Togaviridae, genus Alphavirus , Flaviviridae , and Peribunyaviridae, genus Orthobunyavirus, California serogroup . The principal focus of this study was Inkoo (INKV) and Sindbis (SINV) virus circulating in Norway, Sweden, Finland, and some parts of Russia. These viruses are associated with morbidity in humans. However, there is a knowledge gap regarding reservoirs and transmission. Therefore, we aimed to determine the prevalence of INKV and SINV in blood sucking insects and seroprevalence for INKV in semi-domesticated Eurasian tundra reindeer (Rangifer tarandus tarandus) in Norway.Mosquito-borne viruses pose a serious threat to humans worldwide. There has been an upsurge in the number of mosquito-borne viruses in Europe, mostly belonging to the families In total, 213 pools containing about 25 blood sucking insects (BSI) each and 480 reindeer sera were collected in eight Norwegian reindeer summer pasture districts during 2013–2015. The pools were analysed by RT-PCR to detect INKV and by RT-real-time PCR for SINV. Reindeer sera were analysed for INKV-specific IgG by an Indirect Immunofluorescence Assay  and a Plaque Reduction Neutralization Test .Aedes spp. were the most dominant species among the collected BSI. Two of the pools were positive for INKV-RNA by RT-PCR and were confirmed by pyrosequencing. The overall estimated pool prevalence (EPP) of INKV in Norway was 0.04%. None of the analysed pools were positive for SINV. Overall IgG seroprevalence in reindeer was 62% positive for INKV by IIFA. Of the 60 reindeer sera- analysed by PRNT for INKV, 80% were confirmed positive, and there was no cross-reactivity with the closely related Tahyna virus (TAHV) and Snowshoe hare virus (SSHV).The occurrence and prevalence of INKV in BSI and the high seroprevalence against the virus among semi-domesticated reindeer in Norway indicate that further studies are required for monitoring this virus. SINV was not detected in the BSI in this study, however, human cases of SINV infection are yearly reported from other regions such as Rjukan in south-central Norway. It is therefore essential to monitor both viruses in the human population. Our findings are important to raise awareness regarding the geographical distribution of these mosquito-borne viruses in Northern Europe.The online version contains supplementary material available at 10.1186/s12985-022-01815-0. Togaviridae , Flaviviridae , and Peribunyaviridae  ) p > 0.04, while Tana (2014–2015) had the lowest seroprevalence of 30% with an OR of 0.2 (95% CI [0.1–0.9]) p > 0.03 . Similarly, for the winter season 2015–2016, 30% of the reindeer samples from Tana were seropositive and the highest seroprevalence was detected in Lakselv (85%).The IIFA results illustrated a high average seroprevalence of 67% in Lakselv, Tromsø, and Fosen while the lowest average seroprevalence of 40% was in Tana. There was no statistically significant difference in seroprevalence among the eight different reindeer pastures for the season 2013–2014 (50–73%). However, there was a significant difference between the locations both in 2014–2015  of the reindeer were female, 23% (112/480) were male, and 2% (9/480) of unknown gender  of the samples were confirmed to be negative and had a titre ˂ 20 while 13 (24%) of the remaining samples were positive on neutralization test.Sixty samples of the 480 reindeer sera with strongly positive (25), borderline (25), or negative (10) on IIFA were analyzed by PRNT. Of these 48 (80%) were confirmed seropositive and had neutralizing antibodies against INKV with titre ≥ 20, six samples (10%) were seronegative with titre ≤ 10 and six samples (10%) had no neutralization. Among the seropositives 39% (19/48) had titre ≥ 20–40, 52% (25/48) had titre ≥ 80–160, 8% (4/48) had titre ≥ 320. None of the samples showed any cross-reactivity with TAHV and SSHV.The frequent movement of humans and animals across countries might contribute to the spread of viruses and vectors. Currently, there is no INKV surveillance in Norway. As part of the surveillance analysis of viral infections that may cause encephalitis, an IgG INKV IIFA was performed. We analysed reindeer sera as sentinel animals for the surveillance of possible circulation of INKV-infection in Norway. IIFA can be used in INKV monitoring programs to test antibodies in reindeer as well as for diagnosis of INKV infection in other animal species and humans.Ae. communis mosquitoes feeding on large mammals, such as cattle, reindeer, and moose which show high INKV antibody prevalence [Reindeer have previously been suggested to serve as amplification hosts for INKV . The virevalence . Detectievalence , 45, 46.evalence .IIFA for INKV is known to cross-react with other California serogroups of viruses such as TAHV and SSHV in Finland , 46. NevAedes spp. in Norway, but the methods were not specific enough to identify and confirm the findings as INKV or if they represented other related CE group viruses. Those viruses were isolated from mosquitoes collected from Trandum and Øyern (Akershus), and Masi (Troms and Finnmark) in 1975 and Sjusjøen (Innlandet), Trysil (Innlandet) and Trandum (Akershus) in 1976 [The distribution and prevalence of INKV in mosquitoes in Norway is largely unknown. However, INKV is circulating in Northern Europe including Norway , 16. SimAedes spp. are the biological vector of INKV in Norway, since INKV has been detected previously in adult Aedes (Ae.) communis, Ae. hexodontus and Ae. punctor mosquitoes and in Ae. communis larvae [In the present study, INKV were detected in two out of 213 pools of BSI collected in Fosen and Røros in the summer of 2014–2015, supporting previous findings indicating that INKV may be circulating in Norway , 16. Alts larvae , 15–17. Aedes spp. with lower numbers of Culex spp. SINV has mostly been isolated from Culex mosquitoes in studies from Sweden, and Cx. torrentium has been identified as the main vector of SINV in Scandinavia [Aedes in Norway [Culex. spp. in the present study we cannot rule out the presence of SINV in the studied areas. Also, the presence of migratory birds could affect incidence of SINV. However, SINV could not be detected from the collected BSI pools by RT-real-time PCR in the present study. Increasing the sample size of BSI or pure mosquito pools might have increased the possibility to detect positives.Many mosquitoes collected in this study were identified as ndinavia . Neverthn Norway , 35. Then Norway . The virn Norway , which iThe findings in this study are important for public awareness about geographical distribution of the INKV. This knowledge may help to take necessary preventive measures against INKV infections in the future. In further studies it would be interesting to screen mosquitoes and the human population to determine the seroprevalence of INKV during the mosquito season, as well as whole genome sequencing of isolated INKV to identify which strains are circulating in Norway.There are knowledge gaps regarding the occurrence, distribution, and prevalence of the mosquito-borne viruses, INKV and SINV in Norway. The last study on these viruses was in 1992, reporting circulation of these viruses in the country. Our study is important to establish a baseline and provide new knowledge on the prevalence and distribution of INKV in BSI and reindeer. Moreover, it is important to monitor these viruses in case of an outbreak, and to avoid undiagnosed and unreported human cases. This study indicates that there is a low prevalence of INKV in BSI in Norway. The high seroprevalence of INKV in reindeer may indicate a risk of virus transmission from BSI to humans in these regions. Reindeer are one of the major hosts for INKV-infections and serve as an important sentinel animal for circulation of INKV. The occurrence and prevalence of INKV in vectors, animals, and the human population in Norway and other countries in Northern Europe requires further studies and monitoring.Additional file 1: Table S1. Distribution of IgG antibodies against INKV reindeer sera determined by IIFA for the winter seasons of 2013–2014, 2014–2015 and 2015–2016 with number of total positive/number total tested, seropositivity rate %, standard deviation (SD) and confidence interval (95% CI).


Various strategies have been developed to bypass this limitation and are often time-consuming, low-yielding and labor-intensive. In particular, the synthesis of RNA with internal 2′-azido modifications is restricted to a single approach that employs P(v) chemistry instead of the widely used P(iii) phosphoramidite chemistry. To fill this methodological gap, we present a novel convenient path toward 2′-azido RNA from readily accessible 2′-amino RNA through treatment with the diazotizing reagent fluorosulfuryl azide (FSO2N3). A diazotransfer reaction was established for oligoribonucleotides of different lengths and secondary structures. The robustness of the approach was further demonstrated for RNAs containing multiple 2′-azido moieties and for RNAs containing other sensitive modifications such as thiouridine or methylated nucleobases with a positive charge. The synthetic ease of generating 2′-azido RNA will pave the way for biotechnological applications, in particular for siRNA technologies and for referencing the growing number of RNA metabolic labeling approaches that rely on 2′-azido nucleosides.Azides are versatile bioorthogonal reporter moieties that are commonly used for site-specific labeling and functionalization of RNA to probe its biology. The preparation of azido modified nucleic acids by solid-phase synthesis is problematic due to the inherent reactivity of P( One-step conversion chemistry makes RNA with internal site-specific 2′-azido modifications readily accessible from 2′-amino RNA; it is significantly more efficient compared to thus far used phosphotriester chemistry to generate such RNA. However, the chemical synthesis of azido modified oligonucleotides currently suffers from severe limitations since azides exhibit pronounced reactivity toward P(iii) species (such as nucleoside phosphoramidite building blocks) according to Staudinger-type reactions.10–17For chemical biology, 2′-azido modified RNA is an upcoming tool whose physicochemical properties have been reported earlier.3 modified RNA is provided by the integration of phospho(V)diester building blocks which are manually incorporated by a single coupling cycle of the phosphotriester method during otherwise automated standard oligonucleotide synthesis.10,11,17 Consequently, an expansion of the methodological repertoire for efficient and flexible access to 2′-N3 modified RNA is urgently needed. Therefore, we developed a convenient path starting from readily available 2′-NH2 modified RNA which is converted into 2′-N3 RNA by a diazotransfer reaction  in the presence of divalent metal ions. In 2020, Meng et al.19 demonstrated the rapid and quantitative conversion of a variety of small, organic amines into their corresponding azides in a biphasic solvent system containing potassium bicarbonate. Remarkably, the reaction requires only one equivalent of fluorosulfuryl azide (FSO2N3) and was deemed complete within 5 min at ambient temperature for most of the substrates. Shortly after, Krasheninina et al.20 demonstrated the applicability of FSO2N3 on native RNA and RNA–peptide conjugates containing an aliphatic primary amino group. The reaction conditions were found to be rather similar with the exception that full conversion required two equivalents of FSO2N3 and longer reaction times. To further expand the scope of substrates and to enhance the methodological repertoire specifically for the synthesis of 2′-N3 modified RNAs, the conversion of 2′-NH2-RNAs into their corresponding 2′-N3 analogues has been envisioned here. Because the 2′-position is much more sterically hindered than that in all the previously reported substrates, FSO2N3 may however not be able to approach the reaction centre sufficiently.The potential of diazotransfer reactions on aminoalkylated DNA was first reported by Defrancq and coworkers2 nucleoside phosphoramidites were obtained from commercial sources or were synthesized in-house;21–23 the corresponding 2′-NH2 modified RNA was prepared using standard solid-phase synthesis and deprotection protocols.The canonical 2′-NH2 RNA precursor 1 with FSO2N3 following the conditions described by Krasheninina et al.,20 however, did not lead to any product formation. Only when significantly longer reaction times and increased concentration of the diazotizing reagent were used, we achieved complete conversion  is shown in 2N3 in methyl tert-butyl ether (MTBE) and 24 h reaction time were found to be suitable to achieve full conversion.Incubation of the 2′-NH24 was synthesized and a single 2′-NH2-A was placed in the middle of the stem (13), in the loop region (14) or at the 5′-terminus (15)  at once, and a second hairpin (17) was prepared forming a base pair between 2′-NH2-A and 2′-O-(3-aminopropyl)uridine.12,13,25 The modified RNAs were treated with FSO2N3 for 24 h and clean conversions for the sequences were observed  was equipped with all four standard 2′-NHobserved .3C) was tested   and used e.g. for biotinylation and RNA pull-down, covalent attachment to fluorophores, or photoactivatable-ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP).28–31 Moreover, metabolic labeling-based RNA sequencing methods  have emerged which became indispensable tools for analysing cellular RNA dynamics.26 The 4sU modification, however, can also undergo undesired side reactions like oxidation, dimerization, hydrolysis and degradation. Selective derivatization of 4sU modified RNA that leaves the sensitive 4-thio moiety intact is therefore rare. We show that the treatment of 4sU containing RNA (18) with FSO2N3 indeed results in the selective formation of the desired 2′-N3 modified RNA (18az) preserving the parent 4sU modification. The obtained 4sU and 2′-N3 modified RNA offers two orthogonal conjugation sites to combine the novel RNA sequencing approaches with efficient bioconjugation reactions.Subsequently, the compatibility of the diazotransfer reaction with other sensitive and reactive RNA modifications like 4-thiouridine (4sU) and 3-methylcytidine  . The cor3C) which is another fragile modification due to its positively charged nucleobase. In tRNA, m3C has long been known,32,33 but recently this modification gained increasing attention due to its evidential occurrence in the mRNA of mice and humans.34 Furthermore, the self-methylation activity of a naturally occurring riboswitch scaffold35 and various approaches for detection and genome-wide m3C sequencing36–38 have been reported. Although m3C is prone to nucleobase loss (depyrimidization) and/or hydrolysis to 3-methyluridine  in good yields.Next, we focused on testing the diazotransfer reaction for RNA (19) containing 3-methylcytidine  containing either a single 2′-NH2- or a single 2′-N3 adenosine at the same internal position, flanked by 5′-C and 3′-U  and/or to the larger steric hindrance of 2′-N3 compared to that of the 2′-NH2 group.2′-Amino and 2′-azido nucleotide triphosphates (NTPs) are only modestly accepted substrates for native RNA polymerases; however, polymerase mutants have been described that make such NTPs effective for enzymatic RNA synthesis.and 3′-U . Both mo10,11 being equivalent or even superior to 2′-F and 2′-OCH3 modified siRNAs that have found biomedical and therapeutic applications.42,43 As shown previously, single 2′-N3 moieties were excellently tolerated in the guide strand of the siRNA duplex and caused efficient gene silencing even when the modification was located in the seed region or at the cleavage site.10,11Earlier on, we have pointed out the promising potential of 2′-azido modified RNA for RNA interference,3 RNA synthesis in our hand, we further aimed at demonstrating their siRNA potential by directly comparing with the corresponding 2′-NH2 counterparts and by explicitly placing multiple azides into the modification-sensitive seed region of the siRNA antisense strand. Knockdown of the brain acid soluble protein 1 (BASP1)44 from the DF-1 chicken cell line by transient siRNA transfection was selected as an established model system  did not downregulate.The Northern blot and the corresponding graph depicted in 2 or 2′-N3 installed at C4  in the seed region led to efficient silencing. The picture changed when more modifications were present. For the 2′-amino modified siRNAs U8/A13as (22) and U3/U6/U8/A13as (23), the silencing capabilities were abolished. Interestingly, the two corresponding 2′-azido modified siRNAs U8/A13as (22az) and U3/U6/U8/A13 (23az) still exhibited significant silencing activity, which strongly underlines their virtue for siRNA design and performance. The results were independently confirmed by qPCR analysis polymerases. Additionally, through the manipulation of enzymes in the nucleotide salvage pathway, further azido-modified nucleosides have been applied for RNA metabolic labeling. It has been shown that the overexpression of nucleoside kinase UCK2 or deoxynucleoside kinase dCK enables RNA labeling with 2′-azidouridine46 and 2′-azidocytidine,47 respectively. Moreover, 2′-azidoguanosine has been utilized for AIR-seq (azidonucleoside-incorporated RNA sequencing) which enabled the genome-wide analysis of transcription upon heat stress in Escherichia coli, and hence, represents a new benchmark for metabolic RNA labeling to probe RNA dynamics in bacteria.48 All these novel developments have significantly increased the demand for site-specifically labeled, chemically synthesized 2′-azido RNAs which are needed to reference the approaches. Additionally, 2′-azido RNA is required for siRNA technologies.2′-Azido RNA has attracted much interest recently because of novel opportunities for the cell and tissue-specific metabolic labeling of nascent RNA. Spitale and co-workers demonstrated the efficient metabolic incorporation of 2′-azidoadenosine into cellular RNA.2N3) were investigated. This reagent was previously introduced for NH2-to-N3 conversions on small organic compounds,19,49 and also used on native RNAs and synthetic RNA–peptide conjugates with structurally well accessible primary amino groups.20 For the here demonstrated conversion of the sterically hindered 2′-NH2 groups in RNA into their 2′-N3 modified counterparts, the diazotransfer reaction had to be carefully optimized, and finally was successful for longer RNA containing multiple 2′-NH2 moieties and other sensitive nucleoside modifications, such as 4-thiouridine and 3-methylcytosine. The synthetic ease of generating 2′-azido RNA will pave the way for novel applications in RNA biology, and strengthen existing ones in siRNA technologies and RNA metabolic labeling.Thus, to satisfy the growing demand, a novel synthetic methodology was conceptualized and diazotransfer reactions on readily accessible 2′-amino RNA with fluorosulfuryl azide (FSOThere are no conflicts to declare.OB-020-D2OB01560A-s001


Cutaneous malignant melanoma (CMM) is curable if detected in its early stages. However, the clinical recognition of CMM is challenging. An American research group has shown promising results in detecting CMM based on RNA profiles sampled from suspicious lesions with tape strips. We aim to further develop this technique and validate if RNA profiles sampled with tape strips can detect CMM.This prospective cohort study will include approximately 200 lesions clinically suspected of CMM requiring surgical removal. Tape stripping of the lesions will be performed just before surgical excision. Subsequently, RNA on the tape strips is analyzed using quantitative real-time polymerase chain reaction with TaqMan technology. The results are combined into a binary outcome where positive indicates CMM and negative indicates no CMM. The histopathological diagnosis of the lesions will be used as the gold standard. The main outcome is the results of the RNA test and the histopathological diagnosis, which, combined, provide the sensitivity and specificity of the test.The accuracy of the clinical examination in CMM diagnostics is limited. This clinical trial will explore the ability to use RNA analysis to improve the management of suspicious lesions by enhancing early diagnostic accuracy. Hopefully, it can reduce the number of benign lesions being surgically removed to rule out CMM and decrease patient morbidity.The project was approved by The Committee on Health Research Ethics of the Capital Region of Denmark (H-15010559) and registered at the Danish Data Protection Agency (BFH-2015-065). Worldwide, about 60,000 individuals die from cutaneous malignant melanoma (CMM) each year . CMM is LINC00518 and CMIP [LINC00518 and PRAME, which the group uses in their commercially available test named “pigmented lesion assay” [LINC00518 and PRAME are known to be overexpressed in CMM, and the test result is deemed positive (indication CMM) or negative (indicating absence of CMM) based on the detection of RNA from one or both genes. The test has a reported sensitivity between 91% and 95% and a specificity between 69% and 91% [An American research group supported by DermTech  has proposed a non-invasive RNA test to improve the accuracy of CMM diagnostics using RNA profiles sampled with tape strips from the surface of suspicious lesions –13. The and CMIP . In 2017n assay” , 11. LIN and 91% , 12, 13. and 91% , 16. We GAPDH and CLTC, leaving room for nine disease related RNAs, which we chose from the 17 RNA markers first proposed by Wachsman et al. [We established the RNA analysis method described in the Methods section, which makes it possible to quantify RNA profiles sampled noninvasively with tape. Here, sufficient material on the tape strips is collected to examine the expression profiles of up to 11 genes. To be able to correct for the size of the lesions and amount of collected material, we examine expressions of two housekeeping genes, n et al.  The 11 sThe study is an observational prospective cohort study and does not affect the patients’ treatment which follows standard guidelines. An overview of the study process is provided in (1) Skin lesion suspected of being CMM planned for surgical removal following standard guidelines. (2) At least 18 years of age. (3) Written informed consent for participation in the study.(1) Longest lesion diameter shorter than 3 mm. (2) Lesion with bleeding, ulceration, or serous exudation. (3) Use of systemic steroids or topical steroids on the lesion within 30 days before tape stripping. (4) Allergy to tape. (5) Pregnancy or breastfeeding.Patients are recruited at the Department of Dermatology or the Department of Plastic Surgery and Burns Treatment at Copenhagen University Hospital. Patients are referred to these departments with skin lesions suspected of being CMM. The lesions are examined, and it is decided whether surgical removal is indicated. Patients who need surgical removal of a suspicious lesion are screened according to the eligibility criteria. Eligible patients are given oral and written information about the study. Subsequently, informed written consent for participation is obtained. Sample and data collection are obtained just before the surgical excision. The surgical excision is performed according to standard guidelines without any delay, see Data are collected at Copenhagen University Hospital, the Department of Dermatology or the Department of Plastic Surgery and Burns Treatment. The lesions’ diameters and locations are noted, as are the patients’ unique Danish personal identification numbers , which mThe tape stripping procedure is shown in Gene expression is investigated through the analysis of RNA profiles on the tape strips using quantitative real-time polymerase chain reaction (qRT-PCR). It will be performed blinded by pseudo-anonymization at the Section of Forensic Genetics, Department of Forensic Medicine, Faculty of Health and Medical Sciences, University of Copenhagen. All laboratory personnel are blinded to the histopathological diagnoses. Several aspects of the methods have been optimized, including probe concentrations, temperatures, and timing to achieve as low threshold cycle values as possible. We have established the following method. First, the tape outside the pen mark is removed using a scalpel. No further cuts are made to the remaining tape strip. RNA is extracted from the tape with the miRNeasy Micro Kit  following the manufacturer’s recommendations . No additional steps are performed to remove the biological material from the tape. A sample from one lesion consists of two tape strips. To each of the two tape strips 700 μl QIAzol Lysis Reagent (Qiagen) is added. After addition of chloroform , the suspensions are divided into the aqueous and organic phases by centrifugation. The tape strips are kept in the tubes for the duration of the phase separation when the aqueous phase from each of the two tapes are pooled on one column. The RNA is eluted in 45 μl RNase-free water. The resulting liquid is named the RNA solution and is distributed for examination of the 11 genes.The isolated RNA is then used for cDNA synthesis. Since the expression of the 11 genes differs, but is generally low, each quantification of a gene will be carried out in singleplex, except for the two housekeeping genes that are investigated in the same reaction. For each RNA cDNA synthesis and expression, the analysis is performed in a one-step reaction using TaqMan probes and the EXPRESS One-Step SuperScript qRT-PCR Universal kit  following the manufacturer’s recommendations, but with half the volume: 12.5 μl EXPRESS qPCR SuperMix Universal, 0.05 μl ROX Reference Dye (25 μM), 2.5 μl EXPRESS SuperScript Mix for One-Step qPCR, and 5.7 μl RNAse-free water, 1.25 μl TaqMan probe, and an RNA solution volume depending on the gene analyzed. The probes and corresponding volumes of the RNA solutions are listed in The definitive diagnosis of each lesion is made by routine histopathology by a pathologist at the Department of Pathology, Copenhagen University Hospital, blinded for the RNA analysis. Lesions are classified as benign nevi, CMMs, or another diagnosis. CMMs are subclassified as: superficial spreading, nodular, lentigo maligna, acral lentiginous, amelanotic, spitzoid, desmoplastic, or pigment synthesizing . The thiDemographic and clinical data will be summarized using standard descriptive statistics.S), a type I error rate of 5% (α), a confidence interval size of 10% (W), and an expected prevalence of CMM in subjects of 10% (P), we need 183 patients with complete test results. The main outcome variables are the results of the RNA analysis and the histopathological diagnosis which, combined, will provide the sensitivity and specificity of the RNA test. For our purposes, test sensitivity is the more important factor. Consequently, we chose our sample size to give reasonable precision in estimating sensitivity based on power calculations presented by Buderer . With anThe gene expression levels will be combined into a binary outcome. Positive indicates CMM. Negative indicates the absence of CMM. First, a binary logistic regression using a backward stepwise method will be performed including all 11 RNA parameters using results from all samples to determine significant parameters. Only parameters that are statistically significant after completing the backward regression will be used in the final RNA test. We will plot a receiver operating characteristic (RUC) curve and calculate the area under the RUC curve to summarize the trade-off between the true positive and false positive rates. Afterward, we plan to evaluate the model of the established RNA test using leave-one-out cross-validation. The positive and negative predictive values will be reported. In addition, the study will investigate the impact on the RNA test of the patient’s age, lesion diameter, skin area involved, and storage time of the tape before analysis. Subsequently, we will search for other possible confounders.P-values below 0.05 will be considered significant. All analyses will be done in IBM SPSS statistics .The project was approved by the Committee on Health Research Ethics of the Capital Region of Denmark  and by the Danish Data Protection Agency .The study aims to validate if an RNA test can provide clinicians with valuable additional information when deciding whether to excise a clinically suspicious lesion or not . This wiIt is a serious concern that a sensitivity of about 91% to 95%, as found in the previous RNA test based on two genes , 12, 13,Only the outermost layers of the stratum corneum are removed during the tape stripping procedure. This does not impact the subsequent histological examination of the underlying epidermis , 24, 25.B2M, CLTC, GAPDH, and NAGPA) were investigated as potential housekeeping genes. The overall expression level of NAGPA was low compared to those of the other candidates, and NAGPA could not be detected in several samples. Consequently, NAGPA is not used as a housekeeping gene in the present protocol. We have measured the levels of the chosen RNAs (see We have established the method described in the Methods section by analyzing 118 tape strips after normal human skin sampling in our laboratory at the Section of Forensic Genetics, Department of Forensic Medicine, Faculty of Health and Medical Sciences, University of Copenhagen. Four genes (RNAs see  from norRNAs see  will be To make the test clinically useable, tape strips were stored in a dark and dry place at room temperature for up to 14 days. The pilot study showed that the material could be stored under these conditions improving the ease of shipment from clinic to laboratory. The study of diameter and storage options was performed as a pilot study and must be confirmed in the final study with this protocol.CLTC and GAPDH were equally expressed. B2M that is usually considered a housekeeping gene with similar expression in many tissues was differently expressed in CMMs compared to nevi. Consequently, B2M was excluded as a housekeeping gene. On this basis, we will initiate the study presented in this protocol to investigate the RNAs presented in Likewise, in another pilot study, we identified the RNAs in 54 formalin-fixed paraffin-embedded samples, including 24 benign nevi and 30 CMMs. In these 54 samples, the nine disease-related genes were differently expressed in CMMs compared to nevi. The two housekeeping genes Our study population consists of patients referred for surgical removal of suspicious lesions. Consequently, the study will provide information about the sensitivity and specificity of the RNA test used in this clinical situation. In the future, it could also be interesting to examine how the test performs in a primary care setting or in clinics that follow individuals with numerous atypical nevi showing some of the clinical features of melanoma .


Omics-based technologies, which have developed rapidly over the last few decades, have generated increasing evidence demonstrating pervasive divergent transcription from RNA polymerase II (Pol II) promoters of eukaryotic genome, and indeed have raised considerable discussion as to their potential physiopathological function. Unlike many other long non-coding RNAs (lncRNAs), promoter antisense RNAs (PAS RNAs) were initially considered to be merely passive transcription by-products of active promoters. However, recent studies have begun to reveal their critical importance in a broad spectrum of biological processes. In this Review, I summarize recent technological advances that enable accurate detection of PAS RNA and discuss the mechanisms of PAS RNA biogenesis emphasizing the functional importance of its structure enabling the diverse functions of PAS RNA in transcription and chromatin regulation. Demonstration of a massive lncRNA transcriptional program in the mammalian genome has conceptually challenged our understanding of central dogma in molecular biology and has sparked heated debate about their biological functions . While tIn this Review, I begin with a comparison of the two most powerful methods to detect PAS RNA, and discuss the inherent advantages and disadvantages of each method followed by a review of how PAS RNA is regulated. I also discuss the mechanisms of PAS RNA-mediated gene regulation, primarily focusing on the PAS RNA secondary structure–mediated transcription activation. Furthermore, I propose the potential mechanism(s) that PAS RNA might employ to execute its biological function and emphasize the key challenges for studying the pathophysiological functions of PAS RNA.Almost all the conventional genome-wide transcriptomic techniques only detect stable transcripts in the steady state by purification and enrichment of polyadenylation (poly[A]) tail-containing RNA. Most of PAS RNAs are difficult to identify with conventional RNA sequencing (RNA-seq) approach due to the intrinsic degradation of nuclear RNA exosome complex (details described in the next section: regulation of PAS RNA) . Reverseet al., developed global run-on sequencing (GRO-seq) method to specifically enrich genome-wide nascent RNAs and unravelled a plethora of PAS RNAs from primary human lung fibroblast IMR90 cells , antisense oligonucleotides [ASOs], locked nucleic acids [LNAs]), or edit their genomic sequence. However, these approaches can be confounded by the effects of such perturbations on DNA regulatory elements or transcriptional machinery. Therefore, while previous studies have reported effects of PAS RNAs, whether the PAS RNAs per se are functional is still controversial. In the next section, I will review the alternative approaches we used in our recent study to unambiguously demonstrate that PAS RNAs act as functional RNA molecules to licence ligand-induced transcription activation [In summary, the fate of PAS RNAs is closely monitored and tightly regulated by multiple mechanisms. The precise control of PAS RNAs homoeostasis, combined with unique nuclear retention expression pattern, implies that PAS RNAs, that were previously thought to be transcriptional by-products, may function as molecular entities in gene regulation. Indeed, antisense transcripts and their corresponding biological functions have previously been reviewed in elsewhere ,43. Priotivation .in cis. Hence, there remains a need for new methodology that is able to specifically manipulate PAS RNAs and thus determine the putative function of the promoter derived antisense transcripts.The observation that PAS RNA/mRNA pairs exhibit positive correlated expression changes in response to differentiation or stress stimulation triggers poses an intriguing question as to whether PAS RNA could directly regulate cognate coding gene transcription ,44. A cl2)-induced transcription activation system. Oestrogen Receptor α (ERα) is a ligand-dependent sex steroid-regulated transcription factor that mediates most of the biological effects of E2, acting primarily at the level of gene transcription [2-bound ERα on enhancers causes a global increase in eRNA transcription on the ~1,300 enhancers adjacent to E2-upregulated coding genes that result in increased strength of specific enhancer-promoter looping [2 stimulation results in rapid trans-recruitment of an ERα-dependent MegaDalton sized protein complex, referred to as the MegaTrans complex [2 regulates a large transcriptional program based on Pol II promoter-proximal pause release where a large cohort of PAS RNAs (837 PAS RNAs) and their corresponding coding gene transcripts are robustly induced [Leptotrichia wadei) to selectively degrade PAS RNAs, we have provided direct evidence for the functional importance of induced PAS RNAs in the transcriptional activation of cognate coding genes by E2 in breast cancer cells. Furthermore, to prove PAS RNAs as functional molecules in regulating coding gene expression, we specifically overexpressed several control RNAs and PAS RNAs at selected promoters by using CRISPR-dCas9 -mediated PAS RNA tethering approach. Our data demonstrates that only specific PAS RNA overexpression, but not control RNA overexpression at selected promoters is sufficient to promote coding gene transcription. Therefore, to the best of our knowledge, our endeavour to employ CRISPR-Cas13a- based RNA knockdown strategy and CRISPR-dCas9- based PAS RNA tethering strategy, for the first time, unequivocally proves that PAS RNAs are functional entities in gene regulation.The recent discovery of CRISPR-Cas13 system, a novel type of RNA guided RNA targeting enzymes, adds to the arsenal of versatile RNA manipulation applications . One of cription . E2-boun looping . E2 stim complex . GRO-seq induced ,44. By u2-induced PAS RNAs, acting as functional molecules, regulate Pol II promoter-proximal pause release in a large subset of genes based on their actions to ensure erasure of H3K9me3 (histone H3 tri-methylation at Lysine 9) promoter mark by stabilization of H3K9me3 demethylases KDM4B and KDM4C binding, via a sharing of similar stem-loop cluster RNA structure of PAS RNAs, at promoters. This results in a dismissal of the previously-overlooked H3K9me3 reader proteins HP1α and HP1β assembly that occurs at these euchromatic promoters. We provide evidence that the promoter-bound HP1α and HP1β are capable of stabilizing 7SK small nuclear ribonucleoprotein (snRNP) complex and NELFA, leading to promoter-proximal Pol II pausing, and gene repression. Together, these results indicate that HP1 (including both HP1α and HP1β) stabilizes 7SK snRNP complex and NELFA assembly on paused promoters, and that E2- induced PAS RNA ensures dismissal of HP1-7SK-NELFA assembly for robust coding gene transcription.Transcription, transmitting genetic information from DNA to RNA, plays a fundamental role in nearly all the physiopathological processes and its regulation is precisely regulated by multistep mechanisms . Among tXist enables appropriate X chromosome inactivation (XCI) through its distinct well-ordered structural domains [ABAT PAS RNA, which is an E2- induced promoter antisense transcript, is critical for regulation of Pol II promoter-proximal pause release and the cognate coding gene transcription activation via a stable and compact stem-loop structure. Indeed, while the primary nucleotide sequences of various E2-induced PAS RNAs on promoters exhibiting Pol II promoter-proximal pause release-dependent activation vary widely, here we provide evidence that they share a similar stem-loop cluster, and that this RNA structure is responsible for the recruitment of the H3K9me3 demethylases KDM4B and KDM4C, which serves as the underlying mechanism by which PAS RNAs serve to regulate Pol II promoter-proximal pause release events based on recruitment of H3K9me3 demethylases that release HP1α recruited to promoters (As with proteins, RNA molecules form complex higher-order structures that are indispensable regulators of many biological processes . For ins domains . Recent  domains . Howeverromoters .Figure Rapid ever-evolving genome-wide nascent RNA sequencing technologies have revealed prevalent expression landscape of PAS RNAs across all the investigated genome so far. Despite the pervasiveness of PAS RNAs expression, a full understanding of the mechanisms by which they might regulate gene expression has remained rather enigmatic. Indeed, although accumulating data generated in the last decades points towards diverse functions of PAS RNAs in a myriad of biological processes, direct evidence to prove PAS RNAs themselves, rather than the act of transcription in the upstream antisense direction of promoter regions, in such processes remains inadequate.In this Review, I have described two of the most powerful approaches that enable efficient genome-wide detection of PAS RNAs. I have summarized the most recent understanding of PAS RNA biogenesis and regulation, and have recapitulated our recent work to prove PAS RNAs as functional entities in gene regulation, thus supporting our novel mechanistic insights into PAS RNAs- mediated gene regulation. Nevertheless, it should be noted that although the example of PAS RNA mechanism described above suggests a specific function for PAS RNAs in epigenetic regulation, there remains an uncertainty of the precise roles and mechanisms of most of PAS RNAs. A recent study reports a fascinating model whereby the negatively charged nascent RNAs- mediated electrostatic interaction with positively charged proteins regulates Pol II transcription via a dynamic complex coacervation model , which ccis, it is currently technically difficult to query PAS RNAs in a systemic level. Further efforts are needed to achieve genome-wide perturbation of PAS RNAs. Intriguingly, some studies have also reported an opposite gene repression effect of antisense transcription on coding gene expression. Hence, a subset of PAS RNAs, in specific cellular contexts, may have a direct role in gene repression. Therefore, it is pertinent to dissect whether PAS RNAs have both transcriptional activation and repression function and what determines their functional specification. Additionally, it is necessary to combine CRISPR-Cas13- based PAS RNA knockdown strategy and CRISPR-dCas9- based PAS RNA tethering strategy presented above to distinguish between effects caused by PAS RNA per se and the act of transcription in the upstream antisense direction of promoter region. It is noteworthy that both models underlying PAS RNA functions are not mutually exclusive and may co-exist in some contexts, in terms of the vast number of PAS RNAs in a variety of genomes. It is certainly exciting to investigate the ability of specific structural features or nucleotide sequence elements in PAS RNA biology. Given the increasingly recognized importance of RNA structure in RNA function, it is particularly cogent to test and identify whether there is a ‘structure code’ for PAS RNAs allowing them to establish a dynamic and specific interaction with proteins, RNAs and other macromolecules, thus licencing the formation of ribonucleoprotein complexes essential for key regulatory functions of PAS RNAs. In parallel, attention should be paid to potential modifications of PAS RNAs, which will greatly deepen our mechanistic insights into PAS RNAs. With continually evolving technological advances in nascent RNA sequencing, RNA secondary structure probing and modification approaches, the functional repertoire of PAS RNAs will expand and revolutionize our fundamental understanding of the broader role of these previously neglected transcripts.Ascribed to most, if not all, of PAS RNAs functioning in


Taken together, the use of bioanalytical and computational approaches, involving LC-MS and ligand docking of tertiary structural models, can form a powerful combination to help explain the RNA cleavage behavior of RNases.Knowledge of the cleavage specificity of ribonucleases is critical for their application in RNA modification mapping or RNA-protein binding studies. Here, we detail the cleavage specificity and efficiency of ribonuclease MC1 and cusativin using a customized RNA sequence that contained all dinucleotide combinations and homopolymer sequences. The sequencing of the oligonucleotide digestion products by a semi-quantitative liquid chromatography coupled with mass spectrometry (LC-MS) analysis documented as little as 0.5–1% cleavage levels for a given dinucleotide sequence combination. While RNase MC1 efficiently cleaved the [A/U/C]pU dinucleotide bond, no cleavage was observed for the GpU bond. Similarly, cusativin efficiently cleaved Cp[U/A/G] dinucleotide combinations along with UpA and [A/U]pU, suggesting a broader specificity of dinucleotide preferences. The molecular interactions between the substrate and active site as determined by the dinucleotide docking studies of protein models offered additional evidence and support for the observed substrate specificity. Targeted alteration of the key amino acid residues in the nucleotide-binding site confirms the utility of this  Ribonucleases (RNases) play critical roles in gene expression, RNA maturation, quality control, and microbial defense . These R5C) does not affect its recognition as a substrate, RNA with tandem cytidines is not a substrate for cusativin p[D]) and [m5C]pU are cleaved with high efficiency   of dinucleotide makes it a non-substrate , CpU (80–100%), and UpU (45–60%) bonds , Table 1ctively)  suggestiubstrate .phe revealed additional cleavage for Cp (48–62%) and [m5C]pU (98–100%) bonds, suggesting that methylation in any position of the dinucleotide is not a hindrance to the cusativin-mediated RNA cleavage , CpA bonds as well as UpA (70–90%) , Table 1cleavage .To understand any correlation between the RNA cleavage behavior and the molecular docking scores of ligands bound to the active site, RNase T1 and A models were evaluated initially.A tertiary structure of RNase T1 (GenBank: RMZ48028.1) was generated by homology modeling and docked with a 3′-GMP ligand. This docked ligand exhibited identical overlay with the 3′-GMP bound T1 crystal structures—4GSP  or 1RGA Docking with dinucleotides, GpU, CpU, and ApU revealed interesting observations . The docDocking with a non-substrate, CpU, yielded a low fitness score . Cytosine formed only two hydrogen bonds with Glu46 and Asn98 and the orientation of 3′ uracil was altered. Similarly, docking T1 with ApU revealed the formation of two hydrogen bonds between adenine and Asn43 and Glu46 as opposed to seven bonds for guanine. However, the phosphate group was positioned correctly in the catalytic site, making H-bonds with appropriate amino acid residues, Asn36, Tyr38, and Arg77 . Thus, fewer hydrogen bonds with the 5′ base in the active site could be the reason for negligible or no cleavage of CpU or ApU bonds  under opA homology model of RNase A (GenBank: AAB35594.1) was generated and docked with dinucleotide ligands. The docked model with CpA exhibited a high fitness score  and 12 hydrogen bonds  in the binding site . This doWhen a homology protein model generated with the MC1 amino acid sequence (GenBank—AKU36755.1) was docked with 5′-UMP, it recapitulated a precise match with the 5′-UMP bound B2 site of the MC1 crystal structure (PDB: 1UCD) . Docking2-O-P- is stabilized by Arg150 and Gln164 with hydrogen bonds. Trp37 aligned below the base and ribose in the B1 site to form π-π interactions. Arg181(3.2 Å), Ser44(3.6 Å), and Thr43(3.6 Å) showed weak hydrogen bonds with N4 of cytosine , Asn71 (O2 and N3), and Val72 (O4). Further, Leu73 along with Phe80 made π-π interactions identical to 1UCA, 1UCC, and 1 UCD crystal structures pU bond , and 3′ end digestion products UUGAGA and UUUGAGA (with UU at 5′-end) were observed at high abundance (Uridine monophosphate (UMP), if formed upon cleavage of CpU and UpU in the -CpUpUpUpG- sequence, is not detectable by LC-MS. Its levels, however, could be low as the 5′ end digestion product—UACACCCGUAGGGbundance . Moleculbundance , thereby7G]pU sequences. These observations strongly suggest that the bonding contacts with the 5′ base of the dinucleotide also influence the RNA cleavage. In other words, interactions at the B1 and B2 sites determine the dinucleotide specificity of RNA cleavage by RNase MC1, as they can place or pull the phosphodiester bond from the catalytic site.A similar but more pronounced leftward shift and incorrect positioning of ribose phosphate and uridine in GpU as illustrated by the superimposition with CpU docking solution explain the insignificant cleavage for GpU  or even The semiquantitative LC-MS approach with a customized RNA sequence has documented the cleavage preferences of cusativin (CpA (70–90%), CpG (80–100%), CpU (80–100%), UpA (70–90%), UpU (14–30%), and ApU (14–30%) and no cleavage for the CpC bond) , Table 1Cusativin-mediated cleavage of ApU bond can be explained by the stabilization of adenine (N7 and N3 of purine making H-bond with Ser70 and Arg176) and ribose phosphate in the B1 and catalytic sites. However, the orientation of adenine differs from cytosine in the B1 site  as no H-Higher levels of cleavage at UpA can be attributed to the strong interactions in the active site. Near identical positioning of uracil in B1 sites such as cytosine in CpU, strong H-bond interactions at N1 and N6 atoms of adenine with Asn97 and Gln36, coupled with another H-bond between the C6 amino group and Pro96, led to binding stabilization. Although this seems to be novel, proline is a conserved residue for the T2 family of proteins at this site . AdenineWhen a minimum of >5% cleavage is considered, MC1 cleaved RNA predominantly at ApU, CpU, UpU, and to some extent at CpA (10–20%) , whereas2 = 16) and four homopolymer sequences was purchased from Integrated DNA Technology (IDT). Yeast tRNAPhe was obtained from Sigma, and all other chemicals were procured from Thermo Fisher Scientific. RNase T1 was obtained from Worthington Biochemical Corporation. RNase MC1 [TM (DE3) and BL21 strains of Escherichia coli, as described before.A customized RNA (5′-GCAUCAGAAAUACACCCGUAGGGCUUU-GAGA-3′) with all the dinucleotide combinations (4Nase MC1  and cusaNase MC1  were purSynthetic DNA (651 nt) sequences corresponding to N97S and T100R mutations of cusativin sequence were procured from IDT and cloned into the Bam HI and Hind III sites of protein expression plasmid, pET-22b. Recombinant clones were screened by colony PCR and the gene sequences confirmed by Sanger DNA sequencing at CCHMC  facility. Mutant protein expression and purification procedures were identical to the cusativin wild-type protein preparation .About 50 units of RNase T1 were used to digest 1 µg of RNA at 37 °C for 2 h in the presence of ammonium acetate . In case of MC1 and cusativin, 0.25–1 µg enzyme was used to digest 1 µg of denatured RNA, with 120 mM ammonium acetate (pH not adjusted) at 37 °C and 60 °C for 2 h and dried in speedvac and stored at −20 °C until LC-MS injection.x) and oligonucleotides that represent the undercut sequences (y) as percentage (The semi-quantitative liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) was performed through an ion-pairing reversed-phase liquid chromatography column connected to the Synapt G2-S mass spectrometer. The details of mobile phases, chromatography, and mass spectrometry conditions are described elsewhere ,41. The https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Proteins) (accessed on 5 March 2020). Active site amino acid sequences of select T2 enzymes were aligned by MUSCLE (https://www.ebi.ac.uk/Tools/msa/muscle/) (accessed on 15 March 2020) tool [Ribonuclease sequences that exhibit highest similarities with cusativin were identified by NCBI BlastP (20) tool .https://swissmodel.expasy.org/) (accessed on 5 May 2020) [https://molview.org/) (accessed on 17 August 2020) and saved as a 3D model. These molecules are then optimized for their energy using Avogadro software by choosing the feature, “Fixed atoms are movable” [A model tertiary structure for cusativin was prepared by homology modeling using SWISS-MODEL workspace (ay 2020) . This exay 2020)  as a topmovable” . The premovable” . The defmovable” .https://pymol.org) (accessed on 5 December 2020) for detailed analysis including the ligand alignment between the crystal structures and docked poses.The chemPLP scoring function is used to model the steric complementarity between protein and ligand. Further, the distance- and angle-dependent hydrogen and metal bonding terms from ChemScore  are also considered  to estimIn this work, we detail the cleavage specificity and efficiency of RNase MC1 and cusativin by a semi-quantitative LC-MS analysis using a customized RNA. The sequence information of the observed digestion products reflects the accurate cleavage specificity and efficiency. The cleavage features are further explained by documenting the molecular interactions between the enzyme active site and dinucleotide ligands through homology modeling, docking studies, and targeted mutation studies. These studies support the dinucleotide-specific RNA cleavage nature of MC1 and cusativin. Such RNases serve as bioanalytical tools to locate post-transcriptional modifications in any RNA sequence, including mRNAs, through liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) analysis (or RNA modification mapping) . Knowled


Investigation of RNA- and DNA-binding proteins to a defined regulatory sequence, such as an AU-rich RNA and a DNA enhancer element, is important for understanding gene regulation through their interactions. For in vitro binding studies, an electrophoretic mobility shift assay (EMSA) was widely used in the past. In line with the trend toward using non-radioactive materials in various bioassays, end-labeled biotinylated RNA and DNA oligonucleotides can be more practical probes to study protein–RNA and protein–DNA interactions; thereby, the binding complexes can be pulled down with streptavidin-conjugated resins and identified by Western blotting. However, setting up RNA and DNA pull-down assays with biotinylated probes in optimum protein binding conditions remains challenging. Here, we demonstrate the step-by step optimization of pull-down for IRP (iron-responsive-element-binding protein) with a 5′-biotinylated stem-loop IRE (iron-responsive element) RNA, HuR, and AUF1 with an AU-rich RNA element and Nrf2 binding to an antioxidant-responsive element (ARE) enhancer in the human ferritin H gene. This study was designed to address key technical questions in RNA and DNA pull-down assays: (1) how much RNA and DNA probes we should use; (2) what binding buffer and cell lysis buffer we can use; (3) how to verify the specific interaction; (4) what streptavidin resin (agarose or magnetic beads) works; and (5) what Western blotting results we can expect from varying to optimum conditions. We anticipate that our optimized pull-down conditions can be applicable to other RNA- and DNA-binding proteins along with emerging non-coding small RNA-binding proteins for their in vitro characterization. Gene expression is regulated at the transcriptional, post-transcriptional, and translational levels as a result of changes in interactions between cis-acting regulatory elements of DNA and RNA with specific trans-acting proteins ,2,3. Thehe probe . Along whe probe , force she probe , moleculhe probe , fluoreshe probe , and EMShe probe ,13, end-he probe ,15,16, iThis study shows our pull-down set-up and results for the detection of RNA- and DNA-binding proteins in the human ferritin H gene. Ferritin is the major intracellular iron storage protein composed of 24 subunits of heavy (H) and light (L) chains . Iron hoThe IRE has a stem-loop RNA structure in the 5′-untranslated region (UTR) of several key iron metabolism genes including ferritin H, L, and an iron exporter ferroportin (SLC40A1), while the highly conserved IRE was also found in the 3′-UTR of mRNAs encoding iron transport protein transferrin receptor-1 (TfR1) and divalent metal transporter 1  ,26. All The second cis-acting sequence in the human ferritin H mRNA is an AU-rich element located in the 3′-UTR as reported previously . We alsoThe third cis-acting element is located 4.5 kb upstream from the transcription start site of the human ferritin H gene, where we identified an antioxidant-responsive element (ARE) ,24. Nrf2This work is focused on the in vitro characterization of binding proteins to cis-acting elements by setting up an optimal pull-down condition using biotinylated RNA and DNA oligonucleotides with streptavidin-conjugated resins. Since non-AGO-family RNA-binding proteins are known to directly interact with specific microRNAs and regulate targeted genes ,37, we a32P end-labeled IRE RNA probe [2, 0.2 mM EDTA, 0.5 mM PMSF) pulled down the IRP2-IRE complex much more efficiently  was used for 500 μg of WCLs to pull down the IRP2-IRE complex in The next question was whether this IRP2-IRE interaction is specific or not. To address this important issue, we used a mutated IRE RNA A as a prThe next important questions were (1) what amount of biotinylated IRE RNA is sufficient and optimum to pull down the IRP-IRE complex and (2) whether this pull-down assay is semi-quantitative. To address the first question, we tested 1, 2, 4, and 6 μg (100–600 pmol) of biotinylated wtIRE probe for 500 μg of SW480 WCL in the 200 μL binding buffer A, followed by pull-down with 20 μL of high-capacity streptavidin agarose. As shown in To address the second question of whether the pull-down assay is semi-quantitative or not, we incubated 100, 250, and 500 μg (each duplicate) of WCLs from K562 and SW480 cells with 2 μg of a biotinylated IRE probe for 1 h followed by another 1 h incubation with 20 μL of high-capacity streptavidin agarose to pull down the IRE binding complex. As shown in To corroborate our conclusion in the IRE-IRP pull-down set-up, we prepared WCLs from K562 cells treated with 100 μM FAC (ferric ammonium citrate) or 25 μM iron chelator DFO (deferoxiamine mesylate) for 24 h and tested whether we could detect changes in the binding of IRP2 and IRP1 to the IRE under high- and low-iron conditions. We also tested another streptavidin-agarose  and another type of magnetic bead, Dynabeads M-280 (Invitrogen). As shown in Little is known about binding proteins to the 3′-UTR of ferritin mRNA, partly due to the significant roles of the translational regulation via the IRP-IRE interaction in ferritin expression ,43,44 al2) or 25 μM t-BHQ (tert-butyl hydroquinone) for 14 h  enhancer ,45, to wfor 14 h B, input.for 14 h B. Not onfor 14 h C.We demonstrated here how to set up biotinylated IRE and AU-rich RNA pull-down assays for IRP2, IRP1, HuR, and AUF1 in addition to biotinylated ARE DNA pull-down for Nrf2. In the IRE-IRP pull-down assay, we used 2 μg (200 pmol) of a 5′-biotinylated IRE RNA probe for 500 μg of SW480 or K562 WCL in a total 200 μL of binding buffer A or C, along with incubation with constant rotation for 1 h at room temperature, followed by another 1 h incubation with 20 μL of pre-washed high-capacity streptavidin agarose . Alternatively, 30 μL of streptavidin agarose  worked well to pull down the biotinylated IRE-IRP binding complex. We noticed no clear difference in pull-down efficiency between binding buffer A and buffer C . PBS wasTo confirm the specificity of IRE-IRP interactions, we tested a 5′-biotinylated mutant IRE probe for binding to IRPs, in addition to a pull-down competition assay by adding 4-fold in excess of non-biotinylated wt and mutant IRE competitors F. Like EWe intended that the conditions optimized in this work can be used for RNA and DNA pull-down assays and detection by Western blotting to test (1) whether the binding protein predicted from the defined nucleotide sequences is present or not in the binding complex and (2) whether the protein binding is increased or decreased according to gene expression changes in the same experimental conditions. Protein purities in the pull-down samples are therefore not critical issues for such targeted pull-down assays detected by Western blotting with a specific antibody. However, we also tested the effect of pre-clearing WCLs with a biotinylated mutant IRE and streptavidin-agarose on the protein precipitates subsequently pulled down with a biotinylated wild-type IRE RNA probe. The results showed that the pull-down with a biotinylated IRE RNA probe per se (no pre-clear) can remove the majority of non-interacting proteins . As desthiobiotin binds streptavidin reversibly, HuR bound to an AU-rich RNA was gently eluted with 4 mM biotin-containing elution buffer [We are not sure about the reason for the inability of the magnetic beads to pull down the IRP-IRE binding complex in our assays; however, some key procedures including the biotinylation of an RNA probe and/or binding conditions of the probe with magnetic beads and WCLs may need to be further optimized or modified. For instance, the Magnetic RNA-Protein Pull-Down Kit (ThermoFisher Pierce) uses a 3′-desthiobiotinylated RNA probe (RNA incubated with biotinylated cytidine bisphosphate and T4 RNA ligase) and incubates first with magnetic beads in RNA capture buffer , followed by incubation with WCL in protein–RNA binding buffer  are summarized by Kazimierczyk et.al . For theAccumulating evidence has indicated that non-AGO-family RNA-binding proteins directly interact with specific microRNAs and thereby play important regulatory roles in gene expression and cell physiology  includinIn addition to biotinylation, various methods for RNA labeling to investigate RNA–protein interactions were summarized by Gemmill et al.  and furtConclusions and Future Perspectives: We determined the optimal condition for an IRP-IRE pull-down assay, in which 2 μg (200 pmol) of a 5′-biotinylated IRE RNA probe was used for 500 μg of WCL (either prepared in IP lysis buffer or RIPA) in a total of 200 μL of binding buffer A or binding buffer C. The incubation with constant rotation for 1 h at room temperature was followed by another 1 h incubation with 20 μL of pre-washed high-capacity streptavidin agarose or 30 μL of streptavidin agarose. Mixing all together and incubation for 1–2 h also worked well. To confirm the specificity of IRE-IRP interaction, the inclusion of 2 μg of a 5′-biotinylated mutant IRE probe compared to wt IRE probe in pull-down assays is important along with a competition assay by adding >4-fold in excess of non-biotinylated wt and mutant IRE competitors. This pull-down assay condition was applied for the detection of HuR and AUF1 bound to an AU-rich RNA element as well as Nrf2 bound to an ARE double-strand DNA enhancer element. The pull-down assays optimized in this work will save a fair amount of time for researchers who will need to establish and perform RNA and DNA pull-down assays for their initial characterization of binding proteins anticipated from their RNA and DNA sequences. We expect that this pull-down condition will also help or facilitate the characterization of macromolecules interacting with small non-coding RNAs that regulate the expression of target genes.2 incubator . The culture media and chemicals used in this work are listed in ® system, MilliporeSigma, Burlington, MA, USA) at 100 mM and 25 mM, respectively. Sodium arsenite (NaAsO2) was dissolved in Milli-Q® water at 10 mM. t-BHQ was freshly prepared, first dissolved in DMSO at 1 M and further diluted to 10 mM with Milli-Q® water.All human cell lines  were cultured in a humidified 37 °C CO2, 100 mM NaCl, 1 mM DTT, New England Biolabs, Ipswich, MA, USA) in a microcentrifuge tube, followed by incubation in 300–400 mL of microwaved 90 °C water in a beaker and leaving it at room temperature until gradually cooling down to room temperature. The sequences of human ferritin H RNA and DNA oligonucleotides used in this study (biotinylation at the 5′-end) are as follows:All biotinylated and non-biotinylated oligonucleotides were synthesized by Sigma-Aldrich . Desalt grade RNA oligonucleotides at 0.2 μmol scale yielded approximately 500–700 μg. We experienced that the synthesis scale increased to 1.0 μmol did not increase the yield of IRE RNA oligonucleotides. Desalt-grade DNA oligonucleotides at a 0.05 μmol scale yielded 200–500 μg. All RNA and DNA oligonucleotides were dissolved at 1 μg/μL in TE . 32nt IRE RNA oligos at 1 μg/μL is approximately 100 μM. DNA oligonucleotides were annealed by mixing 100 μL each of 1 μg/μL sense and antisense 5′-biotinylated ARE oligonucleotides together with 20 μL of NEBuffer 3  RNA: 5′-GGUUUCCUGCUUCAACAGUGCUUGGACGGAAC-3′IRE (mt) RNA: 5′-GGUUUCCUGCUUCAAGCUCCGUUGGACGGAAC-3′AU-rich (wt) RNA: 5′-AAACGAGUAUUUGUAUUUAUUAAACUCA-3′AU-rich (mt) RNA: 5′-AAACGAGUACCCGUACCCAUUAAACUCA-3′ARE1 sense DNA: 5′-TCCTCCATGACAAAGCACTTTT-3′ARE1 antisense DNA: 5′-AAAAGTGCTTTGTCATGGAGGA-3′ARE2 sense DNA: 5′-GGAGTGCTGAGTCACGGTGGGA-3′ARE2 antisense DNA: 5′-TCCCACCGTGACTCAGCACTCC-3′Complete sense ARE: 5′-TCCTCCATGACAAAGCACTTTTGAGCCCAAGCCCAGCCTAGCGGAGTGCTGAGTCACGGTGGGA-3′Complete antisense ARE 5′-TCCCACCGTGACTCAGCACTCCGCTAGGCTGGGCTTGGGCTCAAAAGTGCTTTGTCATGGAGGA-3′.2, and 0.5 mM PMSF). Streptavidin magnetic beads  and Dynabeads M-280 Invitrogen, Waltham, MA, USA) were also tested for IRE-IRP pull-down assays. These beads were magnetized with a DynaMag-2 magnetic stand during washing with NEB magnetic beads buffer  or Dynabeads buffer .We used two streptavidin-agarose resins: high-capacity streptavidin-agarose  and streptavidin agarose , which is the same product as currently available SA100-04. The resins were mixed well before being taken (20 μL × sample numbers) into a microcentrifuge tube, washed once with 1–1.5 mL of washing buffer , and resuspended in (20 μL × sample numbers) of binding buffer A  and lysed in IP lysis buffer  or RIPA buffer . There was no difference in our results of RNA-pull down assays when WCL was prepared in IP lysis buffer or RIPA buffer. K562 and other suspension cells (0.5–1 × 106 cells/mL) were centrifuged at 1000 rpm, and cell pellets were washed with PBS and lysed in IP lysis buffer. Protein concentrations in WCLs were 5–25 μg/μL, measured with a protein assay dye reagent . Except for the optimization of input probes and the assessment of semi-quantitative pull-down assays in 2, 20% glycerol, 1 mM DTT). There was no difference in the IRP pull-down efficiency incubated in binding buffer A or C (For the preparation of whole-cell lysates (WCLs) from SW480 and other adherent cells, subconfluent to confluent cells were washed with phosphate-buffered saline  at room temperature for 1–2 h with 4 μg of annealed 5′-biotinylated ARE DNA in 200 μL of binding buffer C. The ARE DNA pull-down assay for the detection of Nrf2 was also performed in PBS as binding buffer B. The reIn IRE-IRP RNA pull-down assays for testing streptavidin magnetic beads  in TM, Beckton Dickinson, Franklin Lakes, NJ, USA) dissolved in 0.1% Tween-20-containing Tris-buffered saline . Incubation with a primary antibody was conducted at 4 °C overnight on a rocker platform , followed by washing with 0.1% Tween20/TBS  and incubation with a secondary antibody at room temperature for 1.5 h. All primary and secondary antibodies were diluted with the same blocking solution (either 1% BSA or 5% skim milk in Tween20/TBS). The PVDF membrane was washed three times, incubated with ECL reagents , and immediately exposed to X-ray films . For incubation of the same membrane with another primary antibody, the membrane was soaked in the stripping solution  for 1 h on a rocker and the incubation was repeated with primary and secondary antibodies.After briefly spinning down heated samples in the microcentrifuge tubes, binding complexes eluted in the 2xSDS-PAGE sample buffer were loaded on a 10% acrylamide SDS-PAGE  mini-gel , along with protein size markers . The stacking gel was 5% acrylamide, 0.1% bisacrylamide, 125 mM Tris, pH 6.8, and 0.1% SDS. The electrophoresis was run at 15 mA per mini-gel for approximately 1.5 h in the SDS-PAGE running buffer . Proteins separated on the SDS-PAGE gel were transferred to the PVDF membrane  at 300 mA for 1.5 h in a mini trans-blot cell  filled with a Western blotting transfer buffer  at 4 °C. The PVDF membrane was soaked at room temperature for 15–30 min (blocking) in either 1% BSA  or 5% skim milk , 500~1000-fold dilution with 1% BSA in Tween/TBS, 4 °C overnight.Anti-IRP2 rabbit monoclonal antibody , 1000-fold dilution with 1% BSA in Tween/TBS, 4 °C overnight.Anti-IRP1 rabbit monoclonal antibody , 1000-fold dilution with 5% skim milk in Tween/TBS, 4 °C overnight.Anti-AUF1 rabbit monoclonal antibody , 1000-fold dilution with 5% skim milk in Tween/TBS, 4 °C overnight.Anti-HuR rabbit monoclonal antibody , 1000-fold dilution with 5% skim milk in Tween/TBS, 4 °C overnight.Anti-Nrf2 rabbit polyclonal antibody , 1000-fold dilution with 5% skim milk in Tween/TBS, 4 °C overnight. As SC-13032 was discontinued, anti-Nrf2 rabbit monoclonal antibody  may be able to be used at 1000-fold dilution with 5% skim milk in Tween/TBS, 4 °C overnight. However, transiently transfected Nrf2 was detected much stronger with SC-13032 than D1Z9C .The HRP-conjugated secondary antibodies used in this study were either anti-rabbit IgG  or anti-mouse IgG  at 5000-fold dilution with the same blocking solution as used for the primary antibody.2 or t-BHQ, cells were washed with PBS, resuspended in 200–500 μL hypotonic buffer , and incubated for 15 min on ice. The 1/20 volume  of 10% NP40 was added, vortexed for 10 s at a high setting, and the suspension was centrifuged at 12,000 rpm for 30 s . The supernatant was transferred to a microcentrifuge tube (cytoplasmic fraction). The nuclear pellet was suspended in 50–200 μL of the nuclear extraction buffer , vortexed for 10 s at high setting, and incubated on ice for more than 30 min. The nuclear pellet was vortexed for 30 s at a high setting (making bubbles is no problem) and centrifuged at 12,000 rpm for 10 min at 4 °C. The supernatant (nuclear fraction) was transferred to a new microcentrifuge tube. Protein concentrations in cytoplasmic and nuclear fractions were measured with the BIO-RAD protein assay dye reagent, usually yielding 5–15 μg/μL.For the DNA pull-down assay to detect Nrf2 binding to the ARE, we used cytoplasmic and nuclear fractions isolated from K562 cells. After treatment with NaAsOWe prepared WCLs from HEK293 cells transiently transfected with pCMVSport6-IRP1  and pRK5-IRP2 plasmids for Western blotting positive control. pRK5-IRP2 was constructed from pCR4-TOPO-IRP2  by two steps: the ligation of IRP2 cDNA (SpeI/NotI) to pCRII SpeI/NotI sites (pCRII IRP2) followed by the isolation of IRP2 cDNA (BamHI/XbaI) and cloning to BamHI and XbaI sites of the pRK5 vector. pRK5Nrf2 was constructed by the ligation of a BamHI- and XhoI-digested Nrf2 DNA from pOBT7Nrf2  into BamHI and SalI sites of the pRK5 vector. These plasmid DNAs were verified by DNA sequencing  and transiently transfected into HEK293 cells by electroporation  as described previously .


Perinatal exposure to smoking has been associated with childhood asthma, one of the most common pediatric conditions affecting millions of children globally. Of great interest, this disease phenotype appears heritable as it can persist across multiple generations even in the absence of persistent exposure to smoking in subsequent generations. Although the molecular mechanisms underlying childhood asthma induced by perinatal exposure to smoking or nicotine remain elusive, an epigenetic mechanism has been proposed, which is supported by the data from our earlier analyses on germline DNA methylation (5mC) and histone marks (H3 and H4 acetylation). To further investigate the potential epigenetic inheritance of childhood asthma induced by perinatal nicotine exposure, we profiled both large and small RNAs in the sperm of F1 male rats. Our data revealed that perinatal exposure to nicotine leads to alterations in the profiles of sperm-borne RNAs, including mRNAs and small RNAs, and that rosiglitazone, a PPARγ agonist, can attenuate the effect of nicotine and reverse the sperm-borne RNA profiles of F1 male rats to close to placebo control levels. MR-070-D) on ice. The spermatozoa were released into the culture media by making 6-8 small cuts to each epididymis with a sharp blade, and the plates were placed in a culture incubator at 37°C for 30 minutes. Following incubation, the medium containing spermatozoa was filtered through a cell strainer  to a 50 mL conical tube, and the filtrate was divided into four 1.5 mL micro-centrifuge tubes. The tubes were centrifuged at 1000×g for 5 minutes, supernatants discarded, and 1 mL lysis buffer (0.05% SDS and 0.005% Triton X-100 in distilled water) added to each tube to gently suspend the pellet. The tubes were kept on ice for 5 minutes to lyse and remove the somatic cell contamination. After confirming the purity of isolated sperms microscopically, the samples were centrifuged at 3000×g for 5 minutes. The supernatants were discarded, and each pellet gently suspended in 1 mL ice-cold PBS. The suspensions from two tubes were pooled and centrifuged at 3000×g for 5 minutes. The supernatants were discarded, and pellets stored at -80°C until RNA isolation and establishment of cDNA library.At culling, each epididymis was isolated by cutting the vas deferens and muscle connections with the testis. After trimming the surrounding connective tissue, the two epididymides from each animal were placed in a tissue culture plate containing 3 mL of HTF culture medium  and subjected to RNA extraction for RNA-seq, as described below. RNA was extracted from cells according to the manufacturer’s instructions using the mirVana miRNA Isolation Kit . The Qubit RNA High Sensitivity Assay Kit  was used to quantify the extracted RNA and measured on the Qubit 2.0 Fluorometer (Invitrogen).Large RNA libraries were constructed using KAPA Stranded RNA-seq Kit with RiboErase  according to the manufacturer’s instructions, as described previously , and seqThe following workflow was used in bioinformatic analyses of the RNA-seq data: QC check (fastQC) ➔ alignment (Hisat2) ➔ featureCounts (subread) ➔ Differential gene expression analysis (DESeq2) ➔ Pathway Enrichment, GO analysis (Bioconductor clusterProfiler). To ensure the quality of RNA-seq data, fastq files were subjected to fastQC  to checkLncRNA information was first obtained with gene symbols by merging two Ensembl releases (release-105 Rattus_norvegicus.mRatBN7.2.ncRNA.fa.gz and release-104 Rattus_norvegicus.Rnor_6.0.104.gtf.gz). The gene symbols were then used in differential expression analyses as keywords to search in Ensemble to obtain the lncRNA information that can be annotated. Finally, those two are combined to obtain all the lncRNA gene symbols. The extracted padj of these lncRNAs were much larger than 0.05.® Small RNA Library Prep Set for Illumina® (Multiplex Compatible)  according to the manufacturer’s instructions, as described previously . Gapdh was used for large RNA expression normalization. Small RNAs were poly(A) tailed using E. coli Poly(A) Polymerase  followed by reverse transcription with LD_CDS primer using SuperScript™ II Reverse Transcriptase. qPCR analyses for small RNA were then performed using TaqMan™ Gene Expression Master Mix  with Illu lib quant probe. U6 was used for small RNA expression normalization. The primer sequences used in this study for qPCR are listed in Datasets S1.cDNAs for large and small RNA were prepared as previously described . Brieflyt-test. Symbols *, ** and *** represent p < 0.05, p < 0.01 and p < 0.001, respectively.All data were subjected to statistical analysis using the SPSS program  and shown as mean ± standard error of the mean (SEM). And statistical differences between two groups were assessed by two samples The RNA-seq data have been deposited into the National Center for Biotechnology Information Sequence Read Achieve database (accession no. PRJNA813596).Adult female rats (F0 dams) received placebo , nicotine , or nicotine  plus RGZ  between E6 and PND21 Figure 1The 3D principal component analyses (3D-PCAs) verified that the differential transcriptomes of placebo control, nicotine-treated (NIC) and nicotine plus RGZ-treated (NR) sperm samples were indeed clustered separately (L-Histidine decarboxylase (Hdc), Fc receptor-like 3 (Fcrl3), Endothelin receptor type B (Ednrb), and Complement C4A (C4a). Hdc encodes a unique enzyme in mammals which catalyzes histamine formation from L-histidine and histamine plays a critical role in the pathogenesis of bronchial asthma. In particular, the level of Hdc mRNA is elevated in asthmatic patients  and Fcrl3_6 (rs3761959) SNPs are protective against asthma in Mexican male patients , Pleckstrin (Plek), and Transcription factor EC (Tfec). Arhgap15 has been called a ‘‘master negative regulator of neutrophil functions’’, and validated as a differentially expressed novel transcript in patients with asthma , PHD finger protein 11 (Phf11), Dipeptidyl peptidase like 10 (Dpp10), Interleukin 4 (Il4), Brain-derived neurotrophic factor (Bdnf), Serine peptidase inhibitor, Kazal type 5 (Spink5), Cd69 molecule (Cd69), etc. Following linkage studies, Adam33  (Ubiquitin-conjugating enzyme E2C (Ube2c) (Adrenoceptor beta 3 (Adrb3) (Coronin 1C (Coro1c) (Sp1 transcription factor (Sp1) (Ras homolog family member B (Rhob) (Serum/glucocorticoid regulated kinase 1 (Sgk1) (BTG anti-proliferation factor 2 (Btg2) (Homeobox D8 (Hoxd8) (Bone morphogenetic protein 4 (Bmp4) (Protein regulator of cytokinesis 1(Prc1)  using TargetScan . Those t, Adam33 , Phf11 (3 (Phf11  and Dpp11 (Dpp10  have beeentation . BDNF maensitive . Studiesf asthma . CD69 wapatients . Further (Hmga2) , Ubiquit (Ube2c) , Adrenoc (Adrb3) , Coronin(Coro1c) , Sp1 traor (Sp1) , Ras homB (Rhob) , Serum/g1 (Sgk1) , BTG ant2 (Btg2) , Homeobo (Hoxd8) , Bone mo4 (Bmp4) , Protein 1(Prc1) , etc.GO term enrichment analyses identified that the affected target genes were mostly involved in the biological processes including embryonic organ morphogenesis, regionalization, epithelial tube morphogenesis, positive regulation of neuron differentiation, telencephalon development, protein localization to the cell periphery, pattern specification process, axonogenesis, regulation of membrane potential, and positive regulation of cell projection organization  can be found in the article/The animal study was reviewed and approved by the Institutional Animal Care and Use Committee at the Lundquist Institute for Biomedical Innovation at Harbor-UCLA Medical Center.VR designed the research. HW and JL performed the experiments. JG performed bioinformatic analyses. HW and WY analyzed the data and wrote the manuscript. All authors contributed to the article and approved the submitted version.This work was supported by grants from the NIH ; the Templeton Foundation (PID: 61174 to WY); and the TRDRP .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.


Lactobacillus delbrueckii subsp. bulgaricus (LdCsm) exhibits robust target RNA-activated DNase activity, which makes it a promising candidate for developing efficient miRNA diagnostic tools. Herein, LdCsm was tested for RNA detection using fluorescence-quenched DNA reporters. We found that the system is capable of specific detection of miR-155, a microRNA implicated in the carcinogenesis of human breast cancer. The RNA detection system was then improved by various approaches including assay conditions and modification of the 5′-repeat tag of LdCsm crRNAs. Due to its robustness, the resulting LdCsm detection platform has the potential to be further developed as a better point-of-care miRNA diagnostics relative to other CRISPR-based RNA detection tools.In cancer diagnosis, diverse microRNAs (miRNAs) are used as biomarkers for carcinogenesis of distinctive human cancers. Thus, the detection of these miRNAs and their quantification are very important in prevention of cancer diseases in human beings. However, efficient RNA detection often requires RT-PCR, which is very complex for miRNAs. Recently, the development of CRISPR-based nucleic acid detection tools has brought new promises to efficient miRNA detection. Three CRISPR systems can be explored for miRNA detection, including type III, V, and VI, among which type III (CRISPR-Cas10) systems have a unique property as they recognize RNA directly and cleave DNA collaterally. In particular, a unique type III-A Csm system encoded by  Developing efficient RNA detection platforms is crucially important for the diagnosis of human diseases such as cancers and infectious diseases caused by RNA viruses. For the latter, the eruption and spreading of human immunodeficiency virus (HIV) , SARS coCurrently, quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) still serves as the traditional gold standard for RNA detection ,12,13, bClustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-associated systems (CRISPR-Cas) provide the prokaryotic adaptive immunity . This imIn addition, Cas12 and Cas13 effectors have been explored for miRNA detection, but each type of detection tool exhibits some drawbacks. For Cas12-based miRNA detection, miRNA targets have to be converted to corresponding DNAs, which activate Cas12 for cleavage of a DNA reporter. The employed methods of conversion include the rolling circle amplification , the catLactobacillus delbrueckii subsp. bulgaricus (LdCsm) that exhibits very strong RNA-activated DNase activity [Recently, we characterized a type III-A Csm system in activity ,49. In tE. coli. To do that, the type III-A cas genes of the L. delbrueckii (Ld Cas module) were cloned into a plasmid expression vector, whereas the RNAs to be detected were employed to design spacers for a mini-CRISPR array, which was then cloned to another plasmid vector  since the latter function as RNA activators to the designed LdCsm effectors, whereas their non-homologous RNAs (unspecific RNAs) cannot  cannot b.The trans-cleavage of ssDNA by type III CRISPR effectors basically works on ssDNAs of any sources. Consequently, this immune response has to be carefully controlled, and indeed, type III CRISPR systems have evolved the spatiotemporal regulation to limit its occurrence at the right place and in the right time. Namely, the immunity is triggered by transcription of the target site, whereas it is switched off by the enzymatic destruction of target RNAs by the nuclease hosted in the large backbone subunits, which is Csm3 for III-A systems. Previously we showed that LdCsm-dCsm3, a mutated effector that is inactivated for backbone cleavage, is also capable of distinguishing a specific target RNA versus an unspecific one. Herein, the same mutated effector was employed for establishing the LdCsm-based RNA detection platform. Reactions were set up with an equal concentration of target RNA S1 (CTR-full) or S10, a non-homologous RNA, in the presence of a fluorescence-quenched polythymidine (Poly-T) reporter and LdCsm-dCsm3. S1 is the activator RNA that facilitates the cleavage of the Poly-T reporter, yielding fluorescent molecules that can be detected and quantified with a fluorometer. As shown in The metal ion dependence of the LdCsm DNase was tested in our previous work where we found that the LdCsm DNase is activated by 10 mM MgCl2, and the addition of 50 mM KCl further increases the DNA cleavage activity . Here, tSulfolobus islandicus III-B Cmr system [The Poly-T reporter was designed based on the preferred sites of cleavage by the r system . While tr system , the nucWe then designed a CT/CC-rich reporter (CT reporter) and tested it for reporting efficiency, along with the Poly-T reporter, in the fluorescence assay . We founLeptotrichia wadei (Lwa) Cas13 protein , a synthetic S1 crRNA, and a fluorescence-quenched RNA reporter, as listed in Cas13 effectors exhibit the target RNA-activated collateral nuclease activity , which iE. coli cells, to a final content of 2 or 10 ng/µL. The resulting mass ratios of the target RNA vs. unspecific RNAs were 1:670–67 and 1:3330–333 for the two series of reaction, respectively. As shown in In clinical diagnosis, samples for nucleic acid detection are usually complex, and the target RNA is often comprised of a very small proportion of total RNAs in the samples. Therefore, it is very important for an RNA detection system to specifically detect the target RNA in the presence of large amounts of unrelated RNAs in the samples, a property that is referred to as “the tolerance to unspecific RNAs”. Herein, the LdCsm-dCsm3 and the Cas13a detection systems were evaluated for this feature. Target RNA of a series of concentrations (0.2–2 nM) was mixed with an unspecific RNA sample, which is the total RNA prepared from The robustness of miRNA detection platforms is another essential feature, which is especially important for developing the POC diagnostics. We assessed the stability of LdCsm-dCsm3 and Cas13a detection systems after storing these Cas enzymes at different temperatures for up to one month including −80 °C, −20 °C, 4 °C, and RT. Aliquots of Cas13a and LdCsm enzymes were prepared and stored at the above specified conditions, and during the storage, aliquots were taken out and tested regularly for the activity of miRNA detection. We found that both LdCsm-dCsm3 and Cas13a retained the full activity during the entire period of storage at −20 °C or −80 °C. However, Cas13a completely lost the detection ability after storage at 4 °C after the same period of storage, and it rapidly lost the activity at RT (after 7 days); in contrast, after storage at 4 °C or RT for 28 days, LdCsm-dCsm3 still retained 83% and 62% of the detection efficiency, respectively b. Thus, Target RNA activation of the DNase activity of LdCsm-dCsm3 has to satisfy two requirements: (1) the target RNA should carry the sequence that is complementary to spacer region of the LdCsm crRNA (this region in target RNA is termed protospacer); (2) the 3′-protospacer-flanking sequence (3′-antitag) of target RNA should show mismatches with the 5′-repeat tag (or 5′-repeat handle) of the LdCsm crRNAs . This kiTo explore the effect of length of target RNA on LdCsm-dCsm3 detection system, activation of the LdCsm-dCsm3 system was tested with RNAs with different lengths of protospacer and/or 3′-antitags , with thAs one of the most common cancer in the world, breast cancer (BC), accounts for over 25% of female cancer cases and causes one of the biggest cancer-related death rolls in women . miR-155miR-155 was synthesized by the Azenta Biotechnology Company  and used as the target RNA to test detection capability of the LdCsm-dCsm3-155-1 and LdCsm-dCsm3-155-2 effectors. As shown in As mentioned above, activation of LdCsm-dCsm3 detection system requires a target RNA carrying a 4 nt or longer 3′-anititag showing mismatches with the corresponding 5′-repeat tag of LdCsm crRNAs c. HoweveTo optimize the detection efficiency of the LdCsm-dCsm3 detection system on miR-155, we chose to introduce single-base substitutions into the repeat sequence of the Ld CRISPR array to generate full mismatches between the 5′-repeat tag of crRNA and the 3′-antitag of miR-155. Examination of the 3′-end of miR-155 revealed that A at position −3 of the 5′-repeat tag is complementary to it in one scenario while the A at position −5 of the 5′-repeat tag is complementary to it in another a. We theLdCsm-dCsm3-155-1-A−3U and LdCsm-dCsm3-155-2-A−5U were successfully purified, and the detection efficiency of them on miR-155 was compared with LdCsm-dCsm3-155-1 and LdCsm-dCsm3-155-2. Compared to LdCsm-dCsm3-155-1, the detection activity of LdCsm-dCsm3-155-1-A−3U on miR-155 was not improved c. In con2O and human sera. miRNA detection with the optimized LdCsm detection system revealed that only miR-155 showed the specific activation of the DNA reporter cleavage, whereas no evident RFU signal was produced in presence of any other miRNAs, including miR-124, miR-149, miR-340, and miR-375  and T. thermophilus Cmr (TtCmr) systems in conjunction with the T. thermophilus Csm6/Csx1 effector proteins [Vibrio metoecus (Vm)Cmr system is combined with VmNucC, a CARF protein derived from the same bacterium that functions in the CBASS antiviral defense [Lactococcus lactis (Ll)Csm for RNA detection in a single assay, including the DNase of LlCsm and the RNase of L. lactis Csm6 (LlCsm6) that is triggered by cOAs, the secondary signal synthesized by LlCsm. In this case, both ssDNA and RNA reporters are employed [Type III CRISPR-Cas10 systems possess two target RNA-activated reactions, i.e., the signaling pathway involving the synthesis of the cOA signal in the Palm domain and the HD-hosted collateral DNA cleavage activity , both ofproteins ,39. In a defense . The immemployed , while tIn this article, we explored the potential of LdCsm-dCsm3-based RNA detection platform on detection of miRNA, a putative biomarker for cancer diagnosis. miR-155, a 24 nt breast cancer (BC)-related miRNA, was successfully and specifically detected by the LdCsm detection system, implying that it is a promising candidate for developing miRNA diagnostic tools. To our knowledge, this is the first type III CRISPR-Cas10 system explored for miRNA detection. Theoretical LoD of LdCsm-dCsm3-155-2-A−5U on miR-155 was determined to be 500 pM-1 nM, a value that is comparable to those obtained from the detection of longer RNAs with the TtCsm or TtCmr detection systems without any isothermal amplification ,39. The For type III CRISPR-Cas10-based detection tools, the limited length of miRNAs renders it impossible to choose a completely mismatching 3′-antitag. By contrast, one has to use the 4-5 nt sequence at the 3′-end of miRNAs as the 3′-antitag sequence. We attempted to engineering the repeat sequence of the spacer, generating new 5′-repeat tags that exhibit full mismatches to the 3′-end of miR-155. We show that the complete mismatch between the 5′-repeat tag and the 3′-antitag of miR-155 increased the efficiency of the LdCsm-based miRNA detection system by 3.9-fold . Since aE. coli DH5α was the bacterial host for DNA cloning. E. coli BL21 (DE3) was the host for expression and purification of LdCsm effector complexes. E. coli strains were cultured in Luria–Bertani (LB) or terrific broth (TB) medium. Incubation was at 37 °C, 200 rpm. If required, antibiotics were added as the following: ampicillin (Amp) at 100 μg/mL, kanamycin (Kan) at 25 μg/mL, and chloramphenicol (Cm) at 10 μg/mL.EcoRI and SalI and then amplified with primers pUCE-repeat-F/R. In this way, the 5′ 18 nt of Ld repeat sequence was added to one end of the pUCE fragment , while the 3′ 18 nt of the Ld repeat was added to the other end of the pUCE fragment (cut by EcoRI). Then, the artificial mini-CRISPR fragments carrying S-155-1/S-155-2 spacer were ligated with pUCE fragments with Ld repeat ends by the Gibson assembly to get pUCE-S-155-1/pUCE-S-155-2 plasmids, which can be used to express crRNA targeting miR-155 specifically. pUCE-X plasmid encodes an artificial mini-CRISPR array carrying an “X” spacer, which transcribes crRNA targeting RNA X specifically. Two spacers named S-155-1 and S-155-2 were designed for targeting breast cancer-related microRNA miR-155. To construct artificial mini-CRISPR fragments of these spacers, fusion PCR amplification was performed using two sets of two primers , respectively, to generate multiple copies of Ld repeats interspaced by multiple spacers. mini-CRISPR fragments of about 1 kb were recovered from agarose gel. pUCE plasmid was linearized by Two sets of fusion PCR primers named miR155-1-A−3U-F/R and miR155-2-A−5U-F/R were designed to introduce an A−3U mutation in the Ld repeat sequence of pUCE-S-155-1 and an A−5U mutation in the Ld repeat sequence of pUCE-S-155-2, respectively. The ligation of fusion PCR products of these primers and pUCE fragments with Ld repeat ends produces pUCE-S-155-1-A−3U and pUCE-S-155-2-A−5U, which transcribe crRNA with an A−3U or A−5U mutation in the 5′-repeat tag, respectively. 600 = 0.8) under the same growth condition. Then, IPTG was added to 0.3 mM and the culture was further cultured at 25 °C for 16 h to induce the synthesis of LdCsm. Cells were harvested by centrifugation at 5000 rpm for 5 min, and cell pellets were resuspended in 50 mL buffer A . The cell suspension was treated by French press at 4 °C, and cell debris was removed by centrifugation at 10,000 rpm for 1 h at 4 °C. The LdCsm complex was then purified in a two-step purification procedure as previously described. LdCsm-expressing strains were obtained by transformation of three plasmids . For purification of each LdCsm effector complex, a few single colonies were inoculated into 20 mL LB medium containing ampicillin, kanamycin, and chloramphenicol and cultivated at 37 °C, 220 rpm overnight. Next, 10 mL of the overnight culture was transferred to 1 L TB medium and cultured to a mid-log phase . The product was further purified by gel filtration with Superdex 200  using the chromatography buffer . Fraction samples were collected and mixed thoroughly with glycerol in a volume ratio of 1:1. Purified LdCsm complex samples were then stored at −80 °C or used for further analysis.2, 50 mM KCl, 0.1 mg/mL BSA, and pH 7.0) containing 50 nM LdCsm complex, 50 nM 5′ FAM-labeled ssDNA substrate, and 500 nM target RNA or unspecific RNA. Nucleic acid cleavage was conducted at 37 °C for 10 min and stopped by addition of 2 × RNA loading dye . Before loading, samples were heated for 3 min at 95 °C and analyzed by denaturing polyacrylamide gel electrophoresis. Cleavage products were visualized by fluorescence imaging with xx.RNA-activated ssDNA cleavage was assayed in 10 μL reactions in the cleavage buffer . The plate was then placed in a fluorescence plate reader  and incubated for 30–60 min at 37 °C, during which the fluorescence signal  was measured for each well every 5/10 min . Background RFU values generated for the reference sample (lacking a target RNA) were subtracted from the RFU values obtained from the reactions containing the cognate target RNA or RNA samples to be detected. The yielding background-subtracted RFU values were used for calculating the RFU generation rate in each reaction, which represented the DNase activity of activated-LdCsm RNP complex or the detection signal of LdCsm system on the indicated RNA samples.DNA reporter reactions were prepared in a total of 20 μL in the cleavage buffer as described above, in which 1 μM Poly-T or CT ssDNA reporter was used as the ssDNA substrate (Sequences listed in v/v) glycerol. A Cas13a crRNA targeting S1 target RNA (sequence listed in 2), 100 nM LwaCas13a, 100 nM Cas13a crRNA, 1 μM RNA reporter, and an indicated concentration of target RNA or unspecific RNA. After the addition of the RNAs to be detected (target RNA), the detection reactions were set up in a 384-well black plate  and placed in a fluorescence plate reader  to measure fluorescence signals. The detection activity was calculated as by the LdCsm detection reaction described above. The Cas13a detection kit contained the LwaCas13a protein , a fluorescence-quenched RNA reporter (5′ FAM-labeled and 3′ BHQ1-labeled, sequence listed in An RNA detection platform based on the LdCsm-dCsm3 system was developed, which solely relies on its target RNA-activated DNase to generate fluorescence from a quenched DNA reporter. Compared to Cas13a detection system, this LdCsm-dCsm3-based detection system has the advantage of tolerance against disturbance from unspecific RNA and shows less dependency on the cold chain transportation. The detection system showed high detection specificity on target miRNA since no fluorescent signal was generated in presence of other miRNAs. The LoD determined for the breast cancer-related miR-155 was 2 nM in clinic mimic samples, and introducing a single-base mutation into the 5′-repeat tag of LdCsm crRNA increased the detection activity by 3.9-fold. Therefore, the LdCsm-dCsm3 system represents a promising candidate for developing a new generation of point-of-care miRNA diagnostic tools. A patent application was filed for optimization of LdCsm DNase-based RNA detection system and for its application in harnessing the optimized system for RNA detection.