Compared with the research on DNA damage, there are fewer studies on RNA damage, and the damage mechanism remains mostly unknown. Recent studies have shown that RNA is more vulnerable to damage than DNA when the cells are exposed to endogenous and exogenous insults. RNA injury may participate in a variety of disease occurrence and development. RNA not only has important catalytic functions and other housekeeping functions, it also plays a decisive role in the translation of genetic information and protein biosynthesis. Various kinds of stressors, such as ultraviolet, reactive oxygen species and nitrogen, can cause damage to RNA. It may involve in the development and progression of diseases. In this review, we focused on the relationship between the RNA damage and disease as well as the research progress on the mechanism of RNA damage, which is of great significance for the pathogenesis, diagnosis, and treatment of related diseases. As the main component of genetic material, DNA damage and repair mechanism has been paid close attention by researchers. However, RNA damage has not been paid enough attention resulting in an underestimated toxic effect of RNA damage on cells and the body. In a cell, RNAs are more abundant than DNAs, accounting for 80–90% of the cell’s total nucleic acid. Antisense RNA can bind to the target site by complementing the target site sequence, or directly prevent its function or change the conformation of the target site to affect its function. DNA post-transcriptional processing and modification usually involve some special RNA. RNA plays an important role in biological evolution. The discovery of ribozyme indicates that RNA is both an information molecule and a functional molecule, and RNA may be the first to appear in the early origin of life . Studiesin vivo , reactive oxygen species (ROS) and reactive nitrogen species (RNS), can cause damage to RNAs . RNA damin vivo . Ultraviage RNAs .At present, although there is an increasing number of studies on oxidative damage of RNA, the mechanism is still unclear. The purpose of this review was to describe the relationship between the RNA damage and disease as well as the research progress on the mechanism of RNA damage, which is of great significance for the pathogenesis, diagnosis, and treatment of related diseases.E. coli and homologous molecule hABH3 in mammals can reverse the damage by direct hydroxylation of methyl groups on DNA or RNA bases damaged by methylation , human cell y-box binding protein 1 (Yb-1) and heterogeneous nuclear ribonucleoprotein (hnRNP) in human cells, which may lead to the silencing of damaged RNA and thus terminate its translation process after binding to damaged RNA (2O2 stimulation was enhanced (8-OHG has been the focus of studies because it appears to be mutagenic . For exaaged RNA . hPNPaseaged RNA . Yb-1 isaged RNA . After ienhanced .Some studies have shown that capillary dilated ataxia mutant protein (ATM), may be involved in RNA damage stress response. ATM is an important DNA damage stress molecule, and the autophosphorylation at serine is a marker of its activation. The activity and signaling pathway of ATM are disrupted after the mutation of the self-phosphorylation site . Recent Unlike AUF1, which recognizes an 8-OHG RNA, PCBP1 recognizes heavily oxidized RNA. This factor does not promote the degradation of target cells but induces cell death. A mutation occurs in one of the two RNA binding zinc finger domain and hnRNPK-homology domain (KH) domains cancel the binding of 8-OHG and inhibit the induced RNA binding activity of apoptosis-related reactions, which is necessary to induce programmed cell death. These observations underscore the ability of cells to use damaged mRNA as a signal of stress. Moreover, this PCBP1-mediated response is activated only in the presence of a large amount of modified RNAs , which aThe damage of genomic coding RNAs can affect gene expression and cause the disorder of proteome expression. About 98% of gene transcripts are non-coding RNAs. Studies have shown that non-coding RNAs have structural functions and catalytic activities. In recent years, a large number of non-coding RNAs have been discovered and identified in the central nervous system. Therefore, the physiological dysfunction caused by RNA damage has a close and complex relationship with the pathophysiology of neurons. Recently, more and more experiments have proved that oxidative RNA damage is related to the pathogenesis of neurodegenerative diseases. Obvious oxidative RNA damage can be detected in vulnerable neurons in the early stage of neurodegenerative disease, indicating that RNA oxidation may contribute to the occurrence and development of the disease . In addiStudies found that patients at the early and middle phase of Alzheimer’s disease  showed up to 50% of mRNA in their frontal cortex had oxidative damage, but in the control group mRNA damage degree is lower than 2%, and further combining with DNA chip analysis found that many mRNAs are associated with the AD . It is wIn addition, several studies also detected a significant increase in RNA oxidative damage in Parkinson’s disease (PD) . ElevateAmyotrophic lateral sclerosis (ALS) is one of the most devastating neurological disorders . Study sIt has also been reported that the intracellular contents of 18 s and 28 s rRNA in atherosclerotic patients decrease with the increase of 8-OHG . OxidatiRecent studies have shown that levels of RNA oxidative damage in the urine of patients with type 2 diabetes can predict long-term mortality. The urinary excretion of the RNA oxidation marker 8-OHG measured shortly after diagnosis of type 2 diabetes predicts the long-term mortality independently of other risk factors. Besides, oxidative damage to RNA may also be related to the development of cancers. Compared to normal cells, cancer cells exhibit an accelerated metabolism and demand high ROS concentrations to maintain their high proliferation rate. ROS is of high significance in cancer treatment and it influences tumor outcome . Recent Investigated RNA oxidation after spinal cord contusion injury in rats. There are findings a significant increase in RNA oxidation in the lesion center immediately after the injury and the level remains high over 3 h, while in the cord segment rostral to the lesion center, RNA oxidation did not increase significantly until 3 h after the injury. Increased RNA oxidation primarily occurs in the neurons around the lesion center at an early stage and in the oligodendrocytes during secondary injury progression, and these cells die later. These results indicate a spreading pattern of the RNA oxidation from the lesion site to the distal areas, and this coincides with the developmental pattern of the secondary injury. These results suggest that RNA oxidation may play an important role in the development of secondary injury .As a common chronic neurological disorder, epilepsy is characterized by recurrent unprovoked seizures. It afflicts more than 50 million people worldwide. Studies have demonstrated that seizure-induced mitochondrial dysfunction and excess free radical production cause oxidative damage to cellular components and initiate the mitochondrial apoptotic pathway , 2002.Oxidatively generated damage to nucleic acids is considered to play a significant role in carcinogenesis, and it has been shown that people with hereditary hemochromatosis are at increased risk of cancer. In this study, they used a new refined liquid chromatography-tandem mass spectrometry method to measure the level of 8-oxo-7 and 8-OHG in the urine of hereditary hemochromatosis patients, and they investigated the effect of treatment on the levels of these modifications. The study was carried out as a classical case-control study of 21 newly diagnosed, never treated hereditary hemochromatosis patients and 21 matched controls. They found that the levels of 8-OHG in the urine of the experimental group were much higher than those of the control group, with a similar level of 8-oxo-7, which returned to normal after treatment . 8-OHG iAt present, the research on the mechanisms of RNA damage oxidation is still scarce. RNA oxidative damage is involved in the pathogenesis of various pathological diseases, such as ischemia, cancer, obesity, diabetes, viral infections, chronic fatigue syndrome, amyotrophic lateral sclerosis (ALS), depression, kidney disease, heart failure, and atherosclerosis. RNA oxidation is a marker of many neurodegenerative diseases as well as an indicator of an increase in oxidative stress which can lead to an exacerbation of diseases. However, the molecular mechanism of RNA oxidative damage in these diseases remains to be explored, the direct cause-and-effect relationship between oxidized RNA and disease pathogenicity is not clear . AlthougZhL, LA, and PL discussed the structure and content of the review. ZhL wrote the manuscript. LA and PL revised the manuscript. XC and ZiL helped to revise the manuscript. WY, LL, LQ, and HD did the final editing. 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.


This increased propensity to mineralize was associated with an increased alkaline phosphatase (Alp) expression (measured by qRT-PCR and activity assay) and an enhanced secretion of the pro-mineralizing IL-6 cytokine compared to control wild-type cells (measured by ELISA). Accordingly, addition of an anti-IL-6 receptor antibody to CD11b KO chondrocytes reduced significantly the calcification and identified IL-6 as a pro-mineralizing factor in these cells. In the same conditions, the ratio of qRT-PCR expression of collagen X over collagen II, and that of Runx2 over Sox9 (both ratio being indexes of chondrocyte hypertrophy) were increased in CD11b-deficient cells. Conversely, the CD11b activator LA1 reduced chondrocyte mineralization, Alp expression, IL-6 production and collagen X expression. In the meniscectomy (MNX) model of murine knee osteoarthritis, deficiency of CD11b led to more severe OA . In conclusion, these data demonstrate that CD11b signaling prevents chondrocyte hypertrophy and chondrocyte mineralization in vitro and has a protective role in models of OA in vivo.Osteoarthritis (OA) is a progressive joint disease that is strongly associated with calcium-containing crystal formation  by chondrocytes leading ultimately to cartilage calcification. However, this calcification process is poorly understood and treatments targeting the underlying disease mechanisms are lacking. The CD11b/CD18 integrin (Mac-1 or αMβ2), a member of the beta 2 integrin family of adhesion receptors, is critically involved in the development of several inflammatory diseases, including rheumatoid arthritis and systemic lupus erythematosus. We found that in a collagen-induced arthritis, CD11b-deficient mice exhibited increased cartilage degradation compared to WT control animals. However, the functional significance of CD11b integrin signaling in the pathophysiology of chondrocytes remains unknown. CD11b expression was found in the extracellular matrix and in chondrocytes in both healthy and damaged human and murine articular cartilage. Primary murine CD11b KO chondrocytes showed increased mineralization when induced  Osteoarthritis (OA) is a long-term chronic degenerative and disabling joint disease  for whicOA is characterized by progressive loss of articular cartilage, osteophyte formation, synovial inflammation and calcification in joint structures such as cartilage . CalciumCell-surface receptors, such as integrins, mediate interaction between chondrocytes and the extracellular matrix (ECM) components, orchestrating its physiological turn-over. Integrins are composed of different combinations of α- and β-subunits, most of which have been found expressed in chondrocytes. In joint physiology, integrins have been demonstrated to play key roles in chondrogenic differentiation  and chonMβ2) is a member of the beta 2 integrin family of adhesion receptors  that is able to measure the time for spontaneous formation of crystalline calcium phosphate deposits. It has also been revealed that CD11b is involved in MyD88/TRIF pathway in SLE, by decreasing IFN-α,β and NF-κB transcribed cytokines, namely interleukin-6 (IL-6)  , which c6 (IL-6) . An addi6 (IL-6) .However, the role of CD11b integrin in calcification has not yet been examined. Based on these observations, we hypothesized that CD11b integrin signaling might be involved in the process of cartilage calcification. Here, we found that both murine and human chondrocytes expressed CD11b integrin. Furthermore, our results indicate that CD11b deficiency in chondrocytes disrupted articular cartilage homeostasis by enhancing chondrocytes calcification, via increased IL-6 production and a switch toward hypertrophic cell differentiation, overall leading to cartilage degradation. Finally, our results identified a newly CD11b-dependent pathway that may be a target in the future development of a successful therapeutic approach in OA.4 cells/cm2 for 7 days in complete DMEM , 1% Penicillin Streptomycin). Medium was changed every 2–3 days. Cells were detached with trypsin-EDTA (Amimed) and counted for stimulation experiments, all performed in complete DMEM medium, with LA1 20uM (ChemBridge) dissolved in dimethyl sulfoxide (DMSO) or equivalent quantity of the vehicle as control, added 30 min before the mineralization experiment. Chondrocytes were stimulated with secondary calciprotein particles (CPP) for 4, 6, or 24 h, or in BGjb medium  with 50 μg/mL ascorbic acid and 20 mM β-glycerol phosphate for 14 days. Cell monolayers were stained with Mmp13 rabbit polyclonal antibody (Abcam 39012) and quantified by ImageJ as percentage of positively stained cells over total cells in each field. Chondrocytes were stimulated with 10 μg/ml rat anti-mouse IL-6R  monoclonal antibody or irrelevant IgG2b antibody control.Articular chondrocytes were isolated from C57BL/6J and CD11b KO mice as described previously . Cells wCPP were generated by preparing 20 ml DMEM containing 10% FBS, 1 mmol/L CaCl2, 3.5 mmol/L inorganic phosphate (1.36 mmol/L NaH2PO4 and 2.14 mmol/L Na2HPO4) . After 7Measurement of lactate dehydrogenase (LDH) as a means of cytotoxicity assay was performed with using CytoTox-ONE Homogeneous Membrane Integrity Assay (Promega) according to the manufacturer’s instructions. Values were calculated as percentage of cytotoxicity using the following formula: LDH release (%) = [ — (background)]/[ — (background)] x100.® Photoshop® after image binarization and related pixel counts. Calcium content was quantified by the QuantiChromTM Calcium Kit (BioAssay Systems) by reading absorbance at 612 nm using the Spectramax M5e reader (Molecular Devices).Chondrocytes were treated with CPP  for 24 h or calcifying medium for 24 h, then washed in PBS and fixed in 10% formaldehyde. Crystals were quantified with Alizarin Red staining with AdobeSupernatants were collected from stimulation experiments, and assayed using murine or human IL-6 and murine MCP-1 ELISA kits (eBioscience) following the manufacturer’s protocol. Results were read at 450 nm by Spectramax M5e (Molecular devices). Synovial fluid was obtained from 24 OA patients at time of joint replacement (average age 69 years old ± 10), and CD11b evaluated in these biological samples by human CD11b ELISA .TM). Quantitative Real Time PCR (qRT-PCR) was performed with gene specific primers (® 480 system (Roche Applied Science). Normalization was performed against Gapdh reference gene, and fold increase of transcripts was calculated against control cells.Cells were treated with TRIzol (500 μl for 1 million cells) and RNA was extracted (RNA Clean and Concentrator 5-Zymoresearch) and reverse transcribed (Superscript II- Invitrogen primers  using thCells were lysed in 0.01% sodium dodecyl sulfate (SDS) and alkaline phosphatase (Alp) activity was measured by use of p-Nitrophenyl Phosphate assay . Lysate was read at 405 nm by spectrophotometric measurement (Spectramax M5e plate reader).®X Mark II Imaging Flow Cytometer  using INSPIRE software. For data evaluation, IDEAS software version 6.0 was used.Flow cytometry was used to assess CD11b expression on chondrocytes. Cells were harvested from confluent cultures by treatment with a non-enzymatic cell dissociation buffer . Cells were re-suspended in fluorescence-activated cell sorting (FACS) buffer  and incubated with PE anti-CD11b , FITC anti-CD45  and FITC anti-F4/80  for 30 min at 4°C in the dark. For intracellular staining, cells were permeabilized with Cytofix/Cytoperm solution (BD Biosciences) for 20 min and then incubated with biotin anti-Collagen II  followed by a streptavidin APC conjugate antibody. Flow cytometric analyses were performed on LSRII cytometer using FACS Diva6 (Becton Dickinson) and FlowJoX software for data processing. The Imaging flow cytometry was carried out with the ImageStreamTM 670 (Invitrogen).Proliferation rate of WT and CD11b KO chondrocyte were analyzed by FACS after 3 days of culture by using Cell Proliferation Dye eFluorad libitum.CD11b KO mice (C57BL/6J background) were provided by Prof. Britta Engelhardt . C57BL/6J purchased from Charles River were used as control mice. All animals were kept in a temperature-controlled environment in a ventilated rack with a 12:12-h light:dark cycle. Food and water were given Control wild-type (WT) and CD11b KO female mice were anesthetized at 12 weeks of age and knee joint instability was induced surgically by partial medial meniscectomy (MNX) of the right knee, and the contralateral knee was sham-operated as control . Animals3), its bone mineral density  and its crystal content (μg). Tibial subchondral bone was analyzed as means of bone mineral density  and trabecular thickness .Scanning of mice knees was performed with a SkyScan 1076 X-ray μCT scanning system , as previously described . New cryFixed knees were decalcified in 5% formic acid, dehydrated, and embedded in paraffin. Frontal sections of 6 μm at different deepness of cartilage were stained with Safranin-O-fast green. OARSI scoring of medial tibial plateau and medial femoral condyle was performed  . FurtherCartilage explants were obtained from 13 human OA patients undergoing total knee replacement surgery from Lausanne University Hospital, Switzerland (average age 71 years old ± 11). Sections of macroscopically undamaged and damaged cartilage were dissected from the femoral condyles and processed for histological analysis from 3 patients. The rest of undamaged and damaged cartilage was cut in small pieces, washed twice with PBS and twice with DMEM. Cartilage was degraded for 10 h in 10% Liberase (Roche) DMEM, and digested tissue passed through a 70 μm filter (BD biosciences) to obtain articular chondrocytes. Cells were plated and cultured as described above.t-test or one-way or two-way ANOVA test, where appropriate. Differences were considered statistically significant for a value of p < 0.05 . Data was analyzed with GraphPad Prism software (GraphPad software), San Diego, CA.All values are expressed as the mean ± SD. Variation between data sets was evaluated using the Student’s Service de la consommation et des affaires vétérinaires du Canton de Vaud,” Switzerland. All efforts were made to minimize suffering and minimize the number of mice needed to assess statistical significance and experimental reproducibility. Human samples were obtained with the approval of the Centre Hospitalier Universitaire Vaudois ethical committee and patients’ written informed consent.Experiments on mice were performed in strict accordance to the Swiss Federal Regulations. The protocol was approved by the “To determine the distribution of CD11b expression in the mouse cartilage, CD11b immunostaining was performed on knee sections from WT and CD11b KO mice. As seen in This data was confirmed by flow cytometry analysis. Indeed, 3–4% of WT primary chondrocytes extracted from articular cartilage expressed the CD11b integrin subunit . CombiniWe next analyzed if CD11b was expressed in human cartilage by CD11b immunostaining analysis. Cartilage was obtained from the knee joint of three OA patients and separated into macroscopically healthy and damaged cartilages. The results in i.e., CD11b KO chondrocytes mineralized more compared to WT control  and mineralization was assessed after 24 h by Alizarin Red. As shown in  control . This ef control .Ank, Anx5, Pit1, Pit2, Pc1 and Alp) by qRT-PCR. Results in Ank, Pit1 and Alp expression were significantly increased in CD11b KO chondrocytes both in unstimulated or calcifying conditions (CPP). Regarding Pit2 and Pc1, no significant differences were observed. We provided further confirmation of the calcifying phenotype of CD11b KO cells by measuring the activity of alkaline phosphatase (Alp) in the cell lysate in non-treated and CPP treated chondrocytes. The Alp specific activity was higher in CD11b KO cells compared to that of WT control  and inhibitors of metalloproteases , whose expression plays a major role in chondrocyte extracellular matrix degradation during OA . LA1 reduced mineralization in six out of eight primary chondrocyte cultures . Moreovein vivo  model as previously described , and sacIn addition, microCT-scan examination was conducted to analyze the crystal deposition . By CT AIn order to rule out an underlying cartilage or bone phenotype in CD11b KO mice, that may predispose them to OA, we analyzed cartilage damage and subchondral bone structure in unchallenged WT and CD11b KO mice. In the first experimental setting we considered knees from sham-operated 20 weeks old WT and CD11b KO mice. OARSI scoring revealed no difference in cartilage degradation between WT and CD11b KO mice . MoreoveIntegrin CD11b/CD18 is a receptor originally described on neutrophils and macrophages, known to support adhesion and molecular cross-talk of these cells with the extracellular matrix proteins. It regulates many functions including phagocytosis, migration and immune tolerance , but it We first demonstrated the expression of the CD11b integrin subunit on primary murine and human chondrocytes and in articular cartilage of both murine and human OA patients by combining flow cytometry, qRT-PCR analysis and immunohistochemistry. CD11b is expressed only on a subset of chondrocytes in the superficial zone of the articular cartilage, and by FACS we confirmed 3–4% of chondrocytes expressing this integrin subunit. In line, in an open access database of single cell RNA sequencing on human OA chondrocytes , the gen2+) and inorganic phosphate (Pi) are uptaken into matrix vesicles (i) is extruded extracellularly via the multipass transmembrane transporter Ank. Extracellular PPi is then hydrolyzed to Pi by the enzyme tissue-non-specific alkaline phosphatases (Alp). Finally, Pi is concentrated in matrix vesicles through the cotransporters Pit1/Pit2 and Ca2+ through annexin 5 (Anx5). Here we demonstrated that CD11b KO chondrocytes, both at basal- and CPP-condition, express higher levels of the pro-mineralizing genes Ank, Alp and Pit1, and have more active Alp enzyme. Down-regulation of these calcification factors could be a potential mechanism by which CD11b exerts its anti-mineralizing properties in chondrocytes. From the literature, we can speculate possible cross-talks between CD11b-dependent signaling pathways such as JNK and NF-kB and these calcification players. Activation of the JNK  mediated activation of CD11b in regulating inflammation in experimental models of inflammatory disorders. By binding to an allosteric pocket in the CD11b subunit, LA1 changes the conformation of the extracellular domain, thereby preventing infiltration of pro-inflammatory monocytes after organ injury and vascular dysfunction . In a stHowever, we cannot rule out the possibility that LA1 can block calcification via a CD11b-independent mechanism, which deserves further investigation.ITGAM gene coding for CD11b and susceptibility to SLE  in the y to SLE . These Sy to SLE . Moreovey to SLE . In addiy to SLE . Finallyy to SLE . HoweverAlthough the contribution of CD11b integrin in calcification is clearly established in the present study, we did not investigate which ligands bind to CD11b in the cartilage. Integrin CD11b is a multi-ligand receptor capable of binding ICAM-1, fibrinogen, fibronectin, factor X and complement factor iC3b, among many others . IntegriEven though our current work unveils a new pathway in OA, it also presents some limitations that have to be considered. The use of global CD11b KO mice does not allow us to determine whether the observed effects are solely due to lack of CD11b expression in chondrocytes and not due to other cells in the surrounding tissues of the joint. This question will be resolved in future by generating mice with a selective CD11b deficiency in cartilage.in vitro. This study offers novel insights into the role of CD11b integrin signaling in regulatory mechanisms that control OA severity and progression, and provides a new target for future therapies, with final aim of improving the quality of life in OA patients.In summary, our study showed for the first time that CD11b signaling plays an important role in regulating cartilage calcification and OA. Deletion of CD11b gene was linked with cartilage degradation in knee joints during OA, mediated by upregulation of the pro-inflammatory IL-6 and MCP-1. Moreover, activation of CD11b with the LA1 agonist decreased the levels of pro-inflammatory cytokines and prevented calcification The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Centre Hospitalier Universitaire Vaudois ethical committee, Lausanne, Switzerland. The patients/participants provided their written informed consent to participate in this study. The animal study was reviewed and approved by “Service de la Consommation et des Affaires Vétérinaires du canton de Vaud,” Switzerland.DE and IB designed, performed and analyzed experiments, and helped with the preparation of the manuscript. VC and MC performed experiments. TH, AS, and LZ provided advice and help with the preparation of the manuscript. NB conceived, supervised the study, and wrote the manuscript. SN performed some experiments, supervised the study and wrote 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.


In comparison with the pervasive use of protein dimers and multimers in all domains of life, functional RNA oligomers have so far rarely been observed in nature. Their diminished occurrence contrasts starkly with the robust intrinsic potential of RNA to multimerize through long-range base-pairing (“kissing”) interactions, self-annealing of palindromic or complementary sequences, and stable tertiary contact motifs, such as the GNRA tetraloop-receptors. To explore the general mechanics of RNA dimerization, we performed a meta-analysis of a collection of exemplary RNA homodimer structures consisting of viral genomic elements, ribozymes, riboswitches, etc., encompassing both functional and fortuitous dimers. Globally, we found that domain-swapped dimers and antiparallel, head-to-tail arrangements are predominant architectural themes. Locally, we observed that the same structural motifs, interfaces and forces that enable tertiary RNA folding also drive their higher-order assemblies. These feature prominently long-range kissing loops, pseudoknots, reciprocal base intercalations and A-minor interactions. We postulate that the scarcity of functional RNA multimers and limited diversity in multimerization motifs may reflect evolutionary constraints imposed by host antiviral immune surveillance and stress sensing. A deepening mechanistic understanding of RNA multimerization is expected to facilitate investigations into RNA and RNP assemblies, condensates, and granules and enable their potential therapeutical targeting. Quaternary structures of biological macromolecules are frequently at the core of functional assemblies that enable life. Such higher-order structures form through a network of intermolecular interactions between individual modules employing recurring interfacial motifs to drive multimerization. Association of identical or different building blocks produce homo or hetero-multimers, respectively. These oligomeric assemblies often lead to overall structural symmetry or pseudosymmetry that can be important for biological function and evolution ,2,3,4. MThe simplest, most common form of RNA-RNA interactions occur through base pairing of complementary single-stranded sequences, forming heterodimeric double-stranded RNA (dsRNA). These specific interactions are widespread in diverse forms of post-transcriptional gene regulation and RNA modification and processing, such as the pairing of small interfering RNAs (siRNA), microRNAs (miRNA), or small nucleolar RNAs (snoRNAs), etc., to their complementary RNA targets. ,10,11,122+ ions and, to a lesser extent, by monovalent cations, such as K+. Mg2+ ions play the dual roles of serving as diffuse counter ions that ameliorate the electrostatic stress from juxtaposing densely charged phosphate backbones, as well as bridging specific tertiary contacts in the form of chelated ions [Consecutive base pairing positions adjacent base planes to stack with each other, either within the same strand or crossing into the opposing strand—termed cross-strand stacking. Notably, stacking can also occur without base pairing as the aromatic stacking itself provides significant favorable enthalpy. These non-covalent, attractive interactions between polarized aromatic nucleobases daisy-chain the base pairs into helices and frequently further concatenate adjacent helices to form long, coaxial helical stacks. In so doing, aromatic-aromatic (or π-π) interactions to a large extent dictate the overall shape of most RNA tertiary and higher-order structures. In addition to base pairing and stacking, other types of tertiary interactions also contribute to RNA-RNA interactions, including dimerization, such as purine-minor groove interactions, ribose zippers, tetraloop-tetraloop receptor interactions, etc. These tertiary and quaternary interactions between discrete RNA elements can be dramatically stabilized by the presence of Mgted ions .In contrast to their apparent biological rarity in cells, RNA multimers frequently emerge in vitro and often present a nuisance in the laboratory. Most of such in vitro multimers are believed to occur via fortuitous pairing of short segments of complementary sequences, when the RNA was heat-denatured and cooled slowly—a process that encourages strand annealing. As such these in vitro multimers are similar in origin to those dsRNAs produced by sense-antisense hybridization events in cells. Various RNA refolding procedures have been developed with an explicit or implicit objective to enrich monomers, while dimers and higher oligomeric states are reduced and usually discarded as artifacts or physiologically irrelevant entities. For instance, many unmodified tRNAs exhibit strong tendencies to dimerize and oligomerize and require a “snap-cool” procedure to refold into monomers . HoweverIn order to understand how RNA dimerizes and multimerizes in general, we survey a representative collection of RNAs that rely on their homodimeric states to exert their biological functions and also those that dimerize fortuitously, such as in crystallo. Interestingly, in several riboswitches, we are able to compare the monomeric and dimeric forms of essentially the same RNA. We maintain a focus on RNA dimerization interfaces and structural motifs used to drive self-assembly with an emphasis on tertiary contacts in addition to base pairing. This meta-analysis revealed that kissing-loop interactions and complementary strand swapping are the predominant dimerization motifs. Since only a handful of recurring RNA structural motifs are responsible for mediating dimerization in most instances, we organize the discussions based on RNA functional classes instead of the dimerization motifs that they employ. In the following sections, we examine instances of RNA dimerization that occur in retroviral genomes, mRNA localization, ribozymes and riboswitches, and fluorogenic RNAs, as well as consequences of RNA multimerization in diseases and immunity.Perhaps the most studied physiologically relevant RNA homodimerization phenomenon is the non-covalent linkage of two viral genomic RNA (gRNA) copies in retroviruses ,29,30,312+ [In the case of human immunodeficiency virus type-1 (HIV-1), a dimerization initiation site (DIS) was identified within the ~ 350 nucleotide DLS ,42. The 2+ ,45,46,472+ ,50,51,522+ . Thus, iThe crystal and nuclear magnetic resonance (NMR) structures b,c of thcis or in trans with the U5 element of the same or the other gRNA copy. It is tempting to speculate that the differences in stability of dimeric gRNAs between immature and mature viral particles could originate from the nature of AUG pairing to U5 (cis vs. trans). The structure of a minimal HIV-1 RNA packaging signal in its monomeric state consisting of the U5, AUG, DIS mutant and splice donor (SD) was recently determined [Lys3 annealing to the primer binding site (PBS) in the DLS was suggested to enhance gRNA dimerization [Conformational changes in the HIV-1 5′ leader regulate the oligomeric state of the gRNA ,57,58. Ttermined ,63. Furttermined . Indeed,termined ,65,66,67rization ,68. Take2  and the closely related Moloney murine sarcoma virus (MoMuSV) ,72,73,742 . Only tw2 h and an 2 h. The NM2 g,i. It iSeveral other classes of viruses also rely on RNA dimerization for specific functions. A palindromic kissing loop in the SARS coronavirus genome was shown to regulate ribosomal frameshifting, thus affecting viral growth . The 3′ In summary, a number of RNA viruses, including retroviruses, rely on kissing loop-loop interactions through palindromic or other complementary sequences to induce genome homodimerization for various essential functions. To buttress these generally short duplexes, single-stranded purines frequently cross-strand stack with both flanks of the intermolecular helices stabilizing them. The stacking of such bases contributes significantly to the stability of the dimeric interface by minimizing exposure of hydrophobic bases to the solvent and by favorable enthalpy changes through the aromatic interactions . This stoskar (osk) and bicoid (bcd) mRNAs to opposite poles of Drosophila embryos [osk or bcd reach their respective destinations [The 3′-UTRs of eukaryotic mRNAs are regulatory hubs for their translation, decay and cellular localization, etc. . Non-uni embryos ,97,98. I embryos ,100 mediinations ,102,103.osk is localization-competent, while unspliced osk is not [osk through intermolecular interactions. A conserved six-nucleotide, GC-rich palindromic loop sequence in the region of 714-827 of the osk 3′-UTR was shown to promote homodimerization in vitro [osk and may not form due to a larger loop flanked by pyrimidines. Double nucleotide substitutions in the palindromic sequence blocked dimerization by preventing kissing interactions. Importantly, dimerization is not required for localization of spliced osk but necessary for hitchhiking by unspliced osk [osk localization or translation activation through dimerization remains unclear, it exemplifies how mRNAs lacking “zip-codes” could still be targeted to specific cellular regions by piggybacking with other RNAs through RNA multimerization.Spliced k is not . Nonethein vitro   [The localization signal of in vitro ,107,108. in vivo ,107. Thi(dsRBDs)  of Staufbcd forms exclusively dimers in vitro, while the isolated domain III assembles into dimers, trimers, and tetramers [bcd quaternary structure. This multimerization mechanism and the ability to form more than one type of multimer is reminiscent of the bacteriophage ø29 prohead RNA (pRNA), discussed below [Interestingly, etramers . It is led below ,112,113 The bacteriophage ø29 pRNA is a component of the packaging motor required for viral genomic DNA encapsulation. pRNA forms dimers in vitro through a kissing loop interaction mediated by four complementary base pairs between two distinct stem-loops ,115. HowRibozymes are RNA catalysts that accelerate various chemical transformations, including self-cleavage, splicing, tRNA aminoacylation and peptidyl transfer, etc. Many naturally occurring ribozymes catalyze post-transcriptional RNA processing, such as backbone cleavage or ligation, and usually proceed through a general acid-base mechanism and produce 2′,3′-cyclic phosphate and 5′ hydroxyl termini ,120. SevNeurospora fungi. It has both self-cleavage and ligation properties and functions in the processing of RNA intermediates of rolling-circle replication [trans-cleaved following self-assembly into a homodimer [2, the largest RNA homodimerization interface described to date (The Varkud Satellite (VS) ribozyme, the largest known nucleolytic ribozyme, was discovered 30 years ago in the mitochondria of lication . It is oomodimer ,123. Itsomodimer . The cry to date . Stem-lo to date a [123,12 to date a. IntrigVeillonella sp. [trans-like symmetric dimer or a more intertwined cis-like pseudosymmetric dimer. In solution, both conformers and potentially others are likely sampled as a result of the apparent flexibility of the 3′ extended tail with the embedded palindromic sequence. An unanswered question is whether this dimerization occurs in vivo and for what purpose, since the palindromic sequence is found in the Hatchet ribozymes of multiple organisms but not strictly conserved [The Hatchet ribozyme is a small ribozyme that was recently discovered in several genes of ella sp.  and showella sp. . The dimella sp. . Interesonserved . cis-acting noncoding elements found in the 5′-UTR of some mRNAs (mostly in prokaryotes) [Riboswitches are aryotes) ,129,130.aryotes)  or tRNAsaryotes) ,133 to aaryotes) . In the Vibrio cholerae glycine apt2 revealed a crystallographic homodimer formed between helix P3 of one protomer and helix P1′ of the other [Fusobacterium nucleatum glycine riboswitch forms an apt1-apt2 intramolecular interface  followed by a single expression platform . Althoughe other   ,149,150.of ~4 nM ,152,153.of ~4 nM . For tanof ~4 nM . This inof ~4 nM ,85. TakeFusobacterium ulcerans ZTP riboswitch, two subdomains formed by P1-P2 and P3, respectively, bind each other forming a P4 pseudoknot. This cis tertiary interface forms the binding pocket for ZTP. Remarkably, in the Schaalia odontolytica ZTP riboswitch structure, this pseudoknot is formed in trans by domain swapping in a crystallographic dimer leading to G12-G15 pairing with C54′-C57′  riboswitch were solved b 154,15,156. In 54′-C57′ b. The twEubacterium siraeum tetrahydrofolate (THF) riboswitch exhibited an unraveled P1 helix that strand-exchanged with the P1′ of another monomer forming a scissors-like structure where the intertwined P1-P1′ forms the hinge  [The recent crystal structure of the operties . The fune (IRES) . AlthougDicistroviridae IRES, the Faecalibacterium prausnitzii preQ1 class III riboswitch bound to its ligand also crystallized in a homodimeric and partially domain-swapped configuration. In its previously elucidated class I and II counterparts, preQ1 binding at the helical junctions between P1 and P2 stabilizes coaxial stacking and directs helical sequestration of the ribosome-binding site  [Like the glutamine-II riboswitch and equence) ,170,171.equence) . The homequence)   riboswitch revealed an intermolecular P1 helix formed by strand swapping between two monomers [The crystal structure of the monomers   creates a six nucleotide palindromic sequence in the D-stem-loop [One primary limiting factor in the evolution of large RNA structures could have been the surveillance by the immune system. Nucleic acid multimerization often triggers innate immune responses ,186,187.tem-loop ,195. As tem-loop . Furthertem-loop ,198. MDAtem-loop . As a reIn addition to antiviral immunity, it is becoming increasingly clear that RNA multimerization is associated with general cellular stress ,18. TranTaken together, it seems that RNA multimerization frequently leads to undesired cellular toxicity or immune responses. These factors could have restricted the evolution and adoption of RNA dimers and higher-order assemblies, at least within the cellular compartments wherein there is active immune surveillance.cis interactions leading to monomer formation and the corresponding trans interactions leading to dimer formation compete with each other. The resulting monomer-dimer distribution can be in a dynamic equilibrium or static and non-interconverting. In both cases, the distribution is modulated by internal parameters, such as RNA concentration and thermal and folding history, as well as environmental parameters, including temperature, pH, ionic strength, divalent cations, crowding agents, osmolarity, etc. [In contrast to the abundance of protein dimers and multimers, there are only a handful of known cases of physiologically relevant RNA homodimers or oligomers. Nonetheless, in the laboratory, RNA multimerization is commonly observed both in solution and in crystallo. Our survey of representative multimeric RNA structures highlights the innate potential and inherent tendency of flexible RNA elements to exchange strands and swap domains to dimerize or multimerize. RNA appears to employ the same sort of interfaces and structural motifs that mediate tertiary structure formation to drive their higher-order assemblies. Occasionally, the ty, etc. ,211,212.bcd mRNA and ø29 pRNA. One exception to the hybridization-centric dimerization motif are RNAs that contain surface-exposed G-quadruplexes such as Corn. The strength of the stacking interactions between these large flat platforms alone is apparently sufficient to drive dimerization without base pairing. It is likely that future investigations will uncover naturally occurring G-quadruplex dimers or multimers in either cellular RNA or DNA. How do RNA multimers form? Our comparative analyses show that long-range kissing-loop interactions and the exchange of complementary strands near RNA termini  mediate most instances of dimerization. Nucleation of RNA protomers during the first encounter is typically initiated by the hybridization of short, consecutive segments of complementary sequences. This is akin to codon-anticodon interactions that occur on the ribosome and T-box riboswitches. Just like the codon-anticodon interactions, initial, sparse base-pairing interactions are usually not sufficiently stable and need to be further stabilized, either by extension of the base-pairing region , or by flanking contacts. These flanking contacts can take the form of cross-strand stacking by adjacent, single-stranded, helix-capping purines (as in the case of codon-anticodon interactions), or coaxial stacking and concatenation of contiguous helices to boost overall stability . Interestingly, these hybridization interactions can be split into two classes: those employing palindromic sequences and those that do not. While palindromic sequences are logically suited to drive dimer formation, non-palindromic but complementary sequences are more versatile. They have been effective in assembling variable numbers of multimers in head-to-tail processions, as seen in the We anticipate that more biologically relevant RNA dimers and multimers will be discovered with the advent of novel crosslinking and RNA-seq-based methods to map RNA-RNA interactions genome wide in vivo ,214,215.Extensive RNA oligomerization or condensation is often associated with cellular stress or immune responses and is normally avoided in the cytosol of healthy cells. The interplay among soluble RNA oligomers and clusters, RNA and RNP condensates, and RNases, helicases, and chaperones is an area of intensive investigation, which is expected to transform our frame of thinking in the coming decade. Such mechanistic understanding will inform the design of molecules that modulate RNA oligomerization and open novel therapeutic avenues.


To verify the efficiency of incorporation of ATP analogs in the presence of ATP, we developed a liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for quantitative assessment of modification frequency based on exhaustive degradation of the transcripts to 5′-mononucleotides. The method also estimated the average poly(A) tail lengths, thereby providing a versatile tool for establishing a structure-biological property relationship for mRNA. We found that mRNAs containing phosphorothioate groups within the poly(A) tail were substantially less susceptible to degradation by 3′-deadenylase than unmodified mRNA and were efficiently expressed in cultured cells, which makes them useful research tools and potential candidates for future development of mRNA-based therapeutics.Chemical modifications enable preparation of mRNAs with augmented stability and translational activity. In this study, we explored how chemical modifications of 5′,3′-phosphodiester bonds in the mRNA body and poly(A) tail influence the biological properties of eukaryotic mRNA. To obtain modified and unmodified in vitro transcribed mRNAs, we used ATP and ATP analogs modified at the α-phosphate (containing either O-to-S or O-to-BH In recent years, in vitro transcribed (IVT) mRNA has emerged as a promising candidate for therapeutic gene delivery . Since p3, which are enzymatically incorporated into RNA by means of RNA polymerases (The poly(A) tail is the stretch of adenylate residues at the 3′ end of most of the mature mammalian mRNAs (except histone-encoding replication-dependent genes ). PolyA and formymerases . HoweverSeveral methods to measure poly(A) tail lengths have been reported, including the polymerase-based poly(A) length assay engaging capillary electrophoresis , ligatioIn this study, to enable the analysis of P-mod mRNAs, we developed a new liquid chromatography–tandem mass spectrometry (LC–MS/MS) method that enabled simultaneous quantification of the number of modified phosphodiester bonds and estimation of the average poly(A) tail lengths in mRNA . SubsequSupplemental Fig. S1). For polymerases that have been studied so far, only the SP isomer of ATPαS has been accepted as a substrate, and the reaction proceeds with inversion of the configuration at the phosphorus atom (18O) within the phosphate were used as internal standards to facilitate quantification. The data were then mathematically processed to determine the absolute concentrations of these nucleotides, the AMP/AMPS ratio, and, if an RNA of known sequence with a 3′ terminal poly(A) tract was analyzed, the average poly(A) tail length was also estimated. We found that the key factor during method development was the optimization of the RNA degradation procedure to ensure complete RNA degradation to (modified) monophosphates, without further decomposition of released nucleotides.To enable systematic investigation of the SAR of modified RNAs, we developed an LC–MS/MS method for quantitative assessment of the incorporation of phosphate-modified ATP analogs into transcripts by RNA polymerases . If ATPαrus atom . To asseSupplemental Fig. S2; 18O) atoms in the phosphate group were synthesized for all tested NMPs and AMPS. Heavy AMPS, AMP, and GMP were used as internal standards for quantification of, respectively, AMPS, AMP, and GMP released during RNA degradation . The predominant fragmentation reaction for AMPS was the 362 → 95 transition, corresponding to the cleavage of the 5′-phosphorothioate bond and release of the PSO2− ion  as a mobile phase reagent . Based on the calibration curves, limits of quantification (LOQ) were determined . The final step of method development was establishing RNA degradation conditions. The goal was to achieve complete RNA degradation within a reasonable time, regardless of the modification content. This task was not trivial, since it has been known that the phosphorothioate modification, in general, stabilizes RNA against nucleolytic cleavage  and human CNOT7 deadenylase. SVPDE, which is a commercially available 3′ → 5′ exonuclease with broad spectrum specificity, releases 5′-mononucleotides . To verify the extent of AMPS desulfurization under optimized digestion conditions, RNA E, which was devoid of an A in the sequence, was polyadenylated with a mixture of deuterated ATP (DATP) and ATPαS D1, following RNA degradation and LC–MS/MS analysis. The amount of undeuterated AMP, which could be derived only from AMPS, was ∼10% . For each kind of RNA, the completeness of the digestion process was additionally verified by gel electrophoresis. If any undigested RNA was observed, the digestion conditions were adjusted individually, which was usually necessary for fully modified RNA.To obtain isotopically labeled internal standards, chemical (thio)phosphorylation of nucleosides followed by hydrolysis with heavy water was applied . To that end, the ratio of AMP and GMP concentrations (rref) in the nonpolyadenylated RNA sequence was experimentally determined. Then, the average poly(A) tail length was calculated based on Equation Supplemental Fig. S4A). The determined AMP to GMP ratio was comparable with the value expected based on the sequence.For polyadenylated RNAs, the average poly(A) tail length was also estimated (SP-ATPαS (isomer D1) will be incorporated into RNA, while RP-ATPαS (isomer D2) will be neither a substrate, nor an inhibitor . High yields of IVT products were obtained in the presence of ATPαS D1 independently of the ATP:ATPαS D1 ratio used in the reaction with SP6 polymerase. In contrast, the presence of higher concentrations of ATPαS D2 isomer resulted in a lower transcription yield. The RNAs were then subjected to degradation by SVPDE and subjected to LC–MS/MS analysis . The determined concentrations of AMP, AMPS, and GMP were plotted as a function of ATPαS D1 or D2 used in the in vitro transcription reaction  and the data were normalized to GMP . The AMPS to AMP ratio corresponded to the ATPαS D1 to ATP ratio , indicating that ATPαS D1 incorporation efficiency was similar to that of ATP . These findings are in good agreement with previous reports on the stereoselectivity of T7 RNA polymerase, which revealed that only isomer D1 is incorporated  were prepared by in vitro transcription. The template for the in vitro transcription reaction encoded a short 5′ UTR sequence, followed by the Firefly luciferase ORF, two consecutive H. sapiens β-globin 3′ UTRs, and a 128-nt long (A128) poly(A) tail  tail A. Consid(A) tail , we used(A) tail B. The trRNA variants with different amounts of phosphorothioate modifications were then evaluated for their translation efficiency in the rabbit reticulocyte lysate (RRL). The lysate, optimized for cap-dependent translation, was programmed with RNA F and the amount of luciferase was determined after 60 min by luminometry. The translation efficiencies of RNA F decreased with an increasing concentration of ATPαS D1 used in the transcription mix C. RNAs oOverall, these results indicated that randomly modifying the entire mRNA sequence with phosphorothioate moieties reduced both the transcription yield and translational activity of mRNA. We envisaged that the most likely interferences with the translation processes resulted from the modifications within the ORF or UTRs, which may impair ribosomal movement. Thus, in the next steps of the study, we investigated properties of mRNAs modified solely within the poly(A) tails.E. coli, a commercially available enzyme that specifically recognized the 3′ end of single-stranded RNA (ssRNA) and performed template-independent synthesis of single-stranded poly(A) tracts using ATP as a substrate. Previous reports have demonstrated incorporation of ATPαS into poly(A) tail using bovine PAP  were performed. To this end, three PAP concentrations  and either ATP alone, ATP:ATPαS in a 1:1 ratio, or ATPαS alone were tested . In the presence of ATP alone, polyadenylated RNAs of similar visual quality and dominant lengths between 300 and 500 nucleotides were formed in the presence of all tested PAP concentrations. In the presence of 1:1 ATP:ATPαS D1, the elongation of RNA was also observed, but the resulting RNAs appeared to be shorter and more homogenous, which indicated either a shorter length, disturbed migration due to modifications, or a smaller length distribution. In the presence of 100% ATPαS D1, the formed poly(A) tails migrated even faster (at the level corresponding to unmodified RNA). In the presence of the ATP:ATPαS D2 mix, the polyadenylation of RNA was clearly visible, and the resulting transcripts migrated only slightly faster than those obtained in the presence of ATP. In contrast, in the presence of 100% ATPαS D2, RNA elongation was not observed. Independently, the obtained transcripts were digested using an SVPDE and CNOT7 mixture and the obtained products were analyzed by LC–MS/MS to determine RNA composition, poly(A) composition, and to estimate the average poly(A) tail length. The amounts of AMP and AMPS released from RNA were initially normalized to the GMP amount . Knowing the concentrations and relative ratios of AMPS, AMP, and GMP in the samples, as well as the template sequence , the average poly(A) tail length was estimated using Equations Supplemental Fig. S8C). The average lengths of poly(A)s in those RNAs (45–95 nt) were comparable or only slightly shorter than those in corresponding RNAs obtained in the presence of ATP, and increased with increasing amounts of PAP . The average RNA lengths were comparable to those obtained in the presence of ATP (To achieve selective poly(A) tail modification of IVT RNAs, we used PAP from vine PAP , but to e tested . The obt 1U, 2U, U and eits of PAP A. In cons of PAP B; howevee of ATP B. RNAs ie of ATP D. This sSupplemental Fig. S6). We found that RNA lengths determined by LC–MS/MS were around three times shorter than the RNA length estimated by electrophoretic analysis, but the correlation between the methods remained linear. Hence, we hypothesized that these differences between the methods arose from large RNA heterogeneity, which caused a significant difference between dominant and average mRNA lengths .The experiments also revealed a notable inconsistency in the lengths of poly(A) tails estimated by LC–MS/MS and PAGE analysis for RNAs obtained in the presence of ATP. To verify whether this arose from significant poly(A) tail length heterogeneity or an inaccuracy in our MS/MS method, we prepared a set of unmodified mRNAs (RNA F2) carrying poly(A) tails of various lengths (different poly(A) tail lengths were achieved by varying the duration of the polyadenylation reaction) and analyzed them using both methods , and in the case of the reference mRNA (RNA I), an A128 poly(A) tail was encoded. To avoid interference from double-stranded RNA (dsRNA) impurities, the IVT mRNAs have been purified by RP HPLC prior to polyadenylation  was polyadenylated by PAP in the presence of either ATP or an ATP:ATPαS D1 mix . The polyadenylation protocol was optimized  to yield RNA G1 with ∼200–250-nt long (based on agarose gel) poly(A) tails containing a phosphorothioate modification as the predominant product. During initial polyadenylation experiments, we noted that high concentrations of ATPαS D1 in the polyadenylation reaction decreased the homogeneity of RNA G1 ; therefore, the 1:1 molar ratio of ATP:ATPαS D1 was not exceeded.To study the effects of poly(A) tail modification on mRNA stability and translation, we prepared unmodified mRNAs (RNA G and RNA H) and enzymatically added a poly(A) tail onto the 3′ end using PAP and phosphate-modified ATP analogs A. We invnylation . This prSupplemental Table S3). The frequency of AMPS in the poly(A) tail, as determined using LC–MS/MS, was ∼60%–70% of the expected phosphorothioate moiety content, according to the molar ratio of ATP:ATPαS D1 used in the polyadenylation reaction . The average poly(A) tail lengths determined by LC–MS/MS were similar to the lengths estimated by agarose gel electrophoresis . Moreover, the sensitivity of LC–MS/MS allowed for the analysis of the average poly(A) tail length of RNA G1, polyadenylated by PAP with a 1:1 molar ratio of ATP:ATPαS D1, whereas the length of the RNA G1 variant was difficult to estimate by electrophoretic analysis, due to heterogeneity .The RNAs were first analyzed by LC–MS/MS to estimate the poly(A) tail composition and average length. The results revealed that ATPαS D1 was efficiently incorporated into the poly(A) tail by PAP . Next, we used this protocol for deadenylation time-course experiments using RNA G1 variants with poly(A) tails synthesized by PAP and selected ATP:ATPαS D1 molar ratios  . We found that RNA G1 variants with poly(A) tails containing phosphorothioate modification were significantly less susceptible to CNOT7 deadenylase than unmodified RNA G1 . RNA G1 with a poly(A) tail obtained at a 9:1 ATP:ATPαS D1 ratio, which contained 6% phosphorothioate moieties in the poly(A) tail , was degraded by CNOT7 at a rate three times slower than unmodified RNA G1 . Further increases in the phosphorothioate content in the poly(A) tail to 13%, 15%, and 23% corresponded to a deadenylation rate reduced by 4.4-fold, 6.7-fold, and almost 20-fold, respectively .To analyze the susceptibility to enzymatic degradation of modified poly(A) tails in mRNA, we set up a deadenylation assay using CNOT7 deadenylase. CNOT7 is one of two catalytic subunits in the human Ccr4-Not deadenylase complex responsible for deadenylation of mRNAs in the cytoplasm . To thisGaussia luciferase as a reporter protein, since this type of luciferase is a secretory enzyme with a long half-life (∼6 d), enabling straightforward monitoring of time-dependent mRNA expression by quantification of luminescence in cell culture medium (Gaussia luciferase activity was measured over a period of 4 d (We next investigated the influence of phosphorothioate moieties present in the poly(A) on mRNA expression in living cells. To facilitate cell culture experiments, we used e medium . Thus, vd of 4 d F,G. In bd of 4 d H,I. Inted of 4 d H,I. It wThese results altogether suggested that (i) using a mixture of ATP and ATPαS D1 for polyadenylation provided access to mRNAs with phosphorothioate-modified poly(A) tails; (ii) phosphorothioate moieties decreased poly(A) tail susceptibility to deadenylation by CNOT7; and (iii) protein expression levels in JAWS II cells did not change with increasing phosphorothioate moiety content in the poly(A) tails of transfected mRNA.3 was chemically unstable  and, thus, could not be used as an internal standard. Furthermore, the main product of boranophosphate RNA degradation was not the expected AMPBH3, but adenosine H-phosphonate (AMPH) . As such, heavy AMPH was used as an internal standard for quantification of both AMPH and AMPBH3 in the samples. AMPH has similar fragmentation pathways to AMPBH3 , which decreased the sensitivity of detection.After demonstrating the utility of the method to analyze RNA with a phosphorothioate moiety, we determined whether it could be adapted to analyze RNAs carrying boranophosphate modifications. The boranophosphate moiety has similar structural properties and net charge to phosphorothioate, and also exhibits nuclease resistance . During o AMPBH3  but is cDATP) was used instead of ATP, or a mixture of DATP:ATPαBH3 (D1 or D2) was used. Polyadenylated RNA E1 was digested using a mixture of SVPDE and CNOT7, and the products were analyzed by LC–MS/MS (3 D1 (1:1 DATP:ATPαBH3 D1 that was used for the polyadenylation reaction), while AMPBH3 was barely observed  tails were ∼10-fold shorter compared to conditions including only ATP. Neither AMPH nor AMPBH3 were detected in samples generated from RNA E1 obtained using DATP and an ATPαBH3 D2 in a 1:1 ratio, which indicated that ATPαBH3 D2 was not incorporated by PAP. Compared to ATPαS D1, ATPαBH3 D1 seemed to be incorporated with lower frequency; ∼40% AMPS  and ∼20% AMPH . RNA E was obtained by in vitro transcription in the presence of T7 polymerase and subjected to a polyadenylation reaction, in which either 8-deutero ATP , and polyadenylated with PAP in the presence of ATP, ATPαBH3 D1, or ATP:ATPαBH3 D1 mixtures  (3 D1 in the reaction mix resulted in shorter poly(A) tails , which was consistent with the results obtained for RNA E polyadenylated with ATPαBH3 D1 , regardless of the ATP:ATPαBH3 D1 ratio used for polyadenylation; therefore, we also prepared an RNA H1 variant with a poly(A) tail synthesized in the presence of 100% ATPαBH3 D1 .To study translational properties of mRNAs with boranophosphate-modified poly(A) tails, RNA H was obtained by transcription in vitro, purified by RP HPLC ( ratios) A. RP HPLPαBH3 D1 . To obta3 D1 were lower than that for ATPαS D1, especially for RNA H1 variants polyadenylated by PAP at high concentrations of ATPαBH3 D1 .To determine the composition and average poly(A) tails lengths, different variants of RNA H1 were enzymatically degraded with SVPDE and CNOT7 ribonucleases, and the mixtures of the obtained NMPs were analyzed using LC–MS/MS B,C. The 0 4.1 nt/min and V0 4.3 nt/min, respectively) , suggesting that the RNA body sequence had no influence on susceptibility to CNOT7. RNA H1, with a boranophosphate-modified poly(A) tail, was more susceptible to deadenylation by CNOT7 than its counterparts containing phosphorothioate moieties at similar frequencies . Interestingly, RNA H1 polyadenylated with 100% ATPαBH3 D1 was degraded faster than RNA G1 polyadenylated by PAP at a 9:1 molar ratio of ATP:ATPαS D1 . These results showed that phosphorothioate modification had greater potential to affect the deadenylation process than a boranophosphate moiety.To evaluate the influence of boranophosphate modification on the poly(A) tail susceptibility to deadenylation, the transcripts were subjected to degradation by CNOT7 and the reaction progress was monitored using agarose gel electrophoresis. Based on the determined poly(A) tail lengths as a function of time, deadenylation rates were determined for each RNA E. The de3 D1 molar ratios, JAWS II and HeLa cells were transfected with RNA H1 variants and RNA I, and the activity of Gaussia luciferase secreted into the medium was measured over time  tail). To test this hypothesis, we first developed a method for quantitative assessment of incorporation of ATP analogs modified at the α-phosphate into RNA by polymerases, which relied on exhaustive degradation of the transcripts by 3′ nucleases followed by LC–MS/MS analysis of the resulting nucleoside monophosphates and their phosphate-modified analogs. The phosphorothioate-containing RNAs released AMPS as the main degradation product, whereas the boranophosphate RNAs released AMPH instead of AMPBH3, which was unexpected, but did not hamper method development. The key factor during method optimization was the adjustment of degradation conditions to ensure complete RNA degradation without desulfurization of AMPS to AMP. The method was successfully used to analyze both short and long RNAs, with the phosphorothioate quantification limit found to be 0.03 µM. The incorporation assessment of ATPαS diastereomers (D1 = SP and D2 = RP) by SP6 and T7 polymerases revealed that only the SP isomer of ATPαS served as the unnatural substrate for these polymerases, albeit not as efficiently as ATP, which was most evident from the decreased transcription yield when ATPαS D1 was used as the only adenine-containing substrate . The RP isomer of ATPαS was neither substrate nor inhibitor of the polymerases. These findings are in good agreement with literature reports on stereo-preferences of RNA polymerases (DATP) and either ATPαS D1 or ATPαBH3 D1 at a 1:1 ratio under optimized conditions revealed less than 10% of nondeuterated AMP (∼7% for phosphorothioate and 2% for boranophosphate), indicating that more than 90% of the modified analyte remained intact in the presence of 5′ nucleases . Following these initial experiments, we set out to evaluate biological properties of mRNAs IVT in the presence of ATP analogs. We found that in the presence of ATPαS D1:ATP mixtures, IVT mRNAs containing template-encoded poly(A) tails were synthesized significantly less efficiently by both T7 and SP6 polymerase than in the presence of ATP, which limited the amount of mRNA available to further biochemical analyses. Moreover, the yields of protein expression (firefly luciferase) per nanogram of RNA measured in RRLs were also significantly lower than those for unmodified mRNAs. This is in contrast to prokaryotic systems, where the detrimental effect of mRNA body modification within phosphorothioate groups on translation of prokaryotic mRNA has been reported only if more than one NTP was replaced with NTPαS  tail impacts mRNA stability and translation efficiency via interaction with PABPs . Recent Gaussia luciferase-encoding mRNAs containing variously modified poly(A) tails in two cell lines: HeLa (cancer cells) and JAWS II . In both cell lines, phosphorothioate-modified poly(A) tails did not affect protein expression, regardless of the phosphorothioate moiety content in the poly(A) tail. This indicated that the phosphorothioate modification was compatible with translation in living cells, albeit it did not provide any evident biological effect, at least for the studied poly(A) tail compositions. In contrast, the presence of a boranophosphate moiety in poly(A) tails clearly correlated with a decline in protein expression levels. Overall, the data from both in vitro and cell culture experiments indicated that only the phosphorothioate modification stabilized the poly(A) tail and efficiently supported translation. It remains to be determined how this modification affects interaction with PABPs. As demonstrated by 11 complex formation is mostly driven by extensive interactions of PABPs with adenines, engaging hydrogen bonds, van der Waals contacts, and base stacking. The sugar-phosphate backbone is less extensively probed by PABP amino acid side chains; therefore, potentially providing steric freedom for substitutions of the nonbridging oxygen atoms of the α-phosphate (To investigate whether poly(A) stability in vitro impacts protein biosynthesis in living cells, we measured the expression of hosphate . Neverthhosphate . FinallyE. coli, L. lactis, and HeLa cells, which might spike even higher interest in the properties of phosphate-modified RNAs in the future  system consisting of an electrospray ion source and triple quadrupole/linear ion trap analyzer. The instrument was coupled with an Agilent Technologies high-performance liquid chromatography (HPLC) system equipped with 1260 Bin Pump and degasser, 1260 ALS autosampler, and 260 TCC column oven. Analyst 1.6 software was used for system control, data acquisition, and data processing.N,N-dimethylhexylamine (DMH) and 97% H218O were purchased from commercial sources. Nucleotide analogs were synthesized by published methods  and starting materials (such as unmodified nucleosides and ATP) and chemical reagents, including  methods . SP6 andIf necessary, the compounds were purified by means of analytical HPLC or semi-preparative reversed-phase (RP) HPLC. Analytical HPLC  was performed using a Supelcosil LC-18-T HPLC column  with a linear gradient of 0%–50% methanol in 0.05 M ammonium acetate buffer (pH 5.9) for 15 min and UV detection at 254 nm. Semi-preparative HPLC was performed on the same apparatus equipped with a Discovery RP Amide C-16 HPLC column  with a linear gradient of 0%–100% acetonitrile in 0.05 M ammonium acetate buffer (pH 5.9) for 120 min with UV detection at 260 nm.−1. The gradient was optimized to obtain sufficient chromatographic separation of all analytes of interest . The optimal gradient was 0%–100% eluent B in 15 min.Chromatographic separation of nucleoside 5′-monophosphates (NMPs) was achieved on an Eclipse XDB-C18 analytical column  equipped with an Eclipse XDB-C18 analytical guard column . The gradient mobile phase contained the DMH ion pair reagent  and compSupplemental Table S1.The analytes in the HPLC eluate were monitored by MS in multiple reaction monitoring (MRM) mode. MS conditions for individual analytes and internal standards were optimized directly from the syringe pump. The optimization was performed using 400 µM solutions of individual NMPs and their respective isotope-labeled internal standards. Optimizations and analyses have been performed in negative ionization mode to maximize the number of fragmentation reactions . The gen2], 5 mM Tris, pH 8,8, and deactivated SVPDE) . Calibration curves were built by fitting the analyte-to-internal standard concentration ratio versus the analyte-to-internal standard peak area ratio using linear regression analysis y = ax + b, where the a parameter is the slope and b is the y-intercept. The limits of quantification are depicted in Supplemental Table S2.The calibration curves were prepared by spiking known concentrations (0.01–50 µM) of NMP and a constant concentration of isotopically labeled internal standard  into reaction solution , 3000 ng/mL SVPDE (Sigma-Aldrich) in SVPDE buffer ; or (ii) 20 ng RNA35 with poly(A) (RNA A1 and RNA E1) or 40 ng (RNA F2 and RNA G1)  mRNA, 9000 ng/mL SVPDE (Sigma-Aldrich), 1 mg/mL CNOT7 enzyme, buffer . The reactions were quenched by snap-freezing in liquid nitrogen, followed by heating at 95°C for 5 min, and snap-freezing in liquid nitrogen again. Samples after degradation were analyzed using LC–MS/MS. To each sample containing 20 ng/40 ng/120 ng of degraded RNA, 1 µM isotopically labeled internal standard was added. The enzyme amount was initially optimized on model RNAs to ensure degradation to single mononucleotides and lack of their further degradation . During optimization, the post-degradation reaction mixtures were analyzed by 15% polyacrylamide gel electrophoresis (PAGE). If any undigested RNA fragments were noticeable, the enzyme amount was increased.A typical RNA degradation reaction (20 µL) was performed at 37°C for 1 h and contained either (i) 20 ng RNACA) were calculated based on calibration curve parameters , internal standard concentration (CIS), and signal intensities ; b was fixed to 0.0  and is the ratio of the concentration of AMP and GMP (rfer = CAMPref /CGMPref). Numbers of adenine nucleotides in the poly(A) tail (nAMPtail) were calculated based on Equation nGMPref):nAMPXtail) were calculated according to Equation Concentrations of analytes (Equation .(1)CA=IThe sequences of RNAs used in different types of experiments are summarized in 2, 1 mM DTT, and 2 mM spermidine), 10 U/µL T7 or 1 U/µL SP6 RNA polymerase (Thermo Fisher Scientific), 1 U/µL RiboLock RNase Inhibitor (Thermo Fisher Scientific), 0.5 mM CTP/UTP/GTP, 0.5 mM different ATP:ATPαX ratios, and 0.4 µM annealed oligonucleotides as a template. Following a 2 h incubation, 1 U/µL DNase I (Thermo Fisher Scientific) was added and the incubation was continued for 30 min at 37°C. The crude RNAs were purified using RNA Clean & Concentrator-25 (Zymo Research). The quality of transcripts was assessed on 15% acrylamide/7 M urea gels, whereas the concentration was determined spectrophotometrically. To remove in vitro transcription side-products of unintended size, RNA samples were gel-purified using PAA elution buffer , precipitated with isopropanol, and dissolved in water.Short RNAs were generated on a template of annealed oligonucleotides: (1) (CAGTAATACGACTCACTATAGGGGAAGCGGGCATGCGGCCAGCCATAGCCGATCA and TGATCGGCTATGGCTGGCCGCATGCCCGCTTCCCCTATAGTGAGTCGTATTACTG) , which c2, 1 mM DTT, and 2 mM spermidine), 10 U/µL T7 RNA polymerase (Thermo Fisher Scientific), 1 U/µL RiboLock RNase Inhibitor (Thermo Fisher Scientific), 5 mM CTP/UTP/GTP, and 1 µM annealed oligonucleotides as a template. Following 3 h incubation, 1 U/µL DNase I (Thermo Fisher Scientific) was added and the incubation was continued for 30 min at 37°C. IVT products were extracted using phenol-chloroform and purified using HPLC on a Clarity 3 µm Oligo-RP C18 column (Phenomenex). To elute the desired products, two eluents were used: Eluent A: 50 mM TEA and Eluent B: 75% acetonitrile in the following gradient: 0–5 min 95% A, 5–20 min 87.5% A, 20–21 min 50% A using a 1 mL/min flow rate. Obtained products were used to obtain short RNAs with a poly(A) tail (RNA E1), as described below.Short RNAs were generated on a template of annealed oligonucleotides (the same sequence as RNA B but with U instead of A) (CAGTAATACGACTCACTATAGGGGTTGCGGGCTTGCGGCCTGCCTTTGCCGTTCT and AGAACGGCAAAGGCAGGCCGCAAGCCCGCAACCCCTATAGTGAGTCGTATTACTG), which contained the T7 promoter sequence (TAATACGACTCACTATA) and encoded a 35-nt long sequence (GGGGTTGCGGGCTTGCGGCCTGCCTTTGCCGTTCT). The in vitro transcription reaction (200 µL) was incubated at 37°C for 3 h and contained: RNA polymerase buffer  tail were generated in a polyadenylation reaction. A typical reaction mixture (20 µL) was incubated at 37°C for 1 h and contained 1 µg 35 nt RNA fragment, 1 mM different ATP([D]ATP):ATPαX ratios, 1 U/µL RiboLock RNase Inhibitor (Thermo Fisher Scientific), and PAP dedicated buffer and PAP (Lucigen) from 0.05 to 0.2 U/µL reaction. The RNAs were purified using RNA Clean & Concentrator-25 (Zymo Research). The quality of transcripts was assessed on 2% agarose gels, whereas the concentration was determined spectrophotometrically.Firefly luciferase transcripts (RNA F) were prepared based on the pJET_T7_FFLuc_128A and pJET_SP6_FFLuc_128A plasmids , 0.33 U/µL SP6 or T7 RNA polymerase, 0.66 U/µL RiboLock RNase inhibitor, 0.5 mM ATP or 0.5 mM mixture of ATP and ATPαS D1, 0.5 mM CTP and UTP, 0.125 mM GTP, 1.25 mM β-S-ARCA  tail: RNA I with a double 3′ UTR and template encoding a poly(A) tail, and RNA H and RNA G without poly(A) tails, and with single or double 3′ UTR sequences, respectively. The pJET_SP6_GLuc_128A plasmid  tail composition, length, stability, and translational properties, three types of mRNA encoding  plasmid , purifie2, 1 mM DTT, and 2 mM spermidine), 2.5 U/µL SP6 RNA polymerase (a second 2.5 U/µL was added after 2 h of incubation), 2 U/µL RiboLock RNase inhibitor, 2 mM ATP, CTP and UTP, 0.25 mM GTP, 2.5 mM ARCA, and 250 ng/µL DNA template. In vitro transcription was followed by removal of the DNA template with DNase I (30 min incubation at 37°C with 0.1 U/µL of the enzyme). The crude transcript was initially purified with NucleoSpin RNA Clean-up XS (Macherey-Nagel), followed by a second purification step using RP HPLC. To isolate the homogenous mRNA fraction sufficient for further polyadenylation, obtained transcripts were purified on an Agilent Technologies Series 1200 HPLC using RNASep Prep (ADS Biotec) at 55°C, as described previously (www.molbiotools.com).A typical in vitro transcription reaction mixture (30–40 µL) was incubated for 4 h at 40°C and contained: transcription buffer  sequence using an enzymatic approach with E. coli PAP (Lucigen). The PAP reaction mixture (5–10 µL) was incubated at 37°C for 45 min (for the preparation of RNA G1 with phosphorothioate-modified poly(A)), while the preparation of the RNA H1 reaction mixture was incubated for 10 min , 12 min (for ATP:ATPαBH3 D1 9:1), 15 min (for ATP:ATPαBH3 D1 5:1), 17 min (for ATP:ATPαBH3 D1 3:1), or 45 min (for ATP:ATPαBH3 D1 1:1 and ATPαBH3 D1 only). The reaction mixture contained PAP buffer , 0.2 U/µL PAP enzyme, 10 U RiboLock RNase inhibitor, 0.5 µM RNA G or 0.2 µM RNA H, ATP, or a mixture of ATP and ATPαS D1 or ATP and ATPαBH3 D1 at a total concentration of 0.25 mM; the reaction was stopped by the addition of EDTA to a final concentration of 25 mM. Polyadenylated mRNA was extracted with phenol:chloroform  and precipitated with 1.25 M ammonium acetate and isopropanol at RT, followed by a washing step with 70% ethanol. Polyadenylated mRNA pellets were resuspended in 10 µL of RNase/DNase free water (Sigma). The absorbance of the mRNA sample was measured spectrophotometrically (NanoDrop 2000c), and the concentration was determined using an extinction coefficient predicted for a particular mRNA sequence by DNA Calculator (software available on www.molbiotools.com). To assess the length of poly(A) tails, polyadenylated mRNAs were electrophoretically resolved in a 1% 1× TBE agarose gel between two RNA ladders  and analyzed using Image Lab Software 6.0.1 (Bio-Rad). Bands were automatically detected and the length of mRNAs was calculated using semi-logarithmic analysis with reference to the two RiboRuler RNA ladders. The poly(A) tail length was estimated as the difference between the length of the polyadenylated mRNA and the transcript without a poly(A) tail, used as a primer for PAP.The 3′ ends of 2, and 10 mM DTT), polyadenylated mRNA (12 ng/µL), and CNOT7 deadenylase (0.2 µg/µL). After 0, 20, 40, 60, or 120 min of incubation at 37°C, 2.5 µL of the reaction mixture was collected and the CNOT7 deadenylase was inactivated by addition of an equal volume of loading dye . Products of the degradation were analyzed by 1% 1× TBE agarose gel electrophoresis. Using Image Lab Software 6.0.1 (Bio-Rad), the degradation rate was estimated as the difference of the mRNA length before deadenylation and its length at a particular time point.The stability of poly(A) tails modified with phosphorothioate (RNA G1) or boranophosphate (RNA H1) was examined by subjecting mRNAs to deadenylation using recombinant CNOT7 deadenylase . A singl2 (0.4 µL of 25 mM solution). After 1 h of incubation at 30°C, 1 µL of the appropriate RNA F dilution  was added and the incubation of the reaction mixture was continued at 30°C for 1 h. The reaction was stopped by freezing in liquid nitrogen. To detect luminescence from Firefly luciferase, 50 µL Luciferase Assay Reagent 2, MgSO4, 0.1 mM EDTA, DTT, ATP, luciferin , and coenzyme A) was added to the sample and the luminescence was measured on a Synergy H1 (BioTek) microplate reader. The results are presented as proportions between the regression coefficients of the linear relationships between the RNA F concentration in the translation reaction  and the corresponding luminescence signal. The relative luciferase activity was normalized to the values obtained with unmodified RNA F, calculated as mean values from three independent experiments ± standard deviation (SD).The RRL (Promega) was used to determine the influence of internal modifications on translation efficiency of RNA F. The reaction mixture (9 µL) contained reticulocyte lysate (4 µL), amino acid mixture without leucine , amino acid mixture without methionine , potassium acetate (1.9 µL of 1 M solution), and MgCl2 and 37°C. HeLa  cells were grown in DMEM (Gibco) supplemented with 10% FBS (Sigma), GlutaMAX (Gibco), and 1% penicillin/streptomycin (Gibco) at 5% CO2 and 37°C. For all experiments, the passage number of studied cells was between 5 and 25. In a typical experiment, 104 per well JAWS II cells were seeded into a 96-well plate on the day of the experiment in 100 µL medium without antibiotics. In the case of the HeLa cell line, 4 × 103 HeLa cells per well were seeded 24 h before transfection into a 96-well plate in 100 µL medium without antibiotics. Cells in each well were transfected for 16 h using a mixture of 0.3 µL Lipofectamine MessengerMAX Transfection Reagent (Invitrogen) and 25 ng mRNA in 10 µL Opti-MEM (Gibco). To assess Gaussia luciferase expression at multiple time points, the medium was fully removed and replaced with fresh media after 16, 40, 64, and 88 h. To detect luminescence from Gaussia luciferase, 50 µL 10 ng/mL h-coelenterazine (NanoLight) in PBS was added to 10 µL cell culture medium and the luminescence was measured on a Synergy H1 (BioTek) microplate reader. Total protein expression (cumulative luminescence) for each mRNA over 4 d was reported as a mean value ± SD normalized to RNA I with a template-encoded A128 poly(A) tail.The murine immature dendritic cell line JAWS II (American Type Culture Collection [ATCC] CRL-11904) was grown in RPMI 1640 medium (Gibco) supplemented with 10% fetal bovine serum (FBS), sodium pyruvate (Gibco), 1% penicillin/streptomycin, and 5 ng/mL granulocyte-macrophage colony-stimulating factor (PeproTech) at 5% COSupplemental material is available for this article.


The emergence of the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a zoonotic pathogen, has led to the outbreak of coronavirus disease 2019 (COVID-19) pandemic and brought serious threats to public health worldwide. The gold standard method for SARS-CoV-2 detection requires both reverse transcription (RT) of the virus RNA to cDNA and then polymerase chain reaction (PCR) for the cDNA amplification, which involves multiple enzymes, multiple reactions and a complicated assay optimization process. Here, we developed a duplex-specific nuclease (DSN)-based signal amplification method for SARS-CoV-2 detection directly from the virus RNA utilizing two specific DNA probes. These specific DNA probes can hybridize to the target RNA at different locations in the nucleocapsid protein gene (N gene) of SARS-CoV-2 to form a DNA/RNA heteroduplex. DSN cleaves the DNA probe to release fluorescence, while leaving the RNA strand intact to be bound to another available probe molecule for further cleavage and fluorescent signal amplification. The optimized DSN amount, incubation temperature and incubation time were investigated in this work. Proof-of-principle SARS-CoV-2 detection was demonstrated with a detection sensitivity of 500 pM virus RNA. This simple, rapid, and direct RNA detection method is expected to provide a complementary method for the detection of viruses mutated at the PCR primer-binding regions for a more precise detection. The emergence of the novel severe acute respiratory syndrome coronavirus SARS-CoV-2) causing the coronavirus disease 2019 (COVID-19) pandemic has brought serious threats to public health worldwide  causing ,4. VirusMany methods based on traditional molecular diagnostics have been developed for COVID-19 detection and more novel methods utilizing nanoparticles as the indicators are under development ,12,13,14Alternatively, a nucleic acid test directly detects the RNA of SARS-CoV-2 in the sample from a patient’s throat or lung lavage fluid, which can better reflect virus infection and offer the earliest detection . Since SHowever, SARS-CoV-2 is an RNA virus, which mutates rapidly from generation to generation. Once a mutation occurs in the sequence of either the RT primer or the PCR primer-binding regions, the primer would fail to bind and lead to a false negative result. Collective genetic data have demonstrated that changes in the viral genome due to nucleotide insertion, deletion, or recombination and interchange among viruses were frequently observed ,22. One Duplex-specific nuclease (DSN) is an enzyme isolated from the hepatopancreas of the Kamchatka crab . It dispIn this work, by taking advantage of the characteristics of DSN in cleaving the DNA strand but leaving the RNA strand untouched, we innovatively developed a DSN-based assay for a SARS-CoV-2 test. m) were estimated by the OligoAnalyzer Tool from Integrated DNA Technologies. Duplex-specific nuclease (DSN) was obtained from Evrogen Joint Stock Company . All other chemicals were purchased from Sigma-Aldrich  and used without further purification. The tips and tubes purchased from Thermo Fisher  were RNase free. No further treatment was performed for RNase inactivation. All synthetic DNA and RNA oligonucleotides (HPLC purified) were purchased from Sangon Biotechnology Co., Ltd. . The DNA strands were dissolved in ultrapure water and RNA strands were dissolved in diethylpyrocarbonate (DEPC) water for a stock concentration of 100 μM. The stock solutions were kept at −80 °C in aliquots of 20 µL. Lower concentrations of oligonucleotides were obtained by serial dilutions from the stock solutions. The sequences of the RNA and DNA oligonucleotides are listed in DSN stock solution was prepared by diluting 100 U lyophilized DSN enzyme in 100 µL DSN storage buffer , and mixed by gently flicking the tube. Subsequently, the tube was spun briefly, and then incubated at room temperature for 5 min. Following that, 100 µL 100% glycerol was added to the tube to make a final concentration of 0.5 U/μL DSN with 50% glycerol. Various volumes of DSN stock solutions were added in a reaction to reach different DSN concentrations. 2, 1 mM DTT), enzyme , DNA probe, and the target RNA. The concentrations of DSN, DNA probes, and the target RNA were tested at various concentrations for an optimized assay. The mixed solution was prepared at room temperature and then transferred to a C1000™ Thermal Cycler  for incubation at various temperatures for 30–60 min. The specific concentrations of probes, targets, and DSN were adjusted in different experiments and are mentioned in the results section. After the reaction, the mixture was transferred to a 384-well microplate and scanned by a SpectraMax M2 fluorescence microplate reader  with excitation wavelength at 515 nm.DSN-based signal amplification was carried out in a 200 μL tube with a volume of 20 µL reaction mixture containing 1×DSN buffer  the RNA-dependent RNA polymerase gene (RdRP gene) in the open reading frame ORF1ab region, (b) the envelope protein gene (E gene), and (c) the nucleocapsid protein gene (N gene). Both the RdRP and E genes have been widely detected with a high sensitivity, whereas the N gene showed lower detection success with a much lower sensitivity ,38. ThisIn this work, we chose the N gene as the model target to demonstrate the DSN-based COVID-19 detection in a proof of principle. The RNA target sequence is listed in m) of about 71 °C, were labeled with a Cy3 fluorescence reporter and a BHQ1 quencher. Cy3, which emits greenish yellow fluorescence, is one of the most popular cyanine dyes and is commonly used to label nucleic acids. BHQ1 has strong absorption range of 480–580 nm, which provides excellent quenching to the Cy3 fluorophores. To verify that the separation of the quencher, BHQ1, from the fluorophore, Cy3, can generate enough fluorescence signal for detection, we designed another two control probes, N22C and N26C, without the label of BHQ1, for a comparison. Both of the probes, N22 and N26, with a similar melting temperature . As shown in The amount of DSN in an RNA detection assay needs to be optimized given the fact that less DSN has lower enzyme activity and more DSN could generate nonspecific junk. In these optimization experiments, 1 μM probe N22, 1 μM probe N26, and 100 nM target RNA were used in the assays. The reaction was carried out at 55 °C for 60 min with 0.1 U, 0.25 U, or 0.5 U of DSN. A control reaction was run in parallel without any DSN included. As shown in Hypothetically, using multiple DNA probes, which can hybridize to different regions of SARS-CoV-2 RNA, could give a higher fluorescent signal, therefore improving the detection sensitivity and accuracy of the DSN-based signal amplification method. To verify this hypothesis, we investigated multiple probes in our assay and compared them with the single probe utilized. In these experiments, 0.5 U DSN, 100 nM target RNA, and two DNA probes (1 μM N22 and 1 μM N26) were added in the reaction mixture and incubated at 55 °C for 60 min. As showed in One thing that needs to be mentioned is that DNase I, a DNA-specific endonuclease that degrades DNA molecules for RNA analysis, may interfere with this assay by cutting the probes and releasing fluorescence. Therefore, DNase I or any other enzymes for DNA degradation should be avoided with this assay. Under the optimized conditions, we investigated the sensitivity of a DSN-based amplification method for SARS-CoV-2 detection. In these experiments, 1 μM probe N22, 1 μM probe N26, and 0.5 U DSN were used in the reaction while the target RNA concentration ranged from 500 pM to 500 nM. The reaction was incubated at 55 °C for 60 min before fluorescence recording. 2 of 0.9994. The limit of detection can be down to approximately 500 pM, comparable to those of miRNA detection methods reported previously, as shown in As shown in To evaluate the selectivity of this approach where the signal only comes up when SARS-CoV-2 RNA is present, we conducted the experiments with miRNA-141 (an RNA sequence with 22 nucleotides) as a control group. No fluorescence signal was generated in the control group a,b, indiThe gold standard method for SARS-CoV-2 nucleotide detection to date is based on real-time RT-PCR. This method needs an additional reverse transcription process and requires specific primers and probes, making the detection process and assay optimization complicated. Besides, changes in the viral genome due to nucleotide insertion, deletion, or recombination and interchange among viruses could easily cause false negative results. In this research, we develop a rapid, simple, and accurate direct RNA detection method with DSN-based signal amplification for SARS-CoV-2 detection. The detection method only needs a one-step reaction for 60 min at 55 °C. The results show that using multiple DNA probes could improve the detection sensitivity and accuracy. The limit of detection can be down to about 500 pM under the optimized conditions. The results also indicate that the more specific probes are in this detection method, the more sensitive and accurate the SARS-CoV-2 RNA detection can be. The method proposed here provides a new option for virus nucleotide detection in pandemic outbreaks.


Varroa destructor, is a leading cause of honey bee population declines. In addition to being an obligate ectoparasitic mite, Varroa carries several viruses that infect honey bees and act as the proximal cause of colony collapses. Nevertheless, until recently, studies of Varroa have been limited by the paucity of genomic tools. Lab- and field-based methods exploiting such methods are still nascent. This study developed a set of methods for preserving Varroa DNA and RNA from the field to the lab and processing them into sequencing libraries. We performed preservation experiments in which Varroa mites were immersed in TRIzol, RNAlater, and absolute ethanol for preservation periods up to 21 days post-treatment to assess DNA and RNA integrity.The honey bee parasite, Varroa mite DNA and RNA libraries were created and sequenced for ethanol preserved samples, 15 and 21 days post-treatment. All DNA sequences mapped to the V. destructor genome at above 95% on average, while RNA sequences mapped to V. destructor, but also sometimes to high levels of the deformed-wing virus and to various organisms.For both DNA and RNA, mites preserved in TRIzol and RNAlater at room temperature degraded within 10 days post-treatment. Mites preserved in ethanol at room temperature and 4 °C remained intact through 21 days. Varroa and at least some of the viruses it vectors.Ethanolic preservation of field-collected mites is inexpensive and simple, and allows them to be shipped and processed successfully in the lab for a wide variety of sequencing applications. It appears to preserve RNA from both  Honey bees are one of the most economically significant agricultural resources, contributing to crop pollination, as well as providing products such as honey, propolis, and beeswax, which also contribute to local economies . The agrVarroa destructor, which jumped hosts from the closely related eastern honey bee (Apis cerana) [Varroa existed, and how it evolved post-host switch remains poorly understood [Varroa mites vector several viruses that impair the honey bee immune system, cause underdevelopment in honey bees, as well as cognitive impairment [Varroa are believed to be the primary driver of declining honey bee populations worldwide, exacerbating parasitism by Varroa mites themselves [Varroa-associated viruses have been identified, most notably deformed-wing virus (DWV) [Varroa and viruses it carries, as standard molecular methods to study them using genomic and transcriptomic tools have not been established [While honey bee declines are multifactorial, they have been accelerated by the pandemic spread of the ectoparasitic mite,  cerana) , 3. Untiderstood , 5. Furtpairment . Virusesemselves . Many Vaus (DWV) , 7–9, acus (DWV) , 9, Kashus (DWV) , 9, and us (DWV) , 9. HoweVarroa mites in RNAlater and its effect on RNA integrity. That study suggested that only when the mites are pierced does RNA remain intact, and did not explore other preservation solutions. Thus, there is limited published research regarding Varroa mite storage conditions and their effect on RNA and DNA integrity. Also, existing protocols for DNA and RNA extraction require pooling several mites to obtain enough material [The majority of honey bee viruses are single-stranded, positive-sense, RNA viruses. These commonly remain dormant, leaving the bees asymptomatic; thus, field diagnosis of honey bee viruses remains challenging , 9. To umaterial , 12.Varroa mite will allow biogeographical mapping to help visualize and track virus trends on a global scale. Here, we explore Varroa mite preservation conditions in TRIzol, RNAlater, and absolute ethanol for storage periods of up to 21 days, and their effects on DNA and RNA quality by mapping to the V. destructor reference genome [We propose a new method to extract both DNA and RNA from a single mite, sufficient in both quality and quantity for downstream analysis, such as next-generation sequencing. This method allows individual analysis, which can be used to map viruses present in each individual rather than pooled samples. Standardizing a method to analyze viruses present in each e genome  and tranVarroa mite DNA exhibited no trend in A260/280 values among the four treatments. In general, this method introduced contaminants into the RNA, with A260/280 values among all samples ranging from 1.76 to 3.35, with a mean of 2.76. High values indicate potential contamination, though they exceeded the threshold of 1.8 that is commonly used in laboratory practice for downstream applications [V. destructor. DNA libraries were successfully produced using a Nextera XT library preparation kit , uniquely indexed for sequencing.Extracted ications . We inspVarroa mite total RNA was extracted with a mean yield of 2,157 ± 2,570 (s.d.) ng. RNA electrophoresis showed that RNA in samples preserved in ethanol remained intact even at room temperature, while RNAlater and TRIzol preserved samples had degraded heavily in both 15- and 21-day samples , uniquely indexed for sequencing.Varroa mite DNA libraries acquired, on average, ~ 231,000 ± 49,000 (s.d.) sequences, whereas RNA libraries averaged approximately 447,000 ± 248,000 (s.d.) sequences per library. Given the short-read length used in sequencing, inspection with FastQC revealed that there were essentially no adapters and that sequence quality was consistently high, so raw reads were not processed further. DNA libraries of mites preserved in ethanol for 15 and 21 days at room temperature and 4 °C aligned to the V. destructor reference genome with a median of 95.49 ± 1.16 (s.d.) % . Kraken2  to classVarroa mite DNA and RNA integrity. Surprisingly, Varroa mite total DNA and RNA libraries mapped well to the V. destructor genome when bees were stored in absolute ethanol at room  when stored in TRIzol or RNAlater . We advise beekeepers and field workers to store mite samples in the refrigerator when possible, and at room temperature when this is not possible. Samples may also be shipped without refrigeration, reducing shipping costs. Additionally, as we observed 21 days of stable storage, shipping delays should be considered if RNA integrity must be maintained for longer periods for high-quality NGS and other downstream analyses. In short, we found that a possible alternative to snap freezing Varroa mite DNA and RNA were adequately preserved in absolute ethanol for up to 21 days, and produced high quality DNA and RNA libraries when sequenced, mapping to the Varroa genome and to other taxa. On the contrary, when Varroa mites were preserved in TRIzol and RNAlater, the mite DNA and RNA degraded within the first 10 days, possibly earlier, likely as a result of poor penetration through the exoskeleton. Ethanolic preservation of Varroa mites is inexpensive and uses a readily available reagent, thus allowing specimens to be shipped and processed for a wide variety of sequencing applications. In addition, ethanol also preserves viruses that Varroa vectors, most notably DWV. We propose that ethanolic preservation can replace cryopreservation, providing a more tractable method for preserving DNA and RNA quality.We found that Varroa mites were collected from managed hives in Onna village, Okinawa by removing honey bees from 2 frames onto a tray with icing sugar, and shaking the tray to remove mites from honey bees  and chilled in liquid nitrogen. A clean, autoclaved pestle  was also chilled in liquid nitrogen to grind each mite in its tube. By visual inspection, ground mite powder that remained on the pestle was used for DNA extraction, ensuring that about half of the mite homogenates remain in the tubes for RNA extraction , according to the manufacturer’s protocol. DNA quantity was determined using a Qubit fluorometer , and quality was evaluated by the A260 / A280 ratio using a NanoDrop spectrophotometer . Eluted DNA was stored at -20 °C until further applications.Mite RNA was extracted using TRIzol according to the manufacturer’s protocol. Due to the small amount of mite present, the protocol was modified by using 50% of specified reagent volumes. Total RNA quality and quantity were evaluated using absorbance ratios of A230/260 and A230/280 on a Nanodrop spectrophotometer.DNA libraries were prepared using a Nextera XT DNA Library Prep kit  according to the manufacturer’s protocol, with optimization for mite DNA, using reagents at 20% of their specified volume. DNA was visualized by running electrophoresis on 1% agarose gels, for 20 min at 135 V.RNA libraries were prepared using an NEBNext Single Cell/ Low-Input RNA Library kit , according to the manufacturer’s protocol. Purified cDNA was indexed with i5 and i7 primers , and then purified and size selected for a range of 400 to 2000 bp using 11% and 11.5% PEG and DynaBeads .Both DNA and RNA libraries, as well as total RNA, were analyzed with a Bioanalyzer 2100  or a 4200 Tapestation . For Bioanalyzer, high-sensitivity DNA kits and RNA pico kits  were used, while for the Tapestation, a high-sensitivity D5000 kit  was used.A MiSeq  was used to perform both DNA and RNA sequencing, at 50 cycles and read with single read-only. We were interested in validating the protocol and estimating mapping percentages, so longer read lengths or higher coverage were not necessary. Raw sequence data from the MiSeq were first analyzed with FastQC  for quic