RNA excited states represent a class of high-energy-level and thus low-populated conformational states of RNAs that are sequestered within the free energy landscape until being activated by cellular cues. In recent years, there has been growing interest in structural and functional studies of these transient states, but the rational design of excited states remains unexplored. Here we developed a method to design small hairpin RNAs with predefined excited states that exchange with ground states through base pair reshuffling, and verified these transient states by combining NMR relaxation dispersion technique and imino chemical shift prediction. Using van’t Hoff analysis and accelerated molecular dynamics simulations, a mechanism of multi-step sequential transition has been revealed. The efforts made in this study will expand the scope of RNA rational design, and also contribute towards improved predictions of RNA secondary structure. RNA molecules exhibit conformational fluctuations between ground states and excited states. Here the authors designed and verified small hairpin RNAs with predefined secondary structure reshufflings. In light of Van’t Hoff analysis and accelerated molecular dynamics simulation, a mechanism of multistep sequential transition has been revealed. Apart from the dominant interests in understanding their structures and functions, the design of synthetic RNAs has received great attention for applications in regulating biological processes such as gene expression5, as well as in developing novel biological devices6, such as RNA switches7, thermometers8, fluorescent probes10, and RNA-based computers11.RNAs represent a class of molecules that are engaged in diverse biological functions, including transcription, alternative splicing, translation, and degradation13. Similar to proteins, RNA molecules under physiological conditions exhibit extensive conformational fluctuations over a broad spectrum of time scales, often linking to their versatile roles in vivo. Such dynamics can be well understood from the perspective of free-energy landscape14. A large number of conformational states pre-exist within different local minima dispersed on the free-energy surface. The interconverting rate between two states is dictated by the height of the energy barrier between them. The conformational states with low free energy are highly populated, referred to as ‘ground states’ (GSs), while others with high free energy, termed ‘excited states’ (ESs), are sparsely populated. These ESs, which are otherwise sequestered in the energy landscape, become functional species in response to cellular cues15. Although predicting alternative conformational states and associated energy barriers remains a major challenge for computational biologists, recent studies have demonstrated the initial success in the rational design of interconverting multi-state conformations of proteins17. However, studies of RNA design have thus far focused on the single state, leaving the rational design of RNA alternative conformations, including ESs, conspicuously unexplored.On the other hand, the ever-growing explorations into the structure and function of RNA have underscored the importance of RNA dynamicsPredefined RNA excited states will beneficially add to the arsenal available for the design of synthetic RNA devices by providing an additional layer of manipulation. The reshuffling between GS and ES can be easily converted into RNA switches as long as the ES is designed to be stabilized by environmental factors such as metabolites. In this regard, an RNA motif with predetermined ES serves as a fundamental building block for RNA devices. In addition, an autonomously reshuffling RNA can be expanded to a molecular machine with parallel processing capabilities, with workloads allocated to alternative states in different proportions by fine-tuning the population of ES.19. These ESs evade detection by most biophysical techniques due to their low population and short lifetime. However, the nuclear magnetic resonance (NMR) relaxation dispersion (RD) technique has proven to be particularly powerful in characterizing such transient states at atomic resolution23. To pursue our goal, we first developed a design protocol to generate candidate RNA constructs using MC-Fold24, a program that has been successfully employed to predict potential RNA ESs in several prior studies28. Next, we employed both the 15N R1ρ experiment30 and the 1HN CEST experiment32 to probe ESs of the designed RNAs, and verified their secondary structures with the aid of a newly developed imino chemical shift predictor33. Finally, by performing RD measurements at varying temperatures, along with accelerated molecular dynamics simulations, we have revealed the details of secondary structure switching through base-pair shift by one or two nucleotides in register.Here we designed an array of hairpin RNAs  base pairs and G ∙ U wobbles can readily form upon structural switching  to filter the resulting secondary structure pool associated with each candidate hairpin, where ∆∆G is the free-energy difference between GS and any alternative structure. For a specific candidate hairpin, if the predefined ES falls into the range of ∆∆G ≤ 3 kcal mol−1, and there exists no alternative ES whose ∆∆G is lower than that of the desired ES, then this RNA construct will be accepted as a qualified candidate. The construct with the lowest ∆∆G in the candidate list, termed ‘T1-short RNA’ (meaning a short version of Type 1 RNA), was chosen for further study.The design procedure is as follows  indicate that both G14 and U21 in ES undergo expected downfield shift relative to GS  and exchange rate kex  for residues of G11, G14, U21, and U23, suggesting a concerted exchange process. The global fitting of RD data yielded an ES with a population of 6.2 ± 0.1% and a lifetime of 2.4 ± 0.1 ms  account for about 70%42. In addition to the GCAA apical loop included in T1 RNA, GAAA and UUCG45 are also among the most frequently occurring tetraloops in RNAs. These tetraloop motifs differ in thermodynamic properties. To investigate the impact of loop stability on the ES, we replaced the GCAA tetraloop of T1 RNA with GAAA and UUCG, respectively, resulting in two constructs: T1-GAAA , the forward transition of T1-GAAA is slightly accelerated (k1 = 30.7 ± 0.8 s−1), while that of T1-UUCG is slowed down (k1 = 13.4 ± 0.4 s−1). Given that the forward rate constant is dictated by the activation free energy between GS and ES (∆G‡GS→ES), which is in turn related to the free energy required to break the tetraloop, the results suggest that the order of stability for these three tetraloops is UUCG > GCAA > GAAA, which is in accordance with prior studies47.Compared to T1 , T1-add1bp shows a somewhat lower exchange rate 49, and hence unlikely arises from the inserted base pair. To confirm this, we designed T1-add2bp by further inserting a G ∙ U base pair in the upper stem of T1-add1bp, and the RD measurement informed a slightly decreased exchange rate kex = 351 ± 11 s−1 , despite fewer base pairs required to break upon the transition. Taken together, these results indicate that elongating or shortening the upper stem by one or two base pairs has little effect on the exchange rate.Compared to T1 . Of note, owing to the combination of faster exchange and lower population of ES, these RD signals are not as pronounced as those detected in other designed RNAs.Next, we designed a Type 3 RNA based on a previously identified ES of the transactivation response (TAR) element RNA of HIV-I. In this ES, bulge residues pair up with residues in the upper stem, causing a two-nucleotide register shift that further propagates to the apical loopES can be accurately predicted, showing an excellent agreement with the experimental values  are in accordance with transitions between U-A and U ∙ G base pairs  and relatively diverse activation enthalpies  match results reported for opening a single WC base pair during the transition to a Hoogsteen base pair in DNA 35. Earlier studies have also found that the opening activation energies of RNA WC base pairs fall in the range of 13–16 kcal mol−1 50. Therefore, unlike the bistable RNAs where the conformational switch requires disrupting multiple base pairs simultaneously48, the RNAs designed in this work most likely break only one base pair at each step during the secondary structure transition, which explains why there is no substantial change in kex upon insertion or deletion of one or two base pairs in the upper stem of T1. It is also noteworthy that the measured activation free energies of T1, T1-GAAA, and T1-UUCG are consistent with their tetraloop stability, as mentioned before.These uniform activation free energies .To explore the details of the RNA reshuffling processes, we embarked on accelerated molecular dynamics (aMD) simulations. Compared to conventional molecular dynamics (cMD) simulations whose timescale is often limited to microseconds nowadays, aMD is capable of sampling motions that occur on a timescale of up to milliseconds or slowerThe first RNA construct for which we successfully observed transitions between ES and GS was T4. For this RNA, we recorded 94 trajectories with lengths ranging from 0.1 μs to 6 μs. In 30 trajectories, we observed 30 ES-to-GS transitions and 2 GS-to-ES transitions. One typical ES-to-GS transition is presented here as the schematic sequential pathway Fig.  and c. Tk−1 rates of these three RNAs.We also observed similar transitions in T1 and T2. Among 161 trajectories of T1, three ES-to-GS transitions  by taking T1 RNA as an example, where the transition from GS to ES proceeds according to a six-state upward pathway Fig. . The frelue Fig. .Fig. 7Ki−1 . This strategy for reducing the energy barrier may have a broader scope of use for RNAs, rather than being limited to several reshuffling modes discussed here. It is likely through dividing a one-step transition into multiple kinetically favorable substeps, a secondary structure rearrangement is allowed to proceed at a reasonable speed that matches its potential functional role.If we assume five base pairs have to be disrupted at the same time, then the energy barrier would be accumulated to a formidable 55.98 kcal mol−1 Fig. , correspers Fig. , the sim −1 Fig. , only sl55. In contrast, little attention has been paid to the design of RNA excited states (ESs). Here, with the aid of a secondary structure prediction tool and NMR relaxation dispersion (RD) techniques, we successfully designed and verified a series of artificial RNAs with predefined secondary structure reshuffling directed towards a low-populated ES on the microsecond to millisecond timescale. Through the replacement of apical loop or alteration of stem sequence, we are able to regulate the population of ESs and, to some extent, the transition rates. Our results demonstrate that such design strategy is robust, and is likely applicable to the design of more complex RNA ESs. The ability to engineer an RNA with predefined ESs will expand the range of functionalities that can be fulfilled by synthetic RNA devices, for example, through coupling these autonomously reshuffling RNAs with existing execution or reporter components of artificial RNA devices58. In addition to acting as integral components of aptamer, actuator, and transmitter of an RNA switch or other devices59, a reshuffling RNA with designed switching mode has the potential to perform multiple tasks simultaneously, thereby gaining applications in the context such as alternative splicing60 or producing miRNA isoforms61. Additionally, the impact of environmental factors, such as monovalent or divalent metal ions, on the thermodynamic and kinetic properties of ESs also deserves further investigation.The rational design of RNA ground states with diverse functions is thus far a blooming research fieldpB and kex. In contrast, elongating or shortening the stem by up to two base pairs has only a limited effect on these parameters. Next, T1, T1-GAAA, and T1-UUCG differ only in the tetraloop. Such a variation affected the exchange parameters to a small extent, especially for T1 and T1-GAAA whose apical loops differ merely by a single nucleotide. This observation is not surprising, since all the three tetraloops belong to stable GNRA or UNCG motif. Another useful comparison is T2-mirror versus T1, in which swapping two chains of the upper stem led to a significant change in pB. We noticed that T2-mirrorES and T1ES have different pentaloop sequences, and very likely it is the pentaloop that accounts for the difference in ∆∆G. Therefore, tweaking the loop region could be a more effective way to tune exchange parameters than modifying the helical stem.An interesting question is how different changes made to the RNAs affect the measured exchange parameters . DNA templates were annealed in 3 mM MgCl2 by heating to 95 °C for 5–10 min and cooling on ice for 30 min. The transcription reaction was carried out at 37 °C for 24 h with in-house purified T7 RNA polymerase using unlabeled (Aladdin) or 13C/15N-labeled nucleotide triphosphates (Cambridge Isotope Laboratories). The samples were purified using 15% (w/v) denaturing polyacrylamide gel electrophoresis (PAGE) in 8 M urea and 1 × TBE buffer, and then eluted by a ‘crush and soak’ procedure in the corresponding buffer . RNAs were subsequently exchanged into phosphate NMR buffer  and concentrated to a final volume of about 250 μL using the centrifugal concentrator (3 KDa cutoff). These samples were then refolded by heating at 95 °C for 5–10 min and rapidly cooled down on ice. Prior to NMR experiments, 0.5 μL of 20 mM 4,4-dimethyl-4-silapentane-1-sulfonic acid (DSS) was added to each sample as a chemical shift reference compound, and D2O was added as well to a final concentration of 8% for the purpose of signal locking.Unlabeled and uniformly Bruker TopSpin 3.2) or 800 MHz (Bruker TopSpin 3.5) spectrometers equipped with a 5 mm triple-resonance TCI cryogenic probe.All NMR experiments were performed on Bruker Avance 600 MHz (1H–15N SOFAST HMQC and 2D 1H–1H nuclear Overhauser effect spectroscopy (NOESY) NMR experiments (acquired using a mixing time of 180 ms) at 10 °C. All data were processed and analyzed using software NMRPipe62 and Sparky .Unless otherwise stated, assignment of imino resonances for all RNA constructs was achieved using a standard set of 2D R1ρ pulse sequence23. Raw data were processed using NMRPipe and autofit script to generate a series of peak intensities. On- and off-resonance R1ρ RD profiles with different offset frequencies were recorded under spin-lock powers (ωSL/2π) ranging from 100 to 500 Hz . The baseline of each CEST profile from B1 at far-off resonance frequency (−12 kHz). A difference 1HN CEST profile was then produced and fitted to extract chemical shifts of the excited state. During the fitting, 15N RD data.All NMR spectra were processed and analyzed using 64,i = 1, −1) represents the forward and backward rate constants of the two-state reaction. T is the temperature in Kelvin. The free-energy change (Further information on research design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Movie 1Supplementary Movie 2Supplementary Movie 3Supplementary Movie 4Supplementary Movie 5Reporting Summary


BEAN1) and thymidine kinase 2 (TK2). Among these repeat sequences, (TGGAA)n repeat was the only sequence segregating with SCA31, which strongly suggests its pathogenicity. In SCA31 patient brains, the mutant BEAN1 transcript containing expanded UGGAA repeats (UGGAAexp) was found to form abnormal RNA structures called RNA foci in cerebellar Purkinje cell nuclei. In addition, the deposition of pentapeptide repeat (PPR) proteins, poly(Trp-Asn-Gly-Met-Glu), translated from UGGAAexp RNA, was detected in the cytoplasm of Purkinje cells. To uncover the pathogenesis of UGGAAexp in SCA31, we generated Drosophila models of SCA31 expressing UGGAAexp RNA. The toxicity of UGGAAexp depended on its length and expression level, which was accompanied by the accumulation of RNA foci and translation of repeat-associated PPR proteins in Drosophila, consistent with the observation in SCA31 patient brains. We also revealed that TDP-43, FUS, and hnRNPA2B1, motor neuron disease–linked RNA-binding proteins bound to UGGAAexp RNA, act as RNA chaperones to regulate the formation of RNA foci and repeat-associated translation. Further research on the role of RNA-binding proteins as RNA chaperones may also provide a novel therapeutic strategy for other microsatellite repeat expansion diseases besides SCA31.Spinocerebellar ataxia type 31 (SCA31) is a progressive neurodegenerative disease characterized by degeneration of Purkinje cells in the cerebellum. Its genetic cause is a 2.5- to 3.8-kb-long complex pentanucleotide repeat insertion containing (TGGAA)n, (TAGAA)n, (TAAAA)n, and (TAAAATAGAA)n located in an intron shared by two different genes: brain expressed associated with NEDD4-1 ( More than 40 refractory neurological diseases are caused by microsatellite repeat expansions, including spinal and bulbar muscular atrophy; Huntington disease (HD); Friedreich ataxia; fragile X syndrome; fragile X–associated tremor/ataxia syndrome (FXTAS); myotonic dystrophy types 1 and 2 ; C9orf72-associated amyotrophic lateral sclerosis/frontotemporal dementia (C9orf72-ALS/FTD); dentatorubral-pallidoluysian atrophy; spinocerebellar ataxia (SCA) types 1, 2, 3, 6, 7, 8, 10, 12, 17, 31, 36, and 37 ; and benSCA31 is one of the autosomal-dominant neurodegenerative disorders showing a relatively pure cerebellar form of ataxia. It is typically a disease of late adulthood, with onset peaking between 60 and 65 years of age . SCA31 iBEAN1 and TK2 genes on chromosome 16q22.1 (SCA31 is caused by a 2.5- to 3.8-kb insertion of complex pentanucleotide repeats containing (TGGAA)n, which is located in overlapping introns of the  16q22.1 . The ins 16q22.1 . Importa 16q22.1 . Among tIn patients with SCA31, marked degeneration of the cerebellar cortex with Purkinje cell–predominant neuronal loss was observed in the cerebellum . PurkinjA neuropathological hallmark of microsatellite repeat expansion diseases is the intracellular accumulation of mutant RNA containing an expanded nucleotide repeat as RNA foci in the nucleus and/or cytoplasm of affected cells. This has emerged as an important disease mechanism for the growing class of such diseases . Previouexp is expressed by BEAN1 transcription. Taking these findings together, TGGAA pentanucleotide repeat expansion gives rise to toxic UGGAA repeat RNAs, which form RNA foci and are translated into poly(WNGME) proteins in Purkinje cells in SCA31.Several lines of evidence indicate that RAN translated proteins are involved in a growing number of microsatellite repeat expansion diseases, including HD, DM1, SCA8, FXTAS, and C9orf72-ALS/FTD . Unlike exp transcripts is RNA-mediated toxicity, as these foci might sequester RNA-binding proteins and consequently alter RNA metabolism, as in other repeat expansion disorders  RNA in compound eyes using the GMR-GAL4 driver caused severe eye degeneration, depending on its expression level, whereas control repeat and short UGGAA22 RNA showed no detectable degeneration.We have established diseases . Eighty RNA foci . The conexp in the nervous system with elav-Gal4 driver exhibited a shorter lifespan and progressive locomotor defects, whereas control repeat and UGGAA22 did not. As expected, RNA fluorescence in situ hybridization (FISH) revealed remarkable accumulation of UGGAAexp RNA as RNA foci in eye imaginal disks of UGGAAexp-expressing flies, consistent with the pathology of SCA31 patients. Fly lines expressing control repeat or UGGAA22 had no detectable RNA foci in the fly tissue. In this model, PPR protein was also detected by Western blot and immunohistochemistry, and its expression level was positively correlated to the severity of eye degeneration in Drosophila. Therefore, both UGGAAexp RNA toxicity and PPR protein toxicity may contribute to the neurodegeneration in SCA31.The expression of UGGAARNA-binding proteins are essential at all levels of RNA processing, including transcription, alternative splicing, stabilization, degradation, and RNA granule formation, in both the nucleus and the cytoplasm. The loss of RBPs influences the processing of these noncoding RNAs and contributes to global RNA dysregulation. Considering that proteins sequestered in RNA foci have been reported to play an important role in gain of toxic function of microsatellite repeat expansion diseases , the intexp in Drosophila, the UGGAAexp-mediated eye degeneration and accumulation of RNA foci were dramatically suppressed. Conversely, the knockdown of TDP-43 enhanced eye degeneration and the formation of RNA foci. Subsequent analysis of atomic force microscopy and circular dichroism spectroscopy demonstrated that the binding of TDP-43 to UGGAA repeat RNA alters its structure. Thus, TDP-43 functions as an RNA chaperone for UGGAA repeat RNA, consistent with the definition of an RNA chaperone , FUS, and hnRNPA2B1. Indeed, FISH analysis showed the colocalization of TDP-43 with RNA foci in the nucleus of human SCA31 Purkinje cells. Surprisingly, when TDP-43 was coexpressed with UGGAAhaperone . Notablyof FXTAS . Interesof FXTAS . Overexpnslation . On the 9ALS/FTD . AlthougInterestingly, these suppressive effects of TDP-43, FUS, and hnRNPA2B1 differ from those of other RBPs that interact with expanded repeat RNAs such as MBNL1 in DM1 and hnRNPA3 in C9orf72-ALS/FTD . For exain vitro and in vivo models of C9ALS/FTD or FXTAS  DNA in pericentromeric heterochromatin also has a modular structure composed of the TGGAA pentanucleotide repeats in common with SCA31  Are PPR proteins necessary or sufficient to induce neurotoxicity? (iii) Do RBPs that bind to other disease-causing repetitive sequences such as C9orf72-ALS/FTD, FXTAS, SCA36, or DM1 indeed play a protective role as RNA chaperones? (iv) Can TK2 transcripts including extended UUCCA repeats cause neurodegeneration with RNA foci formation and RAN translation?  How can UGGAAslation? . It is iTI, YN, and KI designed the manuscript, assessed the literature, and edited the subsequent drafts and revisions. TI wrote the initial draft of the manuscript. All the authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Thermotoga petrophila K4 and Thermococcus kodakarensis KOD1, or a wild-type DNA polymerase from Thermus thermophilus M1. The highest performance was achieved by combining MM4 with the thermostable DNA polymerase from T. thermophilus M1. These enzymes efficiently amplified specific RNA using uracil-DNA glycosylase (UNG) to remove contamination and human RNase P RNA amplification as an internal control. The standard curve was obtained from 5 to 105 copies of synthetic RNA. The one-step real-time RT-PCR method’s sensitivity and specificity were 99.44% and 100%, respectively (n = 213), compared to those of a commercially available diagnostic kit. Therefore, our method will be useful for the accurate detection and quantification of SARS-CoV-2.The general methods to detect the RNA of severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) in clinical diagnostic testing involve reverse transcriptases and thermostable DNA polymerases. In this study, we compared the detection of SARS-CoV-2 by a one-step real-time RT-PCR method using a heat-resistant reverse transcriptase variant MM4 from Moloney murine leukemia virus, two thermostable DNA polymerase variants with reverse transcriptase activity from  The COVID-19 pandemic, caused by the RNA virus SARS-CoV-2, has wreaked havoc on the global economy and many national healthcare systems. Countries with a large number of daily new cases need a reliable and inexpensive diagnostic system. In other countries, there is also an urgent need to stockpile kits for emergency use.Currently, nucleic acid detection and antigen detection are the two primary methods to confirm infection. Real-time quantitative PCR (RT-qPCR) is a highly sensitive and widely used method for detecting SARS-CoV-2 RNA. Fully automated platforms for RT-qPCR have been introduced in large medical institutions and clinical laboratories. However, there is still a global shortage of reagents and consumables. In addition, RT-qPCR involves complicated molecular biological techniques. Moreover, RT-qPCR produces false-positive  or falseThe conventional RT-PCR consists of two steps, the synthesis of cDNA using reverse transcriptase and the amplification of DNA using DNA polymerase. In contrast, one-step real-time RT-PCR is a continuous reaction that performs the two procedures in the same tube. Therefore, it is essential to adjust the reaction buffer conditions so that two or more enzymes remain functional in the same buffer.Thermus thermophilus M1 strain (M1polTth) with a 5ʹ–3ʹ but not a 3ʹ–5ʹ exonuclease domain [L329A) originated from the Thermotoga petrophila K4 strain. K4polL329A has acquired reverse transcriptase activity via mutagenesis; it has a 3ʹ–5ʹ but not a 5ʹ–3ʹ exonuclease domain [Thermococcus kodakarensis. Lastly, we used family I uracil-DNA glycosylase (UNG), cleaving the N-glycosylic bond between uracil and sugar to remove the uracil incorporated in DNA, to remove contaminations from the PCR products [In this study, we applied the widely used primers and probes, reported by the United States Centers for Disease Control and Prevention (CDC), in RT-PCR to detect the SARS-CoV-2 RNA. Previously, we reported a thermostable reverse transcriptase, MM4, harboring 4 amino acid substitutions in the original Molony murine leukemia virus reverse transcriptase . In addie domain  in a Famproducts .https://www.mhlw.go.jp/stf/newpage_11332.html). We aimed to detect 10–105 copies of RNA per test within 45 PCR cycles.Currently, the clinical detection range of the RNA copy number for SARS-CoV-2 varies. The one-step real-time RT-PCR that we developed was evaluated for its detection range compared to that of a commercial SARS-CoV-2 detection kit authorized by the Ministry of Health, Labour and Welfare, Japan, as in vitro diagnostics  approved this study for emergent clinical research. Samples were obtained according to the active epidemiological investigation requested from the Ministry of Health, Labour and Welfare, Japan, under the Japanese law of Act on the Prevention of Infectious Diseases and Medical Care for Patients with Infectious Diseases (the Infectious Diseases Control Law). And the residual RNA was utilized for this study after opting out.RNA was extracted from a total of 213 samples, including at least 35 sputum samples, 124 nasopharyngeal swabs, and 7 saliva samples, using the QIAamp Viral RNA Mini Kit . Uracil-DNA glycosylase (UNG), RNase inhibitor, and deoxyribonucleotides  were purchased from Toyobo Co., Ltd., Osaka, Japan. One-step real-time RT-PCR was performed using the QuantStudio 5 Real-time PCR system (Thermo Fisher Scientific). RNA copy numbers were estimated from the simultaneously tested standard curve.https://www.cdc.gov/coronavirus/2019-ncov/lab/rt-pcr-panel-primer-probes.html), including the 2019-nCoV_N1 Forward Primer (5ʹ-GACCCCAAAATCAGCGAAAT-3ʹ), 2019-nCoV_N1 Reverse Primer (5ʹ-TCTGGTTACTGCCAGTTGAATCTG-3ʹ), 2019-nCoV_N1 probe (5ʹ-FAM- ACCCCGCATTACGTTTGGTGGACC-TAMRA-3ʹ), 2019-nCoV_N2 Forward Primer (5ʹ-TTACAAACATTGGCCGCAAA-3ʹ), 2019-nCoV_N2 Reverse Primer (5ʹ-GCGCGACATTCCGAAGAA-3ʹ), 2019-nCoV_N2 probe (5ʹ-FAM-ACAATTTGCCCCCAGCGCTTCAG-TAMRA-3ʹ), RNase P forward primer (5ʹ-AGATTTGGACCTGCGAGCG-3ʹ), and RNase P Reverse primer (5ʹ- GAGCGGCTGTCTCCACAAGT-3ʹ). RNase P probe was adjusted from the CDC probe for minor groove binder (MGB) coupled with non fluorescence quencher: MoCO RNase P probe 5ʹ-VIC-TTCTGACCTGAAGGCT-MGB-3ʹ (Thermo Fisher Scientific). The synthetic standard RNA for the COVID-19 CDC N1 and N2 primer sets (US-CDC-N1N2-PC)  was purchased. The synthetic standard RNA at the concentration of 107 copies /μL was then serially diluted to 105, 104, 103, 102, 10, and 5 copies/μL in RNA grade TE buffer . The results from our one-step real-time RT-PCR data were compared with those obtained from a commercial one-step RT-PCR kit .Primer and probes for 2019-Novel Coronavirus (2019-nCoV) were synthesized according to the report from CDC , negative predictive value (NPV), and bivariate relationship were analyzed using the JMP 10 software (SAS) or Igor Pro (WaveMetrics).2, Mn(OCOCH3)2, Bicine-KOH (pH 8.2), Tris-HCl (pH 8.3), KCl, and CH3COOK supplemented with purified total human RNA were prepared  in detecting 104 or 103 copies of RNA per test.One-Step real-time RT-PCR was performed by mixing MM4 with the respective DNA polymerases, M1pol buffers . While ws of RNA  by diffeTth were compared. In buffers 1, 3, and 5, the one-step real-time RT-PCR system could detect more than 100 copies of RNA. On the other hand, the method could detect more than 10 copies of RNA in buffer 2 and more than 5 copies in buffer 4 . On the other hand, Ct values at 30 sec were significantly higher than at 5, 10, and 15 min with 104 RNA copies. Therefore, the optimal reverse transcription reaction time was determined to be 5 min  on a total of 213 RNA samples . ClinicaRNase P as an internal standard; the one-tenth of the amount of primers and probes for SARS-CoV-2 was sufficient for RNase P. Our highly sensitive RT-PCR method is expected to be applied to identifying other pathogens and analyzing mutations.Our developed MoCO kit could reproducibly detect at least 5 copies of synthetic RNA , demonstThe MoCO kit was developed to detect SARS-CoV-2 from the extracted RNA samples. Our development kit performed well for emergency research tests with a detection limit of 5 copies of RNA. It is necessary to continue improving the kit by simplifying sample preparation and the diagnosis of viral RNA mutations. Our high-sensitive RT-PCR method is expected to be applied for the molecular detection of various infectious diseases.S1 Table(PDF)Click here for additional data file.


RNA-binding motif protein 39 (RBM39), as a key factor in tumor-targeted mRNA and protein expression, not only plays a vital role in tumorigenesis, but also has broad development prospects in clinical treatment and drug research. Moreover, since RBM39 was identified as a target of sulfonamides, it has played a key role in the emerging field of molecule drug development. Hence, it is of great significance to study the interaction between RBM39 and tumors and the clinical application of drug-targeted therapy. In this paper, we describe the possible multi-level regulation of RBM39, including gene transcription, protein translation, and alternative splicing. Importantly, the molecular function of RBM39 as an important splicing factor in most common tumors is systematically outlined. Furthermore, we briefly introduce RBM39’s tumor-targeted drug research and its clinical application, hoping to give reference significance for the molecular mechanism of RBM39 in tumors, and provide reliable ideas for in-depth research for future therapeutic strategies. RBM39 is characterized by upregulated expression in many cancers, but the precise molecular function in specific cancers may not be thorough enough.RBM39 is indirectly involved in tumor growth and progression by regulating transcription of many tumor-related genes, protein translation, and alternative splicing.RBM39 can be exploited as markers for more accurate diagnosis, as well as improved prognosis and monitoring of treatment response in patients with cancer.RBM39 is identified as a target of sulfonamides, and it has played a key role in the emerging field of small molecule drug development.What is the exact molecular function of RBM39 in normal and cancer tissues?What is the preferential effect of RBM39 degradation in cancer cells?Would SPLAMs be used in more research tests and clinical patient treatments to improve the potential clinical efficacy of SPLAMs?Would SPLAM derivatives recruit new substrates other than RBM39 to join CUL4–DCAF15 to better exert the clinical value of the drug?Cancer has always been regarded as a highly complex disease, which seriously threatens human health and life. People have been committed to the treatment and clinical research of cancer for a long time, so it is necessary to deeply understand the molecular mechanism of cancer progression and research on targeted drugs. Here, RNA-binding motif protein 39 (RBM39), also known as splicing factor HCC1, CAPERα, FSAP59, RNPC2, and CAPER alpha, is an essential serine/arginine-rich (SR) RNA-binding protein, as well as a pre-mRNA splicing factor and transcription coactivator. RBM39 was initially identified as the autoantigen from a patient with liver cirrhosis who later developed hepatocellular carcinoma , and colIn recent years, although there have been relevant reports on the research of RBM39 in tumorigenesis and development, the precise function in specific cancers may not be thorough enough. In addition, due to the limited efficacy of sulfonamides, its research progress in clinical trials is relatively slow, so it is very important to try to reveal its latest research progress in tumors. In this review, we mainly summarized the possible regulation mechanism of RBM39 involved in tumor, and briefly discussed the molecular function of RBM39 in tumors. Furthermore, we outlined the emerging tumor-targeted drug research and its clinical application. Thus, it is highlighting the significance of RBM39 as targets for cancer therapy.RBM39 has significant sequence similarity with splicing factors U2AF65, and RBM23 Table , and conINK and Rb pathways. Therefore, RBM39/TBX3 and UCA1 constituted an important mechanism to regulate the transcription of CDKN2A-p16INK [It has been previously demonstrated that RBM39 is involved in transcriptional regulation of related proteins and various genes Fig. , Fig. 2.A-p16INK . In geneIt is demonstrated that RBM39 binds to mRNAs related to coding and translation proteins and participates in the expression of translation-related proteins , but theAlternative splicing is a highly regulated and coordinated molecular mechanism involving multiple physiological processes . RBM39 hAlternative splicing of RBM39 is an extremely important mechanism, and its regulatory mechanism is similar to that of U2AF65, which can directly bind to RNA or recruit specific splicing factors to regulate selective splicing. It was further revealed that as many as 20% of alternative splicing exons were regulated by both RBM39 and U2AF65 . Mai et A large number of data suggested that RBM39 regulated the transcriptional activity of ERs and PRs and was involved in the progression of human breast cancer , 6, 33. Acute myeloid leukemia (AML) is an aggressive hematological malignant tumor with a high recurrence rate after conventional combined chemotherapy . RegardiRBM39 plays an important role in the occurrence and development of lung cancer. With its tumor-specific immune function, RBM39 has a unique role in regulating the biological functions of tumor cells , 42. KumAs a new oncogene in myeloma, RBM39 is essential for the proliferation of multiple myeloma cells and tumorigenesis. Based on its biological role and clinical significance in tumor progression, RBM39 might become a key prognostic indicator for multiple myeloma patients . Tong etAccording to previous studies, RBM39 is considered to be a novel antigen identified from patients with hepatocellular carcinoma, showing high levels of expression in hepatocellular carcinoma , 14. It Recent studies revealed that SPLAMs, including indisulam, E7820, chloroquinoxaline sulfonamide (CQS), and tasisulam Fig. , are degIndisulam (E7070) is a novel aryl sulfonamide with anticancer activity in vivo and in vitro discovered by Eisai pharmaceutical company in phenotype screening , and itsTargeted protein degradation is an emerging field in the development of small-molecule drugs. Cancer cells are highly sensitive to the cytotoxic effects of proteasome inhibition, making proteasome an important clinical target for targeted therapy of hematopoietic and lymphoid malignancies. As a kind of RNA-binding protein, RBM39 ubiquitination and proteasome degradation are essential for anticancer activity. Owing to its key role in the occurrence and maintenance of hematological malignancies, the degradation of proteasome has gradually become an important way for people to treat cancer. Nothing is more important than the fact that the reduction of RBM39 levels depends on the activity of the proteasome , 54. OncWith the deepening of research, a growing number of scholars have generated a thought that RBM39, as a proto-oncogene, is not only closely related to the occurrence and development of a variety of malignant tumors, but also plays a crucial role in the clinical treatment of targeted drugs. As mentioned above, RBM39 indirectly participates in the growth and progression of tumors by regulating the transcription of many tumor-related genes, protein translation, and selective splicing, which provides new ideas and guiding significance for clarifying the pathogenesis of RBM39 in cancer. Since RBM39 was identified as the target of SPLAMs, the degradation of targeted proteins has gradually become an emerging field in the development of small-molecule drugs. It not only provides novel biomarkers for disease progression, but also affects the splicing of pre-mRNA subsets through the degradation of drugs. Therefore, CRISPR–Cas9-based DCAF15 gene knockout and single amino acid substitution of RBM39 can resist the degradation of RBM39 induced by sulfonamides, suggesting that RBM39 degradation is a key biochemical indicator of the anticancer properties of these compounds. At present, there are still many challenging problems to be solved. First, although the critical role of RBM39 in the occurrence and development of cancer has been supported by many reports, the current understanding of the mechanism of SPLAMs may be insufficient. Furthermore, although the safety of SPLAMs has been confirmed in many previous phase I and phase II clinical trials, it is still unclear whether RBM39 is an effective target of SPLAMs. Hence, it is necessary for future clinical studies to in-depth study the preferential effect of RBM39 degradation in cancer cells based on the spliceosome genotype, and to further determine the exact function of RBM39 in splicing. Although SPLAMs have only shown moderate efficacy in clinical trials so far, the biological basis of sensitivity to these drugs is still unclear, so they have only shown limited efficacy in some patients. Will SPLAMs be used in the treatment and research of more clinical patients in the future to improve the potential clinical efficacy of SPLAMs? Can SPLAM derivatives recruit new substrates other than RBM39 to join CUL4–DCAF15? Furthermore, so many questions are very necessary in the future research field to determine and further clarify the structural basis of CRL4–DCAF15 to identify sulfonamide degradation. The in-depth study of these sulfonamides may expand our understanding of the biological functions of RBM39, thereby guiding us to choose the correct target type that is more sensitive to these drugs for better application in cancer treatment. Taken together, further clinical trials will help determine the use of drugs targeting RBM39 activity in cancer therapy.Abstractcddiscovery-author-contribution-form


Uncovering the structure and function of biomolecules is a fundamental goal in structural biology. Membrane‐embedded transport proteins are ubiquitous in all kingdoms of life. Despite structural flexibility, their mechanisms are typically studied by ensemble biochemical methods or by static high‐resolution structures, which complicate a detailed understanding of their dynamics. Here, we review the recent progress of single molecule Förster Resonance Energy Transfer (smFRET) in determining mechanisms and timescales of substrate transport across membranes. These studies do not only demonstrate the versatility and suitability of state‐of‐the‐art smFRET tools for studying membrane transport proteins but they also highlight the importance of membrane mimicking environments in preserving the function of these proteins. The current achievements advance our understanding of transport mechanisms and have the potential to facilitate future progress in drug design. Recent single‐molecule FRET studies on members of various transporter families reveal the potential of this technique to advance the mechanistic understanding of this protein class. HeA particular challenge has always been the investigation of large proteins and oligomeric transporters. However, recent advances show that the strategic labeling of oligomeric transporters can even be an advantage.[Ph transporter. Here, each subunit was labeled with either a donor, an acceptor, or a surface‐binding tag.Ph transporters in liposomes. Only molecules of the desired orientation, i. e., with the surface‐binding tag on the outside of the liposomes were immobilized whereas molecules that were inserted in the opposite direction were washed off the surface.A particularly successful example is the homo‐trimeric Glt−/proton exchanger CLC‐ec1 from E. coli, where the dynamics of the individual subunits was explored.via point mutations in the gating motifs. After the self‐assembly of the monomers into dimers, only CLC‐ec1 molecules with at least one inactive and surface‐binding monomer were immobilized and only those dimers with a labeled second monomer were detectable in the smFRET experiments. Thus, all dimers contained one active and one inactive monomer, which allowed the authors to selectively monitor the dynamics of only one monomer in the assembly.Another example of smFRET experiments with oligomeric transporters is described in the recent study of the dimeric ClPh.via biotinylated lipids and the subunits of the transporter were labeled with donor and acceptor dyes in a 1 : 2 or 2 : 1 ratio. Afterwards, the different species were separated on the basis of the expected dye‐stoichiometries, using the Alternating‐Laser‐Excitation (ALEX) method.A distinct approach for smFRET experiments with an oligomeric protein was used in another study on Gltet al. The combination with ALEXA proof‐of‐concept study on the novel concept of “caging chromophores”2.2An important factor in the study of transporters is the membrane environment, which is often difficult to realize experimentally.[Ph, dynamics in micelles do not differ significantly from those found in a lipid bilayer.[via liposomes or nanodiscs/nanoparticles.For certain transporters such as GltHowever, creating liposomes of complex lipid composition3Ph[SmFRET has already been used to study transporters from various families. Representatives for each family and their respective substrates are illustrated in Figure 3.1ATP binding cassette (ABC) transporters are primary active transporters.In addition, importers possess PBPs that bind the substrate and deliver it to the transmembrane transporter.[E. coli and OpuA from Lactococcus lactis) and eukaryotic (PgP from Mus musculus and MRP1 from Bos Taurus) ABC transporters.[SmFRET studies were carried out on prokaryotic .[−1 or longer in nanodiscs, the protein was more dynamic in liposomes.A careful comparison showed that the data for McjD are overall consistent between detergent and a lipid environment with only minor differences. The opening of the TMDs in the presence of substrate is slightly wider and the closure of the NBDs with ATP is less tight in liposomes compared to detergent.In contrast to exporters, no spontaneous fluctuations of the NBDs and TMDs were observed for the importer BtuCD. Yet, the time‐resolution in these experiments was 50 ms and faster dynamics may have been missed.Finally, we would like to note that homology modeling had been successfully used to design labeling positions as shown for OpuA.3.22+‐ATPase ortholog LCMA1 from Listeria monocytogenes2+, are rate limiting. In addition, smFRET allowed to monitor so‐called de‐occluded conformations that couple ion transport and ATP hydrolysis. Unfortunately, these states have not yet been described by x‐ray crystallography or other imaging techniques to date.Another well‐studied family of primary active transporters are Phosphorylation‐type (P‐type) ATPase transporters. These membrane proteins are found in all kingdoms of life where they pump cations against their concentration gradients utilizing ATP hydrolysis, which preserves the electrochemical potential across cell membranes.3.3Bacillus cereuss.et al. crystallized the inward‐open conformer in a cross‐linked form and utilized smFRET to verify that this form is indeed similar to the native conformation.Membrane embedded Enzymes IIC (EIIC) are essential components of the phosphoenolpyruvate‐dependent phosphotransferase system that are responsible for sugar uptake. In complex with EIIC‐transporters, sugar is phosphorylated and dissociates.Further smFRET experiments on the native transporter without the crosslinker, identified two conformations in detergent solution out of which the outward‐open conformation was thermodynamically favored, which explains the crystallization bias towards this conformer.3.4The Major Facilitator Superfamily (MFS) is a superfamily of secondary active transporters that is ubiquitous in all kingdoms of life.via an alternating access mechanism.[All MFS transporters share a common fold. It is characterized by 12 transmembrane helices (TMs) that are grouped into two bundles of six consecutive helices each. The bundles are referred to as the N‐terminal and C‐terminal bundle and enclose the substrate binding site.E. coliThe first smFRET experiment on an MFS transporter was carried out on OxlT, an Oxalate:formate antiporter from Subsequent smFRET studies focused on elucidating the conformational states of MFS transporters during the transport cycle, the timescale of transition between those states, and the influence of substrate and pH.[E. coli and the multidrug efflux pump LmrP from Lactococcus lactis adopt inward‐open and outward‐open states in detergent solution.[E. coli on the contrary almost exclusively populate inward‐open states under these conditions.[In its apo state, the multidrug efflux protein MdfA from −1 compared to<10 s−1 for LmrP.[Interestingly, MFS transporters operate at different timescales. In the absence of substrate, MdfA switches conformations with a rate of 0.2 sMost importantly, the studies on DtpA and LmrP revealed that the periplasmic and cytoplasmic side of the transporters are decoupled in detergent,[Finally, it should be mentioned that the conformational state adopted by LmrP and MdfA is influenced by pH. Both proteins preferentially adopt the inward‐open state at low pH[In summary, smFRET studies revealed that MFS transporters are highly flexible proteins and our simplistic model of rigid body movements do not fully describe the transport cycle. Moreover, whereas general transport principles are shared among different transporters, dynamics, conformations, and the response to pH changes or substrate additions can differ substantially.3.5Besides the MFS superfamily, the amino‐acid‐polyamine‐organocation (APC) superfamily is another large superfamily of secondary active transporters.Since the study of BetP (BCCT family) focused mainly on developing smFRET methodology using “caged chromophores”Secondary active NSSAquifex aeolicus, identified conformational variety with opening‐closing dynamics of ∼60 s. The residence times in the outward‐open state were longer in the presence of substrates, thus stabilizing this state and slowing the transport cycle. In addition, Zhao et al. unraveled the molecular basis of the specific TM1a helix and its role in the intracellular gating mechanism using various mutations followed by smFRET measurements. Further smFRET studies+ ion in the second binding site of the transporter. The different substrates and ion modulation in solution from Na+ to Li+ ions, affected the kinetic rates in the transport cycle.Several smFRET studies on LeuT,[+‐binding to the extracellular side of the transporter did not only serve as driving force for transport, but it also provided the required selectivity for a substrate, closed allosterically the intracellular gate, and prevented the premature diffusion of Na+ to the intracellular side. Furthermore, the authors showed that open states on both sides of the membrane exist simultaneously, which deviates from the paradigm of the alternating access model.Another smFRET study of LeuTet al. outline the specific amino acids F259 and I359 in the binding pocket and their action as a “volumetric sensor” that dictates different transport rates according to the chemical identity of the substrates . The study revealed that the substrate size affects the rotamer dynamics of a phenylalanine residue (F259) and is associated with the occupancy of the intermediate conformational state (IO2), which in turn controls the intracellular release of Na+ that was already proven to be pivotal in the rate determining step.The most recent study of LeuTBacillus halodurans with a LeuT‐like structure−1. In the case of the substrate leucine, the second half of the transport cycle, i. e., the return from the apo form to the outward‐open conformation, was found to be the rate‐limiting step with a rate constant of about 1 s−1. But in contrast to the current transporter model for NSS transporter dynamics, the second half‐cycle depended on the transported substrate of the first half of the reaction cycle, suggesting the presence of an extracellular allosteric substrate binding site.The amino acid transporter MhsT is an NSS homolog from + ions increased the transport rate in MhsT while Na+ lowered it.Surprisingly, unlike in LeuT, H3.6Similarly to the NSS family, members of the Excitatory‐Amino‐Acid‐Transporters (EAAT) family are also secondary active sodium‐dependent transporters.Ph from Pyrococcus horikoshi is a suitable model for smFRET experiments because high resolution structures are available for several conformations.[The homolog Glt276,395‐GltPh). Ensemble experiments with this variant but also with the human transporter showed faster dynamics compared to the wild type protein.Ph, were absent in the H276,395−GltPh variant. In addition, smFRET histograms showed that the locked configurations, which are responsible for the quiescent periods, were disrupted in H276,395−GltPh. The inward‐open states of the variant showed an increased donor‐acceptor distance compared to wild type, suggesting an enlargement of the binding pocket in the inward‐open state, which was confirmed using x‐ray crystallography and molecular dynamics simulations.Initial smFRET studiesPh studied the synchronicity between the movements of the homo subunits of the transporter in a vesicle environment. Erkens et al. explored the dynamics that create the symmetric outward‐facing statePh monitored aspartate‐uptake in liposomes using an L‐aspartate‐binding FRET sensor. Surprisingly, uptake rates for single molecules were found to be preserved over multiple uptake cycles, thus exhibiting “molecular memory”, i. e., transporters with slow rates and transporters with fast rates preserve their speed over successive uptake cycles.Another smFRET investigation3.7E. coli. This transporter functions as an antiporter that removes drug molecules from the cell via a coupling to the import of protons.et al. were able to show that EmrE is organized as an antiparallel dimer in which both subunits transport the substrate at the same time but in opposite directions.Another family of secondary active transporters studied by smFRET is the small multidrug resistance (SMR) family. Members of this family are found in prokaryotes where they utilize an electrochemical proton gradient to expel toxic substances such as quaternary ammonium compounds from the cell.[3.8E. coli− ions and simultaneously exports H+ to the environmentThe chloride‐carrier‐channel (CLC) family consists of ion channels and transporters. SmFRET studies of a CLC transporter  family do not require an additional energy source for transport. SWEETs facilitate the transport of mono‐ and disaccharides along the concentration gradient.[A. thaliana provided new insights.A recent x‐ray study combined with smFRET measurements and functional characterizations of AtSWEET13 from 4With the recent advances of high‐resolution techniques,[SmFRET has been shown to complement such structural data with information on alternative conformers and their associated timescales of interconversion. This is a key advantage, not only compared to x‐ray crystallography or cryo‐EM, but also to other ensemble biophysical measurements.To understand transport mechanisms on a molecular basis, data interpretation of smFRET experiments can highly benefit from available high‐resolution structures, combined with MD simulations, mass spectrometry, SAXS and NMR studies. The power of combining smFRET studies with NMR spectroscopy has already been demonstrated on model systems such as EmrE and members of the glutamate transporter family.[In this mini‐review, we discussed recent smFRET results on membrane transporters and highlighted their contributions to advance the mechanistic understanding of this protein class. Technical developments in the field of smFRET spectroscopy, including available commercial setups and the fact that multiple protocols and tools for labeling proteins with fluorescent dyes are available, suggest that this method will evolve to a standard approach for investigating membrane proteins in the future. We expect that it will be possible to record full transport cycles of diverse transporters at a single‐molecule level and to deduce their kinetic characteristics. Such setups not only open the door to understand the impact of different lipids on transport rates and conformational dynamics but they will also be essential in elucidating the function of transporter binding proteins, which have recently been discovered.[The authors declare no conflict of interest.Kim Bartels studied chemistry and biochemistry at the Ludwig Maximilian University Munich (Germany). During her studies she worked on the structure determination of bacterial transmembrane proteins under the supervision of Dr. K. Beis . After receiving her MSc degree in biochemistry, she joined the group of Christian Löw at EMBL Hamburg as a PhD student in 2016. Here, she was using single molecule FRET to study the structural dynamics of proton‐dependent oligopeptide transporters.Tanya (Tatyana) Lasitza Male studied B.Sc. in Chemistry in Tel‐Aviv University (Israel). Afterwards she continued to M.Sc. in the Department of Physical Chemistry (Tel‐Aviv University). Her M.Sc project was guided by Dr. Eli Stern and Prof. Dan Huppert She is currently studying for her Ph.D. in the Department of Chemical and Structural Biology of the Weizmann Institute of Science (Israel), under the supervision of Dr. Hagen Hofmann. She is specializing in single‐molecule fluorescence spectroscopy and dynamics of membrane proteins.Hagen Hofmann (born 1978) studied biochemistry and received his PhD from the Martin‐Luther University Halle‐Wittenberg (Germany). He did his post‐doctoral research in single‐molecule biophysics at the University of Zurich (Switzerland). He then joined the Department of Chemical and Structural Biology of the Weizmann Institute as group leader, where he is working on the dynamics of intrinsically disordered proteins and protein complexes, especially using single‐molecule fluorescence spectroscopy.Christian Löw (born 1979) studied biochemistry at the University of Bayreuth (German) and received his PhD from the Martin‐Luther University Halle‐Wittenberg (Germany). He did his post‐doctoral research in membrane protein structural biology at the Karolinska Institute in Stockholm (Sweden). He then moved as group leader to the European Molecular Biology Laboratory (EMBL) in Hamburg and joined the Centre for Structural Systems Biology (CSSB), where he is studying integral membrane nutrient transporters, mainly using structural and biochemical approaches.


Metastasis causes the majority of cancer-related deaths worldwide. Increasing studies have revealed that circRNAs are associated with the carcinogenesis and metastasis of many cancers. Nevertheless, the biological mechanisms of circRNAs in breast cancer (BC) liver metastasis remain extremely ambiguous.In this study, we identified circROBO1 from three pairs of primary BC and metastatic liver sites by RNA sequencing. FISH assays and RT-qPCR were conducted to validate the existence and expression of circROBO1. The oncogenic role of circROBO1 was demonstrated both in vitro and in vivo. Western blot, ChIP, RIP, RNA pull-down, and dual-luciferase reporter assays were used to confirm the interaction of the feedback loop among circROBO1, miR-217-5p, KLF5, and FUS. Meanwhile, the regulation of selective autophagy was investigated by immunofluorescence, CoIP, and western blot.In this study, upregulated expression of circROBO1 was found in BC-derived liver metastases and was correlated with poor prognosis. Knockdown of circROBO1 strikingly inhibited the proliferation, migration, and invasion of BC cells, whereas overexpression of circROBO1 showed the opposite effects. Moreover, overexpression of circROBO1 promoted tumor growth and liver metastasis in vivo. Further research revealed that circROBO1 could upregulate KLF5 by sponging miR-217-5p, allowing KLF5 to activate the transcription of FUS, which would promote the back splicing of circROBO1. Therefore, a positive feedback loop comprising circROBO1/KLF5/FUS was formed. More importantly, we found that circROBO1 inhibited selective autophagy of afadin by upregulating KLF5.Our results demonstrated that circROBO1 facilitates the carcinogenesis and liver metastasis of BC through the circROBO1/KLF5/FUS feedback loop, which inhibits the selective autophagy of afadin by suppressing the transcription of BECN1. Therefore, circROBO1 could be used not only as a potential prognostic marker but also as a therapeutic target in BC.The online version contains supplementary material available at 10.1186/s12943-022-01498-9. As the most prevalent type of cancer in women, breast cancer (BC) is the leading cause of cancer-related mortality among women worldwide. According to a recent report, the survival rate at 5 years for primary BC is almost 99%. However, approximately one-third of patients with BC present distant nonnodal metastasis, which decreases their 5-year survival rate to 23% . BC mostThe majority of the transcriptome comprises noncoding RNAs, while 2% of the genome can be transcribed into messenger RNAs only . Among tIn our present study, we identified by RNA sequencing that the novel circRNA hsa_circ_0124696 (circROBO1) was upregulated in BC-derived liver metastases and correlated with poor survival. CircROBO1 promoted BC proliferation and metastasis in vivo and in vitro by increasing KLF5 expression, while FUS promoted the back splicing of circROBO1 and was activated transcriptionally by KLF5, resulting in a positive feedback loop. Moreover, circROBO1 could inhibit selective autophagy of afadin, a key biomarker of BC metastasis to the liver, via upregulating KLF5. Therefore, our research revealed for the first time the inhibition of selective autophagy of afadin driven by a positive feedback loop comprising circROBO1/KLF5/FUS, which promoted BC-derived liver metastasis progression and provided novel prognostic biomarkers and antimetastatic therapeutic targets for BC patients in clinical practice.P value <0.01, we found 4735 differentially expressed circRNAs, among which 1941 were upregulated and 2794 were downregulated in liver metastatic tissues  and constructed a circROBO1 overexpression plasmid Fig. a. Then, To evaluate whether circROBO1 affected tumor growth in vivo, human BC xenograft models were established with BT-549 cells and MCF-7 cells stably overexpressing circROBO1, and their corresponding control cells were injected subcutaneously into female BALB/C nude mice. The results revealed that the weight and volume of tumors in the circROBO1 overexpression group were significantly higher than those in the control group for both BT-549 cells and MCF-7 cells Fig. a-d. To fhttps://circinteractome.nia.nih.gov/), the 3′ start flanking intron of circROBO1 has a potential FUS binding region  and TargetScan databases (http://www.targetscan.org). The results showed that miR-217-5p is the only miRNA that has two binding sites for circROBO1 in the intersection between the CircInteractome and TargetScan databases, which indicated that circROBO1 might act as a miRNA sponge in BC , microT (https://bio.tools/DIANA-microT), miRDB (http://mirdb.org) and miRMap (https://mirmap.ezlab.org) databases. The data indicated that KLF5 contains conserved binding sites for miR-217-5p in the intersection of the results of these four databases  revealed that higher expression of KLF5 predicted poor relapse-free survival in BC. Additionally, the data from the TCGA showed that KLF5 expression was higher in BC patients with metastasis than in BC patients without metastasis . Therefore, we speculated that KLF5 upregulated the expression of circROBO1 by increasing the expression level of FUS. To confirm our hypothesis, we performed a ChIP assay and found that E1 (-284~-203) was the most efficient of the three predicted binding sites  in enriching KLF5 protein expression  analysis supported that the correlation between KLF5 and BECN1 was significantly negative . After that, we performed ChIP assays and found that KLF5 was significantly enriched in the predicted binding sites, with E1 (-312~-303) showing the most binding , while MCF-10A cells were cultured in DMEM/F-12 medium . The cell media were supplemented with 50 U/ml penicillin and streptomycin  and 10% fetal bovine serum . All cells were cultured in a humidified incubator  at 37 °C with 5% CO2.All primary BC samples and liver metastasis samples were obtained from patients at the Sun Yat-Sen University Cancer Center (SYSUCC) in Guangzhou, China. All samples were instantly submerged in RNA later upon collection. All samples were collected from patients who provided informed consent under institutional review board-approved protocols and were stored at −80 °C until use.We extracted the total RNA from 3 pairs of fresh frozen BC primary tissues and liver metastatic tissues by TRIzol reagent . Then, the RNA-seq libraries were constructed and sequenced by the Illumina HiSeq2500 platform  according to standard protocols.EdU, colony formation, CCK-8, wound healing, and Transwell migration and invasion assays were performed as previously reported , 18.Formaldehyde (1%) was used for the ten-minute crosslinking reaction, which was terminated by glycine. Next, a Magnetic Bead ChIP Kit  was used according to the manufacturer’s protocols. Finally, the enriched DNA was analyzed by RT-qPCR and separated by agarose gel electrophoresis.The RIP assay was performed using a Magna RIP kit  following the manufacturer’s protocols. The coprecipitated RNA was extracted by TRIzol  and analyzed by RT-qPCR.The biotinylated circROBO1 probe (5′-CGAAGTACTTGAACAGTCAG-3′) and control probe (5′-GAAACTGCTCGGAACGTTAA-3′) were synthesized by RiboBio , while the pre-ROBO1 probes were transcribed using the MAXIscriptTM T7 Transcription Kit . Then, an RNA-Protein Pull Down Kit  was used according to the manufacturer’s guidelines. The abundance of hsa-miR-217-5p was detected by RT-qPCR, while the enriched protein was analyzed by western blot.6 in PBS) were subcutaneously injected into nude mice, which were sacrificed 28 days later. Before this, we intraperitoneally injected 100 µl of D-luciferin (15 µg/µl dissolved in sterilized PBS) . After 15 minutes, images were taken with a Xenogen IVIS Lumina Series II for 1 s, and we analyzed the results with the Living Image 2.11 software package (Xenogen Corp.). The tumor volume was measured weekly and calculated according to the formula volume = width2/2 x length, while the tumor weight was measured after the mice were sacrificed. For the liver metastasis experiments, 1x106 BT-549 cells or MCF-7 cells stably overexpressing circROBO1 with luciferase were injected into the spleens of 5- to 6-week-old female BALB/c mice; corresponding control cells were also injected into other mice. After 60 days, the mice were sacrificed, and livers were removed for further pathological assessment. The number of metastatic nodules of the liver was counted under a microscope. All the procedures were approved by the Sun Yat-Sen University Animal Care and Use Committee.Three-week-old BALB/c female nude mice were purchased from Vital River Laboratories  and housed at the Center of Experimental Animals of Sun Yat-Sen University under standard conditions. BT-549 and MCF-7 cells containing luciferase with stable circROBO1 overexpression or control cells  and GraphPad Prism 9.1 . Student’s t test was used to analyze the differences between groups, while the correlation between the groups was analyzed by the Pearson correlation coefficient. The Kaplan–Meier method was used for survival analysis, and the log-rank test was used to determine the statistical significance. * indicates Metastasis causes the majority of cancer-related deaths worldwide . RecentlIn this study, we investigated the expression profile of circRNAs in primary BC tissues and matched BC-derived liver metastatic tissues by RNA-seq. Then, we focused on the circRNA circROBO1 because it was significantly upregulated in liver metastasis and associated with the poor prognosis of BC patients. Meanwhile, circROBO1 was mostly upregulated in triple negative cell lines such as MDA-MB-231 and BT-549. Next, we found that knockdown of circROBO1 could significantly inhibit cell proliferation, migration, and invasion and suppress oncogenesis and metastasis in vivo, while overexpression of circROBO1 showed the opposite effects.FUS, an RNA-binding protein, is a member of the FET protein family and was recently reported to be a novel regulator of the biogenesis of circRNAs , 21. ForThe functions of circRNAs are correlated with their subcellular location. In our research, we showed that circROBO1 was abundant in the cytoplasm and could function by sponging miR-217-5p based on bioinformatics analysis, RNA pulldown assays, and dual-luciferase reporter assays. MiR-217-5p acts as a tumor suppressor in many cancers –28. The KLF5 is a transcription factor that is overexpressed in the basal subtype of BC . KLF5 haAutophagy, a “self-eating” program, is an intracellular mechanism for protein degradation and the removal of damaged organelles via lysosomes , 36. AltAfadin, an F-actin-binding protein, mediates cellular epithelial polarity, which is located at adherent junctions, forming a complex with actin cytoskeleton and adhesion proteins . Afadin In summary, our research confirmed that the circROBO1/KLF5/FUS positive feedback loop is essential for liver metastasis in BC. We are the first to describe the crosstalk between a feedback loop and selective autophagy of afadin to provide a deeper understanding of the mechanism of circROBO1 in metastasis.Additional file 1. Supplemental Method.Additional file 2: Table S1. Sequences of siRNAs used in this study. Table S2. Primer sequences used in RT-qPCR and PCR analysis.Additional file 3: Supplemental Figure S1.