H-Ras, and numerous studies have shown that p68 RNA helicase is probably involved in miRNA biogenesis, mainly through Drosha and RISC/DICER complexes.DEAD-box RNA helicases catalyze the ATP-dependent unwinding of double-stranded RNA. In addition, they are required for protein displacement and remodelling of RNA or RNA/protein complexes. P68 RNA helicase regulates the alternative splicing of the important proto-oncogene  This study aimed to determine how p68 RNA helicase affects the activity of selected mature miRNAs, including miR-342, miR-330, miR-138 and miR-206, miR-126, and miR-335, and let-7a, which are known to be related to cancer processes.The miRNA levels were analyzed in stable HeLa cells containing p68 RNA helicase RNAi induced by doxycycline (DOX). Relevant results were repeated using transient transfection with pSuper/pSuper-p68 RNA helicase RNAi to avoid DOX interference.Herein, we reported that p68 RNA helicase downregulation increases the accumulation of the mature miRNAs, such as miR-126, let-7a, miR-206, and miR-138. Interestingly, the accumulation of these mature miRNAs does not downregulate their known protein targets, thus suggesting that p68 RNA helicase is required for mature miRNA-active RISC complex activity. Furthermore, we demonstrated that this requirement is conserved, as drosophila p68 RNA helicase can complete the p68 RNA helicase depleted activity in human cells. Dicer and Drosha proteins are not affected by the downregulation of p68 RNA helicase despite the fact that Dicer is also localized in the nucleus when p68 RNA helicase activity is reduced. Herein, we further reported that p68 RNA helicase RNAi induces an increased concentration of mature let-7a miRNA, as shown in Fig. (5). Our results showed that Dmp68 and Dmp68T induce the reduction of HMGA 3’-UTR, as shown in Fig.  that decreases its level. However, we cannot rule out that these miRNAs also require p68 RNA helicase to form the mature miRNA-active RISC complexes containing mature miRNAs. It was found that several other miRNAs also depend on p68 RNA helicase in the Drosha complex to process pri-miRNAs. When we observe mature miRNAs, we may simply be detecting the two major aspects, namely less pre-miRNA formation due to less Drosha complex formation and accumulation of inactive mature miRNAs. This could indicate that helicase should also be necessary for the formation of the mature miRNA-active RISC complexes of the miR-335, miR-330, and miR-342.et al. showed that nuclear retention of fission yeast Dicer is a prerequisite for RNAi-mediated heterochromatin assembly [Schizosaccharomyces pombe) and found that the nuclear accumulation of Dcr1 depends on a short motif that impedes nuclear export promoted by the double-stranded RNA binding domain of Dcr1 [Interestingly, p68 RNA helicase RNAi did not affect Dicer, Drosha, as seen in Figs. (assembly . These a of Dcr1 . The abs of Dcr1 . Herein, of Dcr1 . As a do of Dcr1 , we sugg of Dcr1 -41.In summary, p68 RNA helicase is necessary for mature miRNA-active RISC complex action of certain mature miRNAs. Furthermore, it regulates at least two important miRNAs, namely let-7a and miR-206, which are closely involved in cancer and metastatic processes.


Long non-coding RNAs (lncRNAs) are transcripts longer than 200 nucleotides (nt) that are not translated into known functional proteins. This broad definition covers a large collection of transcripts with diverse genomic origins, biogenesis, and modes of action. Thus, it is very important to choose appropriate research methodologies when investigating lncRNAs with biological significance. Multiple reviews to date have summarized the mechanisms of lncRNA biogenesis, their localization, their functions in gene regulation at multiple levels, and also their potential applications. However, little has been reviewed on the leading strategies for lncRNA research. Here, we generalize a basic and systemic mind map for lncRNA research and discuss the mechanisms and the application scenarios of ‘up-to-date’ techniques as applied to molecular function studies of lncRNAs. Taking advantage of documented lncRNA research paradigms as examples, we aim to provide an overview of the developing techniques for elucidating lncRNA interactions with genomic DNA, proteins, and other RNAs. In the end, we propose the future direction and potential technological challenges of lncRNA studies, focusing on techniques and applications. The term long non-coding RNA refers to RNAs over 200 nt in length that do not encode known functional proteins ,2. EvideCis-acting lncRNAs regulate gene expression in a manner dependent on the location of their own sites of transcription [trans-acting lncRNAs regulate gene expression at locations distant from its own transcription locus [cis and trans regulation involve interactions of lncRNAs with specific DNA target sequences, other RNAs, or RNA-binding proteins (RBPs) including splicing factors, DNA modifiers, histone modifiers, and nucleosome remodeling factors.The Human GENCODE project annotated more than 16,000 lncRNA genes ,23, but cription ,27,28,29on locus ,31,32,33In the past two decades, multiple approaches have been applied to elucidate the modes of action of biologically important lncRNAs. Taking advantage of the rapid development of next-generation sequencing, multi-omics methodologies have acquired an increasingly important role. Generally, ‘RNA-centric’ approaches investigate the possible interactions of DNA or proteins with known lncRNAs  , while ‘Multiple reviews to date have provided excellent discussion on the mechanisms of lncRNA biogenesis, their localization, their biological functions in gene regulation at multiple levels, and also their potential applications ,5,36. Focis and trans regulation, sometimes indirectly due to their affinity for proteins that can bind to RNA and DNA, and in other cases directly by binding DNA in a sequence-specific manner.The functionality of lncRNAs is achieved through their interactions with other RNAs, DNAs, and RBPs. In interactions with DNA, the negative charge of RNA can neutralize the positively charged histone tail, causing chromatin to decompress . TherefoKHPS1 [HOTAIR [PARTICLE [FENDRR [GhDAN1 [LncRNAs have the potential to form heterozygous structures with genomic DNA to affect chromatin accessibility. In one type of direct interaction, RNA can bind within the major groove of purine-rich DNA via Hoogsteen base pairing, forming a triple helical RNA–DNA structure that anchors the RNA to a specific DNA sequence and thereby targets RNA-associated regulatory proteins to distinct genomic sites. A growing number of examples have shown that the ability of lncRNAs to form RNA–DNA triplexes in vivo broadly affects chromatin accessibility . The potKHPS1 ,43, HOTA [HOTAIR , PARTICLPARTICLE , FENDRR  [FENDRR , and GhD [GhDAN1 , indicat [GhDAN1 .TCF21 [TARID lncRNA at the TCF21 promoter to recruit the DNA demethylation factor TET1, resulting in transcriptional activation [A more widely-studied pattern of lncRNA interactions with chromatin based on the principle of complementary base pairing  termed R-loop. R-loop has long been considered a threat to genomic stability. However, the transient properties of the R-loop also make it an ideal regulatory center, and recent studies have re-evaluated its functions in regulating gene expression and coordinating DNA repair ,49. WithTCF21 . GADD45Ativation ,53. BothFAST is transcribed in the antisense direction from the FOXD3 gene and is highly expressed in hESCs, for which it is necessary to maintain pluripotency. Each FAST molecule forms five stem loops that provide a multivalent platform for the E3 ligase β-transducin repeats-containing protein (β-TrCP) and block the degradation of its substrate β-catenin, leading to activation of WNT signaling that is required for pluripotency [NKILA forms two different hairpins, hairpin A and hairpin B, both of which bind to p65. Binding of hairpin B is necessary for NKILA to stably associate with the NF-kB:IkB complex to regulate NF-κB activity [Xist also implies an important relationship of RNA secondary structure with protein chaperone interaction. Xist contains six repeating sequence elements  that mediate its interactions with respective protein partners such as ATRX, which binds Xist repeat A through its N-terminal RNA binding domain (RBD) and C-terminal helicase domain [Xist domains that interact with distinct sets of effector proteins have been revealed, such as DNMT1, CBX3, and phosphorylated CBX3 for the A domain; DNMT1, CBX3, pCBX3, and HNRNPH for the F domain; HNRNPU for the BCD domain; and CHD4, EZH2, SUZ12, DNMT1, and pCBX3 for the E domain [COOLAIR at the Arabidopsis FLC locus [FLC via alternative splicing [COOLAIR, revealing a hyper-variable region gives COOLAIR stronger chromatin binding ability, which leads to down-regulation of FLC expression [In addition to binding to sequence motifs, lncRNAs can also fold into secondary structures that interact with proteins involved in key signaling pathways . As examipotency . The NF-activity . The sece domain . Moreovee domain . DiscretE domain . The lncLC locus ,60 does splicing ,62. Recepression . GeneralMany lncRNAs play regulatory roles through recruitment of specific RNA–RBPs. Ramanathan et al. have systemically discussed the methods for studying RNA–protein interactions , in whicArabidopsis, winter cold triggers enrichment of H3K27me3 in the chromatin of FLOWERING LOCUS C (FLC), a flower inhibitor, which leads to the epigenetic inhibition of FLC. This epigenetic regulation is mediated by the evolutionally conserved inhibitor complex polycomb inhibitor complex 2 (PRC2). The lncRNA COLDWRAP from FLC locus transcribed from the FLC promoter region in the same direction as FLC mRNA. RNA pull-down using biotin-labeled COLDWRAP has been shown to successfully enrich polycomb group CLF protein fused with his-tag [Xist and FIRRE [RNA pull-down a is the  his-tag , PAIR   Figure c and crolization . In detefter RIP . In anottor FOX2 , and alsproteins . Variousproteins , iCLIP [proteins , eCLIP [proteins , irCLIP proteins , GoldCLIproteins , fCLIP [proteins , and Brdproteins  were devproteins . To dateproteins  has probproteins . As an anscripts ,84. The XRNAX (protein-crosslinked RNA extraction) e and OOPTaken together, it is important to select optimal lncRNA–protein interaction approaches for the biological question being addressed. For confident identification of bona fide RNA–protein interactions, RBPs discovered via an lncRNA-centric method should be validated by the complementary protein-centric method, and vice versa, for lncRNAs discovered via an protein-centric method. Thus, to obtain robust supporting data, multiple approaches can be performed in parallel to support each other. In such endeavors, having an antibody with high efficiency is important; to date, available assay-verified antibodies include anti-H3K27me3 (Merck-Millipore), anti-EZH2 (active motif) , and antcis ligation with mRNA at the site of transcription; and, in RADICL-seq, the use of a hairpin adaptor to selectively ligate the bridge adapter that is covalently bound only to RNA and therefore prevent subsequent ligation of sequencing adapters. Ultimately, all four of these methods have confirmed their feasibility by validating previously reported lncRNAs. For examples, the lncRNA MALAT1 has been detected by MARGI, GRID-seq, and RADICL-seq, and roX2 can by GRID-seq and CHAR-seq. Additionally, GRID-seq was applied in Arabidopsis seedlings to identify more than 10,000 RNA–chromatin interactions mediated by protein-coding RNAs and non-coding RNAs, in which inter-chromosomal interactions were found to be primarily mediated by non-coding RNAs [The omics-based approaches for investigating RNA–DNA interactions detect the locations on the genome at which lncRNAs and their associated RNPs are bound; thus, the detections include both direct interactions of lncRNAs and indirect interactions through lncRNA-containing RNPs. Many lncRNAs play regulatory roles through targeting genomic DNAs. LncRNA-centric approaches used to elucidate lncRNA–protein interactions can also determine lncRNA–DNA interactions; for example, ChIRP followed by MS (ChIRP-MS) identifies RBPs that bind to an lncRNA of interest, while ChIRP followed with NGS sequencing (ChIRP-seq) identifies lncRNAs that target genomic DNA sites b. A numbing RNAs . In addiing RNAs . Howevercis- and trans-acting ncRNAs enriched at polycomb- and CTCF-binding sites in human cells, which may be involved in polycomb-mediated gene repression and CTCF-dependent chromatin looping [Two approaches, RedChIP  [Recently, Quinodoz et al. developed RNA and DNA SPRITE to comprehensively map the spatial organization of RNA and DNA  Figure c. The fiilities) . Finallyilities) . There ailities) .PNUTS lncRNA is produced by alternative splicing of the PNUTS pre-mRNA. Bioinformatics predictions indicate that the PNUTS lncRNA contains seven binding sites for miR-205, and incubation of MS2 marker and biotin-labeled PNUTS lncRNA with cell lysates followed by miRNA-specific RT-PCR has shown that the PNUTS lncRNA can pull down miR-205. miR-205 is the mature inhibitor of the transcriptional inhibitors ZEB1 and ZEB2 and is required to maintain epithelial cells. Sponging of miR-205 by PNUTS leads to upregulation of ZEB1 and ZEB2, thereby promoting epithelial–interstitial transformation and breast cancer cell migration and invasion [Unlike protein-coding mRNAs, most lncRNAs are located in the nucleus, and many of them are associated with chromatin . Accordiinvasion . Alternainvasion .RIC-seq (RNA in situ conformation sequencing) is the latest advance developed for the global profiling of RNA–RNA interactions ,106 Figd. GeneraMEG3 is one of the lncRNAs that can be enriched in heterochromatin regions of breast cancer cells by RIP-seq using both anti-EZH2 and anti-H3K27me3 antibodies [MEG3 lncRNA is recruited to the chromatin of the target gene where its GA-rich RNA sequence and the GA-rich DNA sequence form an RNA–DNA triplex, and the PRC2 interacting region of MEG3 promotes PRC2 recruitment to a distal regulatory element, thereby establishing H3K27me3 epigenetic modification to regulate gene expression [MALAT1 (metastasis-associated lung adenocarcinoma transcript 1) is probably the most abundant lncRNA in most cultured cells. MALAT1 localizes to nuclear speckles, as indicated by RNA-FISH assay [MALAT1 interacting proteins. In addition, application of RIC-seq has shown MALAT1 to act as an RNA hub for many highly expressed RNAs. However, characterization using multi-color structured illumination microscopy indicated a nuclear speckle to be a multi-layered compartment at a higher resolution in which nuclear spot proteins such as the splicing factors SON and SC35 are localized in the center of the speckle, whereas MALAT1 are is located on the periphery [MALAT1 indicates it to promote nuclear speck formation and function. The PARIS method, which is based on reversible psoralen-crosslinking and applied for the global mapping of RNA duplexes with near base-pair resolution in living cells, identified MALAT1 to form many long-range structures that may be involved in polyvalent interactions with different RBPs and pre-mRNAs [MALAT1 localize broadly across all chromosomes [MALAT1 lncRNA is a good model for studying lncRNA interactions with RNA and proteins.In many cases, the interaction of lncRNAs with DNA, RNA, or RNPs is not exclusive; there are many that interact directly or indirectly with DNA, RNA, and proteins at the same time. For example, tibodies . MEG3 lnpression . For anoSH assay , fulfillSH assay , ChIRP-MSH assay , and RNASH assay  has ideneriphery . The unire-mRNAs . Additioomosomes . Overallin-cis or trans (‘who’). It was further determined the sub-cellular and sub-organelle localization of lncRNA by single-molecule fluorescence in situ hybridization (smFISH) [Given the diversity of available methodologies applied in lncRNA biology, we summarize a general mind map for lncRNA study . Traditi(smFISH)  combined(smFISH) ,38,129, Recent technical advances in methods for studying lncRNAs have shed light on complex and critical lncRNA–protein, lncRNA–DNA, and lncRNA–RNA interactions, which provided important profiles for lncRNA-mediated gene regulation. However, it is important for researchers to be aware of the strengths and limitations of different methods and to choose among them as best befits their research aims. Challenges to uncovering the biological functions of lncRNAs remain in many respects. Firstly, cutting-edge biochemical technologies will provide advanced sensitivity for versatile modes of action study of lncRNAs in examined biological pathways. It is still challenging to develop methodologies for the ‘all-to-all’ screening and profiling of lncRNAs with concise and precise performance and streamlined bioinformatic workflow. The operability, throughput, sensitivity, and reliability of such approaches still need validation and optimization. Secondly, it will be of great interest to determine lncRNA at the single-cell level, including its dynamic expression by single-cell sequencing technologies, and its localization (sub-cellular and sub-organelle) and dynamic and real-time binding with partner proteins by in vivo biochemical labeling followed by super-resolution imaging technologies. Additionally, single-molecule protein sequencing could provide single-molecule sensitivity detection for lncRNA interacting RBPs . Thirdly


Reoviridae member, with a genome of 10 segments. To address this, we used an RNA-cross-linking and peptide-fingerprinting assay (RCAP) to identify RNA binding sites of the inner capsid protein VP3, the viral polymerase VP1 and the capping enzyme VP4. Using a combination of mutagenesis, reverse genetics, recombinant proteins and in vitro assembly, we validated the importance of these regions in virus infectivity. Further, to identify which RNA segments and sequences interact with these proteins, we used viral photo-activatable ribonucleoside crosslinking (vPAR-CL) which revealed that the larger RNA segments (S1-S4) and the smallest segment (S10) have more interactions with viral proteins than the other smaller segments. Additionally, using a sequence enrichment analysis we identified an RNA motif of nine bases that is shared by the larger segments. The importance of this motif for virus replication was confirmed by mutagenesis followed by virus recovery. We further demonstrated that these approaches could be applied to a related Reoviridae member, rotavirus (RV), which has human epidemic impact, offering the possibility of novel intervention strategies for a human pathogen.How multi-segmented double-stranded RNA (dsRNA) viruses correctly incorporate their genomes into their capsids remains unclear for many viruses, including Bluetongue virus (BTV), a   How viruses correctly package their genomes represents one of the most challenging questions in virology. Genome packaging is a key process in the virus life cycle and must be highly specific, such that, a single genome or a set of genomic segments is correctly packaged into the confined space of the capsid and all other extraneous nucleic acids are excluded. Some viruses use a binding site in the viral capsid protein to recognize a specific sequence of the genome , but thiReoviridae family has 10–12 genomic dsRNAs, all of which must be packaged correctly to generate a viable virus particle, it is therefore unlikely that this family of viruses had adopted a CCMV-like length-dependent packaging strategy. Instead, a specific recognition between genome and capsid seems more likely. This is supported by the fact that, following virus entry, individual ssRNA transcripts leave the icosahedral capsid by separate channels, implying an ordered network of genomic segments primed for transcription  and an inner icosahedral capsid (core) composed of two additional proteins, VP7 and VP3. Within the core, the virus encapsidates 10 dsRNA genome segments (S1 to S10) and two replication related proteins, the polymerase (VP1) and the capping enzyme (VP4). These two core proteins are common across the Reoviridae, but BTV and other members of the Orbivirus genus has an additional protein, VP6, that is essential for RNA packaging (Bluetongue virus (BTV), a member of Orbivirus genus of ackaging ,9.When BTV enters the cell, the outer capsid is removed releasing the inner capsid core into the cytoplasm. Without further disassembly, the released core particle synthesizes and extrudes ssRNA transcripts into the cytoplasm. While some ssRNA transcripts act as templates for viral protein synthesis, others form RNA complexes through RNA-RNA interactions and are packaged, possibly with the help of viral protein(s), into the assembling progeny capsids ,11. GenoReoviridae  share similar assembly processes but do not possess a packaging protein analogous to VP6, understanding any additional interactions between BTV VP3 and RNA may reveal the role of capsid proteins in the RNA packaging of other Reoviridae members.Recent data suggests that a direct interaction between VP6 and VP3 is essential for viral genome packaging and the early stage of virus particle formation . FurtherMost recently, newer techniques such as viral Photo-Activatable Ribonucleoside Cross-linking (vPAR-CL) have shown that it is possible to identify the specific nucleotide sequences that interact with viral proteins . Here, wFJ969719 to FJ969728) and RV SA11 (GenBank accession numbers LC333802 to LC333812) were used for RCAP and vPAR-CL studies. All mutations for reverse genetics and recombinant protein production were generated by site-directed mutagenesis. The sequences of the primers are listed in BTV-1  and the recombinant baculoviruses were generated and selected based on manufacturer's instruction. The VP3 expression was analysed as previously described . PolyacrRecombinant VP6 (rVP6) of BTV-10, which is fully exchangeable with BTV-1 VP6 was used in vPAR-CL. The expression and purification of recombinant baculovirus expressing VP6 in the Sf9 cells have been described previously . The HisP-40). Assembled CLPs were purified from the soluble fraction by centrifugation on a 35% CsCl gradient as described previously , together with the other 9 BTV genome segments that were used to transfect BSR cells or a BSR cell line that stably expresses VP6 (BSR-VP6), as described by Boyce et al. . Cytopatet al.  of 0.01–0.1, and covered by an Avicel overlay as described by Matrosovich et al. . The celin vitro cell-free assembly (CFA) assay has been described previously  with 0.15 J/cm methods . The samFor testing BTV VP6 independently, we used purified rVP6 expressed by baculovirus expression system as described previously  and incuClickSeq libraries of cross-linked and control RNAs were constructed as previously described ,25–27. BThree threshold filters were used to determine the significant and consistent signals for each experiment. In particular, each ‘U’ position has to meet the following criteria to be considered significant and consistent: (i) has a sequencing depth greater than 1000× in both control and crosslink samples; (ii) average vPAR-CL signal of the nucleotide must be greater than the mean signal (of the genome) + 1σ ; and (iii) has an increased U–C transition rate in at least 3/4 trials for BTV core, 2/3 for rVP6 and RV.https://github.com/andrewrouth/vPARCL_call) to extract the nucleotide frequency at each genomic coordinate and to calculate the mismatch rate at each position, including the U-to-C transition rate, as expected in vPAR-CL data. The vPAR-CL signals are defined as the fold change of U-to-C transition rate between the cross-linked sample and the non-crosslinked control, as previously described , and another at residues 500 to 540 (VP3.Region2) . In the VP3.Region 1 contain an arginine, lysine and arginine at position 245–247 that are highly conserved among all Orbiviruses . VP3.RegFor VP3.Region2, four highly conserved positively charged residues  were selected for mutagenesis. Of the 4 mutants, only mutant R502A yielded virus, suggesting that residues K509, R526 and R529 play an important role in virus replication. To clarify whether the failure of virus recovery is due to interference with RNA-capsid interactions, or structural changes of the icosahedral capsid, we verified capsid assembly using the recombinant core-like particles (CLPs) system ,37. In bin vitro cell-free assembly (CFA) assay which allows the generation of infectious core particles with a full complement of 10 RNA segments , which functions as an RNA dependent RNA polymerase (RdRp) and VP4 (644 residues), which modifies the 5’ termini of mRNAs prior to their release from the viral core ,39. HighVP1 is composed of three domains, the N-terminal (aa 1–387), polymerase (aa 388–935), and C-terminal bracelet domain aa 936–1302) (–1302 36. RCAP anO-)-methyltransferase (2’ OMTase) domain (aa 175–377), and the C-terminal guanylyltransferase (GTase) domain (aa 510–644). The atomic structure of VP4 has been solved using BTV VP4 monomers expressed in insect cells , the (guanine-N-)-methylct cells . Howevers Figure , aa 318–s Figure   method  method ,24. In b) method , BSR celThe viral protein-interacting RNA sites are indicated by vPAR-CL signals, which are defined here as the fold change of U-C transition between the cross-linked (4SU+/UV+) experiment and non-cross-linked (4SU+/UV-) control. From the average vPAR-CL signals of four replicates, RNA-protein interacting sites were readily detectable across the entire BTV dsRNA genome, with extensive RNA-protein interactions sites located within the inner core layer Figure . HoweverAmong these, the second largest segment  contained the greatest number of significant sites , which is noticeably more than contained in the largest segment . The smallest segment  contained 17 significant sites (20.7 sites/kb), in stark contrast to other smaller segments such as S8  and S9 . This demonstrated that the encapsidated dsRNA segments of BTV exhibit a fixed topology which provides different levels of RNA-protein interaction inside the core. It is likely that certain segments of the genome  may be organized more centrally and occluded by other genomic segments, since both were observed to contain the least amount of RNA-protein interaction sites. In contrast, the larger segments  and the smallest S10 may be more accessible in order to support an increased number of organized viral protein–RNA contacts inside the core.in vitro, with purified recombinant VP6 (rVP6) and 4SU labelled dsRNA extracted from BTV cores  that could interact with encapsidated dsRNA genome. Among these, VP6 is known to have strong RNA binding affinity ,40. To d methods .in vitro  and rVP6 (483 sites), found that ∼19% of interaction sites in the core are identical to those found in rVP6 (73 sites), and ∼61% sites in the core are adjacent within 10nts) to those found in rVP6   and occurred at multiple locations across different genome segments  were analysed with the ‘STREME’ computational pipeline  to identAmong these sites that potentially contain the specific motif, we tested the sites which had at least 7 matching nucleotides for virus recovery. The nucleotide sequences were carefully changed without altering amino acid coding sequences. Of these mutations, nt523-531 of S1, which was an exact match for the predicted 9 nucleotide motif sequence, was lethal to the virus. Similarly, S3 nt1828-1837, S3 nt2617-2626, S4 nt602-611 and S10 nt538-547, which all matched 7 nucleotides with the proposed motif, were also shown to be critical for virus replication. After altering the nucleotide sequences of S2 nt1305-1314 and S2 nt1704-1713, virus could be recovered, however the mutant virus had a reduced growth rate . Among tWhen a similar motif-finding approach was applied to the vPAR-CL sites bound by rVP6, no sequence motif was identified which passed the set threshold of both p-value and E-value in the dataset. It is noteworthy, that although there is no motif identified from the sites bound to rVP6, the sites matched to the predicted motif are mostly (12 out of 17) identical or adjacent with the sites bound to rVP6 Table , indicatReoviridae family and share many similarities. The inner core/capsid structure of these two viruses are very similar and it was proposed that they share similar RNA packaging mechanism  interacts with genomic RNA. This region is located in the finger's subdomain of the polymerase domain Figure , E, whicSubsequently, we carried out vPAR-CL experiments in RV (SA11 strain) using highly purified RV DLPs, which revealed that across the 11 genome segments of RV, an uneven distribution of vPAR-CL sites was observed . ClusterReoviridae lack nucleoproteins and instead have multi-layered capsids, with the inner capsid layer interacting directly with the viral polymerase and very probably with the viral genome which is present as dsRNA. Furthermore, as the dsRNA genome is multi-segmented, it is plausible that the interaction with RNA will may assist in the packaging process to ensure that all of the RNA segments are properly localized within the capsid.The interaction between capsid proteins and viral RNA are essential for the correct genome packaging of many viruses. Lentiviruses, coronaviruses, and influenza viruses selectively package their genomes with the help of their respective nucleoproteins . Other vin situ atomic structures have revealed that the interaction between VP1 and the VP3 capsid proteins is crucial for the precise localisation of VP1 and the synthesis of ssRNA transcripts. The RCAP data here show that one of the RNA-interacting regions of VP3 is located in its apical domain in close proximity to VP1 within the core. Further, a substitution mutation in this region failed to package RNA in an in vitro RNA packaging assay indicating that this region may also be responsible for RNA packaging. The other RNA-interacting region is located in the carapace domain, near the interface of VP3A and VP3B. Changes in this region perturbed the capsid structure formation, suggesting that this region is important for VP3 structural integrity.BTV is an attractive model system for segmented dsRNA viruses since the mechanism of replication is well-studied and it has an established system of reverse genetics to confirm the influence of targeted mutations on virus replication. Our previous studies established that VP6 is an RNA packaging protein and that it recognizes the BTV genome in a specific manner ,18. FurtThe RCAP analysis of the core also identified three RNA-interacting regions within the VP1 polymerase. The helix bundle of the NTD has been shown by structural analysis to have the potential to interact with RNA termini  whilst tReoviridae with a similar double-layered core structure (DLPs). RCAP analysis revealed that RV VP2, the equivalent of BTV capsid protein VP3, has a major role in interacting with the encapsidated RNA. One RNA-interacting site located in its apical domain and also one in carapace domain, similar to BTV VP3. Further, it has two additional RNA-interacting sites at its N-terminal domain. The RV VP2 N-terminal domain has been reported previously to interact with the RV RdRp  having very limited capsid contacts. we speculate that the larger segments are located closer to the inside surface of the capsid and maybe accessed by the transcription complex more easily. It has recently been demonstrated by cryo-EM that the organisation of the genome within dsRNA virus capsids is ordered ; howeverin silico modelling and/or the prediction of the supramolecular organisation of authentic virus particles.However, despite the importance of the organisation of the RNA genome within the capsid for RNA packaging and virus assembly, the spatial organization of different dsRNA segments, unlike the non-segmented ssRNA viral genomes , remainsin vitro, suggesting that these sites might initiate the packaging process with VP6 before relocating to VP3 upon encapsidation. Further study is required to confirm this theory. Although less investigated, RNA-interacting sites for the RV genome for all three DLP capsids proteins was comparable to that of BTV, with larger segments having more capsid interaction sites per kb than the smaller segments suggesting that the mechanism uncovered here may be conserved in both viruses and possibly other members of the Reoviridae.Since viruses have a limited number of capsid proteins that can interact with the genome, it would be expected that viral proteins would recognise RNAs via similar sequences or secondary structures, as in the case of ssRNA viruses . STREME The sequencing data have been deposited with the NCBI SRA under BioProject ID PRJNA907830.gkad274_Supplemental_FilesClick here for additional data file.


Double-stranded (ds) oligonucleotide probes composedof quencher-dyesequence pairs outperform analogous single-stranded (ss) probes dueto their superior target sequence specificity without any prerequisitetarget labeling. Optimizing sequence combinations for dsprobe designrequires promoting a fast, accurate response to a specific targetsequence while minimizing spontaneous dsprobe dissociation events.Here, flow cytometry is used to rapidly interrogate the stabilityand selective responsiveness of 20 candidate LNA and DNA dsprobesto a 24 base-long segment of severe acute respiratory syndrome coronavirus2 (SARS-CoV-2) RNA and ∼243 degenerate RNA sequencesserving as model variants. Importantly, in contrast to quantifyingbinding events of dye-labeled targets via flow cytometry, the currentwork employs the Förster resonance energy transfer (FRET)-baseddetection of unlabeled RNA targets. One DNA dsprobe with a 15-base-longhybridization partner containing a central abasic site emerged asvery stable yet responsive only to the SARS-CoV-2 RNA segment. Separatedisplacement experiments, however, indicated that ∼12% of thesequencher-capped hybridization partners remain bound, even in the presenceof an excess SARS-CoV-2 RNA target. To examine their quenching range,additional titration studies varied the ratios and spatial placementof nonquencher and quencher-capped hybridization partners in the dsprobes.These titration studies indicate that these residual, bound quencher-cappedpartners, even at low percentages, act as nodes, enabling both staticquenching effects within each residual dsprobe as well as longer-rangequenching effects on neighboring FAM moieties. Overall, these studiesprovide insight into practical implications for rapid dsprobe screeningand target detection by combining flow cytometry with FRET-based detection. One classic paradigm involves molecular fluorescentdonor and acceptor  species with suitable spectraloverlap. Here, each discrete donor–acceptor pair has a one-to-onecorrespondence with each other and a given target species.8 The distance-dependent response involving nonradiative energy transferbetween an acceptor–donor pair enables FRET as a “spectroscopicruler”10 to infer likely atomic spatialarrangements within a secondary structure. While not detailed here,FRET studies often incorporate a different fluorophore instead ofa quencher as the acceptor. In such cases, spectral shifts in emissionevents rather than signal off–on events occur as dynamic conformationalchanges adjust the separation distance between donor and acceptorspecies. Studies of self-folded hairpins or molecular beacons12 and dsprobes typically involve fluorescence spectroscopy of oligonucleotidesolutions initially in a signal-off state. Here, the initial secondarystructure promotes the close proximity of quencher and dye species,resulting in static quenching. Ideally, a specific oligonucleotidetarget hybridizes to the molecular beacon, causing a hairpin-to-duplextransition or to the dsprobe, causing a primary duplex-to-secondaryduplex transition. Both of these transitions can sufficiently separatethe quencher from the fluorescent dye to allow fluorescence to occur.Challenges for molecular beacons include their susceptibility to unfoldingin the absence of target as its self-hybridized stem undergoes spontaneous“fraying and peeling”13 ortransient melting14 resulting in high backgroundfluorescence. To reduce background fluorescence, multiple acceptorscan be conjugated to each molecular beacon.16 To circumvent undesirable molecular beacon sequestration to thenucleus,17 past in vitro studies19 used the tetrameric protein, streptavidin,as a successful carrier to preserve each molecular beacon as a cytosolicprobe. Importantly, excess streptavidin reportedly19 favored occupation of only one of its four biotin-binding sitesby an individual molecular beacon.Reliable and rapid oligonucleotidesensing platforms are key to assuring food and water safety24 but with recent extensions to RNA targets.25 A single-stranded toehold in the original dsprobe favors displacementby enabling the target to nucleate and then complete secondary duplexformation.31 Real-time or quantitative polymerase chain reaction (qPCR) for DNAtargets and reverse transcriptase or RT-qPCR for RNA targets can increasethe copy number of unlabeled targets with accompanying proportionalincreases in fluorescence signal. During qPCR, a longer template sequencehybridizes to a dual-labeled short partner strand up to ∼30nucleotides in length.32 Though separatedby a larger end-to-end distance than self-hybridized molecular beacons,FRET initially still dominates between the molecular quencher on oneend, typically the 3′ end, and the fluorescent dye on the opposingend of the short hybridization partner to maintain a signal-off state.As with molecular beacons, multiple quenchers for a given fluorescentdye can be incorporated to improve FRET efficiency and reduce backgroundfluorescence in these asymmetric duplexes.33 Unlike displacement events involving exchange of partner sequences,the enzymatic degradation of dual-labeled hybridization partners occursduring amplification to allow fluorescence events. Commonly referredto as Taqman probes, these sequences require careful design to, forexample, accommodate sequence variability in viral samples.36 Thus, target oligonucleotide detection through qPCR requires additionalreagents, such as enzymes for amplification and selective probe degradation.For dsprobes, the primary-to-secondaryduplex transition is dueto the displacement of the quencher-capped hybridization partner bythe target. Depending on the dsprobe design, one or more displacementevents can be required prior to reaching the signal-on state, historicallywith DNA targets,37 unfolds toinduce fluorescence. Alternatively, initially quenched, nanoparticle-immobilizeddsprobes release the now fluorescent hybridization partner into thesurrounding solution.39 Solution studies with 200 nMto 1 μM target present and in vitro cell studies39 have reported a ∼2–6-fold increase in fluorescencesignal attributed to RNA displacement activity on gold nanoparticle-based“nanoflares.” These potential cellular probes,40 however, have drawbacks. For example, gold nanoparticle-basednanoflares are susceptible to false positives due to competitive exchangeof thiolated DNA probes with natural biothiols42 such as glutathione.43 Commercial polystyrenemicrospheres, on the other hand, offer covalent45 and noncovalent47 coupling routes for immobilizingprobe sequences, facile centrifugation steps for target capture andseparation, and multiple characterization tools50 to assess hybridizationactivity. In contrast to nanoflares described above, here, flow cytometryof microsphere suspensions measures the fluorescence intensity ofthe detected scattering centers, namely, the microspheres. In paststudies53 of toehold-mediateddisplacement activity in microsphere suspensions, the Milam groupused high throughput flow cytometry to quantify the displacement ofFAM-labeled hybridization partners by unlabeled targets. This signalon–off detection approach allowed for both quantitative endpoint analysis53 as well asin situ analysis55 of displacementactivity. As an alternative approach to this single dye, signal on–offapproach by the Milam group, Liu et al. reported FRET-based detectionof unlabeled DNA targets using microspheres functionalized with initiallyquenched DNA dsprobes.56 Depending on theDNA target concentration (up to 600 nM), their flow cytometry dataindicated up to a 102-fold increase in fluorescence intensity.Here, we further explore this FRET-based approach to uncover how sequencechoices and biotin–streptavidin immobilization affect the stability,selectivity, and sensitivity of microsphere-immobilized dsprobes tovarious RNA targets. While one DNA dsprobe system emerged as highlystable yet specifically responsive to severe acute respiratory syndromecoronavirus 2 (SARS-CoV-2) RNA, clear and surprisingtrends in the resulting fluorescence activity are evident across thesequence parameter space explored here.The above studies all employed soluble probes to detect the presenceof selected target sequences. Particle-immobilized probes, however,provide additional advantages. A metallic nanoparticle, for example,can be employed as an efficient quenching sink for multiple fluorescentdye-capped oligonucleotides. Once target hybridization occurs, theinitially quenched hairpinFigure S1a,b in Supporting Informationto identify optimal probe system(s), each immobilized probe possesseda 6-carboxyfluorescein (FAM) moiety at the 5′ end, and eachhybridization partner possessed an Iowa Black fluorescence quencher(Q) moiety on the 3′ end. Stock oligonucleotides solutionswere prepared and stored in TE pH 8.0  at 100 μM. Prior to coupling to microspheres, probes andtheir hybridization partners were annealed by heating the solutions(2:1 quencher-capped hybridization partner to FAM-labeled biotinylatedprobes) to 94 °C for 2 min and then slowly cooling to room temperature.For microsphere coupling, working suspensions of 1.05 μm diameter and 3.00 μm diameter streptavidin-coated microspheres  wereprepared by diluting stock from 1 to 0.1% w/v in wash buffer 20 mMTris (Sigma-Aldrich), 1 M NaCl (Sigma-Aldrich), 1 mM EDTA , and 0.0005% Triton X-100 (Sigma-Aldrich), per BangsProduct Data Sheet 721. Prior to oligonucleotide coupling, microsphereswere washed 3 times by centrifuging at 14,000g for3 min, aspirating supernatant, and resuspending in wash buffer. Thepreannealed solutions of the dsprobe systems were added to washedmicrospheres for a final solution probe concentration of 1 μMand agitated for 15 min at 22 °C, 800 rpm on a thermomixer. Followingincubation, suspension samples were washed 3 times as described above.For displacement experiments, RNA stock was added to the washed anddsprobe-functionalized microspheres for a final solution concentrationof 1 μM RNA and agitated for 15 min at 22 °C and 800 rpmon a thermomixer. Microspheres were not washed following the RNA addition.To prepare samples for flow cytometry, a 5 μL microsphere suspensionaliquot was added to 1000 μL phosphate-buffered saline (PBS). Singlet populations were gated based on scatteringplots, and 10,000 counts were recorded. Further experimental detailsfor sensitivity, alternate displacement measurements, and varioustitration studies are available in the Supporting Information. Consistent with standard practice for reportingflow cytometry data, fluorescence was quantified in units of moleculesof equivalent soluble fluorochrome (MESF), which was calculated fromthe raw voltage using Bangs Laboratory Quantum FITC-5 MESF standardbeads. The average MESF/μm2 and standard error arereported for three suspensions prepared for each dsprobe system. MESFvalues were normalized to the degree of labeling for each fluorescentsequence, calculated by using spectrophotometry data.All DNA, LNA, and RNA sequenceswere purchased from IntegratedDNA Technologies , with standard desalting. For datain Table S1 in the Supporting Information. This screening focuses on findingone or more dsprobes with sufficient stability prior to target additionyet with sufficient target selectivity to enable fluorescence events,as illustrated in 58 as stronger hybridization partners than DNA.64 Additional rationale for the dsprobe design is provided in the Supporting Information.The current studiesfirst screen through a series of 20 candidate locked nucleic acid(LNA) and DNA dsprobes, shown in LNA N1_FAM or N1_FAM, respectively) perfectly complementary to the 24-base-longRNA target  is immobilized to microspheres.Though popular as a molecular dye, the fluorescence activity of fluoresceinand its derivatives is sensitive to the nucleic acid composition andsecondary structure. For example, these molecular dyes have knownquenching susceptibility to a proximal guanine base66 and even to the hybridized versus unhybridized state of the dye-cappedoligonucleotides.67 Notably, a 2022 studyby Lietard et al.68 of all 1024 permutationsof pentanucleotide sequence segments demonstrated fluorescein activityon either the 3′ or 5′ end of DNA depends on both sequenceand secondary structure. Among all of the guanine-free pentanucleotidesstudied by Lietard et al.,68 5′-FAM-ACCCX sequences exhibited the lowest intrinsicfluorescence intensity. This observation is intriguingly relevantto the N1_FAM sequences shown in First, one of two biotinylated, 24-base-long 6-carboxyfluorescein(FAM)-tagged LNA or DNA sequences -capped hybridization partners listed in Table S1. To find one or more suitable sequence(s),hybridization partners vary in lengths , chemicalmodifications , and sequencefidelity . These dsprobes are then conjugated tostreptavidin-coated microspheres. Consistent with displacement-basedstrategies,71 each dsprobe has a single-stranded or initially unhybridizedsegment ranging from 3 to 15 bases in length next to its prehybridizedrecognition segment. This single-stranded segment is intended to serveas a toehold for SARS-CoV-2 RNA to initiate duplex formation,as illustrated in To form dsprobes in an initially signal-off state, N1_FAM should ensue from a successful toehold-mediateddisplacement event. A heterogeneous mixture of ∼243 similar,yet imperfectly matched, sequences  servedas model variants to assess target specificity  possesses only a few Watson–Crickbase-pair matches with the probe and is thus not anticipated to participatein toehold-mediated displacement events. Flow cytometry analysis inparts a and b of Figure S1 and a more detaileddiscussion of all 20 candidate dsprobe systems in the absence andpresence of unlabeled RNA sequences are provided in the Supporting Information.To complete duplex formation with the recognitionsegment, theunlabeled RNA target must fully displace the quencher-capped LNA orthe DNA hybridization partner. A fluorescence signal from the nowunquenched city see 1. DependN1_FAM:15mQ, exhibitedthe best balance as a stable dsprobe that is also highly and selectivelyresponsive to SARS-CoV-2 RNA. Here, 15mQ forms an imperfect duplex with one symmetric internal loop flankedby two 7-base-long duplex segments. As shown in Figure S1b, its 9-base-long toehold allows for extensive,yet selective, displacement of 15mQ by SARS-CoV-2RNA to induce substantial fluorescence from N1_FAM. On the other hand, little, if any displacement, of 15mQ by var_RNA occurs, as evidenced by the lack of fluorescenceactivity from N1_FAM. Thus, these model variants do notappear competitive as replacements for 15mQ despite each var_RNA sequence having a greater number of total base-pairmatches than 15mQ for N1_FAM probe, includinga continuous 11-base-long match to the recognition segment, as indicatedin N1_FAM:15mQ dsprobe for further studies,we assessed the limit of detection next.One particulardsprobe listed in SARS-CoV-2 RNA, N1_FAM:15mQ-coupled microspheres were incubated with100 pM to 1 μM SARS-CoV-2 RNA. The resulting fluorescencesignals were compared to the background fluorescence of the microspheresprior to RNA addition, as shown in SARS-CoV-2 RNA. At higherRNA concentrations, while saturation of probes by bound RNA is possible,complete displacement of all 15mQ is still not likelyto occur unless excess target is introduced.74 Compared to the 1 μM RNA sample, the 100, 10, 1 nM, and 100pM samples exhibited decreases in fluorescence signal of 9, 78, 95,and 97%, respectively, as shown in To determinethe sensitivity of the optimized dsprobe to 15m displacement by the various RNA sequences,an experiment was conducted using unlabeled N1 hybridizedto the FAM-labeled hybridization partner, 15m_FAM. Incontrast to dsprobes initially in a quenched state depicted in N1:15m_FAM primary duplexes allow fluorescence events to occuruntil an event drives removal of the soluble 15m_FAM sequence.To quantify 15m_FAM removal in the presence of variousRNA sequences, fluorescence was measured before RNA addition and thenafter adding scr_RNA and var_RNA, as illustratedin SARS-CoV-2 RNA. Based on the overall stability of N1:15m_FAM in the presence of either scrambled or imperfectly matched RNA,this marked fluorescence decrease in the presence of SARS-CoV-2RNA must be due to displacement of the shorter, FAM-labeledsequence. These results confirm the hypothesis that while displacementof 15m by SARS-CoV-2 RNA is extensive, itis not complete. Thus, in the dsprobes composed of FAM-capped andquencher-capped duplexes, the potential for longer-range quenchereffects of residual Iowa Black quencher-capped hybridization partnersas well as self-quenching effects between neighboring FAM moietieswas explored next.In order to quantifythe extent of 75 for such self-quenching to occur,typically in a solution in which dye molecules are concentrated butfreely diffusing. Based on the reported76 2 nm separation distance between nearest-neighbor biotin-bindingpockets on streptavidin76 and theoreticalRNA and DNA segment lengths78 illustrated in Scheme S1a,b in the Supporting Information, an estimated separation distance of ∼2–6nm occurs between various combinations of next neighbor sequence possibilities.Examples of nearest neighbors range from two identical upright neighboring N1_FAM:SARS-CoV-2 RNA duplexes to one single-stranded N1_FAM next to a dsN1_FAM:15mQ to mimic incompletedsprobe formation prior to RNA incubation.Homo-FRET or self-quenching is attributed to quenchingof fluorescence events due to the close proximity between identicaldye molecules. For FAM, a Förster radius of 5–6 nm isreportedN1_FAM in thesystem and if this fluorescence response is affected by the hybridizationstate of microsphere-immobilized N1_FAM sequences. Asillustrated in N1 and ssN1_FAM were coupled to the microspheres. Suspensionswere then incubated with var_RNA or SARS-CoV-2RNA to compare the resulting fluorescence measurements afterRNA addition, as shown in N1_FAM inthe system without appearing to approach saturation, indicating thathomo-FRET between nearby FAM molecules is unlikely. The addition of SARS-CoV-2 RNA did not result in any significant change influorescence overall; however, the addition of var_RNA resulted in a proportionally higher fluorescence signal, most evidentlystarting at 50% and higher N1_FAM.Two related studieswere thus conducted in order to determine if the fluorescence responseis directly proportional to the amount of N1 and N1_FAM prehybridized with unlabeled,unquenched 15m were coupled to the microspheres, as illustratedin var_RNA or SARS-CoV-2 RNA to compare the resulting fluorescence activity before and afterRNA addition, as shown in N1 in N1 also continuouslyincreased with increasing concentrations of N1_FAM inthe system. The overlap of the var_RNA series with thepre-RNA series is attributed to the lack of displacement of 15m by var_RNA. Interestingly, the addition of SARS-CoV-2 RNA resulted in lower fluorescence activity comparedto the pre-RNA series, most evidently at 50% or higher N1_FAM. These results, taken with the results shown in N1_FAM probe. For example,MESF values and trend lines do not overlap for imperfect N1_FAM:var_RNA duplexes and perfectly matched N1_FAM:SARS-CoV-2 RNA duplexes in N1_FAM:15m duplexes and blunt-endedsecondary N1_FAM:SARS-CoV-2 RNA duplex products in N1_FAM probe, a separate study was undertakenwith one truncated SARS-CoV-2 RNA and two specific mismatchedRNA sequence variants listed in Table S1. Results and discussion of the varying fluorescence are shown in Figure S2 in the Supporting Information. Notably, Lietard et al. investigated exhaustivesequence segment combinations next to the FAM moiety. In the currentstudy, however, the bases in N1_FAM in each suspensionare the same, and only the hybridization partner varies. While intriguingto pursue in future studies, individually testing each of the 243sequence variants was beyond the scope of the current dsprobe study.For the second titration study, varying ratiosof N1_FAM sequences on the samestreptavidin (see Scheme S2b) falls belowthe reported79 Förster radius of5.4 nm for FAM-Iowa Black quencher pairs. Thus, to determine potentiallonger-range effects of the Iowa Black quencher molecules, N1_FAM was preannealed with varying ratios of quencher-capped 15mQ and quencher-free 15m_noQ. These mixtures were thencoupled to the microspheres and introduced to unlabeled SARS-CoV-2RNA or var_RNA. As the pre-RNA series shows in 15mQ resulted in a 44% decrease in fluorescence compared with the systemwith 0% 15mQ. This particular percentage of quencher-cappedhybridization partners is notable when compared to the results in 15m_FAM by SARS-CoV-2 RNA was 88% complete,leaving approximately 12% of the N1:15m_FAM duplexesintact. Together, these results indicate that residual 15mQ hybridization partners, even at low percentages, can disproportionatelylower the fluorescence signal by quenching additional nearby FAM moleculesin N1_FAM:SARS-CoV-2 RNA duplexes.Whilethe previous subsection focused on fluorescence effects of FAM alone,here, the effects of neighboring Iowa Black quencher species are explored.The estimated ∼2–3 nm separation between various pairsof upright nearest-neighbor var_RNA series does notdeviate from the pre-RNA series, consistent with results in N1:15m_FAM + var_RNA), indicating that 15mQ is notreadily displaced by var_RNA. The SARS-CoV-2 RNA series, however, exhibits a lower fluorescence than the pre-RNAseries at 0% 15mQ. This comparative data point for 0% 15mQ in N1_FAM titrationseries before and after SARS-CoV-2_RNA addition. In 15mQ and higher, however, the SARS-CoV-2 RNA seriesexhibits fluorescence higher than those of both the strongly overlappingpre-RNA and var_RNA series. Moreover, between 0 and 50%quencher in SARS-CoV-2 RNA isadded. This initial decrease, followed by a plateau, is attributedto residual, undisplaced quencher-capped 15mQ sequences.Collectively, the results of these titration studies indicate thatthe quenching behavior of the Iowa Black molecules extends beyondthe static or contact quenching of its own hybridized FAM-capped probe.To further test this hypothesis, static quenching effects are eliminatedin the final titration study by using a dual dsprobe system.The addition of RNA to these dsprobe mixtures hasdifferent effectson the resulting fluorescence, as shown in A24_FAM:B15, a 24-base-long biotinylated, FAM-labeled DNAsequence and its 15-base-long DNA hybridization partner. The otherduplex is unlabeled N1 with varying ratios of quencher-capped 15Q to quencher-free 15_noQ. All hybridizationpartners are perfectly matched to their respective probe and do notexhibit any cross-reactivity (results not shown). A 1:1 mixture ofpreannealed A24_FAM:B15 and dsN1 (with varyingratios of hybridized 15_noQ and 15Q) wascoupled to the microspheres and then interrogated via flow cytometrywithout any RNA additions. As shown in 15Q induced a 28% decreasein fluorescence signal compared to that of 0% 15Q. Asthe percentage of quencher species increases, a continuous drop influorescence activity occurs until at 100% quencher an overall 68%decrease in fluorescence occurs. This quenching by 15Q on A24_FAM confirms that quenching activity of IowaBlack can extend to a neighboring duplex, most likely its nearest-neighborduplex on the same face of streptavidin. Consequently, for the originaldsprobe system, one can infer that any residual quencher species in N1_FAM:15mQ can effectively dim the fluorescence of neighboring N1_FAM:SARS-CoV-2 RNA. Despite this longer-range quenchingeffect, up to a 102-fold increase in fluorescence activitywas observed in these microsphere-based detection platforms upon adding SARS-CoV-2 RNA, as shown in A final titration study was conducted using a dual duplex systemin a 1:1 ratio. The first and otherwise unrelated duplex is 80 Each suspensionsample required ∼30 min of handling time for dsprobe immobilizationon microspheres, followed by RNA incubation. Subsequent flow cytometryanalysis of FRET events took less than 1 min. One particularly responsivedsprobe exhibited both stability and selectivity to RNA of interestwith concentrations as low as 10 nM, inducing an ∼10-fold increasein fluorescence. Our studies revealed evidence of both contact quenchingas well as longer-range quenching effects of residual or undisplacedmolecular quencher species on neighboring FAM-capped probes. Futurestudies may attempt to mitigate these longer-range quenching effectsby using an alternative coupling method to allow for further separationbetween probes. Insight from these studies combining flow cytometrywith FRET analysis hold practical implications for rapid screeningstudies of probe candidates and for facile implementation of microsphere-immobilizeddsprobes for target detection and separation as part of populationsurveillance such as wastewater testing83 or test kits developedin an academic laboratory setting84 wherelocal resources may be limited, but timely results are essential.In this study, we efficiently screenednumerous sequence combinationsof modified and natural oligonucleotides to identify suitable dsprobesfor unlabeled RNA detection using minimal chemical reagents, materialsupplies, and handling steps.


According to the results of the bioinformatics analysis, 589 lncRNAs showed different expressions in the groups. The stacking model had the best classification performance among the applied ensemble learning models. The Acc, B-Acc, Se, Sp, Ppv, Npv, and F1-score values obtained from this model were 90%, 90%, 80%, 100%, 100%, 83.3%, and 88.9%, respectively. lncRNAs with id rna-XR_005492522.1, rna-XR_005492536.1, and rna-XR_005505831.1 with the highest three values according to the variable importance obtained as a result of stacking modeling can be used as predictive biomarker candidates for hepatotoxicity. Conclusions: Among the ensemble algorithms, the stacking technique yielded higher performance results as compared to the bagging and boosting methods on the transcriptomic data. More comprehensive studies can support the possible biomarkers determined due to the research and the decisive results for the diagnosis of drug-induced hepatotoxicity.Background: The first aim of this study is to perform bioinformatic analysis of lncRNAs obtained from liver tissue samples from rats treated with cisplatin hepatotoxicity and without pathology. Another aim is to identify possible biomarkers for the diagnosis/early diagnosis of hepatotoxicity by modeling the data obtained from bioinformatics analysis with ensemble learning methods. Methods: In the study, 20 female Sprague-Dawley rats were divided into a control group and a hepatotoxicity group. Liver samples were taken from rats, and transcriptomic and histopathological analyses were performed. The dataset achieved from the transcriptomic analysis was modeled with ensemble learning methods . Modeling results were evaluated with accuracy (Acc), balanced accuracy (B-Acc), sensitivity (Se), specificity (Sp), positive predictive value (Ppv), negative predictive value (Npv), and F1 score performance metrics. As a result of the modeling, lncRNAs that could be biomarkers were evaluated with variable importance values. Results: According to histopathological and immunohistochemical analyses, a significant increase was observed in the sinusoidal dilatation and Hsp60 immunoreactivity values in the hepatotoxicity group compared to the control group ( The liver is involved in the realization of many chemical reactions, the production of bile, the metabolism of carbohydrates, lipids and proteins, and detoxification . The livMachine learning techniques, which have been widely used in the diagnosis of diseases and clinical decision support systems in recent years, are generally used in the disease prediction process in the health field. Machine learning, which has a very practical application in the health field, constitutes the basic infrastructure of applications, such as early diagnosis of cancer and chronic diseases, and determines the risk factors that cause them to occur ,19. The This study aimed to determine possible biomarkers for the diagnosis/early diagnosis of hepatotoxicity by modeling the transcriptomic data of liver tissues from rats treated with cisplatin hepatotoxicity and without pathology with ensemble learning methods.A total of 20 female Sprague-Dawley rats  were taken from İnönü University Experimental Animal Production and Research Center to detect possible biomarkers of drug-induced hepatotoxicity and to classify hepatotoxicity at the clinical level.Control group (C): The group in which cisplatin vehicle solvent was given intraperitoneally on the first day of the experiment.Hepatotoxicity group (CK): The group in which 7 mg/kg cisplatin was given intraperitoneally on the first day of the experiment.On the 4th day of the experiment, liver tissue samples from rats treated with ketamine (225 mg/kg i.p.) and xylazine (24 mg/kg i.p.) were collected for transcriptomic and histopathological analyses under high-dose anesthesia.Tissues taken at the end of the experimental period were fixed in 10% formaldehyde. At the end of the fixation process, the tissues, which were washed with tap water, were subjected to dehydration and polishing processes, and embedded in paraffin. Sections of 4 µm thickness were taken from the paraffin blocks obtained afterward. The sections subjected to deparaffinization and rehydration procedures were stained using hematoxylin-eosin (H-E) for general evaluation. Stained preparations were examined with a Leica DFC-280 research microscope using the Leica Q Win Image Analysis System .0 (no change);1 (light);2 (medium);3 (severe) .Histopathological evaluations were made by examining ten randomly selected areas in each section for hepatocyte degeneration  and sinusoidal dilatation. Each area examined was scored according to the severity of the changes as follows:Sections subjected to deparaffinization and rehydration processes for immunohistochemical analyses were boiled in 0.01 M citrate (pH 6.0) using a pressure cooker for 15–20 min. To block the endogenous peroxidase enzyme activity on the sections to be examined, 3% hydrogen peroxide was applied for 12 min. Sections washed with Phosphate Buffered Saline (PBS) were subjected to protein blocking for 5 min. Afterward, the sections whose protein blocking process was completed were incubated with primary antibody (Hsp60) for 60 min at 37 °C. Biotin-based secondary antibodies were applied to the tissues washed with PBS for 10 min at 37 °C. At the end of this process, the sections were incubated with streptavadin peroxidase for 10 min at 37 °C. Then, after the chromogen application process was applied to the sections, they were stained with hematoxylin and closed with a water-based sealer. Immunohistochemical staining, the extent of immunoreactivity , and severity  scored semi-quantitatively. The total staining score (H score) and the prevalence x severity was obtained by calculating .Analyses were performed with a Leica DFC-280 research microscope using the Leica Q Win Image Analysis System .Total RNA isolation from tissue samples was performed using kits that provide high-efficiency isolation from low-volume samples. The miRNeasy Serum/Plasma Kit  is designed to purify cell-free total RNA—primarily miRNA and other small RNA—from small volumes of serum and plasma. The used cDNA Synthesis Kit  works with limited sample volumes, making it ideal for reversing valuable RNA into stable cDNA ready for accurate real-time quantification. The RNA amount was measured fluorometrically with Qubit . In addition, the quality of RNAs was checked with Bioanalyzer before sequencing. Samples with RNA integrity number (RIN) ≥7 were sequenced.The lncRNA sequences were prepared using the “TruSeq Stranded Total RNA Library Prep Kit” from Illumina. Compatible with a wide range of samples, including low-quality DNA/RNA and formalin-fixed, paraffin-embedded (FFPE) samples, TruSeq Stranded Total RNA couples all the benefits of TruSeq RNA technology with Ribo-Zero ribosomal RNA reduction chemistry. The product enables analysis of coding and multiple forms of non-coding RNA with precise measurement of strand orientation, uniform coverage, and high-confidence discovery of features such as alternative transcripts, gene fusions, and allele-specific expression.Ribosomal RNAs (rRNAs) were eliminated from total RNA, and the remaining RNAs were purified and fragmented.rRNA elimination was validated with the Bioanalyzer.Then, RNA fragments were reverse transcribed (first strand cDNA synthesis) using random hexamer sequences .Afterward, the RNA template was eliminated, and the second strand of cDNA (blunt ds cDNA) was synthesized .A single ‘A’ nucleotide was added to their 3’ ends to prevent the blunt ds cDNA fragments from binding together during the adapter ligation reaction.Indexing adapters were then added to hybridize the ds cDNA fragments to the flow cell surface.DNA fragments have been enriched.The libraries of the samples were normalized and pooled.Samples of 50M readings were made with the Illumina NovaSeq 6000 platform as a paired-end (PE) 2 × 150 base .The sequences were prepared as follows:t-test was used to compare normally distributed data. The p-value < 0.05 was considered statistically significant. IBM SPSS Statistics 25.0 software was used in the analysis. The TMM  normalization method was employed for the relevant data. In bioinformatic analysis, False Discovery Rate (FDR) was utilized for the assessments.Whether the variables showed normal distribution or not was examined with the Shapiro–Wilk test. The data were presented as median (minimum-maximum) or mean±standard deviation (SD). The Mann–Whitney U test was used to compare non-normally distributed data, and the independent sample The elastic net variable selection method was used as the variable selection method within the scope of the study. R programming language-based RStudio was used in the implementation and variable selection stages of the bagging, boosting, and stacking ensemble learning models planned to be used in the study. This study used Acc, B-Acc, Se, Sp, Ppv, Npv, and F1-score metrics in the model performance evaluation. In addition, the graphics used in the visualizations were made using Excel software and R programming language.p < 0.0001)  B. Histopp < 0.0001). The immunoreactivity evaluation results of the groups are given in Hsp60 immunoreactivity was distinguished by brownish staining in hepatocyte cytoplasm. Accordingly, it was observed that Hsp60 immunoreactivity was mild in the sections of the control group A. It wasDescriptive statistics for the rats used in the experiment are given in Descriptive statistics by the groups (control- hepatotoxicity) are given in The isolated RNA samples were examined with Agilent 2100 bioanalyzer system for quality control. The data whose quality control was completed were sequenced, and the quality control of the raw read data (fastqc) obtained as a result of sequencing was performed using FASTQC* software, version 0.11.8.Quality-controlled raw read sequences were mapped to the reference genome using STAR* aligner software. The reference genome for mapping was taken from Rattus norvegicus (assembly mRatBN7.2) (GCF_015227675.2_mRatBN7.2_genomic.fna) and annotation track GCF_015227675.2_mRatBN7.2_genomic.gff_Lnc_rna. The quantification (count table) of reads mapped to the reference genome was conducted using the HTSeq* tool.The dataset used in the study contains 16,386 expressions. According to the results of the bioinformatics analysis, 589 (FDR < 0.05) lncRNAs showed different expressions in the groups. Of these, 450 showed up-expression (logFC > 1), and 139 showed down-expression (logFC < −1). Data set for bioinformatic analyses is presented in Based on the principal components (PCO) analysis, the distribution of the samples was found to be compatible in terms of lncRNA expression levels in the hepatotoxicity group (C) vs. control group (CK) comparison. Controls and application samples were collected in the same group. In this case, control and treatment samples presented similar expression level changes for similar lncRNAs. The visual figure for PCO analysis is given in The heatmap representation of the fifty most expressed lncRNAs in the two group comparisons is given in In the C vs. CK comparison, it is observed that the application samples exhibited a different expression profile compared to the control, shown in red for the 50 lncRNAs that showed the most variation and in green for the suppressed expression level. The C-4 sample showed a different profile than the application samples.The volcano plot used to visualize differentially expressed genes is given in The data of 16,386 lncRNAs in the data set were obtained by the TMM  normalization method. From these data, 17 lncRNAs were chosen by the elastic net variable selection method from 589 lncRNAs with different regulations (up and down) between the two groups. The data analysis results of these chosen expressions are given in p < 0.05).According to the statistical analysis results in The results of performance metrics obtained as a result of bagging, boosting, and stacking models, which are ensemble learning models using selected lncRNAs, are given in According to the classification performance of the bagging, boosting, and stacking models, the bagging model’s Acc was 85%, B-Acc was 85%, Se was 70%, Sp was 100%, PPV was 100%, NPV was 76.92%, and F1-score was 82.4%. Acc of 85%, B-Acc of 85%, Se of 70%, Sp of 100%, PPV of 100%, NPV of 76.92%, and F1-score of 82.4% were obtained from the boosting model. Acc as 90%, B-Acc as 90%, Se as 80%, Sp as 100%, PPV as 100%, NPV as 83.3%, and F1-score as 88.9% were calculated from the stacking model.The graph of the performance metrics for the stacking model, which gives the best result from the ensemble models used, is given in Liver toxicities based on drugs are known to be one of the leading causes of liver damage . Drug-inIn the present study, transcriptomic and histopathological analyses were performed with liver samples taken from rats treated with cisplatin-induced hepatotoxicity and from rats in the control group.As a result of histopathological analysis, liver damage was examined regarding hepatocyte degeneration and sinusoidal dilatation. In the sections where the H-E staining method was applied, the liver had a normal histological appearance except for mild changes in the control group. In contrast, hepatocyte degeneration was similar to the control group in the hepatotoxicity group, while a significant increase in sinusoidal dilatation was observed. In a previous study, a difference was observed in sinusoidal dilatation in the CIS group after HE staining was performed in the CIS platinum group . AnotherHeat shock proteins (HSPs) are a set of evolutionarily conserved molecules found in almost all living organisms ,35. HSP6This study used transcriptomic data obtained from liver tissues of rats treated with hepatotoxicity and control group rats for the relevant analysis. There are 16,386 lncRNA expressions in the acquired data set. According to the findings of the bioinformatics analysis, lncRNA with the id rna-XR_001840627.2 had a very high gene expression in the hepatotoxicity group compared to the control group. Similarly, rna-XR_005499199.1, rna-XR_005496955.1, rna-XR_005497501.1, rna-XR_001835923.2, rna-XR_005506070.1, rna-XR_005495875.1, rna-XR_005497501.1, rna-XR_005506070.1, rna-XR_005494195.1, and rna-XR_005503371.1, lncRNAs with id had very high gene expressions in the hepatotoxicity group compared to the control group. In an experimental study, lncRNAs were examined in MC-LR-induced hepatotoxicity, and the expression levels of three lncRNAs were found to be significantly increased in all treatment groups . In anotlncRNAs with id rna-XR_005493896.1, rna-XR_005488982.1, rna-XR_005486345.1, rna-XR_005504727.1, rna-XR_005493990.1, rna-XR_005488390.1, rna-XR_005501078.1, rna-rna5490 -XR_005502677.1, and rna-XR_001837692.2 had low gene expression in the hepatotoxicity group compared to the control group.According to the results of the biostatistics analysis performed with 17 lncRNAs selected by the elastic net variable selection method used in the study, a significant difference was found between the two groups for all lncRNAs, and the calculated OR values also support that these lncRNAs may be discriminative RNAs for the two groups. When the performance metrics obtained from the ensemble learning models using the selected 17 lncRNAs as input variables and taking the hepatotoxicity status as the target variable were examined, it was found that the stacking technique produced higher results than the other two methods. Performance metrics results show that the stacking method, one of the proposed models, can correctly classify hepatotoxicity when the high-performance metric values obtained in the classification of the two groups are considered. The rna-XR_005492522.1 (LOC120096269), rna-XR_005492536.1 (LOC120096276), and rna-XR_005505831.1 (LOC102557053) lncRNAs, which have the highest three significance values according to the significance of the variables obtained as a result of the stacking modeling, are for hepatotoxicity as a result of extensive studies and can be used as predictive biomarker candidates.The present study may have some limitations. The results of the current study may illustrate changes in the lncRNA-based regulatory network associated with cisplatin treatment; however, it may have guiding potential for biomarker discovery and personalization of the treatment. From this aspect, it may be a limitation that this research is performed on experimental animals and forms a basis/background for further experimental research. In order to achieve the objectives examined in this study, experimental procedures could be performed on certain experimental animals due to restrictive factors. Therefore, more extensive experimental/clinical studies are necessary for the following periods.Among the ensemble algorithms, the stacking technique yielded higher performance results as compared to the bagging and boosting methods on the transcriptomic data. For the potential biomarkers discovered from the current study to be used in the case of drug-induced hepatotoxicity, the achieved results need to be supported by different extensive studies. Once the accuracy of the obtained biomarker candidates is determined, possible treatment and diagnosis options can be personalized, and potential diagnostic procedures can be performed easily, quickly, and effectively after being confirmed in clinical trials.


The COVID-19 pandemic caused by SARS-CoV-2 has underscored the need for rapid and accurate diagnostic methods. Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP) has emerged as a promising molecular tool in least developed countries due to its simplicity, speed, and sensitivity. Nevertheless, reliable SARS-CoV-2 detection can be challenged by the chain custody of the samples. In this context, carrier RNA can act as a preservative. In this study, we explored the potential of yeast total and transference RNA (tRNA) in the SARS-CoV-2 RT-LAMP. We have found that most optimal conditions are reached with 1 μg/μL tRNA in the RT-LAMP reaction. The emergence and global spread of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) have led to an unprecedented public health crisis, emphasizing the urgency to develop rapid and sensitive diagnostic tools. The standard diagnostic method, quantitative Reverse Transcription Polymerase Chain Reaction (RT-qPCR), has been widely utilized for the detection of SARS-CoV-2 due to its high specificity and sensitivityIn recent years, the RT-LAMP technique has gained increasing interest for its ability to rapidly amplify specific RNA sequences under isothermal conditionsIn the context of nucleic acid-based amplification, the use of additives, including carrier RNA, has been explored in RT-qPCR assays to improve the detection sensitivityInspired by the success of carrier RNA in RT-qPCR, we hypothesize that the inclusion of yeast RNA as an additive in RT-LAMP could similarly facilitate the detection of SARS-CoV-2. Yeast RNA has been utilized as a carrier for various molecular biology applications due to its abundance, stability, and resistance to RNase degradation. However, its potential application in RT-LAMP for viral RNA detection remains largely unexplored. To the best of our knowledge, there is only one previous work in which carrier RNA, namely yeast transference RNA (tRNA), was tested as an additive in colorimetric SARS-CoV-2 RT-LAMPTM9ACTBis often included in diagnostic testsIn this study, we investigated the impact of yeast RNAs as additives on the performance of RT-LAMP for SARS-CoV-2 detection. Our objective was to establish the conditions in which these additives are compatible with colorimetric RT-LAMP, while preserving the overall efficiency and sensitivity of the reaction. To this aim, we set up a SARS-CoV-2 RT-LAMP that was both quantitative and colorimetric. The colorimetric reaction is based on a pH-sensitive indicator that turns from pink to orange/yellow as the pH drops during the liberation of protons, which accompanies the incorporation of nucleotides into the amplified DNAACTB. Little or no amplification was observed when the 200c/75c template was not present . Accordingly, the pre-amp colour tubes were pink in all cases, and turned to yellow when amplification was observed by RT-qLAMP .ACTBalso amplified efficiently from crude nasal swab samples and total RNA extracted from them . Since nasal swab samples came from healthy individuals, N/E primers did not amplify.We first validated the colorimetric and quantitative RT-LAMP with the synthetic 200c/75c template . We found that the time to positivity (Tp) in the quantitative RT-LAMP was 23.4 ± 0.6 min  for N/E, and 16.0 ± 0.6 min  forTM(RNAsec). The highest toRNA concentration (1 μg/μL) turned the pre-amp colour to yellow, so it is incompatible with the overall purpose of the diagnostic . In addition, the RT-qLAMP amplification of N/E was highly inhibited at this concentration . Better results in terms of pre-amp colour were obtained with lower toRNA concentrations (0.1 μg/μL or lower) . The results with tRNA were much better, observing total compatibility with both colorimetric and fluorometric readings even at the highest concentration of 1 μg/μL . In all cases, the combination with RNAsec caused a slight colour shift to orange in pre-amp tubes, advising against this additive in colorimetric RT-LAMP reactions. As for theACTBreaction, neither toRNA nor tRNA gave inhibition . We must bear in mind, though, that this is a LAMP reaction, rather than a RT-LAMP, as the 75c template is human genomic DNA.Next, we included yeast RNAs as additives and performed the colorimetric and quantitative RT-LAMP. Two kinds of yeast RNAs were employed, total RNA (toRNA) and tRNA. Three concentrations were tested: 1, 0.1 and 0.01 μg/μL. In addition, we also determined RT-LAMP performance when combining these carrier RNAs with the RNAase inactivation reagent RNAsecureTM9 fluorescence after adding the yeast RNAs. This was intended to evaluate the sensitivity of this additive as a normalization spike-in during sample handling and preservation. Our results show that a clear pre-amp fluorescence signal was obtained with the highest concentrations .In addition to the RT-LAMP data, we assessed the basal levels of Sytothof the RT-LAMP reaction volume, this implies that clinical samples can be treated with up to 10 μg/μL of yeast tRNA as a preservative and normalization spike-in. As for toRNA, the working concentration in the RT-LAMP reaction must be lowered to 0.1 μg/μL . The difference between tRNA and toRNA may be due to their relative acidic potential. Both RNAs are commonly provided in unbuffered nuclease-free water, but tRNA is expected to have a more neutral pKa because it is highly foldedIn conclusion, we aimed to evaluate the practical use of yeast RNAs as additives to the SARS-CoV-2 RT-LAMP reaction. We conclude that both the colorimetric and the quantitative reactions tolerate well 1 μg/μL for tRNA, which also provides a sufficient baseline for loading normalization. If the sample normally accounts for 1/10The RT-LAMP reaction was performed in a final volume of 10 μL and set as follows: 5 μL of the WarmStart® Colorimetric LAMP 2X Master Mix with UDG, 1 μL of the corresponding 10X primer set, 1 μL of 0.4 M guanidine hydrochloride, 1 μL of 5 μM SYTO™ 9, 1 μL of the template (or water for the negative control), and 1 μL of the corresponding yeast RNA 10X stock when required. For the RNAsec additive, 0.4 μL were added from a 25X stock. Reactions were levelled to 10 μL by adding the required volume of nuclease-free water.The template was 200c/75c, fresh crude mid-nasal swab (resuspended in 1 mL nuclease-free water), or total RNA extracted from the crude sample. For RNA extraction, we used the PureLink™ RNA Mini Kit kit according to the manufacturer. The yield after RNA extraction ranged from 2.7 to 4.9 ng/μL.The primer sets were either N/E or hACTBThe RT-qLAMP was run in a QuantStudio™ 5 Real-Time PCR System (Applied Biosystems™) for 1 h. The amplification conditions were set to constant 65 ºC cycles, measuring fluorescence every 30 sec . After this, the reaction was slowed down by incubating at 4 ºC. The corresponding Ct was then converted to Tp  by dividing by 2. Colorimetric assessment of the RT-LAMP was determined by taking photos over a black background. First, a pre-amp photo was taken right after the reaction setup and before running the reaction in the thermocycler. Then, a second photo was taken after 1 h at 65 ºC (post-amp photo).Graph plots and statistical calculations were performed with GraphPad Prism 10. One-way ANOVA and post hoc tests for multiple comparisons were performed on biological replicates when required, and the meaningful comparisons are indicated in the plots. For plots in panels (f) and (h), a Dunnet’s post hoc test was used, with no carrier RNA and no RNAsec (just 200c/75c) acting as the reference. A Sidak's post hoc was used in panels (d) and (j). No significance (n.s.) is indicated in the figure legend.