compounds which often irreversibly co-precipitate with the RNA. While many methods have been proposed for RNA extraction including CTAB, TriZol, and SDS-based methods, which invariably yield less and poor quality RNA and hence it necessitated the isolation of high-quality RNA suitable for high throughput applications.Isolating high-quality RNA is a basic requirement while performing high throughput sequencing, microarray, and various other molecular investigations. However, it has been quite challenging to isolate RNA with absolute purity from plants like In the present study we made certain adjustments to the available protocols including modifications in the extraction buffer itself and the procedure employed. Our method led to the isolation of clear and non-dispersive total RNA with an RNA Integrity Number (RIN) value greater than 7.5. The quality of the RNA was further assessed by qPCR-based amplification of mRNA and mature miRNAs such as Cs-MIR166c and Cs-MIR396a.The online version contains supplementary material available at 10.1186/s13104-022-06095-z. Crocus sativus is a perennial geophyte with crimson trifid stigmas. The stigmas contain apocarotenoids such crocin, picrocrocin, and safranal, which give saffron its distinctive color, taste, and perfume . It is oThe current scientific evidence supports the significance of microRNAs in regulating essential plant developmental processes as leaf morphogenesis, polarity , 7, florCrocus sativus, it is important to ensure that there is no interference of secondary metabolites, which co-precipitate with the RNA  method. Table S5. Spectrophotometric analysis of RNA isolated from different tissues of Crocus sativus using Chan et al. [13] method. Table S6. Spectrophotometric analysis of RNA isolated from different tissues of Crocus sativus using RNasy Plant kit.


Non-coding RNAs (ncRNAs) offer a wealth of therapeutic targets for a range of diseases. However, secondary structures and high similarity within sequence families make specific knockdown challenging. Here, we engineer a series of artificial oligonucleotide enzymes (XNAzymes) composed of 2’-deoxy-2’-fluoro-β-D-arabino nucleic acid (FANA) that specifically or preferentially cleave individual ncRNA family members under quasi-physiological conditions, including members of the classic microRNA cluster miR-17~92 (oncomiR-1) and the Y RNA hY5. We demonstrate self-assembly of three anti-miR XNAzymes into a biostable catalytic XNA nanostructure, which targets the cancer-associated microRNAs miR-17, miR-20a and miR-21. Our results provide a starting point for the development of XNAzymes as a platform technology for precision knockdown of specific non-coding RNAs, with the potential to reduce off-target effects compared with other nucleic acid technologies. Engineered RNA endonuclease XNAzymes can specifically target individual disease-associated non-coding RNAs (ncRNAs) in vitro with potential as platform technology for precision detection or knockdown of individual microRNAs or longer ncRNAs. Although initially linked to regulation of development, it has emerged that miRNAs are also key players in hepatitis and cardiovascular disease and can act as both tumour suppressors and oncogenes (oncomiRs) in a variety of cancers11. From a clinical perspective, the ability of single miRNAs to pleiotropically regulate multiple disease-associated mRNAs – and thus multiple nodes within a given biological pathway (or indeed multiple pathways) – makes them attractive targets for the development of next-generation therapeutics beyond current strategies that intervene at the protein level, against single targets15.The cell is an ‘RNA machine’; although only 1–2% of the human genome encodes peptides, >70% is transcribed, generating a huge variety of non-coding RNAs (ncRNAs) with myriad roles in biology including regulation of chromatin remodelling, mRNA transcription, editing, splicing, translation, and degradation, and are implicated in a wide range of diseases19, and a variety of functional oligonucleotide technologies: miRNA activity can be enhanced by delivery of miRNA mimics20 into target cells, and can be inhibited by sequestration using transgene-expressed RNAs containing multiple miRNA binding sites (miRNA sponges)22, or by antisense oligonucleotides (ASOs) that compete for their target sites in mRNAs (blockmiRs), or directly bind to mature miRNAs (antimiR/antagomiRs)25. However, in addition to the challenges inherent to any oligonucleotide therapeutic technology , several drawbacks to current miRNA modulation approaches remain32. In principle, such ‘gapmer’ ASOs – oligonucleotides partially modified with synthetic nucleic acid analogues33 – recruit host cell RNase H, leading to cleavage and enhanced degradation of RNA targets28. However, this mechanism is inefficient for mature miRNA targets; antimiR ASOs are thought to principally act by steric blocking and/or sequestration of miRNAs, or inhibition of miRNA processing, rather than induction of RNase H-mediated knockdown37. Indeed, antimiRs have been shown not to reduce the levels of mature miRNAs39. AntimiRs can thus have limited capacity for multiple-turnover, requiring stoichiometric doses for efficacy. Furthermore, as sufficient basepairing with miRNA targets is necessary for inhibition, antimiR function is typically enhanced by ribose modifications that enable invasion of RNA secondary structure, but which can increase off-target binding and cytotoxicity.Current approaches to therapeutic modulation of miRNAs involve small molecule inhibitors or modulators of miRNA processing41. However, whilst targeting multiple related miRNAs or miRNA binding sites may be appropriate in some circumstances, the increased risk of off-target effects by such strategies remains a key challenge for miRNA therapeutics15. Furthermore, the incidence of genetic diseases involving loss of miRNA clusters  demonstrates that blanket inactivation of multiple miRNAs can have a range of deleterious consequences. Alternatively, molecular tools that target individual miRNAs would offer a more precise approach to target single ‘nodes’ in densely interconnected regulatory networks.Typical miRNA-inhibiting approaches involve targeting ‘seed regions’, sequence motifs crucial to mRNA binding shared by miRNA family members49 – and oligo-peptide conjugates52, has been suggested. In principle, such ‘antimiRzyme’/‘antagomiRzyme’ catalysts have the potential to be more specific than ASOs due to the combination of substrate-binding arm complementarity and nucleobase requirements at or close to the substrate cleavage site. Furthermore, their capacity to perform enzymatic multiple turnover RNA cleavage independent of host silencing machinery could enable lower dosing and extension of miRNA targeting to settings in which these are unavailable or challenging to recruit, such as extracellular vesicles or exosomes. However, DNAzyme catalysts capable of cleaving all-RNA substrates have proven to be highly dependent on divalent cations for folding, so at physiological concentrations of free Mg2+ (<1 mM) – unless co-transfected with divalent metal co-factors54 – DNAzymes achieve RNA knockdown by triggering ASO-type effects independent of catalytic activity58, limiting their advantages over other approaches. Extended variants of the hammerhead ribozyme with improved activity under low [Mg2+]61 also offer the prospect of anti-miR catalysts48, however, the poor biostability of RNA must be addressed if these are to be developed as exogenously deliverable molecules rather than expressed from virally-delivered transgenes.To this end, targeting miRNAs using oligonucleotide catalysts – ribozymes and DNAzymes62, including 2′-deoxy-2′-fluoro-β-D-arabino nucleic acid (FANA)63, an artificial polymer chemistry capable of stabilising nucleic acid structures and reducing their dependence on Mg2+ for folding, and offering improved biostability compared with RNA64. We recently identified an artificial modular RNA endonuclease, “FR6_1”, comprising a fully-FANA 17 nt catalytic core flanked by substrate-binding arms Fig. . We ther 42 Fig. . One ins 42 Fig. , which s2+, pH 7.4, 37 °C) choice of cleavage site dinucleotide  and/or (3) binding arm complementarity, in particular at positions directly flanking the FR6_1 catalytic core – in miR-20a and miR-20b, 12 C > U mutations Fig.  with a ph−1)Fig.  comparabily Fig. , and theily Fig. , Fz_miR_ily Fig. . These rily Fig. ) of the U^G Fig. , consistons Fig.  are predons Fig.  with a wons Fig. , which w °C)Fig.  with a sh−1)Fig.  as Fz_mih−1)Fig. , with dih−1)Fig. . Encourah−1)Fig. .R”, containing the same core sequence, was previously found to be active on RNA substrate variants bearing an analogous rC, including one with a C^UA motif65.We also attempted to design a variant of Fz_miR_17 to specifically target miR-106a . Although this variant (“Fz_miR_21”) was found to be only modestly active Fig. , and is h−1)Fig.  and capah−1)Fig. .Fig. 2An48  motif previously shown to improve activity in low [Mg2+]67. However, although DNAzyme miR21dz1 was found to be active under high magnesium conditions (10 mM Mg2+), both DNAzyme RNV541 and ribozyme miR21rz1_TLR were found to be inactive , catalytic activity of DNAzymes miR21dz1 and RNV541B was substantially reduced or entirely abolished at quasi-physiological magnesium concentrations (1 mM Mg2+) exhibited a comparable rate of degradation  as RNV541B (~1.5 h) in 50% human serum at 37 °C, whereas miR21dz1 and miR21rz1_TLRB were degraded within minutes or seconds , miR21dz1 (DNA), RNV541B (DNA) 71.Encouraged by the capacity of the FR6_1 XNAzyme to generate catalysts tailored to specific microRNAs, we wondered whether this could be extended to longer, more structured non-coding RNAs (ncRNAs). Y RNAs are a family of highly conserved molecules involved in assembly of RNA-protein complexes with a variety of roles including DNA replication and RNA stability and quality controlobs = 0.04 h−1)   Fig.  than the/4) Fig. , enablin/4) Fig. .Fig. 3An72. In principle, nanostructure designs can improve biostability by sterically hindering access to 3′ termini by serum exonucleases and promote cellular uptake of nucleic acid catalysts73, and could enable synergistic targeting of multiple microRNAs74.Next, we sought to explore whether multiple ncRNA-targeting XNAzymes could be combined into a single multi-functional artificial enzyme. We have previously found that XNAs including FANA can be engineered to self-assemble into simple nanoscale polyhedra73 that presents three variants of the 17E DNAzyme on single-stranded edges of the nanostructure. The 17E DNAzyme sequences in the component strands  were replaced with those of the three anti-miR XNAzymes and the complete sequence of each component strand synthesised as fully-FANA oligonucleotides  – in principle each component strand contains sequences that hybridise to the other strands, enabling self-assembly into the complete 3D catalytic nanostructure (“TFz_miR3”)  Fig. . Examina3”) Fig.  revealed3”) Fig. . However3”) Fig. .Fig. 4A 3 by removal of the majority (~75%) of any remaining monomers or dimers by centrifugal filtration  and longer (Y RNA) ncRNA families. In contrast with analogous DNA and RNA catalysts, whose activity in vitro is typically only measurable under unrealistic conditions (high concentrations of Mg2+ (10–25 mM) and catalyst (100 μM))49, we show that the anti-ncRNA XNAzymes efficiently cleave their targets under quasi-physiological conditions (1 mM Mg2+) and at modest concentrations (0.5 μM). Observations that anti-miR DNAzyme activity is reduced or abolished under physiologically-relevant conditions in vitro49, as we confirm here for two reported anti-miR-21 catalysts, is consistent with an in vivo knockdown mechanism that does not depend upon catalytic turnover. Our finding that the anti-ncRNA XNAzymes we describe significantly outperform such catalysts (at least in vitro) provides a promising starting point for investigation of their activity in vivo, although we note that dissecting the relative contribution of bona fide catalysis and antisense-mediated effects to intracellular activity can be challenging76.Starting from a previously-described RNA endonuclease XNAzyme, FR6_1obs = 0.04 - 0.5 h−1), with generally reduced activity compared with the parental FR6_1 catalyst (kobs = 1.5 h−1)65 under quasi-physiological conditions, these could in principle be further improved by ‘maturation’ selections or mutation screens (as we demonstrate here in the case of the anti-miR-21 XNAzyme). The rates of microRNA cleavage catalysed by XNAzymes nonetheless compare favourably with the rate of mature microRNA turnover in vivo, which can be very slow 77, suggesting that specific cleavage at kobs ≥ 0.04 h−1 ought to be, at least in principle, sufficient to modulate intracellular miRNA levels.Although the catalytic rates of the ncRNA-targeting XNAzymes were found to be relatively modest . Furthermore, FANA, as we have explored using the parental FR6_1 catalyst, likely stabilises intramolecular pairings in the catalytic core, reducing dependency on divalent cations for folding of the catalytically-active tertiary structure. However, high affinity FANA:FANA pairings in the core may also enhance the proportion of misfolded catalytically-inactive XNAzymes, as suggested by the plateauing of pseudo first-order reactions at 60 - 80% cleavage.The FANA chemistry comprising the FR6_1-derived XNAzymes – a DNA analogue whose fluorinated arabinose sugar enables highly stable homo- and heteroduplex formation87 and/or sugar- or backbone-modified chemistries with advantageous biostability and pharmacodynamics88. The use of nucleic acid analogues capable of stabilising secondary structures is an established approach to improve RNA target accessibility in the context of microRNA-targeting agents; 2′-O-methyl-RNA (2′OMe-RNA), 2′-methoxyethyl-RNA (2′MOE-RNA) and locked nucleic acids (LNA) have proven to be of particular utility for antimiR and antagomiR development91 and likewise enhance the activity of nucleic acid catalysts93. Recently, synthetic genetic systems capable of efficient synthesis and reverse transcription of these chemistries have been established95, and the elaboration of RNA-cleaving catalysts demonstrated using fully-2OMe-RNA polymers95. These systems thus enable the chemical structure space of 2′OMe-RNA, 2′MOE-RNA and LNA to be explored for the development of antimiRzyme / antagomiRzyme XNAzymes with enhanced biostability, RNA structure invasion and cleavage activity in physiological conditions. Furthermore, short LNAs are capable of superior target strand mismatch discrimination97, which has been exploited to reduce siRNA off-target effects98, offering a route to XNAzymes with further improvements in substrate specificity. However, the capacity for multiple-turnover catalysis can be limited by extensive modification with such chemistries99, so careful consideration must be given to optimise this in the context of ncRNA target binding.Future elaboration of catalysts could involve modified nucleobases with the potential to evolve multiple catalytic mechanismsOur results extend the range of targets of artificial RNA endonuclease XNAzymes to include disease-associated non-coding RNA and provide a proof-of-concept demonstration, at least in vitro, of their potential utility as highly specific tools for detection or knockdown of individual microRNAs or longer ncRNAs.100 was kindly provided by Philipp Holliger . HEK293 cells were kindly provided by Jake Barker .DNA and RNA (and chimeric DNA/RNA) oligonucleotides were synthesized by Integrated DNA technologies (Belgium) or Sigma-Aldrich, MilliporeSigma (USA). Polymerase D4K101; single-stranded XNAzymes were subsequently purified by capturing (biotinylated) DNA templates using streptavidin magnetic beads  and eluting (unbiotinylated) XNAzymes using 0.1 N sodium hydroxide, followed by removal of the drP2_Ebo DNA/RNA primer by hydrolysis by incubation in 0.7 N NaOH for 1 hr at 65 °C.FANA XNAzymes were synthesized enzymatically, using DNA/RNA primer “drP2_Ebo”, 3′ biotinylated DNA templates  by incubation at 80 °C for 60 s, then cooled to room temperature for 5 min.Partial alkaline hydrolysis of RNA substrates for use as electrophoresis size standards was performed by incubation at 65 °C in 20 mM sodium hydroxide (pH 12) for 4 min, then neutralized with 1 M Tris pH 7.2 at 37 °C. Cells were detached with StemPro Accutase , resuspended in fresh DMEM, washed twice with PBS, once with ‘Mg-free cell wash buffer’  and the supernatant removed. Cell pellets were lysed by 6 cycles of freeze-thawing in a dry ice ethanol bath and lysate filtered through 0.2 μm Spin-X filtration columns . Lysates were heated at 95 °C for 10 min to inactivate RNases.HEK293T cells were grown in Dulbecco’s Modified Eagle Medium (DMEM)  supplemented with 10% foetal calf serum (FCS)  and 100 U/ml penicillin G, 0.1 mg/mL streptomycin and 2 mM L-glutamine  in 5% CO2) at 37 °C. Reactions were stopped by addition of PAGE gel loading buffer  and analysed by Urea-PAGE using 20% acrylamide gels. Gels containing fluorophore-labelled RNA substrates and 5’ RNA products were imaged without staining using an FLA-5000 scanner ; unlabelled RNA substrates and products were first stained using SYBR Gold stain . Bands were quantified using Fiji102 to calculate proportion of RNA cleaved for each XNAzyme reaction. For SYBR Gold stained RNA products, a coefficient to account for differential staining proportional to oligo length was applied to product band densities. Pseudo first-order reaction rates (kobs) under single-turnover pre-steady-state (Km/kcat) conditions were determined from time courses; samples were taken and reactions stopped at appropriate intervals by snap-freezing on dry ice in excess PAGE gel loading buffer. Quantification data from three independent replicates per time course were fit using Prism 9 (GraphPad), as described previously62.PAGE-purified XNAzymes and RNA substrates were annealed separately as described above and reacted in ‘quasi-physiological buffer’ , “TFz_miR_17”, “TFz_miR_20a” and “TFz_miR_21B”, were prepared from appropriate DNA templates  at 37 °C in 50% human serum  for 48 h. Samples were taken and reactions stopped at appropriate intervals by snap-freezing on dry ice in excess PAGE gel loading buffer. The proportion of undegraded full-length catalyst was determined by Urea-PAGE; gels were stained with SYBR Gold  and imaged as described above.PAGE-purified TFz_miRAll XNAzyme reactions were replicated at least three times using different batches of FANA. Statistical analyses of kinetics experiments were performed on three independent replicates, defined as separate reactions using separately annealed catalysts and substrates.Further information on experimental design is available in the Supplementary InformationDescription of Additional Supplementary DataSupplementary Data 1Reporting Summary-New


Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the COVID-19 pandemic, whereas the influenza A virus (IAV) causes seasonal epidemics and occasional pandemics. Both viruses lead to widespread infection and death. SARS-CoV-2 and the influenza virus are RNA viruses. The SARS-CoV-2 genome is an approximately 30 kb, positive sense, 5′ capped single-stranded RNA molecule. The influenza A virus genome possesses eight single-stranded negative-sense segments. The RNA secondary structure in the untranslated and coding regions is crucial in the viral replication cycle. The secondary structure within the RNA of SARS-CoV-2 and the influenza virus has been intensively studied. Because the whole of the SARS-CoV-2 and influenza virus replication cycles are dependent on RNA with no DNA intermediate, the RNA is a natural and promising target for the development of inhibitors. There are a lot of RNA-targeting strategies for regulating pathogenic RNA, such as small interfering RNA for RNA interference, antisense oligonucleotides, catalytic nucleic acids, and small molecules. In this review, we summarized the knowledge about the inhibition of SARS-CoV-2 and influenza A virus propagation by targeting their RNA secondary structure. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19). On 11 March 2020, the World Health Organization (WHO) declared COVID-19 a global pandemic that was responsible for a lot of infections and deaths all over the world. Moreover, plenty of people struggled with disruptions to health services, travel, trade, and education; in addition, COVID-19 had a negative impact on people’s physical and mental health . In the The SARS-CoV-2 and influenza viruses are RNA viruses ,11. SARSThe RNA secondary structure in the untranslated and coding regions is crucial in the viral replication cycle . It is cFor the influenza A virus (IAV), there are predicted RNA structural motifs ,33,34,35Both the SARS-CoV-2 and the influenza A viruses attack the respiratory tract, cause similar symptoms, and provoke epidemics and pandemics, and it happens that the infections occur simultaneously . FurtherSARS-CoV-2 is a typical member of the betacoronavirus family with a positive-sense, single-stranded RNA that includes a 5′ cap structure and a 3′ poly(A) tail . The genThe secondary structure of 5′ and 3′UTR of SARS-CoV-2 is essential for virus propagation. The model of the 5ʹ-UTR, based on inline probing and enzymatic probing by RNase V1, was determined. This model is highly structured with plenty of accessible loops and bulges that fold into six hairpins, named SL1, SL2, SL3, SL4, SL4.5, and SL5  Figure . All theThe most variable hairpin among the SARS-CoV-2 variants is SL1 , which gThe 3′ UTR of the SARS-CoV-2 RNA contains various domains critical for the regulation of viral RNA synthesis and potential translation . The stuThree main secondary structures have been identified in the 3′ UTR: the bulged stem-loop (BSL), the SL1 loop, and the highly variable region (HVR) . The resNext, a well-known structural RNA motif of SARS-CoV-2 is the frameshifting stimulation element (FSE) that occurs in ORF1ab, which comprises about two-thirds of the coronavirus genome ,73. ORF1RNA interference is a cellular mechanism in which RNA is used to suppress gene expression  Figure . The silExogenous siRNAs are proven to be effective in regulating genes in different molecular studies and novel therapies. SiRNA molecules were also designed to inhibit viral infection by targeting and degrading viral RNA . SeveralTo develop an anti-SARS-CoV-2 strategy using siRNA, Idris et al. designed 18 siRNAs targeting 5′UTR, RNA-dependent RNA polymerase (RdRp), and Helicase (Hel) genes . After sNiktab et al. designed six siRNAs specific to the SARS-CoV-2 mRNAs that targeted the membrane protein (M), an envelope protein (E), the spike protein (S), and the orf3a and orf1ab genes . Before ASOs are single-stranded, chemically synthesized, short DNA or RNA that usually consist of 12–30 nucleotides . ASOs can modify the function of mRNA or other RNA, degrade RNA, or block gene expressions by complementary hybridization to an RNA target . TherefoAntisense oligonucleotides are chemically modified to reduce toxicity and to increase stability and protection from degrading agents. There are three generations of ASOs. The first generation of ASOs consists of phosphorothioate (PS), phosphoramidate (PA), and methyl phosphonate (MP) oligonucleotides. The first generation of ASOs is characterized by replacing the oxygen atoms from the phosphodiester bond with the sulfur, amine, or methyl group. This group of chemical modifications enhances ASOs cellular stability by making antisense oligonucleotides more resistant to nuclease and improves their membrane penetration .The 2′-O-methyl (OMe) or 2′-O-methoxyethyl (MOE) oligonucleotides belong to the second generation of ASOs, which have a higher binding affinity for the target than PS. The antisense mechanism of second-generation ASOs involves the steric blockade of translation when targeting mRNA or other essential functions of targeted RNA. At the same time, recruitment and cleavage by RNAse H are impossible . Finally, the three that are the most studied in the third-generation of ASOs are the locked nucleic acids (LNAs), peptide nucleic acids (PNA), and phorodiamidate morpholino oligomers (PMO). LNA possesses a methylene bridge connecting the 2′-oxygen with the 4′-carbon of the ribose. In PNA, the ribose-phosphate DNA backbone is replaced by the peptide-like N-(2-aminoethyl) glycine-linked nitrogenous bases of nucleic acids via a methyl carbonyl linker. PMO has the phosphodiester bond replaced by a phosphorodiamidate bond and the ribose by a morpholine moiety. With these chemical modifications, the antisense oligonucleotides form a stable hybrid with the target RNA. In addition to the main representatives of the modified ASOs, many laboratories have been working on new modifications, as well as different modifications that are used in one ASO ,108.Zhu et al. designed gapmers with 3–4 LNA modifications at the 5′ and 3′ ends and a DNA-PS window of 9–10 nucleotides . The ASOLi et al. designed and synthesized antisense PMOs to find potential candidates for antisense therapeutics affecting SARS-CoV-2 replication . The PMORosenke et al. investigated the anti-SARS-CoV-2 effect of the peptide-conjugated morpholino oligomers (PPMO) . They deLulla et al. designed six LNA ASOs against the highly conserved structured s2m element from the 3′UTR of SARS-CoV-2 . MoreoveAdditionally, the Vero E6 cells were transfected with gapmers in three concentrations and infected by the SARS-CoV-2 virus. The high content screening (HCS) assay has demonstrated that gapmers 2 and 5 inhibited the SARS-CoV-2 replication the most, in a dose-dependent manner. In addition, at the highest tested concentration, these gapmers reduced virus replication by about 10% compared to the control .Sun et al. used the in vivo click selective 2-hydroxyl acylation and profiling experiment (icSHAPE) technology to determine the secondary structure of the SARS-CoV-2 genome and, based on this finding, to design antivirals.  The cellZhang et al. reported a 3D structure of SARS-CoV-2 FSE RNA by single-particle cryo-EM and tested the inhibition efficiency of several ASOs . Before Effective LNA-modified oligonucleotides were designed based on a careful selection of the structural motif of the viral RNA of SARS-CoV-2 as a target. Two ASOs, LNA-12.8 and LNA-14.3, targeting the RNA motif of N and 5′UTR/ORF1ab, respectively, significantly reduced the viral titer of SARS-CoV-2. The experiments conducted on Syrian hamsters showed that LNA-12.8 prevents or mitigates SARS-CoV-2 transmission, diminishing virus titer in the lungs .Small molecules  are organic compounds that can bind tightly to specific structural motifs of RNAs and proteins due to hydrogen bonding, stacking, and electrostatic and hydrophobic interactions and should meet the Lipinski rules . SMs canAlthough many scientists, especially those who determined or analyzed the RNA secondary structure of SARS-CoV-2, know that RNA motifs could be an excellent target for specific SMs and could lead to the development of a successful drug, there are not many published works on this subject so far ,22,64. NHanif et al. presented an approach to designing small molecules that target an attenuator hairpin (AH) of FSE within the genomic SARS-CoV-2 RNA . The FSESreeramulu et al. selected 15 structural motifs of SARS-CoV-2 genomic RNA that are thermodynamically stable in vitro, exist in vivo and ex vivo, and are conserved between SARS-CoV and SARS-CoV-2 . The autZafferani et al. published dimethylamiloride (DMA) analogs targeting conserved motifs at the 5′-end of the SARS-CoV-2 genome and lowering the virus titer in a dose-dependent fashion in cells . The SMsAn interesting approach was proposed by Zhao et al., searching for possible G-quadruplexes (G4) in SARS-CoV-2 RNA as targets for SMs . Based o50 of 2.6 μM and an EC90 of 12 μM  promoted by an RNA pseudoknot and in such a way disturbed the translation of the open reading frame 1b (ORF1b) . Eight iof 12 μM . The autThe inhibition of SARS-CoV-2 replication by merafloxacin and a small molecule MTDB was shown as a result of searching for the pseudoknot binders as potential therapeutics . From thThe reporter system of SARS-CoV-2 −1 PRF, based on FLuc, was introduced by Chen et al. and used in the high-throughput screening of FDA-approved drugs . The selOther methods of inhibiting viral replication targeting RNA include, for example, the application of antisense PNA. PNA oligomers are single-stranded oligonucleotide analogs in which the sugar phosphate backbone is replaced by an N-aminoethylglycine-based polyamide unit that makes it uncharged. PNA binds to complementary RNA or DNA, forming high-affinity duplexes, or it can also form a triplex. In addition, an amide-based PNA backbone is more chemically, thermally, and enzymatically stable ,111.Li et al. designed and synthesized eight long PNAs covalently attached with cell-penetrating peptides (PPNAs) targeting the 5′UTR, 5′UTR TRS, and the polyprotein 1a/b translation start site (AUG) of the SARS-CoV-2 genome . SubsequSu et al. designed several ASO chimeras that induced the inhibition of SARS-CoV-2 replication . The chiInfluenza virus type A belongs to the Orthomyxoviridae family. The genome is constructed of RNA divided into eight segments that encode viral proteins: hemagglutinin (HA) and neuraminidase (NA); nucleoprotein (NP); two matrix proteins ; the polymerase complex proteins, PB1, PB2, and PA; and four non-structural proteins. About 80% of the virus proteins are hemagglutinin and neuraminidase. Hemagglutinin is the protein that allows the virus to enter the host’s epithelial cells. Neuraminidase cleaves sialic acid residues, which allows the newly formed virions to be released ,112,113.The IAV promoter is never free in the life cycle of the influenza virus and is crucial in two mechanisms, transcription and replication . The proThe secondary structure of (+) RNA plays a significant role in alternative splicing. Based on sequence alignment and chemical mapping experiments, a secondary structure was proposed for the 3′ (+) RNA part of segment 7 (vRNA7). Segment 7 encodes the M1 protein and the smaller M2 protein. The 3′ pre-mRNA region of segment 7 IAV can adopt two different structures: the pseudoknot and the hairpin. It has been proposed that this conformational change in structure may play a role in alternative splicing and affect the production of the M1 and M2 proteins ,121,122.Using bioinformatics tools, Kobayashi et al. also analyzed the structure of segment 7 of IAV RNA in the (+) and (−) strands . They coJiang et al. examined the conserved RNA domain in the intron of influenza virus segment 8 (+) RNA, which encodes the NS1 protein and NEP protein—through alternative splicing . Based oThe correlation between the function and the structure of the influenza virus RNA has been intensively studied for many years. The RNA secondary structures have been studied in vitro for segment 5: vRNA5 (strain A/Vietnam/1203/2004 (H5N1)), strain A/California/04/2009 (H1N1)), (+) RNA5 ); segment 7: vRNA7 (strain A/Vietnam/1203/2004 (H5N1)); and segment 8: vRNA8 (strain A/Vietnam/1203/2004 (H5N1), strain A/California/04/2009 (H1N1)) ,43,44,45The secondary structure and structural motif analysis of the naked segment 7 vRNA of strain A/Vietnam/1203/2004 (H5N1) was proposed by Ruszkowska et al. . The vRNLenartowicz et al. used a chemical mapping method combined with thermodynamics-based prediction and sequence/structure comparison to create a structural model of segment 8 vRNA (strain A/Vietnam/1203/2004) . The secDetailed analysis of the secondary structure of the viral RNA and information on the RNA interactions necessary for the viral replication cycle provide an understanding of the structure–function relationships and can help to predict regions of the influenza virus RNA to be considered in the design and development of potential antiviral tools.The group of Zhang et al. investigated three siRNAs that targeted the polymerase (PA) gene of the avian influenza virus (A/Tiger/HarBin/01/2002 H5N1) for their ability to inhibit the replication of this virus . In crysAn interesting study was conducted by Piasecka et al., who designed siRNAs targeting structural motifs of segment 5 mRNA of influenza virus (coding NP) strain A/California/04/2009 (H1N1) . AvailabJiang et al. used a novel approach to inhibit viral gene expression, protein synthesis, and the production of new virions . The groIn 2008, the group of Duan et al. designed and tested an antisense oligonucleotide (IV-AS) that was specific for a conserved 5′ end sequence found in all eight segments of influenza A virus viral RNA . The effTo inhibit viral proliferation, Lenartowicz et al. designed antisense oligonucleotides to target the internal regions of the viral RNA segment 8 (vRNA8) of influenza A/California/04/2009 (H1N1) . PreviouDetermining the secondary structure preceding the design of sites available for the ASO is also essential for (+) RNA. Soszynska-Jozwiak et al. determined the secondary structure of segment 5 of (+) RNA of the influenza virus . ChemicaThe influenza virus mutates rapidly and can develop resistance to antiviral drugs. The group of Hagey et al. characterized the loop-stalk structure of PSL2 RNA, which serves as a packaging signal for the PB2 segment . First, 50 and EC50. The results showed that DPQ was non-toxic to cells at concentrations up to 500 µM. It inhibited the replication of two different strains of influenza A and one type B, but at a lower degree than amantadine, oseltamivir, or ribavirin. An additional assay as a plaque reduction assay showed that 56 to 500 µM of DPQ inhibited virus growth. The experiments with a modified virus, WSN-Ren luciferase, in modified MDCK-HA cells confirmed the potential of DPQ  as well as two more selected compounds as influenza inhibitors. Taking together the collected observation, the DPQ binding changes the panhandle structure and such a method could interfere with the binding of RdRp and consequently disturb replication [So far, few attempts have found SMs binding to influenza RNA as future potential therapeutics. An elegant approach was proposed by Varani, Pellecchia, and coauthors in two of their papers ,103. Thelication .50, CC50, IC50/CC50, and Kd values using cytotoxicity assays, the WSN-Ren luciferase/MDCK-HA assay, real-time PCR, and NMR spectroscopy. Compounds 7 and 10  of influenza virus strain A/California/04/2009 (H1N1) . The varA different approach was taken by Peng et al., who studied approximately 300 miRNAs derived from human and mouse epithelial cells . They anThe development of bioinformatic and experimental methods for determining the RNA secondary structure allows for the creation of effective methods of inhibiting the multiplication of the SARS-CoV-2 and influenza A viruses. To date, the presence of conserved RNA structural motifs in SARS-CoV-2 and IAV have been identified. ASOs, siRNAs, SMs, miRNAs, and other tools were tested and effectively inhibited SARS-CoV-2 and influenza A virus propagation in cells, and some of them were tested in mice ,139. SimA direct and fair comparison between published inhibitors is not possible because the authors used different systems for validations  in different conditions. However, some general tendencies can be seen and may be a typical feature of a given strategy. Oligonucleotide tools (ASO and siRNA) were successful in a low dosage, even a nanomolar range (the best was 8–50 nM) compared to small molecules, which were used in the µmolar range (the best was 20–50 µM) to achieve maximal effect in cells. In the case of ASO, the design by the authors of the modification of the parent ASO did not always improve it. Clearly, in the field of ASO design a systematic knowledge of the influence of different modifications is lacking. The effectiveness of oligonucleotide is also very dependent on the precise target site, and step-by step screening of some regions gave a better chance of finding the best inhibitors. The discovery of inhibitory SM is mostly based on different types of screening. SM could be specific but proving its non-toxicity and the identification of binding sites are usually very challenging. In addition to target RNA, SM could theoretically bind to different nucleic acids and proteins and such phenomena need to be excluded. The most advanced method of SM binding site determination required its chemical modification, which could diminish SM performance. Despite such difficulties, small molecules remain promising RNA-targeting tools.All kind of inhibitors need to be successfully delivered. Different types of delivery of oligonucleotides were used in the treatment of IAV and the SARS-CoV-2 infection and the knowledge in this field is growing. The delivery of small molecules is not as universal as for nucleic acids, but on the other hand, small molecules do not usually need additional vehicles for cells penetration. One of the problems is solubility in water, and very often, a toxic DMSO needs to be added. The other difficulty when testing SM is confirmation of the proper distribution in the cells. The good news is that in the case of future COVID-19 or flu treatment a simple intranasal delivery of both the oligonucleotide and the small molecule inhibitor was successful in mice.In the usually proposed pathway of inhibitor design and validation, the jump from in vitro to in cells and further in mouse and human research often eliminates promising compounds. On the other hand, it is important to exclude in simpler systems candidates that not working in the designed way. Moreover, it is essential in modern medicine to know a drug’s mechanism of action. Therefore, the research presenting inhibitor specificity for selected targets and proving the method of activity is very valuable. Chimeras of different tools were also proposed against IAV and SARS-CoV-2, but such compounds are complicated and rarely fruitful. From the successful stories of oligonucleotides or small molecule drugs, we can learn that a relatively simple compound can be a good drug when the target, rational modifications, and delivery are all considered and optimally used. The perfect drug needs to be specific, stable in the cellular environment, effectively delivered, active in a low dosage (to minimize possible side effects and reduce price) with no or minimal off-target effect. Additionally, in case of the respiratory tract viruses, a relatively high inhibition is required to obtain a substantially reduced viral titer. Only in that case can an improvement in the clinical condition of the patients during a rapid course of illness be achieved.Because RNA viruses are characterized by high mutation rates , there iThe increased concentration of microRNA-2392 during COVID-19 may be used as an effective biomarker. Therefore, McCollum et al. developed Nanoligomer SBCoV207 with a specific PNA component designed to target microRNA-2392 to treat COVID-19. To test viral replication inhibition with this tool, the African green monkey’s kidney epithelial cells (Vero E6 cells) and human lung epithelial cells expressing human ACE2 (A549-hACE2) were infected with SARS-CoV-2 and treated with Nanoligomer SBCoV207. The viral replication inhibition was measured by immunofluorescence assay for the SARS-CoV-2 nucleocapsid protein (N) and showed a significant reduction in virus titer compared with the control. Moreover, the viral mRNA of TRS-N was measured by qRT-PCR and indicated that the viral mRNA abundance was reduced approximately 10-fold . Targeti