RNA Isothermal Co-assisted and Coupled Amplification) reaction, that consists of a simple one-pot format of ‘sample-in and result-out’ with a primary focus on the detection of low copy numbers of RNA virus directly from saliva without the need for laboratory processing. We demonstrate our assay by detecting 16S rRNA directly from E. coli cells with a sensitivity as low as 8 CFU/μL and RNA fragments from a synthetic template of SARS-CoV-2 with a sensitivity as low as 1740 copies/μL. We further demonstrate the applicability of our assay for real-time testing at the point of care by designing a closed format for paper-based lateral flow assay and detecting heat-inactivated SARS-COV-2 virus in human saliva at concentrations ranging from 28,000 to 2.8 copies/μL with a total assay time of 15–30 min.Simple tests of infectiousness that return results in minutes and directly from samples even with low viral loads could be a potential game-changer in the fight against COVID-19. Here, we describe an improved isothermal nucleic acid amplification assay, termed the RICCA ( In this stream, a simple yet reliable, rapid and field-deployable molecular diagnostic tool for the detection of nucleic acid sequences of infectious viruses is vital for the massive screening of the population and has been greatly needed worldwide during the current global wave of coronavirus disease 2019 (COVID-19). Hundreds of molecular tests have been rapidly introduced but still face an arduous journey to reach mass usage, due mainly to the shortcomings in the existing laboratory-based testing paradigm for RNA-based viral diagnostics3. First, nucleic acid isolation from clinical samples, which is routinely performed in a laboratory for PCR testing, a current gold-standard diagnostic test, constitutes a major bottleneck. This bottleneck becomes more challenging for RNA-based viruses because rapid RNA degradation contributes to poor clinical sensitivity4. Second, the requirements of bulky expensive instrumentation, such as thermal cyclers with fluorometry, hinder the application of current PCR testing in the field.With the emergence of new infectious virus strains that result in outbreaks, the identification and isolation of infected suspects (symptomatic or asymptomatic) constitute the most effective method for preventing human-to-human disease transmission and pandemics9, nucleic acid sequence-based amplification (NASBA) or RNA-specific amplification12, and loop-mediated isothermal amplification (LAMP)13. The advantage of isothermal amplification methods over PCR is that they do not require bulky instrumentation, enable rapid (10–60 min) amplification of nucleic acids at a constant temperature  and therefore improve throughput in situations in which large numbers of clinical samples must be processed and facilitate point-of-care diagnosis. Recent advances in saliva-based isothermal amplification-based assays, such as RT-LAMP or RT-RPA, have encouraged the testing of SARS-CoV-2-containing clinical samples17. Although encouraging, isothermal amplification methods have also been shown to produce more false-positive  results than PCR testing18. Specifically, false-positive signals in RPA, which are more likely to occur due to nontarget-triggered amplification reactions or primer-dependent artifacts, are often exacerbated at low reaction temperatures and are magnified with increases in the reaction time19. To circumvent this issue, the application of probe chemistry20, portable gel electrophoresis21, and CRISPR-based technology23 has been demonstrated to improve the performance of RPA. Noticeably, the problem of nonspecific amplification in RPA generally occurs when the viral loads in the tested samples are low24. Additionally, if the target is RNA, the amplification methods depend on the efficiency of the initial molecular step, reverse transcription, which can greatly affect post DNA-specific amplification efficiency at low concentrations of the RNA template25 and has also been reported to have an effect of apparent false-negative results (from 17% to as high as 48%) due to the role of the SARS-CoV-2 viral load dynamics26. Therefore, to maintain the integrity of RNA in the reaction sample, a method to support reverse transcription and effectively amplify a low viral RNA load is crucial.To obtain PCR-similar molecular testing outside a centralized laboratory, various isothermal (single-temperature) nucleic acid amplification methods have been devised and are in a continuous race to achieve a performance similar to that of PCR tests. The major candidates include recombinase polymerase amplification (RPA)12. In RNA-specific amplification, RT synthesizes promoter-bearing double-stranded (ds) DNA with the help of its RNase H activity. RNA polymerase continues to induce in vitro transcription to produce copies of RNA fragments that are subsequently recycled as RNA templates for the synthesis of promoter-bearing dsDNA. However, the formation of promoter-bearing dsDNA has a possibility of failure if the initial low target RNA template copy number in clinical samples is further removed due to ready degradation by the action of host nucleases and/or metal ions . Noticeably, the half-life of exogenous viral RNA in host saliva is approximately 30 s27. During this failure event, enzymes and primers are consumed in vain; thus, it takes time for trace amounts of template RNA to enter the amplification cycle due to such failure. To circumvent this issue, we proposed to power the RNA cycle by entering the DNA cycle using RPA, a DNA-specific amplification method that can amplify a target DNA sequence at a constant temperature similar to that used in the RNA-specific amplification method using a recombinase enzyme, a single-stranded DNA-binding protein (SSB), and a strand-displacing polymerase. As of now, we have not come across any study that has reported the co-assisted amplification of RNA and DNA cycles using an isothermal amplification method. In this study, we aimed to advance the bioengineering of our earlier studies on RNA-specific amplification12 and RPA21 and then integrate the essentials of both types of isothermal amplification methods into a simple format of ‘sample-in and answer-out’ with a primary focus on the detection of low copy numbers of viral RNA directly from COVID-19 saliva samples without the need for any laboratory handling or sample preprocessing. In this regard, we report the development of a completely homogeneous, isothermal, highly sensitive, and ultrarapid method for detecting virus RNA target sequences for the on-site (low resource settings) molecular diagnosis of COVID-19 and other infectious diseases. We named this method the RICCA  reaction. The current manual prototype concurrently measures samples within 15–30 min with a sensitivity as low as a few copies per microliter of living bacterial cells (E. coli) and heat-inactivated viruses (SARS-CoV-2 and HCoV-229E).RNA-specific amplification specifically amplifies a target RNA sequence at a temperature near 40 °C with reverse transcriptase (RT) and RNA polymerase12, in which the formation of promoter-bearing dsDNA is essential for the template RNA to enter the RNA-specific amplification cycle. This step, however, is the rate-limiting step if the copy number of the target template RNA in the reaction is very low. Furthermore, the possibility of failure in the formation of promoter-bearing dsDNA becomes severe due to the ready degradation of template RNA by the presence of exogenous nucleases in clinical samples such as saliva, which limits the RNA template that can enter the amplification cycle. Therefore, an alternative route for the formation of promoter-bearing dsDNA is essential and is provided in RICCA by the inclusion of RPA, which provides a bypass route and assists RNA amplification, similar to RPA by supplying T7 promoter-bearing dsDNA. Therefore, in RICCA, coupled RNA-specific and DNA-specific amplification reactions co-assist each other by providing a favorable environment  to each other and synergistically improve the efficiency of both amplifications, i.e., RNA-specific amplification provides a DNA template to RPA, and RPA provides sufficient template for RNA-specific amplification. In the RICCA protocol, the clinical sample of RNA viruses is added directly into a reaction tube containing all necessary components for the isothermal amplification of RNA-specific amplification and DNA-specific amplification.A workflow of the RICCA ‘sample-in and result-out’ approach using a lateral flow (LF) device is shown in Fig. E. coli) in the presence of human saliva . However, RICCA with an RNA plus DNA cycle produced amplicons of both RNA and DNA in all reactions, including the lowest template concentration (8.6 CFU/μL). Considering the presence of approximately 20,000 ribosomes in a single cell of E. coli, the detection limit was estimated to be less than 105 copies of template RNA in the presence of saliva. This one-pot reaction confirmed the utility of RICCA for field application with ultrahigh sensitivity and within 10 min of the start of the reaction. Importantly, no false-positive (nonspecific) signal was obtained with the RICCA reaction with the negative control sample (no template).First, we demonstrated the field-deployable application of a one-pot RICCA reaction for ‘sample-in and answer-out’ testing. For this purpose, an initial assessment of the RICCA platform was performed by detecting RNA directly from nonclinical bacterial cells  of SARS-CoV-2 RNA. As shown in Fig. 11 copies) to 20 fg (0.87 × 105) of SARS-CoV-2 RNA standard template spiked into a 15-μL reaction. As shown in Fig. 8) within 10 min of the start of the amplification reaction.To investigate the application of RICCA for the detection of COVID-19, we started by exploring the effect on the efficiency of RNA-specific amplification for amplifying sense or antisense RNA from sense RNA as a template. For this purpose, we compared the sensitivity and specificity of RNA-specific amplification for amplifying the sense or antisense RNA of SARS-CoV-2. We designed sequence-specific primers to target a 134-bp fragment of the nucleocapsid phosphoprotein gene from the SARS-CoV-2 viral genome. As shown in Fig. 6 copies per reaction) to 200 ag (870 copies per reaction or 29 copies/μL) versus longer reaction time. Our results suggested that the yield of a specific amplicon with a lower copy number of template increased with increases in the reaction time but also resulted in nonspecific amplicons; thus, a longer incubation time for the RNA-specific amplification reaction should be avoided, particularly if the sample has a low viral load. Additionally, we noticed that the success of reaction using lower copy number of template (29 copies/μL) was highly depending on the freshly prepared synthetic RNA template and contamination-free experiments since the lower number of smaller size of RNA template can be readily degraded and removed from the system. Subsequently, to examine the primer selectivity for the targeted template of SARS-CoV-2 RNA in RNA-specific amplification, we performed the reaction with a template from a different source (non-SARS-CoV-2). As shown in Fig. To improve the efficiency of the antisense RNA-specific amplification reaction, we optimized (i) the temperature, (ii) the reaction time, (iii) the selectivity for the targeted template, and (iv) the inhibitory effect of saliva on the reaction efficiency. To test the effect of temperature, we performed RNA-specific amplification at 37 °C, 39 °C, 41 °C and 43 °C for 10 and 20 min each. As shown in Fig. 28, which was recently reported to bypass the need for RNA extraction (isolation and purification). Interestingly, we were able to perform successful RNA-specific amplification reactions with the inclusion of a maximum of 10% human saliva using our modified protocol.Then, given the bottleneck issues in the handling of nasopharyngeal swabs and with the current growing interest in testing COVID-19 using saliva, we examined whether the presence of digestive enzymes such as RNases in human saliva can readily degrade intact SARS-CoV-2 RNA. Therefore, to adapt RNA-specific amplification to direct saliva samples, we tested the efficiency of RNA-specific amplification in the presence of human saliva. As shown in Fig. 11) to 20 fg (0.87 × 105). A clear difference between RICCA with RNA cycle only and RICCA with RNA plus DNA cycle could be observed by electrophoretic analysis , we performed a comparative analysis to demonstrate the advantage of the DNA cycle in the RICCA reaction. We sequentially performed reactions by excluding and including a DNA-specific amplification step in the RICCA assay to amplify low template copy numbers ranging from 20 ng  step, and a commercial lateral flow (LF) assay that allows rapid and accurate read-out by the naked eye. For the saliva-to-RICCA reaction, we designed a one-pot assay using sequential combinations of multiple components in a one-pot reaction approach. As shown in Fig. 29. For saliva collection, a simple method with a SalivaBio Oral Swab  was used30. Ten percent human saliva spiked with heat-inactivated SARS-CoV-2 virus was first mixed with RNA stabilizing reagent (cap #1) and heated for 2–5 min at 80–95 °C. Heating at 95 °C demonstrated the remarkable robustness of the procedure. The saliva samples were then mixed thoroughly with RNA amplification reagent (cap #2) by sequentially replacing the caps followed by incubating for 15–30 min at 41 °C. Finally, cap #3 was used to introduce the RNA detection reagent into the reaction mixture. For the LF assay, we designed a pair of digoxigenin-labeled DNA probes and a biotin-labeled DNA probe to enable hybridization with RNA amplicons at the 5′-terminal end and 3′-terminal end, respectively. For high sensitivity, we opted to use an LF assay with a streptavidin patterned line and gold nanoparticle-labeled anti-digoxigenin antibodies for visualization. Subsequently, to demonstrate the RICCA approach for contamination-free  diagnosis31, we aimed to newly design the so-called closed system for the saliva-to-RICCA-to-LF assay, which can avoid aerosol contamination during liquid handling in the LF assay. As shown in Fig. To demonstrate the RICCA approach for lab-free diagnosis, we followed the saliva-direct approachWe introduced and demonstrated a robust isothermal amplification assay, termed the RICCA, that enables a simple one-pot ‘sample-in and answer-out’ format for the ultrarapid detection of low copy numbers of RNA virus directly from saliva, with a total assay time within 30 min and with a sensitivity as low as few copies of SARS-CoV-2 virus per microliter. The RICCA assay requires only a heat block and a tube prefilled with a mixture containing the necessary reagents  to simultaneously perform RNA-specific and DNA-specific amplifications. We estimated that the material cost for in-house reagents for RICCA is very affordable ; thus, RICCA has the potential to decentralize COVID-19 molecular diagnosis to realize a ‘lab-free, lab-quality’ mega-testing platform. In order to evaluate the rapidity of RICCA, a real-time analysis was used to assess the performance of real-time RICCA in a reference with conventional real-time RT-PCR approach  buffer.The sequence of the SARS-CoV-2N gene was identified from GenBank (accession number MN908947), and the 134-nt single-stranded DNA fragment of the nucleocapsid protein gene, corresponding to the RNA sequence at position 28,707–28,840, was synthesized in vitro. A T7 RNA polymerase promoter sequence was added to the 5’ terminal end, and the double-stranded DNA construct was synthesized by overlap extension PCR using DNA oligomers (Table Escherichia coli (E. coli) 16S rRNA was identified from GenBank (accession number J01859.1), and the 204-nt single stranded DNA fragment of 16S rRNA, corresponding to the RNA sequence at position 415–618, was synthesized in vitro. The test strain (NBRC 3301) of nonpathogenic E. coli was obtained commercially  and grown in L broth at 37 °C for 24 h. The cell counts (CFUs per mL) in serially diluted bacterial culture were determined using a JuLI-smart fluorescent cell analyzer and conventional hemocytometer. For isothermal amplification reactions, cells were harvested from 1 mL of culture by 5 min of centrifugation at 15,000×g, and RNA extraction was performed by disrupting the bacterial cells first by sonication and then by treatment with NP40 reagent . The solution was then centrifuged at 15,000×g for 5 min to sediment cell debris. The supernatant was used directly as a template for isothermal amplification reactions or was stored at − 20 °C until use.The sequence of 2, 19.8 μL of 1.9 M Tris HCl (pH 8.6), 0.6 μL of 40 U/μL RNase Inhibitor, 3.3 μL of 100 mM DTT, 41.3 μL of 2.0 mM dNTP (each), 17.1 μL of 58 mM NTP (each) and 4.8 μL of 250 mM inosine triphosphate], 5.0 μL of primer stock solution  and 2.5 μL of RNA template in a PCR tube and incubated at 65 °C for 5 min  and 41 °C for 5 min. The reaction was started by adding 2.5 μL of enzyme stock solution  and continued at 41 °C for 10 to 30 min. The amplified products were analyzed using 1-inch 6% denatured polyacrylamide gel24 and SYBR Gold staining.The reaction mixture was prepared by mixing 5.0 μL of substrate stock solution [17.6 μL of nuclease-free water (NFW), 5.6 μL of 1 M MgCl24 and SYBR Gold staining.The reaction was performed using a TwistAmp Basic kit . Briefly, the reaction mixture for RNA-specific amplification  was mixed with 5 μL of primer mix (10 μM digoxin-modified sense/forward primer and 10 μM biotin-modified antisense/reverse primer) and 29.5 μL of primer-free rehydration buffer. The resultant 49.5 μL of mixture and 2.5 μL of enzyme stock solution were added to the dried enzyme pellet supplied by the TwistAmp Basic kit. The reaction was initiated by adding 2.5 μL of 280 mM magnesium acetate and immediately placed at 41 °C for 10 min. The amplified products were analyzed using a 1-inch 6% denatured polyacrylamide gel24. Ten-well microcassettes with 6% T polyacrylamide gel were prepared using 40 (w/v)%-acrylamide/bis (19:1) , 5× TBE, 10% ammonium persulfate  and tetramethylethylenediamine  and used as a 1-inch (2.5 × 2.5 cm) precast polyacrylamide gel to analyze the RNA-specific amplification or RICCA reaction products. Paper pads (1.0 × 2.0 cm) were soaked in 2 mL of 1× TBE buffer and used as a source of running buffer. One microliter of each sample was loaded in the micro gel wells with 0.5 µL of 6× gel loading dye. A 50-bp DNA ladder was also loaded in one well to confirm the size of the obtained DNA fragments and comigrated with the samples at 100 V for 5–7 min. A 300-μL volume of 10× SYBR gold nucleic acid gel stain (Thermo Fisher Scientific) was poured on top of the gel, and the bands were visualized using the blue LED flashlight installed in the palm-sized electrophoretic device. Images were acquired using a standard camera-equipped cellular phone.A 1-inch electrophoretic device developed in-house was used for gel-based detection32. The composition of the 20-μL reaction volume was as follows: 2 μL of 10 nM MM4 enzyme, 1 µL of 1 mM dNTP mixture, 2 µL of 10 × RT buffer, 0.5 µL of RNase inhibitor (20 U), 1 µL of RNA template, and 3.5 µL of nuclease-free water. The reaction program was 42 °C for 30 min followed by incubation at 65 °C for 5 min. Real-time PCR was conducted using the TB Green™ Premix Ex Taq™ II Kit  according to the manufacturer’s instructions. The composition of the 25-μL reaction volume was as follows: 12.5 μL of 2 × SYBR Premix Ex Taq II (Tli RNaseH Plus), 1 µL of forward primer (10 µM), 1 µL of reverse primer (10 µM), 2 µL of template (reverse transcription product), and 8.5 µL of nuclease-free water. The following thermal program was used: reverse transcription at 55 °C for 15 min followed by 90 °C for 30 s and 35 cycles of amplification .The real-time RICCA or PCR assay was performed with a Thermal Cycler Dice Real Time System (Thermo Fisher Scientific). The real-time RICCA reaction was prepared in a 50-μL reaction volume as described in an earlier section with the addition of 800 × SYBR™ Green I dye (Invitrogen). For real-time PCR, the first reverse transcription reaction was performed using MM4 reverse transcriptaseThe lateral flow dipstick (LFD) was provided by Asahi Kasei Pharma, Japan. The amplification products from the RICCA reaction were added to the sample pad of the LFD strip and allowed to migrate by capillary action, and during process, the biotinylated RICCA product hybridized with a digoxin-labeled DNA probe and complexed with a gold-labeled anti-digoxin antibody. This hybridization product was trapped by a biotin ligand and bound to a lateral flow test strip to form an immune complex bound at the test line (T). The experimental procedure and determination method were as follows: 5 μL of RICCA product was added to 95 μL of NFW, the mixture was added to the sample mat on the test paper strip, and the result was read after 5–10 min.6 TCID50/mL), and the RNA copy number was determined by ddPCR (4.2 × 105 genome copies/μL). Prior to the RICCA reaction, the stock aliquot vial was centrifuged and serially diluted in phosphate-buffered saline (PBS) with replicates of 42,000, 4,200, 420, 42, and 4.2 copies/μL. Two microliters of diluted heat-inactivated SARS-CoV-2 virus was spiked with and without 3 μL of human saliva, which was freshly collected using a SalivaBio Oral Swab . The 5-μL SARS-CoV-2 spiked samples were mixed with 0.5 μL of Proteinase K (Takara) in 0.2-mL nuclease-free and thin-walled microcentrifuge tubes by simple tapping or vortexing followed by heating in a portable heat block  at 95 °C for 5 min. To the lid of the tube, 10 μL of substrate stock solution, 10 μL of primer stock solution and 5 μL of enzyme stock solution were added, and the vial was then spun down the vial and incubated at 41 °C for 15–30 min. Of the 30-μL reaction sample, 28 μL was utilized for the lateral flow (LF) assay, and 2 μL of the sample was used for gel electrophoresis analysis. To prevent possible contamination during liquid handling in the LF assay, we introduced a closed system by designing a device to continuously perform the RICCA reaction and LF assay without opening the RICCA tube. To this end, a prototype is fabricated using a 3D printer that can hold RICCA tubes and LF assay strips. In this design, after a tube is inserted and mechanically forced down by closing the device, a crack forms in the bottom of the tube, and the reaction solution flows out and moves up on the LF strip by capillary action. For the LF assay, 30 μL of the RICCA product was mixed with RNA detection reagents . The concentration of virus in lot #70037781 was estimated by the preinactivation titer (6.45 × 10Supplementary Figures.


The first cluster of coronavirus cases in Europe was officially detected on 21st February 2020 in Northern Italy, even if recent evidence showed sporadic first cases in Europe since the end of 2019. In this study, we have tested the presence of coronavirus in Italy and, even more importantly, we have assessed whether the virus had already spread sooner than 21st February. We use a counterfactual approach and certified daily data on the number of deaths  at the municipality level. Our estimates confirm that coronavirus began spreading in Northern Italy in mid-January. Recent evidence shows sporadic first cases of coronavirus (SARS-CoV-2) in Europe since the end of 2019. For instance, it has been confirmed that a patient hospitalized on 27th December for suspected pneumonia near Paris had coronavirus as claimed in France 24 . LikewisThis paper aims at assessing the plausibility that coronavirus spread in Italy before 21st February and to investigate approximately when. In light of anecdotal evidence, in the main analysis, we focus on Piacenza as a case study, while we report the analysis on other municipalities in the Appendix. We analyze official daily data on the number of deaths made available by the Italian National Institute of Statistics (Istat)The paper is organized as follows. In the next section, we present the identification strategy, while Section To assess if coronavirus was present in Piacenza earlier than 21st February 2020, we adopt a novel counterfactual approach, the trajectory balancing method (TB) developed by Hazlett and Xu . TB is ai such that the weighted average of cumulative deaths and other characteristics .In the main analysis, we use a panel dataset of 33 Italian municipalities , observed for the period 1st January 2020–21st February 2020. Building the ‘synthetic’ Piacenza implies choosing weights for each of the other municipalities With the spread of the coronavirus pandemic, an increase in the number of deaths was observed, higher than that officially attributed to coronavirus [see Report Istat-ISS  on the iPanel (a) of Figure To evaluate the significance of the results, we run an in-space placebo test, i.e., we reassign the treatment to each of the 32 municipalities that make up the ‘synthetic’ Piacenza, where we presume that the coronavirus arrived later. We will deem the effect of the arrival of the virus in Piacenza statistically significant if the estimated effect is large relative to the distribution of placebo effects. We follow Abadie et al.  and showthe treatment date, backdating and postdating the treatment by 5 days;the donor pool, considering all municipalities with at least 10,000 inhabitants in the North of Italy, all regions, and municipalities with at least 10,000 inhabitants in all Italian regions. Besides this, we propose a leave-one-out analysis, i.e., we re-run the trajectory balancing, excluding from the sample one-at-a-time each of the municipalities that contribute to the counterfactualthe predictors of the outcome variable, adding a measure of air quality (PM-10) in 2018the algorithm to assess weights, using the SCMAll robustness checks lead to estimates which are very close to those reported in the main analysis. Moreover, in Figure As suggested in Abadie , we repoOur research aims to analyze not only the presence of coronavirus in Italy sooner than 21st February but, more importantly, to assess whether the virus had already spread in a specific territory. As Italy has been the first European country severely hit by the pandemic, we argue that it must be the first European country where COVID-19 spread, except for a few sporadic cases detected throughout the continent since the end of 2019. To test our hypothesis, we adopt the trajectory balancing approach to analyze certified data on the number of deaths at the municipality level. This allows us to avoid underreporting, which seems to be widespread with official data on coronavirus. We find that Piacenza experienced an unexpected increase in the number of deaths since the beginning of February with respect to the counterfactual. This means that coronavirus had already spread in a specific area of Northern Italy since mid-January (considering the delay between contagion and death) and that by 21st February hundreds of individuals were already infected. This finding might help in the historical investigation of how the virus spread in Italy first and then in Europe. Besides, in the era of big data with the spread of digital health, our evidence underlines the need to invest more in data collection systems’ efficiency and timeliness. An effective information system allows us to spot anomalies in the data and helps policymakers handle emergencies by providing a more precise picture of the situation. Moreover, as highlighted by Birrell et al. , in a pa


Through specific examples, we demonstrate how the structural information can explain the phenotypes of RNA-editing mutants. This work shows how the structural perspective can bridge the gap between sequence and phenotype and provides a framework for the continued analysis of RNA-editing mutants in plant mitochondria and, by extension, in chloroplasts.The last steps of respiration, a core energy-harvesting process, are carried out by a chain of multi-subunit complexes in the inner mitochondrial membrane. Several essential subunits of the respiratory complexes are RNA-edited in plants, frequently leading to changes in the encoded amino acids. While the impact of RNA editing is clear at the sequence and phenotypic levels, the underlying biochemical explanations for these effects have remained obscure. Here, we used the structures of plant respiratory complex I, complex III RNA editing introduces alterations into coding or non-coding regions of RNA molecules after transcription. Alterations include insertions, deletions and changes to the identity of the genomically encoded ribonucleotide, affecting the molecule’s stability, splicing and binding to regulatory factors, as well as the encoded protein sequence in the case of messenger RNA. Thus, RNA editing has significant consequences on an organism’s gene expression.RNA editing may occur in the nucleus, cytoplasm, or DNA-containing organelles. In land plants, extensive RNA editing occurs in mitochondria and chloroplasts ,2,3,4. T2, and complex IV have been obtained [Analyses of the editing sites of mitochondrially-encoded subunits have shown that the edits generally increase the hydrophobicity of the target protein, and that they generally restore the identity of the amino acid to the evolutionarily conserved residue in non-edited plant species ,8. Many obtained ,10,11,12c). The complexes sequentially transfer electrons from reduced metabolites (NADH and succinate) to molecular oxygen while concomitantly pumping protons against their concentration gradient. This generates a proton electrochemical potential that is dissipated by ATP synthase , producing ATP [Respiration occurs through an electron transport chain in the inner mitochondrial membrane composed of four membrane-embedded multi-subunit enzymes (complexes I–IV) and two electron carriers . Next, we mapped each edit to the available structures of complex I  [2  [V. radiata, PDB: 7JRO) [2′s hemes, complex IV’s hemes and putative proton channels), (3) location at an interface with other subunits, (4) change in the biochemical properties of the amino acid , and (5) location relative to secondary structure elements . All of these aspects have structural and functional implications. Lastly, we identified the editing sites that have been linked to detrimental phenotypes in mutant screens.We structurally characterized the RNA-editing sites of mitochondrially encoded subunits of plant respiratory-chain complexes for which there are high-resolution structures, i.e., complex I, complex IIImplex IV B–D. We fB: 7AR8) , complexB: 7JRG)  and compB: 7JRO) . We char2 and 48 in complex IV across 17 plant species. Of these, 65% to 80% are located within the transmembrane region of the proteins, 45% to 65% are edited from hydrophilic to hydrophobic residues, 15% to 25% remove prolines in α-helices or β-sheets, 15% to 40% are at interfaces between subunits and 10% to 50% are close to functionally important residues (2) and half of the edits in COX1 (complex IV) are in the vicinity of key functional residues, such as those involved with quinone binding sites, heme coordination or proton-translocation channels [P. denitrificans) to humans. In NAD7, COX1 and COX3 up to 67% of the edited sites occur at (and restore) highly conserved residues. Overall, only 6 of the 275 total editing sites (~2%) lack any salient structural characteristics . Thus, our analysis revealed that almost all the editing sites in plant complex I, complex III2 and complex IV have implications for the structure, and therefore the function, of the respiratory complexes.Our approach identified 206 sites in complex I, 21 in complex IIIresidues . Notablychannels ,19. AddiTo further assess the functional impact of these editing sites, we examined the existing literature on mutants lacking RNA editing, e.g., due to mutations in PPR proteins. In the next section, we discuss several of these mutants and show how their phenotypic effects can be explained from a structural perspective.MPR25 [SMK1 [PpPPR_79 [DEK605 [MEF9 [A. thaliana subunits, while numbers in complex III2 and complex IV refer to the protein sequences in V. radiata subunits.PPR proteins use protein: RNA interactions to recognize patterns in the mRNA and position the deamination active site. Depending on their cognate pattern, PPRs can have single or multiple targets across the same or different subunits in the same or different complexes. Mutants of single-site PPRs are an excellent tool to link the structural impact of an RNA edit to its functional relevance and illustrate the power of taking a structural perspective. Here, we discuss the effects of mutations in MPR25 , SMK1 [225 [SMK1 , PpPPR_7 [DEK605  and MEF905 [MEF9   and contains a di-copper redox center (CuA). This center accepts the electron from cyt c and transfers it to the hemes and binuclear center in COX1, where the last step of respiration occurs by reducing O2 to water. CuA is coordinated by two histidines, one cysteine and one methionine. If these residues were incorrectly positioned or if the electrostatic environment of the redox center were altered, binding of cyt c and electron transfer would be jeopardized, precluding electron transfer through the whole canonical respiratory chain. COX2-W122 is in the immediate vicinity of the copper-coordinating residues and key cyt c binding residues   D. The ladine-184 D. This wg tunnel E–F, dele2 and complex IV [We used the recently published cryoEM structures of plant respiratory complex I, complex IIImplex IV ,10,11,12mplex IV , the funOur work demonstrates that a structural analysis of RNA-editing sites can provide insight into the functional and physiological effects of RNA editing. Moreover, it provides a framework for the future discussion of PPR phenotypes in plant mitochondria as well as in chloroplasts, where we expect that the same general biochemical and structural principles are in effect. However, not all structures of edited proteins are currently available. We predict that structures of the remaining proteins that are edited will reveal shared principles for the biochemical basis of RNA-editing mutants in plant organelles.


Arum- and Paris-type AMs. As an exception, Solanum lycopersicum (tomato) forms both types of AMs depending on the fungal species. Previously, we have shown the existence of diverse regulatory mechanisms in Arum- and Paris-type AM symbioses in response to gibberellin (GA) among different host species. However, due to the design of the study, it remained possible that the use of different plant species influenced the results. Here, we used tomato plants to compare the transcriptional responses during Arum- and Paris-type AM symbioses in a single plant species. The tomato plants inoculated with Rhizophagus irregularis or Gigaspora margarita exhibited Arum- and Paris-type AMs, respectively, and demonstrated similar colonization rates and shoot biomass. Comparative transcriptomics showed shared expression patterns of AM-related genes in tomato roots upon each fungal infection. On the contrary, the defense response and GA biosynthetic process was transcriptionally upregulated during Paris-type AM symbiosis. Thus, both shared and different transcriptional reprogramming function in establishing Arum- and Paris-type AM symbioses in tomato plants.Arbuscular mycorrhizal (AM) fungi allocate mineral nutrients to their host plants, and the hosts supply carbohydrates and lipids to the fungal symbionts in return. The morphotypes of intraradical hyphae are primarily determined on the plant side into  PHOSPHATE TRANSPORTER 4 (PT4) and AMMONIUM TRANSPORTER 2;3  for symbiotic phosphate and ammonium transport, REDUCED ARBUSCULAR MYCORRHIZA 2 (RAM2) and G-type ABC transporters (STR/STR2) for lipid production and transport, and VAPYRIN (VPY) for arbuscule development ), rinsed twice with distilled water. The fixed root samples were heated at 90 °C for 15 min and neutralized in 2% HCl solution. Subsequently, the root samples were immersed in trypan blue diluted by lactic acid at 0.05% and heated at 90 °C for 15 min. The stained samples were sliced with a scalpel under a SZX16 stereomicroscope , and images were taken using a BX53 light microscope (Olympus) equipped with a digital camera .AM fungal colonization was quantified by staining the root samples with trypan blue and microscopically observing them as previously reported . Brieflyg at 4 °C for 5 min. The supernatant was mixed with 450-μL ethanol and RNAiso Plus (Takara) in a new tube. The genomic DNA-free total RNA was prepared using a Zymo-spin IIICG Column . The column membrane was treated with DNaseI (Takara) before extracting the RNA according to the manufacturer’s protocols. The purity and quantity of total RNA were measured at 260 and 280 nm (A260: A280) using DeNovix DS-11 + . The prepared samples were stored at −80 °C until use.To conduct transcriptome analysis by RNA-sequencing (RNA-seq), we prepared RNA samples from fresh tomato roots. The lateral roots from three seedlings in a nuclease-free tube  containing two 5-mm beads were frozen in liquid nitrogen and then homogenized in a ShakeMan6 ; afterward, 450 μL of Fruit-mate for RNA Purification  was added to each tube. After thoroughly mixing the tubes, the slurry was transferred to another tube and centrifuged at 12,000× https://solgenomics.net/organism/Solanum_lycopersicum/genome, accessed on 2 December 2021) using the STAR program [R. irregularis and G. margarita infections on the expression of AM symbiosis- or SL-related genes, we selected several tomato genes required for the mutualism. To this end, known genes in M. truncatula and Arabidopsis thaliana were used as queries for tBLASTx searches in the Sol Genomics Network using the default setting  using DNBSEQ-G400 and resulted in more than 14 million strand-specific paired-end (2 × 150 bp) reads per sample . The obt program . The res program  to quant program  in the R program . Tomato  setting . Raw nuct-test and analysis of variance (ANOVA) followed by a post hoc Tukey-Kramer test were applied for the colonization rates and shoot fresh weight, respectively. The R package heatmaply was used to draw the heatmaps [To quantify the root colonization rate (%), we considered ten pieces of root fragment from one tomato sample on a microscope slide as one biological replicate. When preparing a sample for RNA-seq, one pool of total RNA extracted from three seedlings was one biological replicate. Finally, we used three RNA samples for the RNA-seq. Statistical analysis was conducted in the R software v4.0.2. Welch’s heatmaps .Arum- and Paris-type AM symbioses in tomato roots. These similarities indicate that the primary and downstream mechanisms for accommodating AM fungi would be common, irrespective of the AM morphotype. In addition, our study demonstrated that the colonization of phylogenetically distant AM fungi differentially affected the defense-related pathways and GA biosynthetic process in tomato roots. These different transcriptional responses would enable tomatoes to fine-tune the mutualism between tomato roots and diverse AM fungi to optimize the host growth. In addition, the upstream regulation underlying Arum- and Paris-type AM symbioses in tomato plants might be differentially modulated by GA. Further investigation of how GA regulates Paris-type AM symbiosis established by tomato roots and G. margarita would improve our understanding of the regulatory mechanisms underpinning AM symbioses.We found some common AM-specific transcriptional programs for the membrane transport, arbuscule development, and SL biosynthetic pathway in


Kidney disease is a multifactorial problem, with a growing prevalence and an increasing global burden. With the latest worldwide data suggesting that chronic kidney disease (CKD) is the 12th leading cause of death, it is no surprise that CKD remains a public health problem that requires urgent attention. Multiple factors contribute to kidney disease, each with its own pathophysiology and pathogenesis. Furthermore, microRNAs (miRNAs) have been linked to several types of kidney diseases. As dysregulation of miRNAs is often seen in some diseases, there is potential in the exploitation of this for therapeutic applications. In addition, uptake of interference RNA has been shown to be rapid in kidneys making them a good candidate for RNA therapy. The latest advancements in RNA therapy and lipid-based nanocarriers have enhanced the effectiveness and efficiency of RNA-related drugs, thereby making RNA therapy a viable treatment option for renal disease. This is especially useful for renal diseases, for which a suitable treatment is not yet available. Moreover, the high adaptability of RNA therapy combined with the low risk of lipid-based nanocarriers make for an attractive treatment choice. Currently, there are only a small number of RNA-based drugs related to renal parenchymal disease, most of which are in different stages of clinical trials. We propose the use of miRNAs or short interfering RNAs coupled with a lipid-based nanocarrier as a delivery vehicle for managing renal disease. Accordihe cause . Taking he cause .Over the years, studies on the diverse functions and mechanisms of action of RNA molecules have enabled its use in therapy. From the discovery of mRNA in 1961  to the aBoth the RNA-based drug development and efficient drug delivery methods have advanced substantially and simultaneously to allow for the clinical use of RNA therapy. Most recently and notably in this regard, are two authorised vaccines used against the coronavirus disease 2019 (COVID-19) ,11. In tIt is widely accepted that RNAs are important molecules involved in nearly all biological pathways . While tOf particular interest here are the RNAs that relate to protein downregulation, which are miRNA and siRNA. As miRNAs and siRNAs have emerged as a class of therapeutics with great potential, especially for the treatment of cancer, there has been some interest in using them as a therapy for renal diseases ,15. BothIn various progressive renal diseases, TGFβ signalling plays an essential role. In human studies, TGFβ1 expression is majorly upregulated in fibrotic tissues . AccumulPodocyte apoptosis has been documented to be associated with diabetic nephropathy, and miRNAs have been implicated in the pathogenesis of diabetic nephropathy. The expression of mmu-miR-29c increased in glomeruli and microvascular endothelial cells in a mouse model . StudiesDownstream effects of TGF-β are mediated by connective tissue growth factor (CTGF), whose expression is upregulated in several renal cells and secreted in the urine in the diabetic state . In addiGlomerulonephritis includes a group of diverse disorders that may present as proteinuria or microscopic haematuria with or without renal dysfunctions. Accumulating evidence has revealed that RNA-related pathophysiology and potential molecules or pathways may be therapeutically useful for treating glomerulonephritis.IgA nephropathy is caused by abnormal O-galactosylation of IgA, resulting in the deposition of IgA complexes in the kidneys and activation of the complement system. Various miRNAs play roles in IgA nephropathy as observed in a genome-wide analysis . MembersSystemic lupus erythematosus (SLE) is a systemic disease caused by abnormal immune system activities. Kidney biopsy analysis of patients with lupus nephritis revealed upregulated miR-146 and miR-198 expression in glomerular lesions and increased miR-638 expression in tubulointerstitial lesions . In lupuFocal segmental glomerulosclerosis (FSGS) is characterised by the obliteration of the capillary lumen and increase in mesangial matrix amount in the involved glomeruli. Podocyte injury is a factor affecting the development of FSGS, and molecules associated with increased permeability of glomerular basement membranes are potentially related to the pathogenesis of FSGS ,49. MicrHypertension is a significant risk factor for coronary artery disease, congestive heart failure, sudden death, left ventricular hypertrophy and stroke ,55,56. HActivation of the renin-angiotensin-aldosterone system (RAS) plays an essential role in the pathophysiology of hypertension. The expression of many miRNAs, including miR-29b, miR-129-3p, miR-132, and miR-212, are upregulated by Ang II in HEK293N cells . In addiOxidative stress potentially induces endothelial cell apoptosis and atherosclerosis development. Overexpression of miR-210 inhibits apoptosis and reduces ROS levels . InhibitIn addition, sympathetic nervous system overactivity has a significant role in hypertension development and persistence. In genetically hypertensive mice (BPH/2J), the roles of the sympathetic nervous system (SNS) and RAS are important. Hypertension in BPH/2J is mediated by SNS during the active period and RAS during the inactive period . During ADPKD is a genetic disorder caused by mutations in either PKD or PKD2 genes, resulting in impaired ciliary functions, leading to renal and hepatic cyst formations. In a rat model of ADPKD, the expression of miR-21 was found to be upregulated . InhibitMutation in genes coding ꭤ3, ꭤ4 or ꭤ5 chains of collagen Type IV leads to abnormal basement membrane formations in the kidneys, eyes, and inner ears. miR-21 is typically expressed in the tubulointerstitial compartment instead of the glomerular compartments in normal mice, while it is expressed equally in both compartments in Col4 ꭤ3-/-mice .The stability of RNA, delivery efficiency, and activation of the innate immune system are the various challenges for the widespread use of RNA therapy. The limitations of RNA-based therapies are attributed mainly to their short half-life and rapid renal clearance . HoweverNanotechnology is an interdisciplinary field that can be defined in various ways. Yet, a reasonable general consensus exists, where nanotechnology is concerned with materials and processes taking place in the nanoscale . AccordiNanocarriers use agents like nanoparticles as a transport carrier. A carrier system is defined as technology that optimises the therapeutic efficiency of a bioactive compound . This isLNPs no doubt aid in the efficient delivery of RNA drugs to their target. Nevertheless, there are still considerations and precautions to be taken. Lipotoxicity has been one of the concerns with using LNPs. Newer generations of LNPs have managed to overcome this issue . AnotherIn the previous sections, we discussed the role of RNA and its types in renal diseases, as well as potential RNA dysregulation. MiRNA-based therapies either inhibit deleterious miRNA or replace a deficient one with a beneficial miRNA. Antagomirs or antimiRs and miRNA antagonists, are single-stranded molecules that can bind to mature miRNAs and block their action. Nanoparticle-based viral vectors express the desired miRNA or siRNAs, which are delivered to the target site . In addiSome phase I and phase II trials are ongoing or have been completed to evaluate the efficacy of RNA-based therapeutic agents for the management and treatment of chronic hepatitis C, diabetes mellitus type 2 with fatty liver and cancers ,91,92. TIn contrast to siRNAs, miRNAs typically bind with partial complementarity and usually promote translational repression by triggering mRNA decay, and different miRNAs can interact with the same transcript by overlapping or non-overlapping sites . ImportaCurrently, only a few RNA therapies are associated with renal diseases. At the time of writing this article, they are at different stages of clinical trials and have not been approved for clinical use. These include QPI-1002 and NOX-E36 (Emamticap pegol). QPI-1002 uses an siRNA to target the p53 gene and is currently in phase I and II trials for acute renal failure and AKI, respectively . NOX-E36It is evident in our time that advancements in medicine and technology go hand in hand. As previously mentioned, the world of medicine and therapeutics have benefitted from both advancements in RNA research and the development of nanocarriers. With the constant evolution and mutation of diseases, treatment methods must likewise keep up with disease progression. Therefore, new approaches for managing current diseases are vital. In terms of nephrology, RNA therapy appears to be a novel approach, opening various avenues for the treatment of renal disease .In this review, we discussed the difficulty in delivering RNA molecules to the intended target. Naked RNAs are unstable in the blood and are rapidly degraded by circulating monocytes and macrophages in the kidneys and liver. Thus, effective delivery methods to prevent miRNA degradation must be developed. The designed delivery vehicles must be non-toxic, have low immunogenicity, and deliver efficiently to the intended targets ,108. WitOur recommendation is that more emphasis and attention be put on the research of combining RNA with LNPs when it comes to renal disease. There are many types of renal disease out there that could benefit from this research. The use of lipid nanocarrier-based RNA delivery holds a promising future for the treatment of renal disease. We expect to see broad applications of this therapeutics across the board in renal disease in the coming years.


Macromolecular complexes of proteins and RNAs are essential building blocks of cells. These stable supramolecular particles can be viewed as minimal biochemical units whose structural organization, i.e., the way the RNA and the protein interact with each other, is directly linked to their biological function. Whether those are dynamic regulatory ribonucleoproteins (RNPs) or integrated molecular machines involved in gene expression, the comprehensive knowledge of these units is critical to our understanding of key molecular mechanisms and cell physiology phenomena. Such is the goal of diverse complexomic approaches and in particular of the recently developed gradient profiling by sequencing (Grad-seq). By separating cellular protein and RNA complexes on a density gradient and quantifying their distributions genome-wide by mass spectrometry and deep sequencing, Grad-seq charts global landscapes of native macromolecular assemblies. In this review, we propose a function-based ontology of stable RNPs and discuss how Grad-seq and related approaches transformed our perspective of bacterial and eukaryotic ribonucleoproteins by guiding the discovery of new RNA-binding proteins and unusual classes of noncoding RNAs. We highlight some methodological aspects and developments that permit to further boost the power of this technique and to look for exciting new biology in understudied and challenging biological models. When about two decades ago high-throughput approaches in molecular biology became a reality, it all began with parts lists. Genomics shed light on the ensemble of an organism’s genes and provided the first idea about what proteins and noncoding RNAs can in principle be there . TranscrExpectedly, next-generation high-throughput approaches, doing the same in a massively parallel way, soon emerged. Genome-wide functional screens were greatly facilitated by modern mutagenesis tools based on random transposon insertion and CRISPR-mediated gene disruption, silencing, or activation . They noOver the last decade, many techniques have been developed to identify partners of specific RNAs and RNA-binding proteins (RBPs) with mass spectrometry-based proteomics and RNA-seq, respectively . Among tRNA–protein associations are commonly subdivided in transient and stable. They differ not only in physicochemical properties but also in biologically relevant modes of action. Transiently interacting macromolecules, epitomized by enzyme–substrate complexes, do the best in fugitive encounters: this keeps the enzyme available for further rounds of reaction while leaving the product to go its way (consider RNases or RNA modification factors as examples). In contrast, as we will see below, stable RNPs can only carry their functions out if the partnership between the RNA and the protein persists for a relatively long time. This long-lasting association is a foundation for quite a peculiar kind of molecular behavior which is more characteristic of biological, as opposed to purely chemical, systems. It creates prerequisites for such biologically important properties as structuration, information flow, state maintenance, and switching. Stable complexes are also naturally more amenable to study, and consequently, we know much more about this group of RNPs. The present review will essentially focus on stable RNPs and the methods to characterize them on a global scale. Transient RNA–protein associations, e.g., most enzyme–substrate complexes, temporary low-affinity interactions between RNA chaperones and their clients, and other weak binding events, fall beyond its scope.–4 s–1 (kinetic stability). We refer the reader to the following publications for examples of these parameters among some well-studied RNPs , and (ii) the “in vitro stability,” when the complex only needs to be intrinsically robust in (effective) isolation from other cellular components  it must have a relatively low dissociation constant, at least in the sub-micromolar range (thermodynamic stability) and/or (ii) its off-rate must be fairly slow, typically <10ied RNPs . It is amponents . This dimponents .Before discussing how this basic physicochemical principle can be converted into working methodology, we will provide an overview of diverse kinds of stable RNPs and explain why they deserve most thorough investigation. Without aspiring to a catch-all classification, we can sort the majority of known stable RNPs in four major classes, based on their biological properties and/or activities and the nature of the interaction between the RNA and the protein components . In factConstitutive RNPs are highly organized, permanent RNA-protein associations where one or both partners provide structural and functional support to each other. Much of our current knowledge on the molecular interplay between the RNA and the protein components of ribonucleoproteins comes from the research on constitutive RNPs . This inBehind all these variations, there is a common architectural theme followed by all constitutive RNPs: both RNA and protein make up a unique and inseparable whole and thereby contribute to the same, shared molecular function. The components of constitutive RNPs are normally bound in an obligate association with each other and only occasionally work outside this context . They arDeinococcus radiodurans, the Ro60-like protein Rsr is tethered via Y RNA to the major bacterial exoribonuclease PNPase and seems to change its substrate specificity by promoting the degradation of structured RNAs , the scanning eukaryotic translation initiation complex, the elongating ribosome, or the translocating transcription termination factor Rho . Of courin vivo” standards. But this offers a time window generous enough for the experimenter to intervene and leverage their hidden “in vitro stability”. Indeed, many processive RNPs have evolved to firmly hold their template and/or the elongated substrate until a termination signal occurs and enables their dissociation. Yet, elongation depends on physiological temperatures and the regular supply of building blocks . If one rapidly cools down the system and dilutes out the building blocks, the bereft processive RNP will stall dead-still and become analyzable. This principle underlies the ribosome profiling, which permits to follow the translational dynamics of all mRNAs in the cell simultaneously  to enable their capture in the same way as described above, i.e., by snap cooling, dilution, and separation from other cellular components  the consequences of such an interaction and whether it involves simple RNA binding or an enzymatic event, class III can be subdivided in “protein–controls–RNA” or “RNA–controls–protein”-type RNPs and inhibitory enzyme–substrate complexes.Stable-state RNPs form the most numerous and actively studied RNP class. Like class I RNPs, they rely on strong RNA-protein interactions. Contrary to the former group, however, these interactions are not obligate and rather represent one of two (or sometimes more) distinct alternative states, each associated with a  different outcome for the partners. Usually very long-lived trans-acting factors boost, mitigate, or override the effects of each other, thereby achieving highly nuanced outcomes , can itself serve as a translational inhibitor of certain mRNAs, including its own , they show rapid exchange dynamics in vivo, where many different RNAs incessantly chase each other from the limited number Hfq hexamers  and “in vivo”  parameters of RNPs . A comprehensive, genome-wide approach actually becomes a necessity when big, transversal questions are raised. One such question is ontological: what stable RNPs exist in the studied biological system under given conditions? Here the profiling nature of a high-throughput technique by far outperforms the serendipity of the traditional “by-chance” discovery. Another advantage of a “bird’s-eye” view on the cellular RNP ensemble is the access to systems-level information: what is the functional state of the cell? How are resources allocated among various RNP-based processes, such as transcription, RNA processing, translation, or degradation? How are the ongoing regulatory programs implemented on the level of persistent RNA-protein associations? Regarding the interactions themselves, genome-wide techniques provide unique biochemical data on the “ of RNPs . And if The logic of the complexomic approach is radically different from that of the traditional bait-prey interactomics . In compEach step of this standard pipeline can be played differently: the last two decades witnessed a flurry of studies employing a wide diversity of complexomic approaches tailored to various biological systems and research questions. For example, Blue-Native (BN) PAGE is traditionally used in microbial and mitochondrial complexomics as it permits to analyze large (up to 30 MDa), soluble or membrane-embedded protein complexes, which cannot be satisfactorily resolved by other techniques . In earlOther separation techniques, such as clear native PAGE, sucrose gradient centrifugation, and size exclusion chromatography, have been successfully used to partition complexes by size and shape . HoweverIn one stunning methodological development, the sucrose gradient fractionation of the cell lysate is coupled to both mass spectrometry and cryo-electron microscopy—to directly classify and visualize complexes present in select fractions, solve their structures, and thereby identify the constituent proteins . PioneerBecause complexomic techniques do not require genetic manipulation of the biological system and provide a large amount of data on the physical organization of the proteome, they have been used to map complexomes not only of model but also of some traditionally “difficult” organisms, including numerous bacteria, archaea, protists, algae, plants, animals, and human subjects .Though largely focused on proteins, some complexomic studies had already reserved a place of honor to major RNPs, most often ribosomes . These wFirst attempts to visualize the RNA component of RNPs relied on low-throughput northern blotting e.g., . The polbona fide in-gradient distributions of individual macromolecules revealed with conventional methods. The result is a complete collection of sedimentation profiles of potentially all expressed proteins and RNAs present in the studied biological model.Grad-seq was introduced in 2016 as a new technique to comprehensively chart the landscape of an organism’s stable RNA and protein complexes without resorting to tagging or enrichment of cellular components , 2017a. Peaks in each distribution indicate the stable assemblies of profiled molecules. Calculation of the sedimentation coefficient for each fraction, based on the behavior of known complexes or standards, enables access to molecular weight estimates for any detected complex . With thAdditionally, if uncharacterized transcripts or proteins co-segregate with other components, whose molecular role has been determined , one can leverage guilt-by-association and predict their involvement in a similar kind of processes or assemblies. Such hypotheses can be verified with the help of accessory techniques, such as RNA and protein pull-downs, to robustly establish the composition of the observed RNPs . TherefoS. Typhimurium  betrays the accumulation of no-stop or no-go translation elongation complexes awaiting resolution  form of RNAP, and the recruitment of σ28 marks the commitment of both cultures to foster motility at the wake of the exponential phase , which platforms the TBK1 kinase within the TLR4-TRIF pathway to induce type I interferon expression -tail of its ligands by ∼3 nucleotides but fails to digest any further  commanding only passing interest it has grown into a major topic of bacterial RNA biology . ProQ hostically , and an stically , 2011. Tstically . Structustically . Howeverstically , that costically , does nostically . The remThe basic Grad-seq pipeline has recently seen two interesting adaptations, marking new important developments in RNA-protein complexomics . In one Salmonella protein complexes on glycerol gradients and revealed a number of RNA-dependent proteins, including Hfq, CsrA, and ProQ, while also spotting an uncharacterized plasmid-encoded FinO/ProQ homolog, FopA, as a potential RBP. RIP-seq and in vitro assays confirmed its specific binding to the Inc sRNA and showed that FopA dramatically accelerates the base-pairing between Inc and the cis-encoded repZ mRNA involved in plasmid replication (Since Grad-seq studies broadly illustrated the dependence of RBP sedimentation on their RNA ligands , three ilication . This stlication , by addiConsidering Grad-seq warts-and-all, next to the advances it brought about in six model species, the method still must face up to a few challenging issues. The most important one is profile matching. A recent critical appraisal exposed a surprisingly high false discovery rate of global complexomic analyses, when complex membership is called by direct matching of protein distributions . This haData mining is another issue. The Grad-seq publications highlighted in this review primarily reported on a few particularly striking findings illustrating the power of the method. But those are merely the tip of the iceberg, and the RNA and especially the protein Grad-seq datasets await deeper exploration to unearth new potentially interesting macromolecular assemblies. This is particularly important if we want to approach the functions of understudied proteins and transcripts.We contemplate many possible avenues for further development of RNP complexomics. It will be exciting to apply Grad-seq family approaches to more exotic and recalcitrant biological systems, such as newly discovered microbes, evolutionarily diverged eukaryotes, and semi-autonomous organelles, like mitochondria and chloroplasts, in search for novel RNA biology . We foreAS designed the study, analyzed data, wrote the original draft, and prepared the figures. MG and JV contributed to the data and reviewed the manuscript. All 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.


We show that the number of G…G (in CGG repeat) or C…C (in CCG repeat) mismatches  dictates the secondary structural choice of the sense and antisense strands of the FMR1 gene and their corresponding transcripts in fragile X-associated tremor/ataxia syndrome (FXTAS). The circular dichroism (CD) spectra and electrophoretic mobility shift assay (EMSA) reveal that CGG DNA (sense strand of the FMR1 gene) and its transcript favor a quadruplex structure. CD, EMSA and molecular dynamics (MD) simulations also show that more than four C…C mismatches cannot be accommodated in the RNA duplex consisting of the CCG repeat (antisense transcript); instead, it favors an i-motif conformational intermediate. Such a preference for unusual secondary structures provides a convincing justification for the RNA foci formation due to the sequestration of RNA-binding proteins to the bidirectional transcripts and the repeat-associated non-AUG translation that are observed in FXTAS. The results presented here also suggest that small molecule modulators that can destabilize FMR1 CGG DNA and RNA quadruplex structures could be promising candidates for treating FXTAS.CGG tandem repeat expansion in the 5′-untranslated region of the  Such a catastrophic DNA damage has consequences within many biological processes, such as replication, transcription, repair, and recombination processes, leading to several neurological disorders. When the number of trinucleotide repeats exceeds the threshold, it forms an unusual nucleic acid conformations4. One such example is CGG trinucleotide repeat expansion in the fragile X mental retardation-1 (FMR1) gene.The eukaryotic genome comprises ubiquitous repetitive sequences, namely microsatellites. Although microsatellites can be tracts of repetitive nucleotides with the lengths varying between 1 and 6, certain trinucleotide microsatellite is prone to undergo expansion, resulting in a variety of genetic disordersFMR1 gene encodes for fragile X mental retardation protein (FMRP), which is an RNA binding protein and is essential for the brain development5. The 5′-untranslated region (5′ UTR) of the FMR1 gene has CGG tandem repeats, and when the repeats expand beyond 200, this leads to fragile X syndrome (FXS). The prevalence of FXS is approximately 1 in 4000 males and 1 in 8000 females, and it populates ~ 30% of all X-linked disorders6. Nonetheless, when the CGG repeat number lies between 55 and 200 in premutation carrier individuals, it leads to fragile X-associated tremor/ataxia syndrome (FXTAS)7. One in 150–300 and 400–850 women and men, respectively, in the general populations are found to be the carriers of the FMR1 premutation state9. Among the premutation carrier individuals, about 40–75% and 16–20% of males and females, respectively, develops FXTAS at an older age10.11, complex RNA misprocessing mechanisms can be observed in FXTAS12. A bidirectional transcription of the FMR1 gene and concomitant repeat-associated non-AUG translation (RAN) are among these misprocessing mechanisms. The unusual secondary structural choice of CGG (sense strand) and CCG (antisense strand) repeats at both the DNA and RNA levels could be traced to this RNA misprocessing. However, there are controversial evidences on the secondary structural preference of DNA and RNA strands consisting of CGG repeats16. Although some studies suggest a hairpin structure formation17, the others favor quadruplex formation18, wherein 4 guanines engaged in a Hoogsteen base pairing stack onto each other in a helix. Similarly, the complementary CCG repeat can favor a four-stranded i-motif structure19, wherein the cytosines are engaged in C+…C (at acidic pH) or C…C (at non-acidic pH) base pairing in an intercalating fashion. However, the secondary structural preference for CCG repeats in the context of a number of repeats remains elusive22. Coincidently, fragile XE syndrome (FRAXE), an X-linked disorder, is caused by the abnormal expansion of CCG triplet repeats that are present in the 5′ UTR of FMR2  gene25. The protein encoded by the FMR2 gene acts as a transcription factor that is essential for the cognitive development5. The number of CCG repeats in the FMR2 gene occurs between 60  and 200 in the premutated state, whereas it occurs above 200 in the full mutated state26.Although the total inhibition of FMRP is seen in FXSIn the current study, we investigate the secondary structural choice of CCG and CGG repeats from the perspective of addressing the molecular basis of FXTAS by employing molecular dynamics (MD) simulations, circular dichroism (CD), and electrophoretic mobility shift assay (EMSA). The results show the preference for a quadruplex by both the CGG sense strand (DNA) and the sense transcript (RNA). Interestingly, although the antisense CCG strand favors the hairpin structure, the antisense transcript prefers the i-motif/i-motif conformational intermediate structure. Such a noncanonical secondary structural choice may be the underlying molecular cause for the RNA misprocessing in FXTAS. The mechanism proposed here, which is based on the secondary structural choice of CGG (quadruplex) and CCG  repeats, explains the neurotoxicity observed in FXTAS.MD, EMSA, and CD investigations have been carried out to explore the association between the repeat number and secondary structural preference for the DNA and RNA sequences consisting of the CGG and CCG repeats Table .Table 1C27)  to form intramolecular quadruplex structures . In fact, the migration speed of RG5 is intermediate to that of RG1 (duplex) and RG6 (quadruplex), indicating that RG5 may take up an intermediate conformation. This result supports the CD data, which indicate that RG1, RG5, and RG6 take up A-form, intermediate, and quadruplex geometries, respectively  can be tolerated in the CCG DNA duplex  and 6 (DC6) C…C mismatches, respectively, show stable B-form geometry over the 100 ns simulations. The RMSD value calculated with respect to the initial model stays ~ 4 Å during the entire simulation  also supports the formation of B-form geometry with a positive peak around ~ 285 nm and a negative peak around ~ 260 nm, irrespective of pH   that possesses the canonical base pairs indicate the presence of B-form conformation at various concentrations of KCl and NaCl. This can be seen by a positive peak at ~ 270 nm and a negative peak at ~ 250 nm , d(T)15 (lane 2), and d(T)10 (lane 3), which is indicative of the formation of an intramolecular-folded conformation at pH 5  at pH 5. Thus, more number of C…C mismatches can be tolerated in a B-form geometry without inducing much structural distortion in the helix, as seen in the MD simulations  and RNA  sequences. The results reveal that DC6 lane 4) and DC5 (lane 5) migrate faster than the single-stranded d(T) & DC6 anH 5 Fig.  at pH 5, and DC5 FMR1 gene leads to neurodegenerative disorders such as FXS , FXTAS, fragile X-associated primary ovarian insufficiency (FXPOI), and fragile X-associated diminished ovarian insufficiency (FXDOR)40. The occurrence of CGG repeats in the range of 55–200 (premutated state) and above 200 (full mutated state) in the noncoding region of FMR1 gene result in FXTAS/FXPOI/FXDOR and FRAXA, respectively41. Further, CGG expansion in the intronic regions of the Zinc finger protein 713 (ZFN713) and AF4/FMR2 family member 3 (AFF3) genes leads to fragile site 7A (FRA7A) and fragile site 2A (FRA2A), respectively41. In the FRAXA (the full mutation state), hypermethylation of CpG islands43 switches off the transcription and translation of the FMR1 gene44, resulting in the loss of gene function.CGG repeat expansion associated with the 5′ UTR region of the FMR1 gene are nonmethylated42, and complex mechanisms are shown to be involved in the pathogenesis of FXTAS. Neuropathology of FXTAS predominantly includes altered RNA processing, such as bidirectional (sense and antisense) transcription of the CGG repeat region45, aberrant RNA splicing12, formation of repeat RNA foci through the sequestration of RNA-binding proteins (RBPs)49, RAN translation to produce homopolypeptide aggregates corresponding to both sense and antisense transcripts50 and reduced51 translation of the gene product (loss of gene function). For instance, toxic mRNA gain-of-function takes place in FXTAS, as revealed by the elevated expression of FMR1 mRNA46, along with the diminished expression of FMRP11. FMR1 mRNA intranuclear inclusion is also found in brain tissue isolated from the post-mortem of FXTAS patients 46 and in mouse models48. In addition, the antisense FMR1 CCG mRNA is shown to have elevated expression in FXTAS patients, which is similar to the sense CGG mRNA45. RAN translation of both sense and antisense transcripts of FMR1 mRNA produce toxic poly P, poly R, poly A, and poly G aggregates as ubiquitin-positive inclusions50. Indeed, poly G and poly A aggregates produced due to RAN translation in the FMR1 gene are found in Drosophila, cell cultures, and mouse models, as well as in FXTAS patient’s brain as ubiquitin-positive inclusions55.In sharp contrast, in the FXTAS (the premutation state), the CpG islands in the FMR1 sense and antisense strands and their mRNA transcripts in the above-mentioned biological alternations, there is no precise information about their secondary structural choice. In the current investigation, we are exploring the influence of the number of noncanonical base pairs on the secondary structural preference of CGG and CCG repeats to provide a structural basis of FXTAS by employing CD, MD, and EMSA techniques.Although one can envisage the role of unusual secondary structural preference by the expanded CGG/CCG repeat in 13 and hairpin15 structures. For instance, one of the earlier studies on d(CGG)n=2,4,8,16 repeats shows the formation of a quadruplex structure at higher concentrations of K+ ions17. Both quadruplex56 and hairpin 14 structures are observed for RNA sequences with CGG repeats in the range of 17 and 20. Yet another biophysical study shows that RNA sequences that contain 19 to 45 CGG repeats can form stable hairpin structures in the presence of an AGG interrupt57. Until now, 6 crystal/solution structures of CGG repeat(s) have been deposited in the PDB. These include one DNA (PDB ID: 4HIV) and four  RNA structures that have 1 to 3 CGG repeats and are shown to form a hairpin structure. In contrast, DNA sequences that have 2 CGG repeats connected by 3T’s (loop) are shown to form a bimolecular antiparallel G-quadruplex structure (PDB ID: 1A6H). Thus, the influence of the repeat number in deciding the secondary structure of the expanded CGG repeat still remains unclear.CGG sequences are shown to take quadruplex4C2)4 sequence in an earlier study, which is indicated by the presence of positive peaks at ~ 265 nm and ~ 200 nm58. The inability to form any stable conformation at low concentrations of KCl and in the presence of NaCl  forms a parallel quadruplex structure  as also confirmed by CD spectra . Thus, the reluctance to take up a duplex conformation by CGG sequences with more number of CGG repeats perhaps due to the sequence effect rather than the nonisostericity of G…G base pairs with the flanking canonical base pairs. It is noteworthy that CD spectra corresponding to the canonical base pairs (DWCa & RWCa) show the formation of B-form and A-form geometry, respectively, for the DNA and RNA in the presence of KCl and NaCl 17 forms a hairpin structure, which is the least stable among all the CNG  repeats14. Similarly, an earlier study suggests that RNA sequences with 2 CCG repeats are prone to form a hairpin structure21. The CD spectra show that d(CCG)12 takes up a B-form conformation, but it changes to a Z-form duplex in the presence of aluminum ions68. Apart from the hairpin/duplex structure20, the CCG repeats can also favor i-motif structures at acidic pH22. The i-motif structure consists of two intercalating C…C base pair mismatches that are formed by 4 different strands at acidic pH70. This four-stranded i-motif structure has been reported for a d(T(CCG)3A) sequence that is stabilized by C…C+ and G…G mismatches22. In contrast, d(CCG)272, d(GCC)373, d(CCG)1520 are prone to adopt an ‘extrahelical’ structure in the minor groove side of the duplex, the so called e-motif structure. In fact, structural studies of short oligonucleotides that contain CCG repeats report the preference for duplex , e-motif (PDB ID: 1NOQ with 2 CCG repeats in a DNA sequence), and i-motif (PDB ID: 4PZQ with 3 CCG repeats in a DNA sequence) structures. However, the above studies do not clearly pinpoint the structural basis for the conformational choice of CCG repeats. One can envisage that the number of C…C mismatches can play a role in deciding the secondary structure of CCG repeats. Thus, to investigate the tolerance for the maximum number of C…C mismatches in a DNA duplex and an RNA duplex, MD simulations carried out for duplexes with one to six C…C mismatches  and 6 (RC6&RC6a) C…C mismatches are left unpaired because of the movement of the cytosines toward the major groove or the minor groove ove Fig. B, S7C an75 and for RC5 18 at pH 5, 7, and 9  forms a parallel quadruplex conformation. In line with this, earlier studies have shown that bimolecular quadruplex telomeric DNA-binding protein 42 (qTBP42) and unimolecular quadruplex telomeric DNA-binding protein 25 (uqTBP25) recognize and destabilize d(CGG) tetraplex83. Similarly, cationic porphyrin TMPyP4 is found to destabilize d(CGG) tetraplex84. Such a quadruplex formation in the FMR1 gene  A2 or CArG-box binding factor A (CBF-A) (CGG quadruplex destabilizing proteins) significantly raises the efficacy of (CGG)99 mRNA translation in HEK293 cells, while the mutants of hnRNP A2 or CBF-A that lacks quadruplex-disrupting activity does not promote (CGG)99 mRNA translation56. Strikingly, hnRNP A2 is one among the protein found in the FXTAS inclusion91 along with the FMR1 mRNA itself46. Interestingly, TMPyP4, which can unfold an extremely stable quadruplex92, is shown to cooperate with hnRNPs and increase the translational efficiency of fragile X premutation mRNA93. These clearly support in vivo quadruplex formation in the premutated CGG toxic RNA. FMRP, which is shown to bind to the parallel G-quadruplexes94, is also shown to recognize its own CGG mRNA95. Further, quadruplex formation may result in the aberrant translation of FMR1 mRNA and may lead to RAN translation of polyG, polyA, and polyR, which are found in the ubiquitin-positive inclusion in the human brain of FXTAS patients96. A study has revealed that piperine, a known quadruplex-binding compound97, is shown to be effective in improving r(CGG)-associated splicing and RAN translation in a FXTAS cell model system98. Considering this, it is evident that quadruplex formation in FMR1 transcript may be a cause for FMR1-premutation-associated diseases. Indeed, G-quadruplexes are generally found in a high density in the 5′ UTRs and play a regulatory role in post-transcriptional events99. In line with this, CGG repeats are found in the 5′ UTR of FRM1 gene, which upon expansion forms G-quadruplex structure. One can envisage that such a quadruplex formation may thus lead to aberrant post-transcriptional events and may be the cause of the RNA misprocessing events observed in FXTAS. Although some studies have shown that both RNA and DNA CGG repeats can form a hairpin structure, one cannot rule out the possibility that 2 such hairpins can come together and form an antiparallel quadruplex structure, as found in the atomic structure of DNA (PDB ID:1A6H) quadruplexes. Here, the quadruplex is stabilized through CGCG and GGGG quadrats instead of CCCC and GGGG quadrats, which are found in the parallel/hybrid quadruplex conformations  has also been prone to form a quadruplex Fig. C. The foFMR1 mRNA and antisense mRNA leading to RAN translation of polyG, polyA, and polyP, which are found in the ubiquitin-positive inclusion in the human brain of FXTAS patients55. Although d(CCG) favors the hairpin structure, the formation of bi/multimolecular i-motif structures cannot be ignored in the FMR1 premutated state, as reported earlier22. Thus, the pathogenic mechanisms presented here for FXTAS provide a convincing rationale for the molecular basis for FXTAS, as illustrated in Fig. FMR2 (AFF2) gene, which is associated with FRAXE syndrome, is shown to exhibit RAN translation in the premutated state in the Drosophila model100. Thus, the results presented here could be extended to FRAXE as well.The results presented in the current study also reveal the formation of the i-motif conformational intermediates structure by the antisense transcript. Similar to a quadruplex, such an i-motif or i-motif conformational intermediates secondary structure may also facilitate RNA foci formation and RAN translation Fig. B′–E′. ThFMR1 gene and the corresponding sense transcript form a quadruplex structure instead of a hairpin/duplex structure. Further, the corresponding antisense strand (CCG) has been shown to prefer a hairpin structure, and the antisense transcript is shown to prefer i-motif conformational intermediates structure due to its intolerance to more number of C…C mismatches in an A-form duplex. As quadruplex and i-motif structures are shown to be involved in transcriptional regulation, these secondary structural preferences reported here may have a role in altered the RNA processing and RAN translation seen in FXTAS. Combining the results presented here with the existing in vivo and in vitro data, we have presented here a convincing model that explains the neuropathology of FXTAS.The results presented here illustrate that CGG repeat expansion in the www.pymol.org, Schrödinger, LLC). The sequences were designed in such as a way that the mismatch containing CCG repeat should be flanked by equal number of CCG repeats on both the sides. This can be visualized from the sequences given Table 101 they were eventually the same. Thus, to further capture the precise information about the influence of 4 and 6 C…C mismatches, an additional scheme (RC6a), an extension of RC6 scheme was designed. The scheme RC6a was designed in such a way to have an additional CCG repeat on both the sides of the helix to capture the pure effect of 6 C…C mismatches. All the sequences used in the MD simulations were designed in the perspective of capturing the influence of number of C…C mismatches. However, such a variety of sequences were not considered in the case of CCG DNA since there was no significant structural deformation observed between different schemes (DC1 and DC6 which were designed to have different number of C…C mismatches) during the MD simulation. The modeled duplexes were refined using constrained-restrained molecular geometry optimization using XPLOR-NIH102. Subsequently, the duplexes were solvated with a TIP3P water box and net-neutralized with Na+ counter ions. MD simulations were carried out under isobaric and isothermal conditions with SHAKE (tolerance = 0.0005 Å) on the hydrogen, a 2 fs integration time, and a cut-off distance of 10 Å for the Lennard–Jones interaction using the AMBER 12 suite103. The simulation was carried out at the neutral pH. The FF99SB force field  enabled through FF99SB option) was used for the simulation. The systems were initially equilibrated for 50 ps, following which the production runs were extended to 100 ns individually for the DNA and RNA duplexes, as given in Table 104. These duplexes were subsequently subjected to MD simulation following the protocol mentioned above. See Supplementary file for the details.The initial models for the various DNA and RNA CCG duplexes Table  were man105 of AMBER 12 was used to post-process the MD simulation trajectories of the various DNA and RNA duplexes considered for the current investigation (Table www.mathworks.com) software was used for plotting the graphs. Note that the two terminal residues at the 5′ and 3′ ends of the duplex were not considered for the analyses.The Ptraj and cpptraj modules†” in Table 106.HPLC grade DNA and RNA oligonucleotides with CCG and CGG repeats . The data were collected in triplicate in the wavelength region of 200–320 nm and the baseline correction was done with respect to the appropriate buffer. All CD spectra corresponding to the triplicate average are reported here.For the CGG samples, polyacrylamide gel electrophoresis (PAGE) was carried out using a 14% gel. The electrophoresis was carried out at 60 V for 3.5 h under cold conditions (4 °C). 1X TAE buffer was used to prepare the gel and the running buffer. Both the DNA and RNA samples were prepared with different concentrations of KCl (0.05 M to 3 M) and 50 mM Tris–HCl buffer (pH 7.4). Subsequently, a 25 µM concentration of the CGG RNA and DNA samples were mixed with 25% glycerol and loaded into the well. After running the electrophoresis, the PAGE gel (pretreated with ethidium bromide (EtBr)) was photographed under UV light using chemiDoc™ XRS from Biorad.To run the electrophoresis for the DNA and RNA CCG samples, 10% polyacrylamide gel was prepared using 1× TAE buffer . Both the DNA and RNA samples were prepared in 50 mM NaCl and 50 mM Tris–HCl (pH 7 and 9) or Tris–acetate buffer (pH 5). As before, a 25 µM concentration of the CCG RNA and DNA samples was mixed with 25% glycerol and then loaded into the well; 1× TAE buffer  was used as the running buffer. Stains All (sigma) dye was used to stain the gel and photographed under a normal white light digital camera.Supplementary Information.