Myocardial infarction (MI) is one of the most common illnesses seriously harmful to human health. Notwithstanding, the systems of its pathogenesis are as yet not totally demonstrated. CircRNA is one sort of non-coding RNA, and late distributed information proposes that circRNAs assume a significant part in heart diseases; however, their expression profiles in the peripheral blood of patients with MI are not yet totally characterized. Therefore, RNAs from peripheral blood were recruited for high-throughput RNA-seq analysis. A total of 3,862 circRNAs were distinguished to be remarkably different, including 2,738 circRNAs being upregulated and 1,124 circRNAs being downregulated. circTMEM165, circUBAC2, circZNF609, circANKRD12, and circSLC8A1 were reconfirmed by RT-qPCR in the cell model. ROC curves uncovered that they have great sensitivity and specificity in the determination of MI. Besides, circRNAs are associated with cell metabolism and function by directing complex networks among circular RNAs, microRNAs, and messenger RNAs. In outline, our study portrayed the specific articulation profiles of circular RNA in patients with MI. The outcomes showed that circRNAs might fill in as a sort of ideal biomarkers for MI diagnosis. Further exploration of these circRNAs may enrich our understanding of MI etiology and progression. Cardiovascular diseases (CVDs) are the most common cause of death globally. The number of deaths from CVD accounted for about one-third of all global deaths . MyocardRecently, circRNAs have become the focus and the front field of RNA research based on advanced RNA sequencing technology and bioinformatics analysis. circRNA is a kind of non-coding RNA, which contains a closed continuous loop structure and is broadly expressed in multispecies genomes , 13. cirThe peripheral blood involves numerous leukocyte subpopulations, for instance, T cells, B cells, mononuclear cells, and natural killer cells. Studies have shown that key genes in leukocytes are associated with alterations in gene expression and the onset of disease .To date, the associations between circRNAs in peripheral blood and MI are not fully understood. Therefore, we explored the expression profiles of circRNA in patients with MI and healthy controls by high-throughput circRNA sequencing. We collected the volunteers’ peripheral blood and constructed the cell model to confirm the dysregulated circRNAs. ROC and combined ROC curves were created for demonstrating the biomarkers of MI. The roles of circRNAs in MI were analyzed by bioinformatics. Moreover, we discovered a possible complex endogenic RNA regulatory network among circRNAs-miRNAs-mRNAs, with a view to providing some references for related research in this field.All testing programs were performed according to the protocols approved by the Medical Ethics Committee of the Affiliated Hospital of Qingdao University. All sample providers were informed of their blood samples, clinical data were used for this study, and informed consents were signed.The peripheral blood samples from 80 patients with MI were collected before percutaneous coronary intervention surgery between March 2018 and January 2019 in the Affiliated Hospital of Qingdao University . Fresh pvia the TRIzol method . This experiment was performed according to the kit’s recommendations. RNA samples were stored at −80°C.The samples were used for RNA extraction ® Spectrophotometer . RNA concentration was detected by Qubit® RNA Assay Kit in Qubit® 2.0 Flurometer . RNA integrity was measured by the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system . RNA degradation and contamination were monitored on 1% agarose gels.The purity of the extracted RNA was tested by a NanoPhotometer™ rRNA Removal Kit  and ethanol precipitation. Subsequently, the linear RNA was digested with RNaseR . The sequencing libraries were produced by NEBNext® Ultra™ Directional RNA Library Prep Kit for Illumina® .A total of 5 μg RNA per sample was used for the RNA library preparations. First, rRNA was removed by Epicenter RibozeroThe cluster generation of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq PE Cluster Kit v3-cBot-HS. After that, the libraries were sequenced on an Illumina Hiseq 4000 platform. These experiments were performed following the manufacturer’s instructions.First, clean data were gained by wiping off reads containing adapter and low-quality reads from raw data. The clean data with high quality were used for downstream analyses. Index of the reference genome was created using Bowtie2 v2.2.8, and paired-end clean reads were united to the reference genome using Bowtie . The cirThe junction site part of circRNAs was confirmed by polymerase chain reaction (PCR) with divergent primers. Convergent primers were used as the control. The products of the PCR amplification were authenticated by 1% agarose gel electrophoresis. The primers for circRNA validation are listed in The dysregulation circRNA was reconfirmed on a CFX96 Real-Time PCR Detection System (Bio-Rad). Total RNA was extracted using Trizol reagent. Reverse transcription reactions were performed by using the TransScript II One-Step gDNA Removal and cDNA Synthesis SuperMix  to make cDNA according to the manufacturer’s guide. TransStart Green qPCR SuperMix (TransGen) was used for quantitative PCR (qPCR) analysis, and the procedures were performed in accordance with the kit instructions. The levels of dysregulation circRNA analyzed by RT-qPCR were normalized to that of GAPDH . The priThe ROC curves were created to estimate the diagnostic value of circRNAs for MI. An ROC curve was calculated, and the specificity and sensitivity of predictive power were measured by the AUC. The AUC was used to assess the diagnostic value of the circRNAs in the peripheral blood of patients with MI . The comP-value less than 0.05 were considered to be significantly enriched by differential expressed genes. As a database resource for understanding high-level functions and utilities of the biological system  enrichment analysis for host genes of differentially expressed circRNAs was carried out by the GO seq R package, and the gene length bias was corrected during this procedure . GO terml system , Kyoto Epathways .via binding to an miRNA  incubated at 37°C for 12 h in a complete culture medium. The cells were collected for RNA extraction and dysregulated circRNA reconfirmation.AC16 cells were cultured in DMEM (Gibco) with 10% fetal bovine serum (TransGen), and 100 U/ml penicillin and 100 mg/ml streptomycin (Invitrogen) in a humidified 5% COApoptosis was determined by the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) using a kit from TransGen. The detection procedures were in accordance with the kit instructions.Mitochondrial staining was performed as described earlier with modifications . BrieflyImmunoblot was carried out as described earlier . Brieflyt-test with Welch’s correction for two groups. Statistical analyses were completed with GraphPad Prism 7.0 . P < 0.05 was regarded as statistically significant.The results are expressed as mean ± SEM of at least three independent experiments. The statistical comparison between different groups was completed by one-way ANOVA for multiple comparisons or The circRNAs were identified by find_circ and Bowtie2 based on the high-quality clean data. A total of 3,862 circRNAs were recognized in five pairs of MI and healthy controls samples. The size of the circRNAs extended from ≈100 to 2,500 nt . The difFirst, we screened candidate circRNAs by comparing our results with the previous study on human heart tissue sequencing sample results  31) in  in 31) iTo verify the RNA-seq data, five intersectional circRNAs  were selected and validated by RT-qPCR using 80 MI samples and 80 healthy individuals. The general information of selected circRNAs shown as To reconfirm the dysregulation of these circRNAs in the cell model, we used the myocardial cell injury model induced by reactive oxygen species (ROS) to simulate cell damage during MI . The datTo evaluate the potential diagnostic value of selected circRNAs for MI, we computed and created ROC curves  for RT-qThe calculated optimal cutoff point for circTMEM165 level in the peripheral blood was 1.6089505 (Youden index = 0.55), while the sensitivity and specificity were 21.3% and 23.7%, respectively. The calculated optimal cutoff point for circUBAC2 level was 1.968652 (Youden index = 0.663), while the sensitivity and specificity were 82.5% and 83.8%, respectively. The calculated optimal cutoff point for circ ZNF609 level was 1.947015 (Youden index = 0.638), while the sensitivity and specificity were 86.3% and 77.5%, respectively. The calculated optimal cutoff point for circ SLC8A1 level was 1.5414885 (Youden index = 0.737), while the sensitivity and specificity were 82.5% and 91.25%, respectively. The calculated optimal cutoff point for circANKRD12 level was 1.4872795 (Youden index = 0.575), while the sensitivity and specificity were 87.5% and 57.5%, respectively. The optimal cutoff point of the combination was 0.06, while the sensitivity, specificity, and Youden index were 0.863, 0.937, and 0.8, respectively. The results revealed that the AUC of these circRNAs at least reached 0.8073, and the AUC of recombination circRNAs reached up to 0.9289 , indicatP = 2.892E-46), intracellular part , and protein binding , respectively.The source genes of the validated differentially expressed circRNAs were uncovered by using GO annotation, as shown in The data of the KEGG pathway study of the circRNAs genes are shown in The miRNA binding sites exist in circRNAs, which can function as miRNA sponges. To further explore their possible roles in patients with MI, we predicted the circRNA-miRNA-mRNA network and analyzed using miRanda and RNAhybrid. The top five predicted miRNAs for these upregulated circRNAs are shown in Moreover, we noted the potential function of these regulatory networks. For example, circTMEM165 is predicted as “sponge” to hsa-miR-548a-3p, which can bind to mRNA GREM1, which is responsible for regulating organogenesis, body patterning, and tissue differentiation. The network of circZNF609-hsa-miR-221-5p-WDR7 may be involved in the low-density lipoprotein (LDL) metabolism. The regulatory pathway of circANKRD12-hsa-miR-876-5p-MYLK may promote myosin interaction with actin and facilitate to produce contractile activity. circANKRD12-hsa-miR-130b-5p targets both ACSM2A and RNF213, which regulate mitochondrial acyl-coenzyme A synthetase and ATPase activity, respectively. These networks indicate the possible interactional RNA regulatory relationships between circRNAs, miRNAs, and mRNAs in MI.2O2 after the knockdown of circSLC8A1 expression. We found that the number of apoptotic cells was significantly decreased in the si-circSLC8A1 group (via Western blot analysis, and our results revealed that the expression levels of Caspase3 and cyto c were markedly decreased (As circSLC8A1 has the best ROC in the verification of clinical samples, we should further explore its mechanism of action in MI. To further clarify the underlying mechanism of circSLC8A1 in MI, we first knocked down circSLC8A1 expression in the myocardial cells through circSLC8A1-siRNA. The AC16 cells were treated with 100 μM HA1 group , and theA1 group . To inveecreased . These dTaken together, these data demonstrated that knockdown of circSLC8A1 decreased intracellular oxidative stress, kept the balance of mitochondria dynamics, and then reduced apoptosis during oxidative stress.2O2. The results indicated that overexpression of circSLC8A1 remarkably promotes mitochondria fission (To understand the role of circSLC8A1 in myocardial cell apoptosis, we overexpressed circSLC8A1 in AC16 cells using circSLC8A1-plasmid. After transfection, the cell was treated with 100 μM H fission , and the fission . In cell fission . We also fission .Altogether, our results demonstrated that the overexpression of circSLC8A1 increased the intracellular oxidative stress, and it sensitized the myocardial cells to apoptosis by further increasing mitochondrial fission under the stimulation of oxidative stress.Myocardial infarction is a kind of multifactorial disease, which causes complex pathogenesis and leads to high mortality. It is hard to diagnose and prompt the intervention of MI before the disease occurs. As a consequence, there is an earnest for early detection markers for MI diagnosis. circRNAs are single-stranded, covalently closed molecules  that areRecent reports have demonstrated that circRNAs are more constant, varied, and conserved when compared with other kinds of RNAs, including mRNAs, miRNAs, and lncRNAs . circRNAEarly diagnosis can guide clinical medication to effectively treat MI and significantly improve their prognosis. circRNAs have already been confirmed as effective biomarkers for numerous diseases, such as cancer , acquireRecently, several studies have indicated that circRNAs might be key regulators in CVDs. For instance, Wu et al.  showed tWe also used circRNA-seq data to predict circRNA-miRNA-mRNA networks and aimed to find the possible roles of circTMEM165, circUBAC2, circZNF609, circANKRD12, and circSLC8A1 in the peripheral blood of patients with MI. We observed the possible target mRNAs of these circRNAs including GREM1, WDR7, MYLK, ACSM2A, and RNF213. Moreover, the predicted target mRNAs played a key role in regulating organogenesis, body patterning, tissue differentiation, LDL metabolism, and myosin interaction with actin, mitochondrial acyl-coenzyme A synthetase, and ATPase activity, respectively. All of them are highly correlated with CVDs.To further clarify the potential mechanism of circSLC8A1 in MI, we tested the cell function through knockdown and overexpressed circSLC8A1 based on the cell model. Uncontrolled mitochondrial fission contributes to apoptosis , which pA few limitations of this study ought to be mentioned. First, we only verified 10 circRNAs that were upregulated in MI, and still, a lot of differentially expressed circRNA needed to be verified. Second, although we use a set of filter methods to get the more accurate results, the sample size of this study is still limited, which will increase the false positive rate while analyzing RNASeq data. Third, we only explore more upregulated circRNAs, and the downregulated circRNAs are also worth studying in depth. Moreover, further experiments are needed to explore the regulatory effects of circRNAs in the occurrence of MI in patients with CVD.To the best of our knowledge, this study is the first to report the expression profiles of circular RNA in the peripheral blood of patients with MI compared with healthy individuals. Bioinformatics analysis indicated that the upregulated expressions of circTMEM165, circUBAC2, circZNF609, circANKRD12, and circSLC8A1 were highly correlated with MI. Moreover, all of these five circRNAs may be used to improve the diagnosis accuracy of MI. Besides, the bioinformatics analysis found that these circRNAs could be involved in the regulation of hundreds of mRNAs, and numerous of these mRNAs may affect the occurrence of MI. Although our results revealed that these circRNAs are highly expressed in the peripheral blood of patients with MI, their predicted functions still need to be confirmed by further experiments.The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by Ethics Committee of Affiliated Hospital of Qingdao University. The patients/participants provided their written informed consent to participate in this study.QL, JW, and YG conceived and designed the experiments. QL, YW, and YA collected and analyzed the data. QL and YW wrote this manuscript. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Enteroviruses (EVs), especially coxsackieviruses B (CVB), are believed to trigger or accelerate islet autoimmunity in genetically susceptible individuals that results in type 1 diabetes (T1D). Therefore, strategies are needed to fight against EV infections. There are no approved antiviral drugs currently available, but various antiviral drugs targeting viral or host cell proteins and vaccines have recently shown potential to combat CVB infections and may be used as new therapeutic strategies to prevent or reduce the risk of T1D and/or preserve β-cell function among patients with islet autoantibodies or T1D. Picornaviridae family [Enterovirus A–D and Rhinovirus A–C) and over 250 serologically distinct viruses. Enterovirus A–D include more than 110 serotypes, the best known of which are the polioviruses, enterovirus A71, echoviruses, coxsackieviruses A and coxsackieviruses B [Enteroviruses (EVs) are small (25 to 30 nm diameter) non-enveloped, positive-sense single-stranded RNA genome viruses which belong to the e family . The genThey replicate primarily in the gastrointestinal tract or in the upper respiratory tract and can spread to various target organs through the lymphatic system and the bloodstream . They arSeveral epidemiological, clinical, and experimental data support the hypothesis that CVB are the most incriminated environmental factors linked to the development of islet autoimmunity or onset and progression of T1D ,8. Despipro, 2B, 2C, 3A, 3B, 3Cpro, and 3Dpol) [The EVs genome is approximately 7400 bases and contains a single open reading frame flanked by an untranslated region at 5′ and 3′ ends . After vd 3Dpol) . Viral ppol (a RNA-dependent RNA polymerase) [Pleconaril is one of the most studied antiviral drugs that was initially developed for the treatment of common cold or prevention of asthma exacerbation due to picornavirus infections . Pleconaymerase) . Antivirymerase) . Remdesiymerase) . Umifenoymerase)  has showymerase) .Some repurposed drugs such as fluoxetine (a selective serotonin reuptake inhibitor used as antidepressant drug) targeting non-structural viral 2C protein ,28,29, i+ channel and the cellular Na+/H+ exchanger) strongly inhibit CVB3 RNA replication but emergence of amiloride-resistant mutants with amino acid substitutions within the 3Dpol was reported [Enviroxime, PIK93, and GW5074 are kinase inhibitors that can inhibit CVB replication by targeting the host factor, phosphatidylinositol 4-kinase type IIIβ, involved in the positive-strand RNA synthesis ,38. Envireported . Golgicireported . Hsp90  is an autoimmune endocrinological disorder characterized by chronic hyperglycemia caused by selective destruction or dysfunction of β cells in the pancreas leading to severe endogenous insulin deficiency. According to a recent meta-analysis the incidence of T1D is currently estimated to be 15 per 100,000 people per year and the prevalence was 9.5% worldwide  with an The precise etiology and the mechanisms that trigger autoimmunity against islet antigens are not fully understood but growing evidence support that the disease process is initiated by environmental factors in genetically susceptible individuals . SeveralIt has been estimated that 80% of T1D could be prevented or cured by eliminating the effect of diabetogenic viruses . TherefoLactobacillus plantarum can inhibit CVB4 infection in vitro [Syzygium brazzavillense can inhibit CVB infection of cells in vitro [Research into alternative treatments based on natural products from bacteria and/or their metabolites and plant extracts may provide an alternative to synthetic molecules. For example, it has been reported that lipoproteins from bifidobacteria and in vitro ,71. Few in vitro  and thisin vitro .The high mutation rates of RNA viruses such as CVB lead to the emergence of resistant variants ,75 oftenInterferon (IFN) response markers are often identified in islets of T1D patients and are associated with EV persistent infection ,87,88. AIn conclusion, fighting EVs, especially CVB is possible. Vaccines are now being developed and offer hope for the prevention of T1D induced or aggravated by EVs, in particular by CVB. Various approaches to inhibit EVs infection, especially CVB, have been reported . However


Caenorhabditis elegans muscle provides an excellent model to study integrin adhesion complexes. In Caenorhabditis elegans, UNC-112 (kindlin) binds to the cytoplasmic tail of PAT-3 (β-integrin) and to PAT-4 (ILK). We previously reported that PAT-4 binding to UNC-112 is essential for the binding of UNC-112 to PAT-3. Although there are crystal structures for ILK and a kindlin, there is no co-crystal structure available. To understand the molecular interaction between PAT-4 and UNC-112, we took a genetic approach. First, using a yeast 2-hybrid method, we isolated mutant PAT-4 proteins that cannot bind to UNC-112 and then isolated suppressor mutant UNC-112 proteins that restore interaction with mutant PAT-4 proteins. Second, we demonstrated that these mutant PAT-4 proteins cannot localize to attachment structures in nematode muscle, but upon co-expression of an UNC-112 suppressor mutant protein, mutant PAT-4 proteins could localize to attachment structures. Third, overexpression of a PAT-4 mutant results in the disorganization of adhesion plaques at muscle cell boundaries and co-expression of the UNC-112 suppressor mutant protein alleviates this defect. Thus, we demonstrate that UNC-112 binding to PAT-4 is required for the localization and function of PAT-4 in integrin adhesion complexes in vivo. The missense mutations were mapped onto homology models of PAT-4 and UNC-112, and taking into account previously isolated mutations, we suggest a surface of PAT-4 that binds to UNC-112.Integrin plays a crucial role in the attachment of cells to the extracellular matrix. Integrin recruits many proteins intracellularly, including a 4-protein complex .  Caenorhabditis elegans muscle used for locomotion is located in the body wall and consists of 95 spindle-shaped mononuclear cells arranged in interlocking pairs that run the length of the animal in 4 quadrants. The myofibrils are restricted to a narrow ∼1.5-μm zone adjacent to the cell membrane along the outer side of the muscle cell. The thin filaments are attached to dense bodies , and the center of the bundle of thick filaments (A-bands) are attached to M-lines. Moreover, all of the dense bodies and M-lines are anchored to the muscle cell membrane and extracellular matrix, which is attached to the hypodermis and cuticle. This allows the force of muscle contraction to be transmitted directly to the cuticle and allows movement of the whole animal , we examined muscle localization of mutant PAT-4 with and without suppressor UNC-112. After sustained expression (24 h of heat shock) of these mutant proteins, we examined the organization of muscle adhesion complexes. We found that mutant PAT-4 proteins that cannot bind to UNC-112 fail to localize to muscle attachment structures. However, upon co-expression of suppressor mutant UNC-112 proteins that restore the binding, localization of mutant PAT-4 proteins to attachment structures is restored. We also found that overexpression of a mutant PAT-4 protein results in the disorganization of adhesion plaques at the muscle cell boundary, and co-expression of a suppressor mutant UNC-112 alleviates this defect. The missense mutations were mapped onto homology models of PAT-4 and UNC-112, and taking into account previously isolated mutations, we suggest a surface of PAT-4 that binds to UNC-112.We have suggested a model for the assembly of integrin-associated proteins previously , especia+ colonies. LEU2 was the marker for the prey plasmid pACT-PAT-4; URA3 was the marker for the bait plasmid pGBDU-PAT-6; and HIS3 was the first marker for the detection of the yeast 2-hybrid interaction. His+ selection ensured that the mutagenized PAT-4 could still interact with PAT-6. This step was essential for eliminating clones with premature stop mutations or with many other mutations. Because His+ selection yields a high level of false positives, ADE2 was used as a second marker for the detection of the yeast 2-hybrid interaction. Thus, His+ colonies were streaked onto an −Ade plate and screened for His+Ade+ colonies. After streaking on a 5-fluoroorotic acid plate to eliminate the URA3 marker bait plasmid (pGBDU-PAT-6), prey clones were isolated from yeast and amplified in Escherichiacoli. From a total of 48 His+Ade+ yeast colonies, 47 mutagenized clones were isolated. These prey clones were transformed separately into PJ69-4A carrying either pGBDU-PAT-6 or pGBDU-UNC-112 (full-length) to confirm interaction with PAT-6 and screen for the lack of interaction with UNC-112. Among 47 mutagenized PAT-4 clones, all 47 showed binding to PAT-6, but 16 of these clones could not bind to full-length UNC-112. DNA sequencing of these clones revealed the mutations . These vectors contain 2 different heat shock promoters. pPD49.78/83-HA-PAT-4  were mixed with pTG96 (SUR-5::NLS::GFP) as a transformation marker (sfEx56), Q308H , and I432F ] and pTG96 were established by picking GFP-positive worms using a GFP dissection microscope. Expression of the HA-tagged PAT-4 proteins  was induced by incubation of the transgenic worms at 30°C for 2 h (heat shock). We prepared worm lysates  and PAT-4-XhoI (CGC CTC GAG TCA TAA TAT CAT TCT CTC TAA) and cloned into the HA-tag vector, pKS-HA(Nhex2). From these pKS-HA (Nhex2) clones, NheI fragments (containing HA-tagged PAT-4 cDNAs) were cloned into the NheI site of + colonies were 151. Among these 151, 54 clones were isolated and tested for binding to PAT-4 (Q308H). Among 54 isolated clones, 8 clones retained their ability to bind to PAT-4 (Q308H), and the DNA sequences of these clones were determined . Among 54 isolated clones, 14 clones retained their ability to bind to PAT-4 (I432F), and the DNA sequences of these clones were determined , CAA TCT TTG CTT CTC AAA CGA ATC TTG ATA TGC GC and GCG CAT ATC AAG ATT CGT TTG AGA AGC AAA GAT TG; for UNC-112 (Q27R), CAC CGA TTT GAA TAT TCG AAG AAG CAT CTC TGT CC and GGA CAG AGA TGC TTC TTC GAA TAT TCA AAT CGG TG; and for UNC-112 (F117S), TTT CTC GGT AAA TTC ATC CAA AGC AAC AAA GAA AT and ATT TCT TTG TTG CTT TGG ATG AAT TTA CCG AGA AA. UNC-112 N-terminal fragments with site-directed mutations were confirmed by DNA sequencing, then cloned into pGAD-C3 and tested for binding to PAT-4 (Q308H) and PAT-4 (I432F) by the yeast 2-hybrid system.We utilized random mutagenesis by PCR, as described previously  or pPD49.78/83-HA-UNC-PAT-4 (I432F)/pPD49.78/83-myc-UNC-112 (F301S)  and pTG96 were established by picking up GFP-positive worms using a GFP dissection microscope. Expression of HA-tagged PAT-4 (I432F) and myc-tagged UNC-112 (wild type or F301S) proteins was induced by incubation of the transgenic worms at 30°C for 2 h (heat shock). We prepared Laemmli-soluble protein extracts  ; sfIs15, for pPD49.78/83-HA-PAT-4 (I432F); sfIs16, for pPD49.78/83-HA-UNC-PAT-4 (I432F)/pPD49.78/83-myc-UNC-112 wild type; and sfIs17, for pPD49.78/83-HA-UNC-PAT-4 (I432F)/pPD49.78/83-myc-UNC-112 (F301S). For The extrachromosomal arrays containing pPD49.78/83-HA-PAT-4 (wild type) with pTG96, pPD49.78/83-HA-PAT-4 (I432F) with pTG96, pPD49.78/83-HA-UNC-PAT-4 (I432F)/pPD49.78/83-myc-UNC-112 wild type with pTG96, and pPD49.78/83-HA-UNC-PAT-4 (I432F)/pPD49.78/83-myc-UNC-112 (F301S) with pTG96 were integrated into the genome by ultraviolet irradiation  with somsfIs14, sfIs15, and sfIs17) were exposed to 30°C for 24 h. Worms were fixed using the method described below and immunostained with anti-PAT-6 .To investigate the effect of expression of PAT-4 wild type, PAT-4 I432F, and PAT-4 I432F/UNC-112 F301S on muscle organization, wild-type worms and worms containing integrated arrays  and UNC-112 (kindlin), we isolated missense mutations in PAT-4 that result in lack of binding to UNC-112 using the yeast 2 hybrid system. Using an error-prone PCR approach for generating random mutations, we isolated 16 mutant clones . We founC. elegans muscle, we created transgenic animals expressing HA tagged wild type or mutant PAT-4 proteins. Expression of HA tagged PAT-4  was confirmed by western blot, and was strictly controlled by the heat shock promoters of our constructs  co-expression in body wall muscle of HA-PAT-4 I432F and myc-UNC-112 wild type and (ii) co-expression in body wall muscle of HA-PAT-4 I432F and myc-UNC-112 F301S. Heat shock induced expression of each protein in the 2 transgenic lines was demonstrated by western blot using antibodies to HA and myc . Myc-UNCThe PAT-4 I432F mutant protein cannot bind to UNC-112 but can bind to PAT-6 . In addiThe goal of this study was to obtain more information about an interacting surface between PAT-4 and UNC-112. Previously, we identified extragenic suppressor mutations in PAT-4 that restore binding to UNC-112 D382V [see As we noted in Results, the I432F mutation results in nonbinding to UNC-112, whereas the next amino acid residue A433 when mutated to serine, restores binding to UNC-112 D382V . The previous data and data shown here expand the region and the residues of kindlin family proteins that are likely involved in the interaction with ILK.We have reported previously that the UNC-112 N-terminal half containing the FERM_N and part of the FERM_M domains binds to PAT-4 and that a missense mutation in the FERM_M domain (D382V) abolishes binding to PAT-4 . In factpat-4 and unc-112 are Pat/embryonic lethal  protein that cannot bind to PAT-4, it was shown that PAT-4 binding to UNC-112 is required for UNC-112 to localize to IACs (M-lines and dense bodies)  embryonic lethal . In addition, the effect of PAT-4 I432F overexpression was limited to muscle cell boundaries and dense bodies (somewhat), but not to M-lines. Our previous study suggests that the adhesion plaques at muscle cell boundaries are more dynamic than dense bodies or M-lines : first, the Unc allele, unc-112(r367), is a missense mutation, T85I (unc-112(kq715), L715E, near the end of protein, which shows a defect in the migration of the distal tip cell (unc-112 and pat-4 shown in C. elegans.Worm and yeast strains, and plasmids are available upon request. The authors affirm that all data necessary for confirming the conclusions of the article are present within the article, figures, and tables.G3 online.jkac117_Supplemental_Figure_1Click here for additional data file.jkac117_Supplemental_Figure_2Click here for additional data file.jkac117_Supplemental_Material_LegendsClick here for additional data file.


A worldwide shortage of molecular biology consumables is in surge. This includes filter tips, nucleic acid purification kits, polymerases, reverse-transcriptase, and different types of reagents which are included in viral diagnostic kits. In developing countries, the problem is even worse, since there is few capital enterprise to adopt this kind of industry. So, our aim is to develop a suitable, functional, comparable to commercial ones, and affordable in-house protocol to purify viral RNA. We sought some published and commercial RNA purification solutions to set-up an in-house protocol for viral RNA extraction. Solution was prepared accordingly. Also, LPA (linearized polyacrylamide) carrier was evaluated. The whole setting of in-house solutions with addition of LPA carrier was compared to QIAamp viral RNA minikit solutions. Our results showed that linearized polyacrylamide (LPA) carrier in homemade solutions is comparable to poly A carrier which is used in the most commercial kit. In addition, the whole setting of RNA purification solutions did achieve the purpose of viral RNA purification. Also, the result was confirmed using sputum of a Sars-Cov2 infected patient. Our experiments did end up with an affordable homemade solutions for viral RNA purification. According to Worldmeters data, more than 200 million people were infected with SARS-CoV2 worldwide with 4.4 million death cases . Washing buffer 1 is  and washing buffer 2 is   carrier and it was prepared as Gaillard and Straus protocol which is approximately one hour experiment (Gaillard and Strauss EZ-10 spin columns (Cat.#: SD5005) was purchased from biobasic, Canada.2.In the present study, Vero cells infected with measles virus  was used to optimize RNA purification protocol. Vero cell line is ATCC cell line (cat# CCL-81) and was obtained through a distributing company for biological products & vaccines (VACSERA), Cairo. The cells were grown in DMEM with 10% FBS and 100 IU/ml pencillin/streptomycin. Cells were propagated as a monolayer culture at 37 °C under humidified atmosphere of 5% COVero cells is permissive for cultivation of measles virus and suitable for viral isolation and replication. Cytopathic effect (CPE) of Measles virus infection into vero cells is characterized by formation of syncytium with spikes as it causes cell fusion together and make cells appeared like giant.Therefore, vero cells is used for many applications for measles virus like: laboratory diagnosis of measles virus, viral titration to identify TCID50 using Reed & Maunch equation and in antiviral assays  using MeV210 (GCT ATG CCA TGG GAG TRG GAG TGG) and MeV217 (CAA TGA TGG AGG GTA GG) primers previously published . Qiagen poly A carrier was substituted by LPA carrier and all Qiagen solutions (Lysis and washing buffers) were kept to assess whether changing the carrier is influencing the PCR amplification or not. This experiment was carried out in parallel with Qiagen poly A carrier.From our results, we found that LPA carrier is compatible with Qiagen solutions. Also, the performance of our carrier was comparable to Qiagen carrier. Then, we did proceed to test our solutions with LPA carrier. We did the same as Qiagen protocol as the following with very small modifications:First, adding the LPA carrier to the Lysis buffer contains was prepared by the following equation which was stated in Qiagen handbook:560 μl prepared lysis buffer containing carrier RNA was added into a 1.5 ml micro-centrifuge tube.140 μl of the sample  was added to the lysis buffer + LPA carrier in microcentrifuge tube and mixed by pulse-vortexing for 15 s.The mixture was incubated at room temperature for 10 min.It was centrifuged briefly to remove drops from the inside of the lid.560 μl Ethanol (96–100%) was added to the sample, and mixed by pulse-vortexing for 15 s, followed by brief centrifugation to remove drops from inside the lid.g for 1 min. The collection tube was replaced, the tube containing the filtrate was discarded.Up to 700 μl of the solution from step 6 was applied to spin column (in a 2 ml collection tube), followed by centrifugation at 8000×Step 8 was repeated.g for 1 min. The flow-through was discarded.500 μl of wash I was added to the spin column. Then was centrifuged at 8000×g) for 3 min.500 μl of wash II was added. Then it was centrifuged at full speed . The thermal profile was 30 min at 50 °C, 15 min at 95 °C, forty cycles of amplification , and final extension at 72 °C for 10 min.To visualize PCR products, 2% agarose gel electrophoresis was performed. 50 bp Generuler DNA ladder (Thermoscientific) was used as a standard. The electrophoresis pictures were analyzed using ImageLab software version 6.1 (Biorad).6 copies/reaction to 101 copies/reaction of synthetic fragments of the N gene in the FAM channel and E gene in the ROX channel and the both genes were detected at 10 copies/reaction.A sputum sample was collected from a volunteer with positive coronavirus. RNA extraction was performed using QIAamp viral RNA extraction kit in comparison to our in-house solutions. The purified RNA concentration was measured using Nanodrop spectophotometer. The viral RNA was detected by real-time PCR assay using Vitro S.A One-Step RT Kit. Standard curves were constructed using Serial dilutions from 10According to our objective, the solutions were prepared and tested to execute the RNA purification experiment in the cheapest format, but at the same time with quality comparable to commercially available kits. So, firstly, LPA carrier was tested with QIAamp viral RNA mini kit. Then, the experiments were executed in a workflow as shown in Fig. The main expensive components in RNA purification kit are the carrier and spin-columns. Therefore, in order to make in-house affordable protocol, we did synthesize linearized polyacrylamide carrier in our lab. To test efficiency of the LPA carrier, solutions of QIAamp viral RNA mini kit were used and the poly A carrier was substituted with LPA carrier. Our control was QIAamp viral RNA mini kit whole reagents. After the purification, one-step RT-PCR for measles virus was performed to assess the RNA purification process. Then, amplification of N-450 of Measles virus was visualized by gel electrophoresis and bands intensities were measured using ImageLab software version 6.1 (Biorad) Fig. . AccordiFollowing step of validation was to test LPA carrier with the homemade solutions. Hence, we did follow the protocol as written in the materials and methods section. The RNA purification of measles virus was tested using one-step N-450 and MeV210 and MeV217 primers RT-PCR, and then visualized by 2% gel electrophoresis. Both Qiagen QIAamp viral RNA mini kit  and in-house solutions were carried out in parallel to control the experiment. The captured pictures of electrophoresis were analyzed using ImageLab software version 6.1 (Biorad).In comparison to Qiagen QIAamp viral RNA mini kit, band intensity was approximately 80% of commercially available Qiagen kit as shown in Fig. For further sensitivity testing, Vero cells was diluted before RNA purification to 1:1, 1:10, 1:100, and 1:1000. Also, RT-PCR for N-450 was performed for the purified RNA. The PCR amplicon was detected in all samples with different dilution as shown Fig. A sputum sample from covid-19 positive volunteer was subjected to RNA purification using our in-house solutions in comparison to Qiagen kit. The RNA concentration was measured as shown in Table In SARS-CoV2 pandemic, screening of suspects is highly recommended to reduce the disease transmission FDA . Thus, tSilica-based RNA purification are the most common in both research and clinical diagnostics labs due to their advantage of efficient saving of time and high purity of the eluted nucleic acid (Nicosia et al. To make in-house viral RNA purification buffers for silica-based extraction is to use a type of carrier to increase the RNA yield, especially with those samples having low copy numbers of viruses/genetic materials. Most of the commercially available kits include poly A carrier RNA. However, it is too expensive for the current pandemic situation especially in poor labs. LPA carrier is a very cheap, easy to prepare, and efficient neutral carrier for precipitating picogram amounts of nucleic acids with ethanol and doesn’t interfere with downstream application (Gaillard and Strauss et.al. (Ambrosi et al. In COVID-19 pandemic, several kit were compared with different performance as published by Amrosi In conclusion, our results are giving alternative homemade solutions to purify viral RNA. The method is affordable alternative for researchers during the crisis of materials shortage in the market and comparable to the commercial kit. Moreover, using LPA carrier is novel in RNA purification which is a big advantage over poly A carrier in the cost and easy preparation. With further future modification, it could be used in diagnostics as well. Also, we are now working in synthesizing spin-column which will make the protocol more affordable in low-income countries.


Accumulating evidence indicates that RNA methylation, as the most common modification of mRNA, is of great significance in tumor progression and metastasis. Colorectal cancer is a common malignant tumor of the digestive system that seriously affects the health of middle-aged and elderly people. Although there have been many studies on the biological mechanism of the occurrence and development of colorectal cancer, there are still major deficiencies in the diagnosis and prognosis of colorectal cancer. With the deep study of RNA methylation, it was found that RNA modification is highly related to colorectal cancer tumorigenesis, development and prognosis. Here, we will highlight various RNA chemical modifications including N6-methyladenosine, 5-methylcytosine, N1-methyladenosine, 7-methylguanine, pseudouridine and their modification enzymes followed by summarizing their functions in colorectal cancer. With the fourth modality rate among all diseases, colorectal cancer (CRC) is one of the most commonly seen tumors of the gastrointestinal tract, and its incidence is the highest in developed countries . At presEpigenetic modifications are dynamically reversible and heritable, including DNA methylation, histone modifications, chromatin remodeling, microRNAs (miRNAs) and noncoding RNAs (ncRNAs) which are of great importance in the progression and metastasis of colorectal cancer . RecentlRNA modifications play a key regulatory role in gene expression . In receIn this review, we will summarize the biological characteristics of various RNA modifications, including m6A, m5C, N1-methyladenosine, 7-methylguanine, pseudouridine and their roles in the occurrence and development of colorectal tumors by providing a basis for further search for biomarkers and therapeutic targets.m6A methylation modification is a common RNA methylation modification on the 6th N of adenine. It is a dynamically reversible post-transcriptional modification that is most abundant in mRNA and noncoding RNA . The modIn recent years, m6A modification has gradually become the hot field in studying colorectal tumorigenesis, development and metastasis. m6A modifications have widely altered the expression of certain colorectal tumor-associated genes. Abnormally expressed writer protein, eraser protein, and reader protein, by regulating the level of m6A in different RNAs, affects the function of downstream pathways, such as the classical pathway related to colorectal tumorigenesis and apoptosis, the Wnt pathway and the Hippo pathway, and plays pro- or anti-cancer effects .It was found that METTL3 expression was increased in tumor tissues of colorectal cancer patients, and the higher its expression, the worse the prognosis of patients . METTL3 + T cell infiltration  modifications in mRNA and lncRNA is about 30%–84% . PseudouRNA modification plays an important role as a key post-transcriptional regulator in gene expression, and researchers gradually realize that the functional network of its interaction involves many fields such as metabolism, epigenetics, chromatin remodeling, as well as the immune system. Although research on the epitranscriptome has made great progress, most studies have only focused on the biological functions of m6A modifications in mRNA, and the epitranscriptome includes more than 170 chemical modifications that modify coding and noncoding RNAs. Therefore, the association of hundreds of other RNA modifications with coding and noncoding RNAs remains to be explored. To explore the specific biological functions of various RNA modifications, it is first necessary to develop systematic methods and tools for rapid and quantitative detection of RNA modifications. Most existing sequencing methods are based on second generation sequencing technology, which cannot accurately identify chemical modifications on RNA. When researchers detect specific RNA modifications, they often need to detect RNA modifications using specific antibody-based immunoprecipitation techniques or indirect methods such as chemical labeling and the unique base modification properties of RNA pairings. Although these methods have good feasibility, its reproducibility is still low due to the reasons such as technology and incomplete algorithms. Therefore, future detection technologies and methods for various RNA modifications still need further research and exploration.To date, treatments for colorectal cancer are still very limited. The rapid development of experimental technologies based on high-throughput sequencing and proteomics provides more possibilities for a deep study of the mechanism of RNA modification in colorectal cancer. In colorectal cancer, the complex of methyltransferase modified by m6A methylation, including METTL3, which plays a major catalytic role, has been thoroughly studied and is involved in the regulation of multiple classical signaling pathways in tumor development and apoptosis. However, there are still few studies on other writer molecules, and the roles of WTAP and METTL16, which can act independently, in colorectal tumors are still unclear. Similarly, eraser and reader proteins have been shown to contribute to the occurrence, development, metastasis and drug resistance of colorectal cancers by regulating classical signaling pathways, such as the Wnt pathway and the PI3K/AKT pathway, as well as changing the m6A modification level of noncoding RNAs, but there is still wide room for improvement in the transformation from basic to clinical. Although m6A methylation modification is abundant at the mRNA level, tumor-associated noncoding RNAs  are also regulated by m6A methylation and act in the disease progression of colorectal cancer. circRNAs have recently been found to encode polypeptides. Whether m6A modification plays a key role and the specific mechanism has attracted the attention of researchers. Therefore, it is also essential to pay attention to tumor-associated noncoding RNAs. In addition, few eraser proteins have been found and only two more abundantly studied molecules, FTO and ALKBH5. ALKBH3 has been confirmed to have the function of demethylase, but there are still many areas that we have not studied in the Alk homolog family. It is still a research direction to explore whether more members of this family can function as demethylases. There are many types of reader proteins, and it is still unknown that reader proteins specifically read m6A modifications on different RNAs and play different downstream functions.Various RNA modifications including m6A, m5C and ψ function in the regulation of stem cell function and cell survival under stress. The regulation of these RNA modifications may be of great value in reducing tumor cell chemotherapy resistance and tumor recurrence. The identification of precise epitranscriptomic biomarkers in a specific cell type or tumor microenvironment and the identification of aberrantly modified oncogenic or tumor suppressor effects are of great significance for finding precise molecular targets and developing highly selective and effective therapeutic approaches . In the


In recent years, various newly emerged and re-emerged RNA viruses have seriously threatened the global public health. There is a pressing need for rapid and reliable nucleic acid–based assays for detecting viral RNA. Here, we successfully developed a highly sensitive, easy-to-operate G4-ThT-NASBA system to detect viral RNA that no need for labeled primers and probes. Next, we tested the system for detecting the Classical Swine Fever Virus (CSFV), an RNA virus that causes a highly contagious disease in domestic pigs and wild boar and easily causes huge economic losses. Results showed that the system, integrated the G4-ThT fluorescent biosensor and NASBA (Nuclear acid sequence-based amplification),is capable to detect as little as 2 copies/μL of viral RNA without interfering by other swine viral RNA. Moreover, we were able to detect CSFV RNA within 2 h in serum samples taken from the field in a real-time mode. These findings indicate that the G4-ThT-NASBA system is a rapid, high sensitivity and easy-to-operate technique for RNA detection. The method also has the real-time detection capability which may be easily integrated in a highly automated system such as microfluidic chips. Early virus detection methods mainly used the isolation and culture of the virus from the tissues, blood, etc. of infected individuals. The identification of the virus usually took at least a week2. The method is not only time consuming, but also easy to contamination and misdiagnosis. The diagnosis based on Nucleic Acid Amplification Technology (NAT) can overcome these disadvantages. RT-qPCR has been widely used in RNA virus diagnosis due to its unique advantage. But it mainly disadvantage is in the need for expensive thermal cyclers and fluorophore-labeled probes3. The emergence of nucleic acid isothermal amplification technology and new biosensors could overcome these disadvantages.As one of the biggest threats to global public health, viruses have received widespread attention. In which RNA viruses have brought many challenges to the existing detection and prevention system due to their high mutation rate and frequent recombination4. This isothermal amplification reaction involves three enzymes: AMV Reverse Transcriptase (RT), RNase H and T7 RNA polymerase. This method is suitable for RNA analysis, such as mRNA, rRNA or genomic RNA. NASBA has been used in viral nucleic acid detection due to its advantages such as the combination of reverse transcription and amplification processes and the needless for temperature-changing instruments. And it has shown high sensitivity and accuracy6. Moreover, NASBA also shows some advantages in cooperation with more sensors, including Enhanced Chemiluminescence (ECL) 7, Enzyme-linked Oligonucleotide Capture (EOC)8, Molecular Beacons (MB) 9 and so on.NASBA is a sensitive transcription-based isothermal amplification system (TAS) for the specific in vitro amplification of nucleic acids11. It has been found that the combination of G-quadruplex and Thioflavin T can not only promote the folding of the G-quadruplex structure, but also increase the fluorescence intensity of ThT by more than 1700 times12. Compared with other G-quadruplex sensors, this sensor significantly reduces the dependence on K+ which is the limitation of G-quadruplex as a detection sensor. Moreover, this kind of sensor has more fluorescence response in G-quadruplex RNA than DNA13 and is more suitable for construction of NASBA-based detection methods. In this study, we developed a fluorescence detection method combined with the NASBA and G4-ThT fluorescent sensor which can easily detect viral RNA sensitively and specifically.In recent years, G-quadruplex as a specific nucleic acid structure has been used for molecular diagnosis14. In this study, we took CSFV as the detection object by using the system integrated the G4-ThT fluorescent biosensor and NASBA. It can be achieved sensitive and specific diagnosis of viral RNA in serum within 2 h. Meanwhile, we have also verified the possibility of this method for real-time fluorescence detection, which further increases the practicability of this system. This study provides a new solution for rapid, convenient and instant field diagnosis of viral RNA.Classical Swine Fever Virus (CSFV) is a single-stranded positive RNA virus that can cause highly infectious animal diseases. CSFV has long existed in developing countries and often causes huge economic lossesFigure In order to verify the feasibility of G-quadruplex as a biosensor, we added different G4 RNA or DNA and non-G4 RNA or DNA to the NASBA reaction system to detect its fluorescence spectra. As shown in Fig. + in the system. Three NASBA R primers and five F NASBA primers with different G-quadruplex reverse complementary sequences were optimized. In Fig. To achieve the best performance of the detection method, we have optimized some key factors in the detection process, including the primers, the concentration of ThT and KThe concentration of ThT significantly affect the detection sensitivity of the system. As shown in Fig. + generally promotes the folding of the G-quadruplex. The results are shown in the Fig. + will not have a significant effect on the background fluorescence in the negative product. In the positive product, we found that too much K+ not only does not increase the fluorescence value, but also produces some inhibitory effects. After a series of optimization experiments, we finally selected F2-2 GG29 as the forward primer for the NASBA reaction. The ThT concentration was selected as 4 μM and no additional K+ for the following experiments.The presence of K15, we found that the G4-ThT-NASBA system achieved a lower detection limit.To test the sensitivity of the system, we explored the fluorescence response with different concentrations of CSFV-E2 RNA. In Fig. To test the specificity of the system, we selected BVDV, PRRSV, PPV, PCV2 and PRV and other porcine viruses for analysis. In Fig. We conducted research on real-time fluorescence detection using G4-ThT-NASBA system. The results shown in Fig. We used this system to detect viral RNA in CSFV-infected PK15 cells and serum samples. The results are shown in Fig. Figure 16. In this study, we developed a novel label-free G4-ThT-NASBA system that could specifically detect CSFV viral RNA. Results showed that this system could detect viral RNA at a minimum of 2 copies/μL and without interfering by other porcine viral RNAs. We also tested the possibility of this system for real-time fluorescence detection. Results showed a sensitivity and specificity equivalent to that of the endpoint method which further expands the application scenarios of this system.New and re-emerging viruses are responsible for many recent epidemics. Among these viruses, RNA viruses have brought many challenges to virus detection, prevention and control due to their high mutation rate and high adaptability17, colorimetry18 or fluorescence19. However, these methods need label various tags to nucleic acid sequences to make photoelectric biosensors, which are not only expensive but also difficult to store and thereby limit their application in field detection. As a specific nucleic acid structure, G-quadruplex has been confirmed to realize the nucleic acid detection without chemical label. The split G-quadruplex probe was used to carry out the signal output of the oxidation reaction through the peroxidase properties of the complete G-quadruplex20. Regardless of whether it is based on G-quadruplex peroxidase enzyme activity or fluorescence characteristics, most of the current methods rely on the form of split probes or molecular beacons22. In this study, the detection method of G-quadruplex generated during isothermal amplification has not been reported before.At present, most specific nucleic acid detection methods rely on the hybridization between the probe DNA and the target sequence, and then signal amplification and detection by electrochemistry23. We use the feature to construct and optimize a new biosensor G4-ThT, and combine the sensor with the isothermal amplification method NASBA to develop a new viral RNA detection system. In the past, the detection of CSFV, an RNA virus, mainly relied on virus isolation and serological diagnosis. At present, molecular diagnosis such as RT-qPCR have also been used in CSFV detection. However expensive thermal cyclers and labeled probes are often required, which increases the cost of detection and is not convenient for detection in remote areas24. In addition, Abdolahzadeh et al. used the combination of nested NASBA and Mango aptamers to construct an RNA detection method25. But this method needs two NASBA reactions, which is not very convenient and may be easy contaminated. The G4-ThT-NASBA detection system in this study could overcome these disadvantages very well.The latest research showed that the fluorescent complex ThT could promote the folding of different G-quadruplex DNA or RNAIn conclusion, the G4-ThT-NASBA system provides a more convenient, economical and wider application scenario for the detection of viral RNA. This method is more suitable for transplantation on microfluidic chips and other automated systems due to its simple amplification steps and real-time detection mode.26 mentioned in the previous study, the PCR and NASBA amplification primers designed according to the primer design principle are shown in Table According to the comparative analysis of the CSFV-E2 sequence on NCBI, combined with the reverse complementary sequence of the G-quadruplexThe CSFV E2 DNA is derived from the full-length infectious clone of CSFV pT7SM plasmid deposited in our laboratory. Using the CSFV E2 F and R primers in Table The cells infected with 1 CSFV Shimen strains, 1 BVDV strain (NADL), 1 PRRSV strain (Hubei), 1 PRV strain (HB), 1 PPV strain (HN99), 1 PCVII strain (HBZX) were provided by Hubei Academy of Agricultural Sciences. Clinical swine serum samples infected with CSFV are preserved in our laboratory.Trizol  method was used to extract total RNA from cell and serum samples, according to the manufacturer’s protocol. The purity of the extracted total RNA was determined by measuring the absorbance ratio at 280 nm and 260 nm, and the RNA concentration was based on the absorbance at 260 nm using a Nanodrop 2000c spectrophotometer . RNA samples with a ratio of 260 nm/280 nm of 1.9–2.1 were used for NASBA templates. Ten-fold serial dilutions of RNA from virus isolates were prepared.Different G-quadruplex RNA and DNA probe sequences were sent for synthesis, and the sequences are shown in Table 27. The final volume of the reaction mixture was 25 μL. First, assemble 17.4 μL volume of pre reaction mixture comprising: 50 mM Tris/HCl(pH 8.5), 10 mM MgCl2, 90 mM KCl, 10 mM DTT, each dNTP 1 mM, each NTP 2 mM, 10% (v/v) DMSO, 0.4 μM NASBA primer F and 0.4 μM NASBA primer R. Add 2 μL target RNA, the mixture was incubated at 65 °C for 5 min to unfold the secondary structure of the target RNA, and the reaction mixture was transferred to 41 °C for annealing for 5 min. Finally, add 5.6 μL enzyme mixture, 6 U AMV-Rt, 0.1 U RNase H) to the tube and obtain total volume to 25 μL. The mixture was incubated at 41 °C for 2 h for isothermal amplification of RNA targets. The amplified product can be directly fluorescence detected by ThT in the next step.The NASBA procedure used in this study was previously described by kievits For CSFV RNA fluorescence detection, the above NASBA reaction product was denatured at 80 °C for 3 min, slowly cooled to room temperature, and the final volume was adjusted to 71 μL with DEPC water. 4 μL of 100 μM ThT was then added to the system and incubated at room temperature for 10 min. The mixture was placed in a 96-well black microplate, and the fluorescence value at 490 nm absorption light was detected using the SpectraMax i3x Platform  at 425 nm excitation light.2, 90 mM KCl, 10 mM DTT, 1 mM each dNTP, 2 mM each NTP, 10% (v/v) DMSO, 0.4 μM NASBA Primer F and 0.4 μM NASBA primer R, add ThT at a final concentration of 6 μM to the reaction premix. After adding target RNA, denaturation at 65 °C and annealing at 41 °C, the enzyme premix was added and incubate at 37 °C for 0, 20, 40, 60, 80, 100, 120 min. The products of different reaction times were added to 384-well microplates, and the fluorescence value under 490 nm absorption light was directly detected under 425 nm excitation light.In addition to the required 50 mM Tris/HCl (pH 8.5), 10 mM MgClwww.graphpad.com.The fluorescence value results of the added reactants  were excluded from the effect of the fluorescence value of the microplate without the added reactant. Each experiment was carried out 3 times, and the data were expressed as mean ± standard deviation. Column analysis of different experimental groups, standard error analysis of the mean value of three replicate experimental data, and Fit spline/LOWESS analysis of real-time fluorescence numerical curve were performed using GraphPad Prism version 8.0.2 for Windows, GraphPad Software, San Diego, California USA, Supplementary Figure S1.Supplementary Figure S2.Supplementary Figure S3.Supplementary Figure S4.


The noncoding RNA score ≥ −0.336  was also independently predicted the sICH, the addition of the noncoding RNA score to the clinical variables improved discrimination and reclassification as well. The noncoding RNA score was also associated with the infarct volume and symptom improvement at 7 days after rtPA treatment. In conclusion, a higher neutrophilic noncoding RNA score provides predictive value to identify AIS patients with worse outcomes after rtPA treatment. miRNA-23a, miRNA-99a, and lncRNA H19 are worth further investigation for their effects in thrombolysis after AIS.There’s no evidence demonstrating the association between noncoding RNAs levels before IV recombinant tissue plasminogen activator (rtPA) administration and the outcomes of acute ischemic stroke (AIS). 145 AIS patients received rtPA treatment were recruited at the stroke center from 2018 to 2019, and 103 patients were included in this study. A panel of noncoding RNAs  were measured in the circulating neutrophils of AIS patients before rtPA treatment. Endpoints included excellent outcome  or poor outcome (mRS > 1) at 3 months and symptomatic intracerebral hemorrhage (sICH) after rtPA treatment. Among the eight noncoding RNAs detected in circulating neutrophils of the 103 participants, miRNA-23a levels were associated with the stroke severity on admission and symptom progression at 24 h after rtPA treatment. A noncoding RNA score composed of miRNA-23a, miRNA-99a, and lncRNA H19 was screened to predict the functional outcome at 3 months and the incidence of sICH after rtPA treatment. In the logistic regression analysis, the noncoding RNA score ≥ −0.336 (OR = 2.862 [1.029–7.958],  Recombinant tissue plasminogen activator (rtPA) is the standard thrombolytic agent approved for recanalization to salvage the penumbra after acute ischemic stroke (AIS) . IdeallyNoncoding RNA (ncRNA) is one of the most potential biomarkers for diseases , and linThe aim of this study, therefore, was to investigate the association between the eight potential noncoding RNA candidates  in the circulating neutrophils of AIS patients before rtPA treatment and stroke outcomes as assessed by symptomatic intracerebral hemorrhage (sICH), and modified Rankin Scale (mRS) at 3 months.From November 2018 to May 2019, 345 AIS patients were enrolled in the stroke center of Xuanwu Hospital of Capital Medical University. A total of 145 patients received intravenous thrombolysis with rt-PA. The inclusion criteria were as follows: age ≥ 18 years old; patients presented with neurological deficits and admission within 4.5 h after symptoms onset; a confirmed diagnosis of ischemic stroke  by brainThe primary outcome was defined as an excellent outcome  anbaseline - NIHSS24h) and 7dΔNIHSS (NIHSSbaseline - NIHSS7days) were then recorded (ΔNIHSS ≥ 4 was considered as an improvement of symptoms). The functional outcome was evaluated with the mRS via the face-to-face visit or the telephone interview at 3 months conducted by trained neurologists. All patients and investigators involved in the present study were blinded to the noncoding RNA measures.Baseline variables included demographic characteristics, stroke characteristics and treatment, medical history, stroke etiology, lesion location, and clinical and laboratory findings collected from clinical interviews and neurologic examinations by board-certified neurologists. Stroke severity was assessed by the NIH Stroke Scale (NIHSS)  on admisn = 72), and infarct volumes were calculated on DWI sequences as described previously  administration was conducted according to the recommendations of the Circulating neutrophils were obtained from the blood sampled before rtPA treatment in all patients, preserved in TRIzol Reagent  and then stored in the −80°C refrigerator until detection. All samples were handled according to the same protocol throughout the study.® Ⅱ 1st Strand cDNA Synthesis Kit . Total cDNA was followingly prepared for RT-qPCR with the Hieff® qPCR SYBR Green Master Mix  in a QuantStudio™ Real-time PCR System . The expression of universal U6 was used as the control for miRNA, and GAPDH was used as the control for lncRNA. 2−ΔΔCT method was used for relative quantification of noncoding RNA expression. Each quantitative PCR assay was performed in triplicate independently. The sequences of the primers used in the present study were listed in file e1. All samples were tested by a professional technician blinded to the clinical assessments.Total RNA was extracted from the neutrophil samples with TRIzol Reagent, and then quantified with nanodrop. According to the manufacturer’s instructions, 1 μg of total RNA in a final volume of 20 μl was used for miRNA First Strand cDNA Synthesis (Tailing Reaction)  and lncRNA reverse transcription was conducted with a Hifairp < 0.05 was considered of statistical significance.All the data were analyzed with SPSS 26 , R studio 3.6.1 , and GraphPad Prism 8.4.0 . 2 test or Fisher exact test. We assessed the normality of data distribution with the Kolmogorov-Smirnov test.In the first step, we compared the baseline characteristics and noncoding RNAs levels of the patients according to the stroke severity on admission . Specifiβ0 + βA * noncoding RNAA + βB * noncoding RNAB + βc * noncoding RNAC, etc., where βA, βB, βc, etc represented regression β-coefficients for noncoding RNA A, noncoding RNA B, and noncoding RNA C, respectively, and β0 represented an intercept from the LASSO regression. The noncoding RNA score was also applied to the analysis of the secondary outcome of the patients.In the second step, to predict the outcomes based on joint consideration of all eight noncoding RNAs simultaneously and reduce possible overfitting, we constructed the noncoding RNA score with a penalized regression approach . In detaIn the third step, the predictive value of the noncoding RNA score for stroke outcomes was calculated with the receiver operating characteristics (ROC) curve. The noncoding RNA score was cut off by the index p < 0.05 was set as the criterion for statistical significance.The prediction of the mRNA targets of the noncoding RNAs was performed based on Starbase. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genome (KEGG) enrichment analysis of the noncoding RNAs in the noncoding RNA score was conducted using the Database for Annotation, Visualization, and Integrated Discovery (DAVID). The study design was complied with the principles of the Declaration of Helsinki, and approved by the Ethics Committee of Xuanwu Hospital, Capital Medical University. Written, informed consents were obtained from all enrolled patients or their legal representatives.p = 0.047, for patients with mild stroke and moderate-to-severe stroke, respectively), and a less onset-to-treatment time  were associated with the mild stroke on admission  of the patients had symptomatic intracerebral hemorrhage after stroke and 62 (60.2%) of the patients had excellent outcome at 3 months .p = 0.005) was associated with the mild stroke on admission (p = 0.045) was associated with the 24 h symptom improvement after rtPA treatment , miRNA-99a , and miRNA-494   and miRNA-let-7a ; increased NLR was correlated with decreased miRNA-193a  and miRNA-128a   ; increas < 0.05) .p = 0.009) had a lower incidence of excellent functional outcome at 3 months.To find the association between the noncoding RNAs and outcomes, we constructed the noncoding RNA score using the LASSO regression. miRNA-23a, miRNA-99a, and lncRNA H19 were screened as ideal variables among the eight candidate ncRNAs for the noncoding RNA score. A cutoff point for the noncoding RNA score of −0.336 had 48.8% sensitivity and 69.4% specificity for the prediction of the excellent outcome at 3 months . Patientp = 0.045) was independently predictive of functional outcome at 3 months. After adjustment with clinical variables including age, admission NIHSS score, a history of hypertension, coronary heart disease, atrial fibrillation, cardioembolic stroke, onset-to-treatment time, neutrophil-to-lymphocyte ratio, and total cholesterol (p = 0.044) was still an independent predictor of functional outcome at 3 months , as well as the net reclassification   .p = 0.038) was an independent predictor of sICH with the adjustment of clinical variables , as well as the net reclassification .As the comparisons between the two predictive models shown in p = 0.038) was associated with the symptom improvement at 7 days after rtPA treatment (n = 72) with available infarct volume, the noncoding RNA score was significantly correlated with the infarct volume  , a higher neutrophilic miR-23a level was associated with a higher lncRNA H19 level , and a higher neutrophilic miR-99a level was associated with a higher lncRNA H19 level   . Among t= 0.044) .To investigate the possible enriched biological processes of the noncoding RNA score, we performed bioinformatic analysis of the miRNA-23a, miRNA-99a, and lncRNA H19 in the noncoding RNA score, the top ten significantly enriched biological processes included “Positive regulation of transcription by RNA polymerase II”, “Negative regulation of transcription by RNA polymerase II”, “Regulation of transcription, DNA-templated”, “Regulation of transcription by RNA polymerase II”, “Negative regulation of cell population proliferation”, “Negative regulation of transcription, DNA-templated”, “Positive regulation of cell population proliferation”, “Positive regulation of cell migration”, “Cell junction” and “Positive regulation of autophagy” ; and theIn the present study, we demonstrate an association between the noncoding RNAs of circulating neutrophils and the outcomes of AIS patients received rtPA treatment. A higher noncoding RNA score composed of miRNA-23a, miRNA-99a, and lncRNA H19 is related to sICH after rtPA treatment and a poor outcome at 3 months. Of note, after adjustment for confounders, we find that the noncoding RNA score is an independent predictor for the poor outcome at 3 months and sICH after rtPA treatment, with an additional 2.16-fold risk to have a poor outcome and an additional 5.25-fold risk to develop sICH for noncoding RNA score ≥ −0.336, respectively. Additionally, the noncoding RNA score adds prognostic value to the clinical model for predicting the poor outcome and sICH after rtPA administration. Particularly, the noncoding RNA score is positively correlated with the infarct volume after rtPA treatment, and a lower noncoding RNA score is associated with symptom improvement at 7 days after rtPA treatment.Noncoding RNA is one of the most promising biomarkers for early diagnosis, risk stratification, and prognosis of stroke . A largeA prospective cohort study involved 84 AIS patients received thrombolysis or bridging therapy found three circulating miRNAs detected at 24 h after treatment were associated with the unfavorable outcomes at 3 months . ConsistNoncoding RNA targets multiple genes and proteins and plays a key role in various aspects of the complicated pathological processes of ischemic stroke , which mNeutrophils shape the progression of the immune response in the ischemic brain , thus paThe strength of our study is that we generate the pretreatment noncoding RNA score to establish the prediction model for the functional outcome at 3 months and sICH of AIS patients after rtPA treatment, which takes into consideration of the incremental effect of the noncoding RNA interaction network. There are also some limitations in our study. Firstly, the predictive value of the noncoding RNA score for sICH and poor outcome after rtPA treatment needs to be validated in a larger sample size. Secondly, the levels of noncoding RNA may be dynamic, and the temporal changes of neutrophilic noncoding RNAs levels both pre- and post-rtPA treatment in AIS warrant further study.


Utilizing this property, we developed a method named Split Activators for Highly Accessible RNA Analysis or ‘SAHARA’ to detect RNA sequences at the PAM-distal region of the crRNA by merely supplying a short ssDNA or a PAM containing dsDNA to the seed region. Notably, SAHARA is Mg2+ concentration- and pH-dependent, and it was observed to work robustly at room temperature with multiple orthologs of Cas12a. SAHARA also displayed a significant improvement in the specificity for target recognition as compared to the wild-type CRISPR-Cas12a, at certain positions along the crRNA. By employing SAHARA we achieved amplification-free detection of picomolar concentrations of miRNA-155 and hepatitis C virus RNA. Finally, SAHARA can use a PAM-proximal DNA as a switch to control the trans-cleavage activity of Cas12a for the detection of both DNA and RNA targets. With this, multicomplex arrays can be made to detect distinct DNA and RNA targets with pooled crRNA/Cas12a complexes. In conclusion, SAHARA is a simple, yet powerful nucleic acid detection platform based on Cas12a that can be applied in a multiplexed fashion and potentially be expanded to other CRISPR-Cas enzymes.CRISPR is a prominent bioengineering tool and the type V CRISPR-associated protein complex, Cas12a, is widely used in diagnostic platforms due to its innate ability to cleave DNA substrates. Here we demonstrate that Cas12a can also be programmed to directly detect RNA substrates without the need for reverse transcription or strand displacement. We discovered that while the PAM-proximal “seed” region of the crRNA exclusively recognizes DNA for initiating  Upon infection, the invading DNA sequences are captured and integrated into the host genome between an array of repeat sequences. The captured DNA sequences are called ‘spacers’ and they provide a genetic memory of prior infections3. For prokaryotic immunity, the CRISPR locus is transcribed and processed to generate multiple mature CRISPR RNAs (crRNA), each encoding a unique spacer sequence. Cas (CRISPR-associated) proteins are RNA-guided endonucleases that when complexed with the CRISPR RNAs (crRNAs) can enable the cleavage of nucleic acids that are complementary to the crRNA sequence.CRISPR  is an adaptive immune system encoded within prokaryotes that have evolved to counter invasion by foreign nucleic acids such as bacteriophages and plasmids5. Among these, Cas12a is a class II, type V RNA-guided DNA endonuclease6. Since its discovery, it has been widely used for genome editing as well as molecular diagnostic applications11. Structural and biochemical studies have shown that Cas12a can catalyze the cleavage of DNA substrates14 but there are no reports of targeted RNA cleavage by Cas12a. Recently there has been a report of a novel enzyme named Cas12a2 that sometimes co-occurs with Cas12a systems in bacteria and can use the Cas12a crRNA, but recognizes an RNA target instead of a double-stranded DNA15. A special characteristic of the type V Cas12 family of enzymes is their ability to initiate rapid and indiscriminate cleavage of any non-specific single-stranded DNA (ssDNA) molecules in their vicinity after target-specific recognition and cleavage17. This unique catalytic property, known as trans-cleavage has been harnessed to engineer CRISPR-based diagnostic tools that rely on the cleavage of FRET reporters upon target recognition18.There are several diverse naturally occurring CRISPR/Cas systems found in prokaryotes21. Recently, we discovered that CRISPR-Cas12a can tolerate DNA/RNA heteroduplexes, but only when RNA is located at the non-target strand22. We and others utilized the system to detect RNA targets with Cas12a by simply creating a heteroduplex using a reverse transcription step without amplification23. A reverse-transcription step is inconvenient because it adds to the time, cost, error, and complexity of the assay. The other alternative is to use an RNA-targeting enzyme such as Cas12a215, Cas12g24, or Cas13a-d27; however, these systems can only detect RNA.So far, Cas12a-based tools have been limited to the detection of DNA substrates, unless they are coupled with additional steps involving reverse transcription or strand displacement6. It has been previously shown that DNA-cleaving enzymes such as Cas9 can be manipulated to also cleave RNA through the addition of a PAMmer sequence28. However, similar approaches have not yet been investigated with trans-cleaving Cas enzymes like Cas12, primarily because the PAM recognition mechanism is very different between Cas9 and Cas12 enzymes29. For instance, unlike Cas9, Cas12a can recognize and even trigger trans-cleavage activity using a PAM-less ssDNA. To date, there is no single CRISPR-Cas system identified that can innately tolerate both DNA and RNA substrates to trigger trans-cleavage.Cas12a orthologs are known to require a short protospacer adjacent motif (PAM) to be present on the target DNA to initiate recognition and cleavagetrans-cleavage activity. In essence, we have found that while the PAM-proximal seed region of the crRNA strictly tolerates DNA substrates, the PAM-distal end of the crRNA can tolerate both RNA and DNA substrates with multiple Cas12a orthologs. Thus, by merely supplying a short ssDNA or a PAM-containing dsDNA at the seed region of the crRNA we can detect RNA substrates at the 3’-end of the crRNA. We harnessed this unique property to develop a tool for RNA detection using Cas12a named Split Activators for Highly Accessible RNA Analysis (SAHARA).In this report, we discovered that Cas12a can also tolerate RNA substrates at the PAM-distal end of the crRNA and initiate trans-cleavage activity of Cas12a, and we have utilized them to develop SAHARA, a valuable and versatile tool that can simultaneously detect both DNA and RNA substrates.We achieved reverse transcription (RTx)-free detection of picomolar levels of DNA as well as RNA without amplification using SAHARA and applied it for detecting clinically-relevant targets including hepatitis C virus (HCV) RNA and microRNA-155 (miR-155). We showed that compared to conventional CRISPR-Cas12a, SAHARA has improved specificity and can be performed at room temperature. We demonstrate that its activity can be turned ON or OFF using the seed region binding DNA activator as a switch. We took advantage of this switch to perform multiplexed and simultaneous detection of different DNA and RNA targets. We also coupled SAHARA with Cas13b to perform multiplexed detection of different RNA targets. These key findings provided insights into the substrate requirements for the Lachnospiraceae bacterium ND2006, Acidaminococcus sp. BVL36, and Eubacterium rectale are simply referred to here as Lb, As, and Er, respectively33 . The mature crRNAs for each ortholog are 41–44 nt in length, containing ~19–21 nt of scaffold and the remaining ~20–24 nt of spacer6.LbCas12a, AsCas12a, and ErCas12a are orthologs of Cas12a nucleases that are derived from the trans-cleavage activity of Cas12a . For this, we analyzed the activity produced by truncated activators shorter than 14-nt in a combinatorial fashion  as well as PAM-deprived (-PAM) dsDNA activators complementary to either the Pp or the Pd . We observed the split activator system to have the best activity when a +PAM dsDNA binds at the Pp and the -PAM dsDNA binds at the Pd. We also observed that there is no activity when a -PAM dsDNA binds at the Pp-end, indicating that a PAM is crucial for dsDNA binding at the Pp-end.22. We were curious to check if these modified crRNAs (termed ENHANCE or EN) would have any significant effect on the trans-cleavage activity produced by the split-activator system. We analyzed the detection of ssDNA and ssRNA substrates using the split-activator system with both the wild-type (WT) and EN crRNAs. We observed that both ssDNA and ssRNA had significantly greater activity with the EN crRNA for LbCas12a, while AsCas12a and ErCas12a were unaffected by the different crRNA designs. This is consistent with previous observations with ENHANCE. We observed ErCas12a to have the highest activity with the split-activator system irrespective of the crRNA design being used.  or 8-nt of the Pd region of the crRNA to DNA (crRNA-12R8D) . However, our results indicated that the chimeric DNA-RNA hybrid guides performed poorly in the detection of split activators .Chimeric DNA-RNA hybrid guides have been previously used to increase the sensitivity and reduce the off-target effects for both Cas9 and Cas12 nucleasesSplit Activators for Highly Accessible RNA Analysis) a tool for direct RNA detection with Cas12a. The design of SAHARA was such that a short (12-nt), synthetic PAM-containing dsDNA activator (named ‘S12’ due to its length) would bind to the Pp-end of the crRNA, while any target RNA sequence can be detected at the Pd-end. While it was clear that short RNA activators could be easily detected by SAHARA, this study aimed to aid in the optimization of detection for a variety of targets and lengths. Therefore, we designed two RNA targets: a short RNA (20-nt) and a longer RNA (~730-nt) containing the same 20-nt target sequence . While all three crRNAs tested displayed some level of RNA detection, the 24-nt crRNA had the best activity for RNA detection with SAHARA.While a fully complementary RNA activator is not recognized by Cas12a, we have shown that RNA can be recognized at the 3’-end of the crRNA if DNA is bound at the seed region. Therefore, somewhere between the seed region and the 3’-end of the crRNA protospacer, RNA recognition starts being tolerated. To examine the exact position at which RNA detection begins, we designed increasingly long RNA activators of length ranging from 6–14 nt that are complementary to the Pd region of the crRNA, and their corresponding DNA activators of different lengths binding to the Pp region . We tested these DNA-RNA combinations in tandem and observed that while RNA activators of length 6–10 nt are recognized by Cas12a, increasing the length of the RNA activator beyond 10-nt stops any trans-cleavage activity. This suggests that the Pp 10-nt nucleotides of the crRNA protospacer can exclusively recognize DNA targets while the Pd 10-nt can detect both RNA and DNA.While the catalytic activity of Cas12a enzymes is optimal at 37°C, the short (12-nt) DNA activators that are necessary for SAHARA to function, can bind more stably at temperatures lower than 37°C, due to their lower Tm. To study this, we tested the detection of an ssDNA and ssRNA target each at 37°C and room temperature (RT) . We observed that both targets can be detected at RT. However, the activity drops by 3- to 5-fold depending on the Cas12a ortholog being used.2+ ions are routinely used in Cas12a-based applications, Mn2+ ions have also been shown to work well37. To study the effect of different metal ions on the activity of SAHARA, we tested a range of different metal cations  and discovered that most metal ions including severely inhibited the trans-cleavage activity of SAHARA .Cas12a effectors are metal-dependent endonucleases and therefore, the type and concentration of metal ions used in the reaction can have a significant effect on their activity. While Mg2+ ions displayed the highest activity in-vitro, consistent with the literature. Therefore, we characterized the effect of increasing the concentration of Mg2+ ions on the activity. By varying the amount of Mg2+ ions in the Cas12a reaction, we observed a significant increase in activity with increasing concentrations up to 15 mM. Increasing the Mg2+ concentration beyond 15 mM slightly decreases the activity, suggesting 15 mM is the optimal concentration of Mg2+ for SAHARA .Among the different divalent metal ions tested, MgFinally, the pH of the reaction can have a notable effect on the charge of the enzyme and can subsequently affect its activity. We screened the activity of SAHARA in different buffers with pH ranging from 5.5–9.25. We observed that SAHARA has optimal activity at pH = 7.9 and the activity drops sharply upon further increasing or decreasing the pH . There was no observed activity at pH = 9.25 for any of the three orthologs tested.38. For HCV, we synthesized a short target RNA resembling a polypeptide precursor gene that is conserved across multiple HCV genotypes. For this RNA, we designed three different SAHARA guide RNAs each targeting it at either the 5’-end (head), 3’-end (tail), or in the middle (mid)  .40. Therefore, reliable detection of miR-155 is important for early diagnosis. The mature miR-155 is ~23-nt in length. We synthesized the mature miRNA target and designed two SAHARA guides targeting it at either 12-nt at the 5’-end or 11-nt at the 3’-end, keeping the S12-binding region constant.The miRNA-155 (or miR-155) is known to play a crucial role in breast cancer progression and is overexpressed in breast cancer tissuestrans-cleavage, we observed significant variability in the activities produced by different guides. For miR-155, the head targeting guide showed an almost 5-fold higher activity than its tail targeting counterpart. Similarly, for HCV, the tail targeting guide showed as much as a 6-fold increase in activity as compared to head-targeting and the mid-targeting guide . Targets with a high amount of secondary structure are more inaccessible to bind to the Cas12a-crRNA complex, and are therefore harder to detect, while targets with relatively low or no secondary structure are detected easily. For miRNA-155, the fact that the tail guide only binds to 11-nt of the target as opposed to 12-nt binding in the head guide might play a role in the reduced activity.41. We rationalized that a similar approach would also work with SAHARA. In concordance with the previous reports41, SAHARA also displayed a higher activity with pooled crRNAs for both miRNA-155 and HCV targets.It has previously been shown with Cas13a enzyme that pooling together multiple crRNAs enhances the level of detection42.Finally, we tested to check the sensitivity of SAHARA. For this, we created dilutions of miRNA-155 and HCV targets ranging from 25 nM - 10 fM and tested for the detection of each target at different concentrations with their corresponding pooled crRNAs. We obtained a limit of detection of 132 pM for HCV and 767 pM for the miRNA-155 RNA target ,f. Thesetrans-cleavage activity for Lb and Er , with unique targets and S12 activators, and pooled them all together. To demonstrate that we can simultaneously detect DNA and RNA with SAHARA, we used an ssDNA target A for crRNA-a while targets B and C were ssRNAs. We then performed the detection of each of the three targets with the pooled crRNAs and different Cas12a orthologs, in a combinatorial fashion, in the presence or absence of the corresponding S12. We also used a control wherein no S12 sequence was supplied to the crRNA-Cas-target mix.We could leverage the switch-like function of the seed-binding S12 DNA activator to selectively turn ON the trans-cleavage activity with any of the target combinations despite the crRNA-Cas complex and the target being mixed, further reinforcing the idea that S12 is critical for activity with SAHARA. In the presence of different S12s, only the mixtures with the corresponding target and crRNA displayed trans-cleavage. For instance, in the presence of S12a, only the reactions where target A was available  were active, whereas the reactions without target A  were inactive despite the Cas, crRNA-b, crRNA-c, and their respective targets being mixed. The data here shows strong evidence that the S12 DNA can be used to selectively activate specific crRNAs and can be used to control the trans-cleavage activity of CRISPR-Cas12a, thereby enabling simultaneous and multiplexable detection of both DNA and RNA targets.In the no S12 control, there was no 26. We used DNA and RNA reporters consisting of orthogonal dyes to differentiate the signal obtained from SAHARA and Cas13. This is similar to the multiplexed detection with Cas13 and Cas12 demonstrated before26, but here we aimed to detect RNA substrates with both Cas12 and Cas13, and not DNA. To test this, we used Lb, As, and Er Cas12a orthologs in conjunction with PsmCas13b to detect two distinct RNA targets (T1 and T2). We designed Cas12 and Cas13 guide RNAs such that the guide for Cas12 was complementary to only the T1 target while the guide for Cas13 was complementary to only T2. We also used a FAM-containing DNA reporter and a HEX-containing RNA reporter to distinguish the signals of SAHARA and Cas13b.Next, we postulated if we could perform multiplexed RNA detection by combining SAHARA with Cas13btrans-cleavage activity only in the presence of the T1 target, while Cas13b produced trans-cleavage only in the presence of T2  + 6-nt (Pd) shows higher activity than the combination of 6-nt (Pp) + 14-nt (Pd) despite having identical total length and sequence. These observations reinforce the idea that the seed region of the crRNA plays a vital role in modulating the trans-cleavage activity of Cas12a orthologs.We discovered that truncating the length of ssDNA activators binding to the crRNA drastically diminishes the trans-cleavage, the PAM-distal end of the crRNA could tolerate RNA along with DNA. The exception here was AsCas12a, which was able to tolerate RNA even at the PAM-proximal end, unlike Lb or Er Cas12a. We also observed that using a PAM-containing dsDNA at the Pp-end greatly increases the trans-cleavage as compared to using an ssDNA for two of the three Cas12a orthologs (As and Er).Furthermore, upon changing the truncated activators from ssDNA to dsDNA or RNA, we observed that while the PAM-proximal seed region of the crRNA exclusively tolerates DNA for initiating Surprised by our results, we wondered if we could detect longer lengths of RNA sequences at the PAM-distal end of the crRNA. To enable this, we designed long crRNAs containing 24-nt of the spacer. Within this spacer, 12-nt at the Pp end was complementary to a PAM-containing dsDNA that we call S12, while 12-nt at the distal end was complementary to the target of interest. We named this split-activator-based method of RNA detection with Cas12a as SAHARA. Upon testing the detection of short and long RNA substrates with WT CRISPR-Cas12a and SAHARA, we observed that WT Cas12a could not detect RNA at all, or in the case of As, had a very small level of RNA detection. With SAHARA, however, all three cas12a orthologs displayed robust RNA detection activity for different lengths of RNA. ErCas12a was observed to have the strongest activity for the detection of long RNA sequences.trans-cleavage activity and that changing the PAM sequence eliminates the activity for Lb and As Cas12a while reducing it for Er. Changing the GC content of the S12 activators seemed to have negligible effects on the activity of all three orthologs, suggesting that a wide range of GC content can be used for the S12 activator. Remarkably, we found that the S12 concentration as low as 50 pM was sufficient to trigger the trans-cleavage of a 30 nM crRNA-Cas complex for detecting a 25 nM target. These results indicated that very low levels of the S12 DNA binding to the crRNA are sufficient for detection; however, the fluorescence intensity output is increased at higher concentrations. Notably, in the absence of the S12, there is no trans-cleavage activity with any of the three Cas12a orthologs, implying that the S12 can be used as a switch to control the trans-cleavage of Cas12a.Furthermore, we found that the presence of a PAM sequence on the S12 is necessary for robust We applied SAHARA for the detection of clinically relevant targets such as Hepatitis C virus (HCV) and miRNA-155. We found that the secondary structure of the target played an important role in the RNA detection activity of Cas12a. Increased secondary structure in the RNA target makes it more inaccessible to bind to the crRNA and reduces the activity. We also observed that pooling together multiple crRNAs targeting different regions of the activator enhanced the detection capability, as previously reported for Cas13. Using this knowledge, we were able to detect picomolar levels of both miRNA-155 as well as HCV with SAHARA.trans-cleavage activity of a single crRNA from a pool of different crRNAs. We were also able to combine SAHARA with Cas13b for multiplexed detection of different RNA targets using DNA and RNA reporters containing orthogonal fluorescent dyes. Thus, SAHARA can be used as a unique diagnostic system that can detect both DNA and RNA targets simultaneously and in a multiplexable fashion.We also found SAHARA to be highly specific to mutations at certain positions along the target. Mutations at positions closer to the interface of the S12 DNA and the bound target seemed to be especially detrimental in inhibiting activity. Finally, we used the switch-like behavior of the S12 activator to turn ON the 46. Briefly, plasmids expressing LbCas12a and AsCas12a were obtained from Addgene (a gift from Zhang lab and Doudna lab) and directly used for protein expression. For ErCas12a, a plasmid containing the human codon-optimized Cas12a gene was obtained from Addgene, then was PCR amplified using Q5 Hot Start high fidelity DNA polymerase , and subcloned into a bacterial expression vector . The product plasmids were then transformed into Rosetta™(DE3)pLysS Competent Cells  following the manufacturer’s protocols.Plasmids expressing Lb, As, and ErCas12a enzymes were constructed as described in Nguyen et.alFor protein production, bacterial colonies containing the protein-expressing plasmid were plated on an agar plate and grown at 37°C overnight. Individual colonies were then picked and inoculated for 12 hours in 10 mL of LB Broth . The culture was subsequently scaled up to a 1.5 L TB broth mix and grown until the culture reached an OD = 0.6 to 0.8. The culture was then placed on ice before the addition of Isopropyl β- d-1-thiogalactopyranoside (IPTG) to a final concentration of 0.5 mM. The culture was then continued to grow overnight at 16°C for 14–18 hours.2+ was stripped off and recharged with Co2+) pre-equilibrated with Wash Buffer A  connected to BioLogic DuoFlow™ FPLC system (Bio-rad). The column was eluted with Elution Buffer B . The eluted fractions were pooled together and transferred to a 10 kDa – 14 kDa MWCO dialysis bag. Homemade TEV protease (44) was added to the bag, submerged in Dialysis Buffer  and dialyzed at 4°C overnight.The overnight culture was pelleted by centrifuging at 10,000 × g for 5 minutes. The cells were then resuspended in lysis buffer . The cell mixture was then subjected to sonication followed by centrifugation at 39800 × g for 30 minutes. The cell lysate was filtered through a 0.22 μm syringe filter  and then ran through into 5 mL Histrap FF  before injecting into 1 mL Hitrap Heparin HP column pre-equilibrated with Wash Buffer C operated in the BioLogic DuoFlow™ FPLC system (Bio-rad). The protein was eluted from the column by running a gradient flow rate that exchanges Wash Buffer C and Elution Buffer D . Depending on how pure the protein samples were, additional size-exclusion chromatography may have been needed. In short, the eluted protein from the previous step was run through a HiLoad® 16/600 Superdex® . Eluted fractions with the highest protein purity were selected, pooled together, concentrated using a 30 kDa MWCO Vivaspin® 20 concentrator, snap-frozen in liquid nitrogen, and stored at −80°C until use.The protein mixture was taken out of the dialysis bag and concentrated down to around 10 mL using a 30 kDa MWCO Vivaspinin-vitro transcribed using the HiScribe T7 High Yield RNA synthesis kit (NEB #E2040S) following the manufacturer’s protocol. The transcribed product was treated with DNase I for 30 min at 37°C and then purified using RNA Clean and Concentrator Kit (Zymo Research #R1016).All DNA and RNA oligos as well as the chimeric DNA/RNA hybrid crRNAs were obtained from Integrated DNA Technologies (IDT). Single-stranded oligos were diluted in 1xTE buffer . Complementary oligos for synthesizing dsDNA were first diluted in nuclease-free duplex buffer  and mixed in a 1:5 molar ratio of target:non-target strand. Both strands were then subjected to denaturation at 95°C for 4 mins and gradient cooling at a rate of 0.1 °C/s to 25°C. For generating the 730-nt long GFP target sequence, Addgene plasmid pCMV-T7-EGFP (BPK1098)  was obtained and PCR amplified using Q5 Hot Start high fidelity DNA polymerase  from position 376–1125. The PCR amplified product was 4+, Rb+, Mg2+, Zn2+, Co2+, Cu2+, Ni2+, Ca2+, Mn2+, and Al3+) were diluted to a final concentration of 10 mM. The pH of the buffer was adjusted to 7.9 by adding 1M NaOH.The different metal ion buffers were prepared by first creating a master mix of the following components: 50 mM NaCl, 10 mM Tris-HCl, and 100 μg/ml BSA. To this master mix, chloride salts of different monovalent, divalent, and trivalent cations , the