Enterococcus faecalis and Enterococcus faecium are major nosocomial pathogens. Despite their relevance to public health and their role in the development of bacterial antibiotic resistance, relatively little is known about gene regulation in these species. RNA–protein complexes serve crucial functions in all cellular processes associated with gene expression, including post-transcriptional control mediated by small regulatory RNAs (sRNAs). Here, we present a new resource for the study of enterococcal RNA biology, employing the Grad-seq technique to comprehensively predict complexes formed by RNA and proteins in E. faecalis V583 and E. faecium AUS0004. Analysis of the generated global RNA and protein sedimentation profiles led to the identification of RNA–protein complexes and putative novel sRNAs. Validating our data sets, we observe well-established cellular RNA–protein complexes such as the 6S RNA–RNA polymerase complex, suggesting that 6S RNA-mediated global control of transcription is conserved in enterococci. Focusing on the largely uncharacterized RNA-binding protein KhpB, we use the RIP-seq technique to predict that KhpB interacts with sRNAs, tRNAs, and untranslated regions of mRNAs, and might be involved in the processing of specific tRNAs. Collectively, these datasets provide departure points for in-depth studies of the cellular interactome of enterococci that should facilitate functional discovery in these and related Gram-positive species. Our data are available to the community through a user-friendly Grad-seq browser that allows interactive searches of the sedimentation profiles (https://resources.helmholtz-hiri.de/gradseqef/). Enterococcus faecalis and Enterococcus faecium were found to cause life-threatening infections , and that this RBP might have an RNA processing function. Our Grad-seq data sets, available in a user-friendly browser (https://resources.helmholtz-hiri.de/gradseqef/), provide a global resource to better understand transcriptional and post-transcriptional regulation in E. faecalis and E. faecium, the two pathogenic species of the Enterococcus family.Here, we applied Grad-seq to chart the landscape of RNA–protein and protein–protein complexes in E. faecalis V583 and E. faecium AUS0004, gift from Jean-Christophe Giard, were grown at 37°C on M17 agar plates (Oxoid) supplemented with 0.5% glucose. Single colonies were inoculated each time liquid cultures were needed. 8.3 ml of M17 medium supplemented with 0.5% glucose were inoculated in a 25 ml glass tube, in order to keep the final proportions of one-third of media and two-thirds oxygen. Cultures were grown overnight at 37°C and back diluted 1:100 into fresh media and grown without agitation to late logarithmic/early stationary phase at an OD600 of 2.0. For the glycerol gradient sample preparation, 200 OD600 of bacterial culture was used for each strain. 1 ml of the overnight culture was diluted in 100 ml of GM17 and grown to OD600 of 2.Bacteria, g at 4°C for 20 min in 50 ml falcon tubes. Pellets were pooled in order to have one tube per strain. Three washes with 25 ml of ice-cold 1x TBS were applied and the pellets were resuspended in a final 1 ml 1x TBS, transferred into a 2 ml tube and centrifuged at 13,000rpm, 4°C, during 2 min. After removing all the TBS, pellets were resuspended in 500 µl of lysis buffer . An equal volume of 0.1 mm glass beads was added, and cells were vortexed for 30 s followed by 15 s cooling on ice. This lysis step was repeated 10 times.Once the desired OD was reached, the cultures were decanted into prechilled 50 ml falcons and incubated on ice for 15 min with rotation of the tubes every 3–5 min to ensure fast and uniform cooling. The cells were harvested by centrifugation at 4000 2, 1 mM DTT, 1 mM PMSF, 0.2% Triton X 100, 10% (w/v) glycerol) and the 40% (w/v) glycerol solution  glycerol). Clean ultracentrifugation tubes were divided into two. The first half of the tube was filled up with the 10% solution and using a 70-mm injection needle and a syringe, the 40% solution was then placed underneath the 10% solution until the interphase reached the middle of the tube. The loaded gradients were centrifuged at 100,000 g  for 17 h at 4°C in a precooled ultracentrifuge. The gradients were then manually fractionated into 590 µl fractions. Following this method, 20 fractions and the pellet were collected for each gradient .The lysate was then cleared by centrifugation at 13,000 rpm, 4°C during 10 min. Without disturbing the pellet, all supernatant was collected. A couple of aliquots were saved  for subsequent analysis of proteins and RNAs. A volume of 200 µl of the very top part of the gradient was removed and 200 µl of the cleared lysate was gently placed. The gradient used here was a linear 10%–40% (w/v) glycerol gradient. To prepare the gradient, two solutions were made. The 10% (w/v) glycerol solution . KhpB was detected by the αKhpB antibody  SABC-serum)  and a secondary anti-rabbit-HRP (goat) conjugate antibody . The rest of the samples was prepared for mass spectrometric analysis as previously described , 40 min at −80°C, centrifuged at 13,000rpm 4°C for 30 min, washed with 350 µl 70% EtOH, and centrifuged at 13,000rpm 4°C for 10 min. The pellets were air-dried and dissolved in 35 µl nuclease-free water. 10 µl of each sample was mixed with 10 µl of GLII RNA loading buffer.From each collected fraction (minus the 90 µl taken for protein preparation), 50 µl of 10% SDS was added (or 25 µl for the pellet as the volume was expectedly lower in the sample). After shaking by hand vigorously for about 20 s, 600 µl of acidic P:C:I was added to each fraction (300 µl for the pellet). 400 µl of chloroform was added also to the Trizol-dissolved lysate and shaken by hand for about 10 s. All the samples were then vortexed for 30 s and let rest for 5 min at room temperature. The samples were centrifuged at 13,000 rpm at 4°C for 15 min. The aqueous phases were collected in 2 ml tubes and 1 µl of Glycoblue, together with 1.4 ml ice-cold 30:1 EtOH:3 M NaOAc pH 6.5 were added. Tubes were put at −80°C for 40 min in order to let the RNA precipitate. The samples were then centrifuged at 13,000rpm, 4°C for 30 min, pellets were washed with 350 µl of ice-cold 70% EtOH and centrifuged again for 10 min at 13,000 rpm, at 4°C. After air-drying the pellets, 40 µl nuclease-free water was added. DNase treatment was performed. For one entire gradient, a master mix was prepared: 115 µl of DNase I buffer with MgCl32P-ATP end-labeled oligodeoxynucleotide probes  and subjected to library preparation for next‐generation sequencing (Vertis Biotechnologie) following the protocol that has been previously described  were conducted in READemption 0.4.5 , as described previously  with default settings  with C-terminal 3C-cleavage site, 3 × FLAG-tag, and 6 × His-tag. The E. coli BL21-CodonPlus (DE3)-RIL cells . Cells were grown in LB to OD600 0.6 at 37°C, cooled to 18°C and induced with 0.5 mM IPTG overnight. Cells were resuspended in lysis buffer  and disrupted by sonication . The lysate was cleared at 15,000 g for 15 min at 4°C. Supernatant was loaded onto an Ni-NTA column  and washed with 5 column volumes. Protein was eluted with 200 mM imidazole and desalted  in 3C-cleavage buffer . Cleavage of the tag was performed in 2–3 ml with 20 µl 3C protease  added and incubated over-night at 4°C. The sample was loaded again on an Ni-NTA column for reverse-IMAC and the cleaved product collected in the flow through. Unfortunately, KhpB remained bound to the column, and required to be washed, and eluted again. The sample was diluted with 10 volumes anion exchange buffer A  and loaded onto an anion exchange column . Protein was eluted in a gradient up to 1 M NaCl. KhpB eluted as a second peak ∼0.5 M NaCl. The peak was pooled and concentrated by centrifugal filtration  rebuffered in storage buffer  and flash frozen in liquid nitrogen. The concentration of KhpB was estimated by absorption measurement at 280 nm using the extinction coefficient of 10,430/mM/cm. A total of 0.8 mg protein was used for raising two antibodies against KhpB in rabbit .For expression pMiG-034 was transformed into 2, 1 mM DTT) and incubated for 1 h. Beads were washed two times with lysis buffer. A total of 60 OD of E. faecalis cells at OD 2 were resuspended in 1 ml lysis buffer , 5 µl RNase inhibitor, 5 µl DNase I), and were lysed via FastPrep with lysing matrix E at 6 m/s for 40 s.A volume of 300 µl polyclonal anti-KhpB serum (SY0916 (MA11009) SABC-sérum for experiment 1 and SY0917 (MA11010) for experiment 2), or corresponding preimmune sera for negative control, was added to 75 µl of prewashed protein A sepharose beads (Sigma-Aldrich) together with 700 µl lysis buffer  SDS and one volume PCI. The aqueous layer was re-extracted with chloroform and the RNA was precipitated with 0.1 M sodium acetate and three volumes of ethanol. RNA was pelleted, dried, and solubilized in water. DNA was digested with DNase I in reaction buffer (Thermo Scientific). RNA was re-extracted by PCI, and the concentration was determined by absorption at 260 nm. Library preparation and sequencing was conducted at Vertis Biotechnologie AG. RNA-sequencing was analyzed as described previously. Read quantification between both experiments with independently generated antibodies against KhpB was merged and normalized in deseq2 by geometrical averaging and P-values were calculated using the Wald-test.The lysate was cleared for 10 min at full speed at 4°C. Beads were added to the lysate and incubated at 4°C for 2 h with rotation. Beads were pelleted at 300 Met, tRNAIle, tRNASer, the 5’UTR of tRNAIle and tRNASer, the sRNA070, sRNA010, and sRNA156 are listed in The organic layer was precipitated with 10 volumes methanol and LS-MS/MS analyzed for precipitated proteins at the MS core unit . RNA extracted from supernatant, the flow-through with either the preimmune serum or with the anti-KhpB serum and the elution with either the preimmune serum or the anti-KhpB serum were used to validate KhpB targets by northern blot assays with a similar protocol described in the previous session “RNA/RNA-seq sample preparation.” The probes used against tRNAE. faecalis and E. faecium, we performed Grad-seq  in M17 rich media. We chose this growth phase based on a previous study  fractions in which free proteins and small complexes accumulate. In the high-molecular weight (HMW) fractions, two characteristic peaks around fractions 10 and 15 are observed, which correspond to the small (30S) and large (50S) ribosomal subunits, respectively; this pattern is very similar to the ribosome peaks observed in the closely related Gram-positive species, S. pneumoniae , tmRNA, M1 RNA (rnpB), or sRNA_0869, an sRNA previously identified in E. faecalis  is present in the Uniprot database. Overall, the total number of transcripts and proteins for which we could establish sedimentation profiles via Grad-Seq in E. faecalis and E. faecium is comparable to previous Grad-seq experiments in others species, such as C. difficile, E. coli, or S. pneumoniae  were found in fractions 4–6 Fig. , many ot faecium  than we lis Fig. . This suE. faecium than E. faecalis, is present in fractions 9–12; i.e. these sRNAs occur in the 30S ribosomal fractions. There are two tempting possible explanations for this sedimentation profile: firstly, these sRNAs might be associated with translated mRNAs as part of their regulatory activity; alternatively, these sRNAs themselves might be translated, because they contain small open-reading frames (ORFs), perhaps with noncanonical start codons that have been overlooked so far. The latter phenomenon has recently been established in E. coli through Grad-seq analysis. RyeG, presumed to be a noncoding sRNA, was found to peak in the 30S fractions of the gradient and was shown to contain a previously unnoticed ORF of 48 amino acids (aa) starting with a GUG start codon  that are present in the 30S fraction  complex—PdhA, PdhB, and AceF—cluster together, and so did many ribosomal proteins   suggested a potential conservation of protein function , which encodes a protein translocase; rnpA, which encodes the RNA component of RNase P; and mnmE, which encodes a tRNA modifying GTPase. One characteristic feature of KhpB proteins is their shared domain architecture showing conserved Jag-N, KH and R3H RNA-binding domains . The experiment was performed twice using one of the two independent α-KhpB antisera per IP. Preimmune sera were used as a negative control , determined by deseq2; P-value < 0.05 ), we recovered cDNA reads for 54 RNAs, showing a nearly even distribution across coding, noncoding, and UTRs. A quantitative distribution based on RNA ligand abundance indicates relative enrichment of tRNAs, sRNAs, and 3'UTRs, with tRNAs being most enriched.  and a tRNA modifying GTPase (mnmE)  Fig. .Our RIP-seq data also predicted KhpB to interact with sRNAs and UTRs Fig. . To valiS. pneumoniae and C. difficile  and to manually dissect the profiles for positions and tailing. In addition, candidates belonging to a similar cluster of sedimentation profiles can be displayed automatically. A comparison is possible by adding items to a list, and visualization is provided via bars, plots, and heatmaps. The user can, therefore, compare protein and RNA sedimentation profiles in order to identify correlated sedimentation and obtain an indication of stable complexes.We have facilitated access to the enterococcal Grad-seq data via an interactive online browser Fig. . The broE. faecalis and E. faecium.Grad-seq is a powerful method to study native RNA–protein complexes. Here, we have used this approach to provide a global resource for the identification of stable RNA and protein complexes in the human pathogens E. faecalis and E. faecium have previously been implicated in stress responses and virulence phenotypes (Fouquier d'Hérouel et al. E. faecalis and E. faecium based on dRNA-seq data (Michaux et al. Several sRNAs of Enterococcus sRNAs, like the sRNAs in other bacterial species, interact with other RNAs or with proteins. Like most Gram-positive bacteria, neither E. faecalis nor E. faecium encode any of the well-known sRNA chaperones such as Hfq and ProQ. Regarding putative new sRNA-related proteins, our Grad-seq revealed similar sedimentation between KhpA, KhpB, and many sRNAs. In addition, both proteins were previously shown to interact with sRNAs in S. pneumoniae and C. difficile (Lamm-Schmidt et al. E. faecalis KhpB to interact with tRNAs, sRNAs, and 3'UTRs. While sRNAs are common interaction partners of KhpB in all bacterial species studied so far, C. difficile KhpB was not seen to interact with 3'UTRs. This may be because C. difficile possesses Hfq and ProQ, both known to bind to the 5' and 3' ends of mRNAs. Streptococcus pneumoniae does not encode Hfq, and the KhpB interactome featured UTRs, like the Enterococcus KhpB interactome. This might imply that in bacterial species that lack Hfq and ProQ, UTRs are targeted by KhpB.Interestingly, while sRNAs are enriched in the LMW fractions, they also show a broader distribution in HMW fractions (Gerovac et al. Tyr and tRNAPro were also found to be RNA ligands of KhpB in S. pneumoniae (Zheng et al. C. difficile, despite the much broader interactome of C. difficile KhpB (Lamm-Schmidt et al. Our data also suggest that KhpB might have a role in tRNA and sRNA processing, but future work is required to establish such a function. Curiously, both tRNAE. faecalis and E. faecium. We are confident that this data will aid in the investigation of post-transcriptional regulation in these opportunistic pathogens, an area of molecular microbiology, that is still in its infancy. Of note, we recently expanded the molecular toolkit for the global identification of RNA–protein interactions by developing SEC-seq, a method that couples size exclusion chromatography with RNA-sequencing and mass-spectrometry (Chihara et al. E. faecalis and E. faecium by performing SEC-seq on these species.Overall, we believe that our Grad-seq data provides a powerful resource and ample starting points to identify RNA–protein complexes of uqac027_Supplemental_FilesClick here for additional data file.


S-adenosylmethionine (SAM) analogue and catalyses the transfer of a propargyl group to a specific adenosine in the target RNA. Almost quantitative conversion was achieved within 1 h under a wide range of reaction conditions in vitro, including physiological magnesium ion concentrations. A genetically encoded version of the SAM analogue-utilizing ribozyme (SAMURI) was expressed in HEK293T cells, and intracellular propargylation of the target adenosine was confirmed by specific fluorescent labelling. SAMURI is a general tool for the site-specific installation of the smallest tag for azide-alkyne click chemistry, which can be further functionalized with fluorophores, affinity tags or other functional probes.Post-transcriptional RNA modification methods are in high demand for site-specific RNA labelling and analysis of RNA functions. In vitro-selected ribozymes are attractive tools for RNA research and have the potential to overcome some of the limitations of chemoenzymatic approaches with repurposed methyltransferases. Here we report an alkyltransferase ribozyme that uses a synthetic, stabilized  S-adenosylmethionine analogue to a specific adenosine on the target RNA within cells.Ribozyme-mediated post-transcriptional RNA modification is a powerful method for site-specific RNA labelling and analysis of RNA functions. Now, an alkyltransferase ribozyme—termed SAMURI—has been shown to catalyse the transfer of a propargyl group from a stabilized synthetic  Chemoenzymatic strategies with repurposed methyltransferase (MTase) protein enzymes and S-adenosylmethionine (SAM or AdoMet) derivatives have emerged as powerful methods for targeted RNA modification6. Several types of chemically modified SAM analogue have been developed, including variants for the transfer of alkyne8 or azide tags10, photoreactive groups11, fluorophores12 or biotin13. The installation of bioorthogonal functional groups allows further derivatization via click reactions for several purposes, including affinity purification8, chemical degradation14 or fluorescent labelling15 of target RNAs. In some examples, modified SAM analogues have been applied for target modification in the intracellular context by using a cascade reaction of methionine adenosyl-transferase (MAT) and MTases18. Such metabolic approaches are designed to give global modifications of DNA, RNA or proteins19, but often lack target specificity, because many cellular MTases maintain a common SAM recognition domain. To address specific RNA targets beyond the mRNA cap20, either structure-specific tRNA MTases21, or C/D box snoRNPs with sequence-specific guide RNAs7, have been used in vitro.Site-specific chemical modification methods that enable precise post-synthetic or post-transcriptional functionalization of RNA provide robust means to probe and manipulate RNA functions in vitro and in cells22. Usually, the catalytic core is flanked by binding arms that hybridize to a particular target sequence by Watson–Crick base-pairing, placing the target nucleotide into the active site for reaction with a specific, non-covalently bound cofactor. Thus, these trans-active ribozymes combine both functions—the guide and the catalyst—in one RNA molecule that interacts with the RNA target. Several combinations of ribozymes and cofactors for RNA labelling have been identified by in vitro selection. For example, ribozymes catalyse the attachment of fluorescently labelled nucleotides24 or acyclic nucleotide analogues25 to a specific internal 2′-OH group of the target RNA, or at the 3′-end26. Other classes of ribozymes work in cis, that is, they catalyse their own modification, for example, via self-alkylation with electrophilic iodoacetyl groups28 or reactive epoxide probes30. The most recent addition to the repertoire of RNA-catalysed reactions concerns site-specific RNA methylation. The in vitro-selected methyltransferase ribozyme MTR1 uses O6-methylguanine (m6G) as cofactor32, and a split riboswitch uses an engineered ligand O6-methyl pre-queuosine (m6preQ1)33 to methylate their RNA targets at the Watson–Crick base-pairing face to produce m1A (ref. 31) and m3C (ref. 33), respectively. Self-methylation at the Hoogsteen edge to install m7G was achieved by an in vitro-selected copper ion-dependent ribozyme that uses native SAM as cofactor34. In analogy to chemoenzymatic MTase engineering, these recently reported methyltransferase ribozymes have high potential to be repurposed for RNA labelling. However, only MTR1 has so far been addressed to a variety of RNA target sequences, including tRNAs, and m6preQ1- and SAM-dependent ribozymes suffer from a severely restricted sequence scope.Ribozymes have gained increasing momentum as an alternative sequence-specific approach for post-synthetic RNA modification2+ concentrations. This feature enables the intracellular site-specific RNA modification upon expression of the ribozyme in living human cells.In this Article we report a ribozyme that catalyses the sequence-specific transfer of a propargyl group from bioorthogonal SAM analogues to the minor groove side of a specific adenine in various RNA target sequences. We explore the scope of the bioorthogonal SAM analogue-utilizing ribozyme and find that the catalytic activity is retained even at low Mg36. Double-activated cofactor analogues contain an unsaturated bond at the β carbon to stabilize the transition state by hyperconjugation and to rescue the reduced reaction rates compared to the transfer of a methyl group37. To further increase the reactivity for the nucleophilic attack, the sulfur centre was replaced by selenium, and propargylated adenosyl selenomethionine  has become a heavily investigated cofactor for MTase protein enzymes39. A similar approach using artificial SAM analogues would be attractive for alkyltransferase ribozymes, but the rather poor chemical stability of ProSeAM and its analogues limits their utility as reagents for in vitro selection of catalytic RNA. Several pathways contribute to the spontaneous decomposition in aqueous environment40. Propargylated SAM undergoes rapid hydration of the triple bond at physiological pH, probably via an allene intermediate, and the major decomposition pathway for ProSeAM at physiological pH involves an intramolecular nucleophilic attack of the carboxylate group, resulting in breaking the weak selenium–carbon bond19. This intramolecular lactonization of the 2-aminobutyrate moiety affords the by-products homoserine lactone and Se-propargyl-5′-selenoadenosine   was used as the nucleoside, with the aim to increase the binding affinity between cofactor and ribozyme by introducing an extra hydrogen-bond donor. The chemical synthesis of ProSeDMA involved four steps, starting from the protected α-aminoamide alcohol 1, via transformation to the selenohomocystine amide 3  was purified by reversed-phase (RP) HPLC. Analogous cofactors with Se-methyl  and Se-allyl groups  were also prepared, as well as the N,N-dimethylamide 8, in which the amide group of ProSeDMA was further blocked by alkylation  Fig. . The nuce 3 Fig. . After rion Fig. . In addiSe-propargyl-5′-selenoadenosine after 1 h. In contrast, ProSeDMA was much more stable, with less than 5% loss after 1 h, and still more than 60% of the intact material remaining after 5 h of incubation at pH 7.0.The chemical stability of ProSeDMA was compared to ProSeAM Fig.  under th37. The modification efficiency was evaluated by enzymatic digestion with the restriction enzyme R. EcoR1 after MTase treatment  and MeSeDMA (6) were performed in parallel. The digestion pattern of the ProSeDMA-containing sample was almost the same as in the experiment without cofactor. This result suggests that ProSeDMA was not recognized as a cofactor by EcoR1 MTase. By contrast, ProSeAM, and surprisingly also MeSeDMA, showed similar activity to SAM. Thus, the combination of propargyl and amide modification provided sufficient structural change to escape from MTase recognition, and ProSeDMA is a potential candidate to serve as a bioorthogonal cofactor for intracellular propargyl modification of RNA with a suitable ribozyme.Next, we investigated the bioorthogonality of ProSeDMA, expecting that the terminal amide substitution and 2-aminoadenosine modification probably prevent recognition by native MTases. We chose the bacterial MTase M.EcoR1 and plasmid DNA for enzymatic modification, as previously described31. Two independent selection experiments were performed using ProSeDMA (5) and its α-hydroxy analogue 9 as co-substrates. After RNA folding and incubation with the cofactor at pH 7.5 and 37 °C for 1 h, the transferred propargyl group was reacted with biotin azide under Cu-catalysed azide-alkyne cycloaddition (CuAAC) reaction conditions. The biotinylated RNA molecules were then separated via streptavidin affinity purification on magnetic beads. The captured RNAs were amplified by reverse transcription and polymerase chain reaction (PCR). The generated dsDNA template was transcribed by T7 RNA polymerase, and the enriched RNA library was used in the next round of selection. After eight rounds, the enriched library was cloned and sequenced, and individual ribozyme candidates were screened for activity by a streptavidin gel shift assay . Strategic mutations in the binding arms that fixed mismatched base pairs or bulged out nucleotides indicated the localization of the reacting adenosine (A5), which was further confirmed by primer extension assays and by re-targeting (‘transplanting’) the ribozyme to a different adenosine (A9) in the RNA sequence  or 7-deazaadenosine (c7A) showed similar reactivity to unmodified adenosine. The RNA with 2′-deoxyadenosine (dA) showed two products of lower intensity (31% and 24%). By contrast, the RNA containing 3-deazaadenosine (c3A) did not give any modified product. These results strongly suggest that the N3 nitrogen is essential for catalytic activity, whereas N1 and N7 are dispensable.For detailed characterization, SAMURI was hybridized to the target RNA with two 8-nt binding arms and showed over 90% AGE Fig. . The reaAGE Fig. . The preAGE Fig. . The sub41, and the second product observed with the dA-containing substrate is consistent with the formation of an abasic site. This mechanism is exploited by DNA targeting natural products and minor groove-binding cytotoxic agents42. In contrast, N3 alkylation of adenosine in RNA has barely been investigated, despite its biological importance43 and the mechanistic involvement of adenine-N3 in natural ribozymes44. Thus, to further characterize the product produced by SAMURI, the modified RNA was analysed by MS , which was overlapped with the guanosine peak. The masses of the direct N3-alkylation product 10 (m/z 306.12) and the formyl intermediate 11 were not found after the digestion. For reference, we also recorded LC-MS spectra of stable isomers of 3-propargyladenosine 10, namely 1-propargyladenosine (p1A), 2′-O-propargyladenosine (pO2A) and N6-propargyladenosine (p6A), which showed the expected EIC at m/z 306.12 at three different retention times. Based on the combined results of atomic mutagenesis and MS, we conclude that SAMURI catalyses the transfer of the propargyl group from ProSeDMA to the N3 nitrogen of the unpaired adenosine A9 in the target RNA.To further confirm these mechanistic hypotheses, the PAGE-purified propargylated RNA was digested with snake venom phosphodiesterase (SVPD) and alkaline phosphatase enzymes, and the mononucleoside mixture was analysed by liquid chromatography-mass spectrometry   Fig. .Fig. 3Ch8 reacted almost as efficiently as the parent cofactor 5, suggesting that the terminal amide is not strictly recognized within the catalytic core. In contrast, replacement of the α-amino group by an α-hydroxyl group (cofactor 9) decreased the activity of the ribozyme, suggesting that the protonation state of the α-amino group is important for interaction. Together, these results suggested that the selenomethionine amino-acid side chain of ProSeAM may also be tolerated. Indeed, 60% propargylation was observed with 10 µM ProSeAM within 1 h  showed comparable RNA methylation levels when used at 3 mM . As expected, the ProSeDMA dose-dependent Cy5 signal (red) colocalized with the DFHBI-1T signal (green), indicated by the merged yellow colour in the multi-channel image of Cy5 and DFHBI-1T in Fig. The cSAMURI-expressing cells were treated with increasing concentrations of ProSeDMA in growth medium (from 100 µM to 3 mM). Cell viability was not decreased under these conditions, suggesting that ProSeDMA is not strongly cytotoxic in this concentration range Fig. . We alsonce Fig. . The Cy5trans-active SAMURI variants to target endogenous RNAs, similar to approaches recently shown for F30-MTR1 targeting Escherichia coli transfer RNA31, and circular ADAR-recruiting guide RNAs (cadRNAs)49 to target mRNAs.For comparison of the labelling efficiency, total RNA was also extracted from transfected cells that produced cSAMURI but were grown in the absence of ProSeDMA. The isolated RNA was then treated with the cofactor in vitro, followed by click labelling with Cy5. Using the normalized Cy5/DFHBI-1T ratio, we estimate that ~17% of cSAMURI was propargylated in the cell Fig. . Compari2+ concentrations. Using atomic mutagenesis and MS, we fully characterized the RNA-catalysed reaction of SAMURI and found that the modification occurred at N3 of an unpaired adenosine between the binding arms. Although N3-alkylated adenosines are usually prone to depurination, we found that the enhanced electrophilicity at C2 provides a pathway for the favourable formation of a stable N-alkylated aminoimidazole nucleoside. Thus, SAMURI introduces the propargyl group site-specifically as a small clickable tag for RNA labelling. Moreover, SAMURI is permissive to ProSeDMA analogues, and can even use native SAM to synthesize m3A in RNA, which is a challenging chemical modification that cannot be prepared by solid-phase synthesis. This work expands the known repertoire of RNA-catalysed RNA modification reactions and suggests that appropriately designed cofactors and fine-tuned in vitro selection strategies are likely to create additional RNA-modifying ribozymes in the future. Desirable new ribozyme and cofactor combinations could then also enable strain-promoted click chemistry for applications in which the need for copper ions with the here-described propargyl groups is of any concern. Given the increasing importance of RNA technologies, we envision SAMURI to become a useful tool for RNA labelling and for studying the biology of damaged nucleobases in RNA.In summary, we have reported SAMURI as an alkyltransferase ribozyme in combination with ProSeDMA as a bioorthogonal and stabilized SAM analogue. The replacement of the terminal carboxylic acid by an amide improved the chemical stability of ProSeDMA compared to previously reported propargylic SAM and SeAM derivatives. The catalytic activity of SAMURI was demonstrated in vitro and in living cells. The successful intracellular application was possible due to the low toxicity of ProSeDMA and the high activity of SAMURI under physiological MgO-TOM-protection on controlled pore glass (CPG) solid supports50. RNA and DNA sequences are shown in Supplementary Tables 1A (ref. 51) and c3A (refs. 53), were prepared by following published procedures55. Other modified phosphoramidites were purchased. RNA oligonucleotides were deprotected with ammonia and methylamine (AMA), followed by 1 M tetrabutylammonium fluoride in THF, desalted and purified by denaturing PAGE. The quality of RNAs (purity and identity) was analysed by anion-exchange HPLC  and 6 M urea; solvent B was 25 mM Tris-HCl (pH 8.0), 6 M urea and 0.5 M NaClO4; the gradient was linear, 0–40% solvent B, with a slope of 4% solvent B per column volume, flow rate 0.5 ml min−1, UV detection at 260 nm) and HR-ESI-MS . Measured and calculated masses are listed in Supplementary Table RNA oligonucleotides were prepared by solid-phase synthesis using phosphoramidite chemistry with 2′-2, pH 6.0, 7.0 or 8.0) or with cell lysate generated from HEK293T cells with RIPA buffer . The reaction mixtures were incubated at 37 °C, and 20 μl aliquots were taken from the samples to 40 μl of H2O + 0.1% trifluoroacetic acid (TFA) solution every 1 h. The collected samples were analysed by RP–HPLC . The analysis was run with a linear gradient: B conc. 5–7% (0–15 min), 7–70% (15–30 min); solvent A was H2O + 0.1% TFA; solvent B was MeCN + 0.1% TFA; and flow rate was 0.7 ml min−1 at 30 °C with UV detection at 260 nm.1 mM ProSeDMA was mixed in a total volume of 100 μl of reaction buffer  by overlap extension using Klenow fragment DNA polymerase. We used 500 pmol template DNA for in vitro transcription with T7 RNA polymerase in reaction buffer , 4 mM NTP, 30 mM MgCl2 and 2 mM spermidine). For the first selection round, the 5 nmol RNA pool  was annealed in selection buffer . Cofactor 5 or 9 was added together with MgCl2 and the reaction filled with water to 1 ml, to give final concentrations of 5 μM RNA, 5 μM cofactor and 10 mM MgCl2. In further selection rounds, 1 nmol RNA was used in 200 µl total volume for the selection step. After incubation at 37 °C for 1 h, an excess amount of cofactor was removed by ethanol precipitation. The reacted RNA was labelled with biotin azide via CuAAC methyl)amine (TBTA) in 10 μl of H2O:DMSO:tBuOH (5:3:1)). The biotinylated RNAs were captured using either neutravidin- or streptavidin-coated magnetic beads , washed according to the manufacturer’s instructions and eluted with formamide and amplified by PCR with reverse transcription (RT–PCR) with Ready-to-Go RT–PCR beads (Illustra). The second PCR was done with a primer containing the T7 promoter. In vitro transcription was performed , followed by PAGE purification to prepare the enriched RNA library for the next selection rounds. After eight rounds of selection, the library was cloned using the TOPO-TA cloning kit, and colonies were randomly picked up for sequencing.The initial library was prepared by in vitro transcription from double-stranded template DNA made from two synthetic DNA oligonucleotides  buffer) after 1 h incubation with cofactors 2) at 37 °C for 1 h and analysed by anion-exchange HPLC on a Dionex DNAPac PA200 column . The analysis was run with a linear gradient of B: conc. 0–48% (0–20 min). Solvent A was 25 mM Tris-HCl (pH 8.0) and 6 M urea; solvent B was 25 mM Tris-HCl (pH 8.0), 6 M urea and 0.5 M NaClO4. The flow rate was 0.5 ml min−1 at 60 °C with UV detection at 260 nm. For the digestion assay, 10 pmol unmodified and modified RNA were treated with 0.5 U of RNase T1 in 10 μl of reaction solution  for 5 min at 37 °C or in 10 μl of alkaline hydrolysis solution (25 mM NaOH) for 3 min at 95 °C. For MALDI–TOF analysis, unmodified, modified and isolated RNAs were applied on a matrix (20 mg ml−1 3-hydroxypicolinic acid and 10 mg ml−1 diammonium hydrogen citrate). After drying the matrix, mass data were collected by negative-mode ionization. For LC-MS analysis, the 500 pmol unmodified and modified RNA were digested by 6.0 U of bacterial alkaline phosphatase and 1.0 U of SVPD in reaction buffer . After extracting the digested nucleoside mixture with 100 μl of chloroform, the aqueous layer was concentrated by lyophilization, and the residue was dissolved in 70 μl of 5 mM NH4OAc and analysed by LC-MS, using an Synergi Fusion RP column . The analysis was run with a gradient of 0–5% (0–15 min) and 5–72.5% (15–45 min) of solvent B. Solvent A was 10 mM NH4OAc (pH 5.3), solvent B was MeCN and the flow rate was 0.2 ml min−1 at 25 °C with UV detection at 260 nm and online MS in a microTOF-Q III system in positive-ion mode.For anion-exchange HPLC analysis, the substrate RNA was incubated with ProSeDMA in reaction buffer . Each time-point sample was analysed by denaturing PAGE (20% polyacrylamide), and the band intensities were quantified by fluorescence imaging using a 695/55-nm emission filter. The yield versus time data were fitted to Y = Ymax(1 − exp(−kobst) using Origin (2019). All kinetic assays were carried out as three independent replicates. For determination of apparent Km values, kobs was determined with different concentrations of ProSeDMA and the kobs versus concentration data were fitted using the Michaelis–Menten equation.We mixed 10 pmol of Cy5-labelled RNA and 100 pmol of SAMURI in reaction buffer . After an annealing step (3 min at 95 °C and 10 min at 25 °C), 10 mM MgClcis-active SAMURI was prepared by overlap extension of amplicon from previously reported plasmid31 and synthetic DNA oligonucleotides . The sequence of the insert and successful ligation into the plasmid were confirmed by Sanger sequencing.The insert of 5 HEK293T cells (ATCC) were cultured in 1 ml of 1× Dulbecco’s modified Eagle medium (Life Technologies) with 10% fetal bovine serum, 100 U ml−1 penicillin and 100 μg ml−1 streptomycin in a 12-well culture plate. Cells were plated for transfection using FuGENE HD (Promega), according to the manufacturer’s instructions, with 2 μg cSAMURI plasmid in OptiMEM I reduced serum medium (Gibco). RNA expression was checked by fluorescent microscopy  with DFHBI-1T. After 48 h of incubation, total RNA was collected from the cultured cells by mixing with Trizol LS reagent (Invitrogen).1 × 10−1 penicillin and 100 μg ml−1 streptomycin in a 24-well culture plate. After 24 h, the medium was replaced by 0.5 ml OptiMEM containing 0.1–3 mM ProSeDMA and 50 mM HEPES (pH 7.5) and the cells were incubated for 6 h. The cells were then extensively washed with PBS and mixed with 0.4% trypan blue solution (Thermo Fisher Scientific) in a 1:1 ratio. After 5 min of incubation at room temperature, total and stained cells were counted and cell viability was calculated as ) × 100 (%).HEK293T cells were cultured in 1 ml of 1× Dulbecco’s modified Eagle medium with 10% fetal bovine serum, 100 U ml−1 actD and 50 mM HEPES (pH 7.5) and incubated for 1 h. The incubated cells were washed three times with 1× PBS and suspended in 200 μl of 50% methanol + 0.1% formic acid. The suspended cells were completely disrupted by sonication. The resulting cell lysate was extracted with 200 μl of chloroform and the water phase was evaporated. After evaporation, the residue was dissolved in 100 μl of H2O containing 0.1% formic acid. After filtration, 50 μl of the sample was injected into an LC/MS system using an RP-column . The analysis was run with a linear gradient of B: conc. 3% (0–15 min), 3–23% (15–30 min); solvent A was H2O + 0.1% formic acid, solvent B was MeCN + 0.1% formic acid and the flow rate was 0.2 ml min−1 at 25 °C with UV detection at 260 nm. The micrOTOF-Q III with an ESI ion source was operated in positive-ion mode , with a capillary voltage of 4.5 kV, end plate offset of 500 V, nitrogen nebulizer pressure of 1.4 bar, dry gas flow of 9 l min−1 and dry temperature of 200 °C. Data were analysed with Data Analysis software DA 4.2 . For the calibration curve, multiple concentrations of ProSeDMA  were spiked into cell lysate generated from cells that were not treated with ProSeDMA, and the peak areas of the ProSeDMA extracted ion chromatograms were plotted against the ProSeDMA amount injected. The slope was determined and used for calculation of the concentration of ProSeDMA in the lysate of cells that were fed with ProSeDMA in the medium.HEK293T cells were seeded in a 12-well culture plate and grown for 24 h. The medium was replaced by 1 ml of OptiMEM containing 0, 0.1, 0.5, 1 or 3 mM ProSeDMA, 5 μg ml−1 actD (RNA transcription inhibitor) and 50 mM HEPES (pH 7.5). After 6 h of incubation, the cells were extensively washed and total RNAs were collected by Trizol LS reagent. The collected RNA was labelled by the CuAAC reaction . Labelled RNAs were analysed by 5% denaturing PAGE, and the gel was imaged in the Cy5 channel before staining with DFHBI-1T and SYBR Gold. The band intensities were quantified and the intracellular activity was calculated from the Cy5/DFHBI-1T ratio, relative to the in vitro labelling efficiency of total RNA extracted from cells that were not treated with ProSeDMA. This control was prepared by labelling in the test tube .After transfection, cells were cultured for 48 h and then the medium was replaced by 1 ml of OptiMEM containing 0.1–3 mM ProSeDMA, 5 μg mlFurther information on research design is available in the Any methods, additional references, Nature Portfolio reporting summaries, source data, extended data, supplementary information, acknowledgements, peer review information; details of author contributions and competing interests; and statements of data and code availability are available at 10.1038/s41557-023-01320-z.Supplementary InformationSupplementary Tables 1–4, Fig. 1, methods with schemes 1–3, NMR spectra, HR-ESI-MS spectra and HPLC chromatograms.Reporting SummarySource Data Fig. 2Unprocessed full size gels for Fig. 2c,e.Source Data Fig. 2Statistical source data for Fig. 2e.Source Data Fig. 3Unprocessed full size gels for Fig. 3a,b,d,f.Source Data Fig. 3Source data for plots in Fig. 3d,e,f,g.Source Data Fig. 4Unprocessed full size gels for Fig. 4d.Source Data Fig. 4Statistical source data for Fig. 4c,e.Source Data Extended Data Fig. 1Unprocessed gels for Extended Data Fig. 1b,c.Source Data Extended Data Fig. 3Unprocessed gels for Extended Data Fig. 3.Source Data Extended Data Fig. 4Unprocessed gels for Extended Data Fig. 4.Source Data Extended Data Fig. 8Source data for plots in Extended Data Fig 8.


Our strategyallows any RNA binding small molecule to be converted into a degrader,empowering RNA binders that are not potent enough to exert a phenotypiceffect on their own. PINAD raises the possibility of targeting anddestroying any disease-related RNA species, which can greatly expandthe space of druggable targets and diseases.Nature has evolvedintricate machinery to target anddegrade RNA,and some of these molecular mechanisms can be adapted for therapeuticuse. Small interfering RNAs and RNase H-inducing oligonucleotideshave yielded therapeutic agents against diseases that cannot be tackledusing protein-centered approaches. Because these therapeutic agentsare nucleic acid-based, they have several inherent drawbacks whichinclude poor cellular uptake and stability. Here we report a new approachto target and degrade RNA using small molecules, proximity-inducednucleic acid degrader (PINAD). We have utilized this strategy to designtwo families of RNA degraders which target two different RNA structureswithin the genome of SARS-CoV-2: G-quadruplexes and the betacoronaviralpseudoknot. We demonstrate that these novel molecules degrade theirtargets using  in vitro, in cells, and in vivo.An approach to convert RNA-binding moleculesinto RNA degradersis described. Reported molecules are found to affect the SARS-CoV-2genome  For example, tetracyclines bind the16S rRNA on the 30S subunit of prokaryotic ribosomes, thus blockingthe tRNA docking onto the A-site and interfering with the translationof prokaryotic proteins, which is the underlying mechanism behindthe bacteriostatic effect.3 Strikingly,mutation of a few key bases on 16S rRNA results in the loss of bindingcontacts against tetracyclines leading to resistance against thisclass of molecules. While tetracyclines exert their mechanism of actionby sterically blocking access to the ribosome, for most RNA effectorsa binding event might not be sufficient to modulate the functionsof the targeted RNA. This issue may be circumvented by convertingRNA binders into bifunctional molecules which can alter the functionsof their target RNAs, e.g., by degrading or editing them.RNA is a structured biomolecule, and certainRNA structures canbe attributed to pathologies. Moreover, the majority of the humangenome is transcribed into RNA, whereas only ∼3% of RNA transcriptsget translated into proteins, meaning that for each pathology, thereare potentially many more disease-relevant RNA species than proteins.4 In the RIBOTAC approach, a small molecule RNAbinder is appended to a ligand of ribonuclease . The two distinctparts of this bifunctional molecule bring together the target RNAand a ribonuclease, which results in target RNA getting degraded.Thus, RIBOTACs act akin to siRNAs but have several caveats which insome cases can limit their therapeutic potential.5 First, activities of RIBOTACs described thus far dependon endogenous concentrations of ribonuclease L which is not evenlyexpressed across different tissues; thus, not all cell types wouldbe compatible with this approach. Second, for RIBOTACs to function,the RNA and RNase L ligands need to have spatial orientation compatiblewith bringing the two biomacromolecules together, the RNA and an enzyme.Moreover, RIBOTACs conjoin two small molecule ligands having largemolecular weights, which might negatively affect their physiochemicalproperties. It is also worth noting that RIBOTACs disclosed so farhave a limited effect on their target RNA, with degradation usuallycapping at 50–60%. However, this might be a consequence ofthe fast turnover of RNA species targeted rather than an inherentfeature of RIBOTACs. Altogether, this calls for alternative approachesto small molecule-induced targeted RNA degradation.Degradationof RNA utilizing small molecule RNA binders has beenpreviously attempted in the form of RIBOTACs .In our design, PINADs are composed of three components—a smallmolecule RNA binder, a long flexible linker which can reach multiplepositions on the targeted RNA, and an imidazole—the RNA degradingmoiety present on many ribonucleases. We have previously shown thatit is sufficient to covalently attach imidazole moieties onto RNAto result in their degradation; we thus postulated that bringing animidazole into proximity will have comparable results.7 The Duca group has recently reported a similar approach,in which they weaponized the aminoglycoside antibiotic neomycin withan amino acid histidine.8 For our studies,we developed two series of PINADs using imidazole warheads conjugatedto flexible PEG chains, targeting two different structural elementsof the genome of SARS-CoV-2, and demonstrated that these moleculesare active using in vitro, in cellulo, and in vivo infection models of SARS-CoV-2  and two in the S gene .11 It was demonstratedthat the presence of the G4 structure in the N genereduces the efficiency of the nucleocapsid phosphoprotein (N protein)translation, suggesting that this RNA structure is utilized to maintainthe optimal ratio of viral proteins.11 Thebetacoronaviral pseudoknot is a more complex and unique structure,consisting of three stem-loops.12 Thisstructure has a well-defined role in −1 frameshifting—aprocess in which the ribosome shifts by one nucleotide to the 5′direction resulting in a shifted reading frame; this process is usedby SARS-CoV-2 for translation of RNA-dependent RNA polymerase Nsp12.13 Since this enzyme isvital for replication of this virus, disruption of −1 frameshiftingcan severely hamper the rate of replication for SARS-CoV-2.14 This is a likely explanation for why the sequencecorresponding to the pseudoknot is so well conserved—it isuniform throughout different SARS-CoV-2 variants of concern, and thehomology between the pseudoknots in SARS-CoV and SARS-CoV-2 is 99%(difference of a single nucleotide), much higher than that of theoverall genome. The rapid mutation rates of viral genomes make targetingof the conserved regions highly desirable.15As a proof ofconcept, we elected to test the PINAD approach againstthe genome of SARS-CoV-2 because it possesses genomic RNA with multiplecharacterized structural features and is disease-relevant, being thecarrier of genetic information of the virus that causes COVID-19.RS-CoV-2 1b,c. ThePDS), a well-explored ligand with a good selectivityand KD in the nanomolar range.16 For the pseudoknot, we chose MTDB, a pseudoknot ligand identified through an in silico screen and demonstrated to affect the −1 frameshifting,17 reported to bind the pseudoknot in the uppermicromolar range.18 For both series ofcompounds, we functionalized the binders with alkynes so they couldbe readily exposed to a copper-catalyzed alkyne–azide cycloaddition(CuAAC) reaction. We made PDS derivatizes with degradersof two different lengths so we could investigate how the length ofthe linker affects the efficiency of degradation. We also functionalized PDS with a carboxy group as a control (CBX-PDS), shown previously to selectively bind RNA . AUTODOCK4.220 was used for the calculations (see Methods for details). As inferred from thesecalculations, both ligands can interact with several regions of thepseudoknot . One of the bestposes for MTDB-imi6  featured a hydrogen bond between the thiazole ring andU45 (the numbering of the nucleotides used for the cryo-EM structure21 was applied in this manuscript), which was absentin TDB-imi6. Additionally, the amide group of MTDB-imi6, the tertiary amine, and one of the oxygen atoms of the PEG linkerwere engaged in hydrogen bonding interactions with A76, C17, and C71,respectively . The stabilizinginteraction between the thiazole ring and the receptor has been identifiedas a key intermolecular interaction21 thatcould contribute to the efficiency of the degradation process. Thebinding energy calculated by AUTODOCK 4.2 for the best ranked posesof MTDB-imi6 and TDB-imi6 was −8.02and −5.23 kcal/mol, respectively . Interestingly, the best binding pose suggested thatour calculations, which are based on the cryo-EM structure of thepseudoknot,21 differ from what was proposedby Park and co-workers,17 who used a modelof the pseudoknot to predict the 3D structure of the complexes. However,overall findings of this study suggested that exchanging an esterinto an amide will not abolish binding and that the MTDB-derived degrader has a higher affinity to the pseudoknot comparedto the TDB-derived degrader.To gain insight into how an attachment of a linker and a degradercould affect the ability of MTDB-imi6 (pose #3) and the pseudoknot (see Methods for details) was then subjected to extensive moleculardynamic (MD) simulations in the presence of explicit water moleculesand ions . According to thesesimulations, the complex is stable through the trajectory but exploresseveral regions of the receptor. In addition, the PEG linker was quiteflexible, allowing interactions between the imidazole degrader anddifferent sites of the pseudoknot. The flexibility of the complexresulted in several structures that exhibited different transienthydrogen bonds, especially between the triazole moiety or the oxygenatoms of the linker and the RNA molecule, which could explain thehigher affinity of MTDB-imi6 compared to MTDB. The most important hydrogen bond (population ∼20%) engagedthe carbonyl of the amide group and G46. The complex was also stabilizedby transient π-staking interactions between the phenyl groupof the ligand and several bases of the pseudoknot. These findingssuggest that the long linker present on the MTDB-imi6 enables the degrader moiety to reach many nucleotides on this structure.The complex between PDS is a known fluorescence-quencher, and thus we utilizeda fluorescence quenching assay to evaluate compounds derived fromthis scaffold.22 In brief, PDS and its derivatives were incubated with a Cy5-functionalized oligomercorresponding to a G-quadruplex in NRAS 5′UTR, a well-establishedG4 model. As fluorescence quenching is proximity-induced, the signalcorresponding to Cy5 is quenched upon PDS binding. Wefound all the compounds functionalized on the central pyridine coreto have a similar binding affinity, close to 200 nM, with the parentcompound PDS having a stronger affinity with KD of 26 nM  reader . In an MST workflow, a 5′-Cy5-labeledoligomer corresponding to the coronaviral pseudoknot is incubatedwith one of the binders. With the observation that the fluorescencewas affected by bound ligand, the binding curve could be obtainedin a concentration-dependent manner by an initial capillary scan.We found that MTDB exhibited a KD value of 56.1 μM . However, it cannot be discarded that some degradation could betaking placing during the measurement, which would influence the observed KD value. We also attempted to measure the bindingaffinity of TDB by using the same approach, but we werenot able to establish a KD due to limitedsolubility of the molecule.The two degrader-functionalized MTDBderivatives, ectively3b. This MTDB-imi6 relative to MTDB,we investigated whether the degraderalone could bind to the G4 or the pseudoknot. Because the degraderitself does not influence the fluorescence of the RNA constructs,we used the thermophoresis curves of the MST approach to estimatebinding affinities. For both systems, binding was observed only atmillimolar concentrations, and thus we could not establish KD values ,with the results observed with the pseudoknot further validated viagel electrophoresis. Briefly, for the LC-MS assays we ensured thatthe relationship between injected oligonucleotide concentration andmass signal was linear ,and then we used the method to quantify oligonucleotides after incubationwith an appropriate PINAD . We also considered using UV–vis spectra for quantification,and although it showed good evidence for degradation, we elected notto use it due to high noise levels and the necessity in applying anoise subtraction algorithm, which biases numerical data .Confident in the abilityof our molecules to bind their targetstructures, we tested their capacity for degradation PDS by incubating them with the G4-forming NRAS oligonucleotide at 37°C in either potassium-containing (promotes G4 formation by stabilizingthem) or lithium-containing (does not stabilize G4 structures) buffer,followed by LC-MS analysis. In the potassium buffer, we observed asignificant amount of degradation with both PDS-imi6 and PDS-imi4 relative to CBX-PDS, with the PDS-imi6 being a more efficient degrader, which shows thatan appended imidazole can indeed act as a degrader, and for this molecule,the longer linker performs the best, whereas in the lithium-containingbuffer little to no degradation was observed, as would be expectedin a system where the population of formed G4 structures is not promotedby present cations . The 3′overhangs allowed for a better visualization of the pseudoknot degradationcompared to the non-extended oligonucleotide. No significant degradationwas observed when these molecules were incubated with the perturbedpseudoknot, demonstrating their selectivity toward the folded betacoronaviralpseudoknot . To assess how well these observations translate to a cellularmodel, we treated VERO–CCL-81 cells infected with SARS-CoV-2either with MTDB-imi6 or a vehicle control, followedby RNA extraction and qPCR analysis of 15 loci of the SARS-CoV-2 genometo get a full genomic coverage and reveal which parts are affectedthe most. This validation was in broad agreement with Nanopore sequencing,demonstrating that MTDB-imi6 affects the flanks of thepseudoknot area but not any other region of the tested subgenomicSARS-CoV-2 RNAs  of the viral culture .We found that PDS-imi6 had no effect on the viabilityof VERO-CCL-81 cells, and thus the observed effect cannot be explainedby cell death , suggesting retention of G4 structuresbetween these variants .We tested the antiviralactivity of the two families of PINADs by using SARS-CoV-2 infectedVERO-CCL-81 cells as a model of SARS-CoV-2 infection. First, we testedthe inhibition of viral replication in a dose-dependent manner. Viralloads were determined using qPCR on either the E (Envelope) or N (Nucleocapsid)gene. We found that nfection 6a,b. Sim culture S5a.We f culture S5a.We fMTDB-imi6, on theother hand, is onlyknown to targetthe coronaviral pseudoknot and its RNA–RNA interactome andthus is a better PINAD for demonstration of specificity. As for the PDS-imi6, we tested the effect of the MTDB familyof molecules on viral replication and evaluated it using qPCR, inthis case probing the E gene and the pseudoknot region itself. Weobserved that low-micromolar concentrations of MTDB-imi6 exhibited marked antiviral effects with a significant reductionof coronaviral RNA in cells treated before or after infection, havinglower IC50 values when treating the cells postinfection. Moreover, to furtherdemonstrate the anti-SARS-CoV-2 impact of MTDB-imi6 treatment,we examined the status of two robust SARS-CoV-2 infection biomarkers.Indeed, treatment with MTDB-imi6 significantly reducedthe phosphorylation of MAPK2 (p-MK2 and T334) and led to a significantdown-regulation of the mRNA of the key cytokine IL6, with both biomarkers known to be dictated by the p38/MAPK signalingpathway and elevated during SARS-CoV-2 infection .30 We found thatnone of the compounds were cytotoxic to host cells, indicating thatthe observed effect on viral replication was not a result of celldeath . Additionally, we investigated whether the MTDB family of compounds could affect the SARS-CoV-2 virions alone byexposing them to these compounds in a cell-free environment . No virucidal effects were observedwhen cell free virus was incubated with MTDB-imi6, MTDB, and TDB-imi6, providing evidence that MTDB-imi6 indeed only affects the virus postinfection, whenits genomic RNA is exposed to the cellular media and small moleculestherein. Finally, we found that MTDB-imi6 also inhibitedthe replication of the SARS-CoV-2 Alpha and Delta variants of concern, which was expected giventhat the pseudoknot is conserved across all the SARS-CoV-2 variants.Overall, the antiviral drug assays show that MTDB-imi6 exhibits antiviral activity against SARS-CoV-2, is specific againstthe betacoronaviral three-stemmed pseudoknot, and affects them withan irreversible impact, and thus the PINAD approach could be usedin the development of therapeutics against SARS-CoV-2 infection.We also observed that the ability of the virus to recover followinga 24-h drug exposure was compromised in MTDB-imi6invivo in a SARS-CoV-2 mouse model of infection . Intranasally infected mice were treated1 h before and 3 h after the infection with one of the molecules fromthe MTDB series (MTDB-imi6 (at 25 mg/kg) showed a significant reduction of lung viral loadrelative to the vehicle control group by plaque assay, unlike thecontrol molecules ,a class of bifunctional small molecules which bind and then degradenucleic acids in a proximity-induced manner. To exemplify, we havedesigned two series of PINADs against structural elements of the SARS-CoV-2genome—PDS-degraders which degrade G-quadruplexes and MTDB-degraderswhich degrade betacoronaviral pseudoknots. We achieved this by functionalizingthe parent binder molecules with imidazole moieties, which act asRNA-degrading warheads, attached via long and flexible PEG linkers.We have demonstrated that these molecules bind and degrade their targetRNAs 31 mRNAs of disordered proteins,32 long noncoding,33 and micro-RNAs34 are all valid therapeutic targets that cannotbe subjected to protein-centered approaches; they can instead be targetedas RNAs. In some cases the RNA binding event alone can result in thedesired phenotype.3 However, using a PINADrather than a binder provides a number of advantages: RNA degradationcan result in a different, stronger phenotype, whereas the effectof the binder alone might be too weak to influence the phenotype;weak binders can be transformed into potent degraders as exemplifiedby MTDB-imi6; finally, taking advantage of the linkerto modulate the solubility of small molecules is especially relevantsince many binding pockets in RNA are hydrophobic35 and require a hydrophobic binder, which might exhibit poorsolubility in aqueous media, as is the case with MTDB.Indeed, targetingRNAs with small molecules can greatly expandthe druggable genome. For example, many viral genomes,37 Combininghigh-throughput screening with a PINAD or a different approach totargeted RNA degradation holds potential for a rapid drug discoveryplatform, the need for which was affirmed during the COVID-19 pandemic.Viruses are capable of rapid mutation as a part of their immune evasionstrategy, which greatly complicates antiviral drug discovery campaigns.15 The ability to target and degrade highly conservedviral RNA structures, such as the betacoronaviral pseudoknot, canprevent these issues and result in efficient antiviral therapies.Another group of diseases which can be tackled by targeted RNA degradersare pathologies hallmarked by protein misfolding, such as Alzheimer’sdisease, Huntington’s disease, or type II diabetes.38 These misfolded proteins do not have well-definedstructures and thus are extremely difficult to target with small molecules.One way to circumvent this issue is to target mRNAs of correspondingproteins.32 Thus, the PINAD approach holdsthe promise for rapid development of therapeutic modalities againstdiseases impossible to tackle using well-established methods.The recent advances in RNA binder screening hint that discoveryrate of selective RNA binders is not that different to that of proteinbinders, which suggests that many more selective RNA binders willbe within reach in the near future.


Introduction: The application of RNA-sequencing has led to numerous breakthroughs related to investigating gene expression levels in complex biological systems. Among these are knowledge of how organisms, such as the vertebrate model organism zebrafish (Danio rerio), respond to toxicant exposure. Recently, the development of 3′ RNA-seq has allowed for the determination of gene expression levels with a fraction of the required reads compared to standard RNA-seq. While 3′ RNA-seq has many advantages, a comparison to standard RNA-seq has not been performed in the context of whole organism toxicity and sparse data.Methods and results: Here, we examined samples from zebrafish exposed to perfluorobutane sulfonamide (FBSA) with either 3′ or standard RNA-seq to determine the advantages of each with regards to the identification of functionally enriched pathways. We found that 3′ and standard RNA-seq showed specific advantages when focusing on annotated or unannotated regions of the genome. We also found that standard RNA-seq identified more differentially expressed genes (DEGs), but that this advantage disappeared under conditions of sparse data. We also found that standard RNA-seq had a significant advantage in identifying functionally enriched pathways via analysis of DEG lists but that this advantage was minimal when identifying pathways via gene set enrichment analysis of all genes.Conclusions: These results show that each approach has experimental conditions where they may be advantageous. Our observations can help guide others in the choice of 3′ RNA-seq vs standard RNA sequencing to query gene expression levels in a range of biological systems. Danio rerio), a model vertebrate organism , alignment to a genome, or  RNA-seq . A laterrequired . Another3′ RNA-seq offers a potential major advantage over standard RNA-seq in that it can often provide critical information on gene expression levels with fewer input reads. However, when to use either 3′ or standard RNA-seq to gain the best advantages of each to answer biological questions, particularly in whole animal, vertebrate systems is still being examined. Here, we applied each of these sequencing approaches to samples collected from whole zebrafish larvae exposed to a control condition or elevated perfluorobutane sulfonamide (FBSA). We set out to determine, for each method, 3′ or standard RNA-seq, how well alignment took place to the genome, the overlap of DEGs detected in both approaches, how this held up under conditions of sparse data and how many enriched functions were detected, which could be interpreted as the response of this organism to the toxic effects of FBSA. The current study differs from previous analyses in that we are more focused on the quality of enriched functions detected by the two methods, not merely the number and overlap of differentially expressed genes, as well as the application of each method when examining sparse data and the use of complex multicellular model organisms for chemical hazard assessment.Danio rerio) were maintained following protocols approved by Oregon State University’s Institutional Animal Care and Use Committee (IACUC-2021-0166) at the Sinnhuber Aquatic Research Laboratory . Adult zebrafish were maintained on a light:dark cycle of 14:10 h and at densities of 400 fish per 50-gallon tank on a recirculating filtered water system supplemented with Instant Ocean salts   . GEMMA M States) , and kep at 28°C .Perfluorobutane sulfonamide (FBSA) was obtained from SynQuest Laboratories . As previously published, a 22 mM stock solution was prepared in 100% methanol , shaken on an orbital shaker overnight, and analytically measured using high-performance liquid chromatography and triple quadrupole mass spectrometry (LC-MS/MS) .®, product number: 353227; Glendale, AZ, United States), 100 μL EM and a single larvae in each well, using an automated placement system (At 4 h post fertilization (hpf), larvae were dechorionated enzymatically with pronase and an automated dechorionator . Larvae t system . At 8 hpAt 48 hpf, prior to the onset of morphological effects that were confirmed to occur at 120 hpf, mRNA was collected from a subset from both experimental conditions. Ten zebrafish larvae were pooled per replicate (n = 3) into 1.5 mL safe-lock tubes and euthanized on ice . To homogenize the samples, excess water was immediately removed, 200 µL RNAzol  and 100 µL 0.5 mm zirconium oxide beads were added. Homogenization was performed with a Bullet Blender  on setting 8 for 3 min. An additional 300 µL RNAzol was added to each tube prior to storage at −80°C.Total RNA isolation was performed using a Direct-zol RNA MiniPrep kit , and the optional DNase I digestion step was included. Following isolation, the RNA concentration was measured using a SynergyMix microplate reader and Gen5 Take3 module  quality of the RNA samples was assessed with an Agilent Bioanalyzer 2100  by the Center for Quantitative Life Sciences (CQLS) at Oregon State University, and all RINs were greater than 9.For standard RNA-seq, library preparation and RNA sequencing were performed by the Beijing Genomics Institute . The DNBseq platform entailed the purification and fragmentation of mRNA using oligo (dT)-attached magnetic beads, cDNA synthesis and processing, followed by amplification and further purification. Library quality was confirmed an Agilent Bioanalyzer 2100. Amplified products were then circularized to form the final library, amplified and formed into DNA nanoballs, loaded into patterned array, and sequenced with the BGISEQ-500 (100 bp paired end). For 3′ RNA-seq, library preparation and RNA sequencing were conducted by the CQLS at Oregon State University. Library preparation was performed using the Lexogen QuantSeq 3′ kit for Illumina. The details for this kit can be found there but importantly this library preparation does contain a step where polyA-tailed RNA transcripts are amplified with oligo (dT) primers, so both 3′ and standard RNA-seq enrich for mRNAs. The final 3′ RNA-seq final library was then sequenced using a HiSeq (100 bp single end).All fastq files were first examined with FastQC. Standard RNA-seq data had a slightly higher quality score (measured by Phred) compared to 3′ RNA-seq but both approaches gave high quality data with Phred scores above 22   was carried out using gProfiler  algorithm was performed by averaging the log2 fold changes across the 3 replicates for each condition and using these values as input scores to GSEA, which was performed using the fgsea package (http://geneontology.org/), as well as KEGG terms (https://www.genome.jp/kegg/) were used as input gene sets.Output files of HTSeq were used to determine alignment rates as well as alignment to features features . Note thfeatures  with defProfiler . Output  package . The GO 3′ and standard RNA-seq show nearly identical genome alignment percentages but differing alignment to annotated or non-annotated regions.Our initial viewing of the data showed that the samples from 3′ RNA-seq had a greater number of reads compared to standard RNA-seq . This isp-value  of less than 0.05. There was Jaccard overlap of 0.32 when comparing standard to 3′ RNA-seq with regard to DEGs. Standard RNA-seq identified 143 DEGs and 3′ RNA-seq 116, with 64 DEGs in common between the two methods. We used these shared DEGs to examine sample-sample correlation and hierarchical clustering of samples  may be different in 3′ or standard RNA-seq. As mentioned above, on average, DEGs detected by 3′ RNA-seq showed higher overall expression compared to standard RNA-seq but this was not statistically significant.We next examined whether 3′ and standard RNA-seq identified the same DEGs when comparing control sample to those from FBSA-treated fish . Here, w samples . We foun of 0.94 . Howevervs. standard RNA-seq when the gene counts were iteratively reduced from 100% (the complete data) down to 1% of the original gene count levels  and 3′ RNA-seq and standard RNA-seq rarely identified the exact same number of DEGs, we also used Gene Set Enrichment Analysis (GSEA) with both approaches to examine how functions may be identified when there is no cutoff used to designate a DEG. With GSEA analysis the results between 3′ and standard RNA-seq were much more similar . There was also strong overlap between the two approaches with all functions identified by 3′ RNA-seq also identified in the standard RNA-seq dataset as well as six additional functions. As in q-value) was higher in the Standard RNA-seq datasets (4.21) compared to the 3′ RNA-seq dataset (3.67) with the p-value of this comparison just slightly over significance at 0.11 using a Student’s t-test.To determine whether the large difference between standard and 3′ RNA-seq regarding identified functions was an effect of this particular pair of DEG lists, we took advantage of the sparse data sets we generated earlier . Here ag similar . Using tde novo transcriptome assembly is needed. This advantage of standard RNA-seq against unannotated genomes was also found in another study that examined whole organism samples  and 3′ RNA sequencing (based on enriching for 3′ ends of the transcript and using this as a proxy for expression of the whole gene) to determine under which experimental conditions each approach demonstrated advantages or disadvantages. Rather than definitively identifying one or the other of these approaches as superior, our results suggest that each technology should be applied specifically when certain experimental parameters are present. 3′ RNA seq is far better at alignment to annotated regions of the genome compared to standard RNA seq. We had the opportunity to study this aspect because zebrafish has a very well annotated genome . However samples .We also found that 3′ RNA-seq is well suited to identifying DEGs under conditions of data sparsity. A low number of reads is a common issue in transcriptomics especially in the case of metatranscriptomic studies where many species are being examined simultaneously in a mixed community or in cases where costs mean that many samples must be multiplexed in the same sequencing run. In cases with microbiome studies, 50-100 million reads must be split across thousands of genomes. Many of these genomes will be covered at a level that allows for some determination of gene expression but not at levels that are ideal for robust analysis . Under svs. 3′ RNA-seq show some differences in alignment, the number of DEGs detected and how this may change when data becomes sparse with each approach having advantages in certain situations. However, standard RNA-seq was by far the superior method when examining the number of enriched functions identified from DEG lists as well as the statistical significance of the enrichment of these functions. Interestingly, this was not the case when, instead of DEG input lists, gene set enrichment analysis (GSEA) was used. GSEA does not rely on user defined cutoffs to generate DEG lists and instead uses global expression levels from all genes. This suggests that the same general set of pathways are detected as activated when either standard or 3′ RNA-seq is used, with part of the reason for the differences in functional enrichment lying in whether genes examined by 3′ or standard RNA-seq have reach user-defined thresholds of a differentially expressed gene . Standard RNA-seq generally finds a greater number of DEGs which could lead to a greater number of statistically-enriched functions. However, in our analysis of sparse data certain datasets led to standard and 3′ RNA-seq identifying virtually the same number of DEGs. In these cases, standard RNA-seq still identified more functions so the absolute number of DEGs identified is likely only part of the answer. Regardless of the outcomes of functional enrichment with gProfiler the results of GSEA show that both standard and RNA-seq identify activation of many of the same functions, though standard RNA-seq still does have a slight advantage in the number of functions detected and the statistical significance. We chose to include both functional enrichment with a set of DEGS and GSEA as both methods are still widely used to interpret RNA-sequencing data. However, it is known that relying on an arbitrary definition of a DEG  can miss key genes and thus pathways. Other researchers have also pointed out this flaw and encouraged the use of GSEA, which does not rely on DEG definitions and instead uses expression data from all genes  and 3′ RNA-seq identified 26 functions. Two functions (∼3%) were found to be in common between the standard RNA-seq function list and the function list from our earlier study. In contrast, no functions were found to be in common between the 3′ RNA-seq list and our earlier study. Again, differences between the analyses lead to different functions being enriched. It should also be noted that in many cases very similar functions were found between this study and the earlier study but did not count towards an overlap due to not having the exact same GO term . We also found strong overlap in the actual DEGs identified by both 3′ and standard RNA-seq with the previous study (which as mentioned earlier used the same RNA samples we sequence here). For standard RNA-seq 424/547 of the DEGs identified (77%) were also identified as DEGs in the previous study, for 3′ RNA-seq this overlap was 189/252 genes (75%). For all analyses it should be noted that this is a single study comparison of two conditions. Conclusions here should not be taken as definitive answers of how 3′ or standard RNA-seq behaves generally across all experiments. Rather, these findings should be added to the growing list of analyses contrasting these two methods in other publications. By taking this approach we can address how reproducible the conclusions presented here are. We have found that 3′ RNA-seq has a distinct advantage in alignment when using annotated genomes and in working with sparse data. These aspects have also been found in additional studies mentioned above. The fact that these characteristics have been reproducibly found in several studies suggest that they should be given considerable weight when choosing RNA-seq methods in future experiments. In contrast, the enrichment advantages of standard RNA-seq found in this study have not yet been examined in detail in other systems. Therefore, that aspect of standard vs. 3′ RNA-seq should be taken as a preliminary conclusion generally until other studies confirm it.In addition to a greater number of functions identified by standard RNA-seq with DEG input lists, there was also stronger overlap between this study and a previous, independent analysis using the same RNA samples . We compAs new RNA sequencing technologies emerge, the best way to use them will be in circumstances where they give the most advantage. Here, we directly compare standard and 3′ RNA-seq and find that 3′ RNA-seq is better with annotated genomes and with sparse data. In contrast, standard RNA-seq is better at finding functions using DEG lists and with more significant enrichment scores, which are important for the evaluation of dose- and time-dependent mechanisms of toxicity . This ad


Youth with disabilities have the same right to sexual and reproductive health (SRH) as their peers without disabilities. However, their needs and rights are often ignored. Little is known about the knowledge, needs and access barriers related to SRH information among youth with different types of disabilities in China.A cross-sectional survey was carried out among 473 unmarried youth aged 15–24 with visual, hearing, physical disabilities in both urban and rural areas in China.Out of a maximum possible score of 100, respondent’s median score ranged from 30 to 50 for knowledge related to sexual physiology, STIs/HIV/AIDS and contraception. For these three categories of knowledge, respondents with hearing and physical disabilities or from rural areas scored lower than their counterparts with visual disabilities or from urban areas. The multivariate analyses showed that the residential area and education level were strong correlates of knowledge among respondents with visual and hearing disabilities. Other significant correlates were age for respondents with visual impairment and physical impairment, and single child status in the family and father’s education level for respondents with hearing impairment. Sources of and barriers and preferences in accessing SRH information differed by type of disabilities, residential areas and gender. In general, school teachers were the primary and most preferred sources of SRH knowledge, followed by the Internet, peers/friends and parents. Unaware of where to get accurate information and feeling embarrassed to seek information were the two most frequently mentioned barriers in accessing SRH information.Respondents had poor knowledge of SRH and limited access to SRH information, especially those from rural areas. Efforts should be made to promote school and family-based sexuality education tailored for youth with different types of disabilities. Youth with disabilities have the same sexual and reproductive health (SRH) needs as their peers without disabilities and the equal right to attain the highest standard of SRH. However, their SRH needs and rights are often overlooked or neglected. Studies on SRH among unmarried youth with disabilities are very limited in China. This study used data collected from 473 unmarried youths with different types of disabilities from both urban and rural areas in China, to understand their SRH knowledge and its associated factors, as well as barriers and preferences in accessing sexuality-related information. This study demonstrated that unmarried youth with disabilities lacked knowledge of SRH, especially those with hearing or physical disabilities and those from rural areas. In general, residential area and education level were significant correlates of knowledge among respondents. Sources of and barriers and preferences in accessing SRH information varied across types of disabilities, residential areas and gender. In general, school teachers were the primary and most preferred sources of knowledge, followed by the Internet, peers and parents. Unaware of the available sources of accurate information and feeling embarrassed to seek information were the most frequently mentioned barriers in accessing SRH information. The findings of the study highlight the need to reduce the barriers to sexuality education for unmarried youth with different types of disabilities. Efforts should be made to promote school and family-based sexuality education tailored for youth with different types of disabilities and empower them to seek information proactively. The Programme of Action (PoA) of the International Conference on Population and Development (ICPD), held in Cairo in 1994, explicitly called for governments at all levels to consider the needs and rights of all people in achieving sustainable development and affirmed that sexual and reproductive health (SRH) and rights are fundamental to people’s survival, health and wellbeing. The PoA also gave high priority to adolescents' SRH and rights and drew attention to the elimination of specific forms of discrimination that persons with disabilities may face regarding reproductive health and rights . The ConPersons with disabilities have the same SRH needs as other people and equal rights to attain the highest standard of SRH. Despite the above-mentioned commitment, persons with disabilities face more barriers than their peers without disabilities in accessing SRH information and services, mainly due to discrimination and misunderstanding by the people around them rather than their disabilities –6. For eChina has a population of 85.64 million living with disabilities , 25. As The cross-sectional survey was conducted among unmarried young people aged 12–24 with visual, hearing and physical disabilities. Considering this was the first study in China in terms of sexuality and disability, focus group discussions and in-depth interviews with young people with disabilities and in-depth interviews with their teachers and parents were conducted to inform the design of the quantitative survey. The results of qualitative interviews were published elsewhere –30. All Respondents of the survey were recruited from both urban areas  and rural areas  in China. These sites were selected in consideration of the level of support from local Disabled Persons’ Organizations (DPOs), communities and schools. In China, young people with disabilities are either enrolled in regular schools, special schools and rehabilitation training institutions, working in welfare-oriented or disability-friendly enterprises, or staying at home, depending on their level of disability, the local availability of special schools, and welfare-oriented or disability-friendly enterprises. Given the challenges of conducting studies on sexuality among unmarried young people with disabilities, convenient sampling was used to recruit participants. Participants with visual and hearing disabilities were recruited from five special education schools including three from urban and two from rural areas, while respondents with physical disabilities were recruited from two special education schools, two communities and one sports training center for the disabled in urban areas, and welfare-oriented or disability-friendly enterprises and regular schools and communities in rural areas. All eligible respondents in the selected schools, enterprises and communities were recruited for the study. A total of 699 respondents participated in the survey, with 126 of them aged 12–14 years and 473 aged 15–24 years. This paper is based on the analysis of the data concerning the sub-sample of 473 respondents aged 15–24 years, including 158 youth with visual impairment, 207 with hearing impairment and 108 with physical disability.The study used an anonymous structured questionnaire to collect data. The questionnaire was initially designed by two age groups (12–14 and 15–24), and was revised and finalized following a face validity study and a pilot test involving male and female young people with different types of disability of both age groups. The survey was administered in an accessible way to the respondents and the data were collected from May to June 2015. For respondents with visual impairment, those with low vision filled a self-administrated paper questionnaire in big fonts and those who were blind filled a self-administrated electronic questionnaire via computer software or an interviewer-administered questionnaire by listening to tape recorders. The respondents with hearing impairment answered a self-administered paper questionnaire with the help of interpreters and interviewers. Most of the respondents with physical disability answered a self-administrated paper questionnaire while those with difficulties in completing the questionnaire on their own answered an interviewer-administered questionnaire. Trained investigators were present to assist participants who had difficulties with filling out the questionnaire and to check and ensure the completion of the questionnaires. This study was reviewed and approved by the institutional review board of Shanghai Institute of Planned Parenthood Research (PJ2014–04). Informed consent or assent was obtained from the participants, parents as well as school teachers before data collection.Socio-demographic characteristics Respondents were asked about their gender, date of birth, residential area, student status, education level, disability onset date, single-child status, cohabitants, parents’ education level and disability status.SRH knowledge Respondents were asked three categories of SRH knowledge—sexual physiology , sexually transmitted infections (STIs)/HIV/AIDS (transmission modes and prevention methods) and contraception (contraceptive methods and where to get contraceptives). The three categories of knowledge were assessed by 9, 16 and 14 single-choice questions respectively. For each question, 1 point was awarded to the correct answer and 0 point to the incorrect or “do not know” answer. The sum of the points for each category or all three categories was then converted into a scaled score ranging from 0 to 100 (100 is the highest possible score), with higher scores indicating higher levels of knowledge.Sources of, barriers and preference in accessing SRH Knowledge Respondents were asked about their main sources of SRH knowledge, barriers in accessing sexuality-related information, and their preferred sources, contents and ways when acquiring sexuality-related information.2-test, Wilcoxon rank sum test and Kruskal–Wallis H rank sum test as appropriate. Multivariate ordinal logistic regression model was used to examine the association between socio-demographic variables and SRH knowledge level using quartiles or tertiles as the cutoff points to divide the data set into four or three equal groups depending on the test of the proportional odds assumption. For each variable included in the regression models, odds ratio (OR) and its 95% confidence interval (95% CI) were calculated. All analyses employed an alpha level for statistical significance of 0.05 (two-tailed).The SAS 9.3  was used for statistical analysis. The data was analyzed by type of disability. Subgroup differences were analyzed using χAround half of the respondents with hearing and physical disabilities were male, but more than 70% of the respondents with visual impairment were male. More than half of the respondents were from urban areas, with the percentage for respondents with hearing impairment 61.4%) significantly higher than that for the visually impaired (41.8%). The mean age of respondents was 18.89 (± 2.41) years old with more than half of them aged 15–19 years. All respondents with visual and hearing impairment were students and most of them had an education level of senior high school or above, while the corresponding percentage among the respondents with physical disability was only around fifty percent. Less than half (46.5%) of all respondents were the only child in the family with the percentage of the visually impaired (39.2%) and physically disabled (38.9%) much lower than that of the hearing impaired (56.0%). More than two-thirds of the respondents were living with parents and more than 60% of their parents had an education level of junior high school or above. Nearly half of the respondents were born with a disability, with the percentage of the visually impaired (61.4%) significantly higher than that of the hearing impaired (41.1%) and physically disabled (44.4%). More than 70% of the respondents reported their parents were without disabilities  and parents (35.2% vs. 26.4%) and less access barrier as demonstrated by a lower percentage of them responding “unaware of the available sources of accurate information” (24.5% vs. 40.9%). The findings from in-depth interviews with the teachers of special education schools and regular schools in urban and rural areas revealed that schools in rural areas were more likely to face challenges than those in urban areas in delivering sexuality education, due to lack of teaching materials and tools, lack of professionally trained teachers, lack of awareness of the importance of sexuality education and lack of support from parents . In ruraNo gender difference in SRH knowledge was observed by this study, which was also in line with the study conducted in Ethiopia. Age wasThe relationship between the level of education and SRH knowledge has been documented by many studies , 39. In As shown in this study, although differences in the needs and preferences in accessing SRH information were observed across disability types, residential areas and gender, respondents with disabilities had similar SRH information needs and preferred sources. Puberty changes and health care, friendship/love/marriage, sexual harassment/abuse/self-protection and STIs/HIV prevention were the most frequently mentioned topics. School/teachers and the Internet were the most frequently mentioned preferred sources. In the traditional Chinese society, it remains a taboo to talk about sex in public or between different generations . Even ifSexuality education is essential to the health and wellbeing for youth with disabilities. Understanding their needs and access barriers to sexuality education is the first step to address their unmet needs. To the best of our knowledge, this was the first study about the understanding of SRH knowledge, as well as barriers and preferences in accessing sexuality-related information among unmarried youth with different types of disabilities in both urban and rural areas in China. In addition, this study sheds lights on how to address the barriers to sexuality education for youth with different types of disabilities living in the Chinese culture. First, there is an urgent need to raise awareness among relevant government officials, school teachers and educators as well as parents about the importance of providing sexuality education for youth with disabilities, particularly in rural areas. Raising awareness about the SRH needs and rights of youth with disabilities is necessary to overcome stigma, discrimination and misunderstanding. Second, school and family-based sexuality education should be promoted and tailored to the needs of youth with different types of disabilities, with more priority given to school-based sexuality education. Third, policies, systematic supervision and evaluation, disability-friendly teaching and learning materials and relevant curriculum standards should be improved, developed or implemented to support school-based sexuality education. Efforts should be made to support capacity building among teachers and parents. Finally, youth with disabilities must be empowered to seek information proactively.The study has some limitations. First, given the difficulties in reaching enough respondents with disability, the convenient sampling method was used to recruit participants. Findings in this study may not necessarily represent the overall population of youth with disabilities in China. For example, all of the participants in this study were literate, while only 72.7% of disabled children aged 6–14 received compulsory education nationwide , suggestUnmarried youth with visual, hearing and physical impairment and disabilities, especially those from rural areas, lacked knowledge about sexual physiology, contraception and STIs/HIV/AIDS, and had limited access to SRH information through schools and families. Limited sources of information and the embarrassment in seeking information under the influence of the Confucian culture deeply-rooted in the Chinese society were the two most common barriers they faced in accessing SRH information. Despite limited access to SRH information through school, the school teachers were still the most preferred source of SRH knowledge. This study highlights the barriers faced by youth with disabilities in accessing SRH knowledge and information in the Chinese culture and sheds some light on the potential strategies to reduce the barriers. To address their unmet needs for SRH information, there is an urgent need to raise awareness among relevant government officials, school teachers, educators and parents about the importance of providing sexuality education for youth with disabilities. Furthermore, efforts should be made to support the delivery of sexuality education by schools and families and empower young people with disabilities to seek information proactively.