For a subset of 31 cases, we integrated the findings from RNA sequencing (RNA-seq) analysis to include fusion gene detection and classification based on gene expression. Compared with RNA-seq, WGS was sufficient to detect and resolve recurrent genetic subtypes; however, RNA-seq can provide orthogonal validation of findings. In conclusion, we demonstrated that WGS can identify clinically relevant genetic abnormalities missed with SoC testing as well as identify leukemia driver events in virtually all cases of B-other ALL.Genomic profiling during the diagnosis of B-cell precursor acute lymphoblastic leukemia (BCP-ALL) in adults is used to guide disease classification, risk stratification, and treatment decisions. Patients for whom diagnostic screening fails to identify disease-defining or risk-stratifying lesions are classified as having B-other ALL. We screened a cohort of 652 BCP-ALL cases enrolled in UKALL14 to identify and perform whole genome sequencing (WGS) of paired tumor-normal samples. For 52 patients with B-other, we compared the WGS findings with data from clinical and research cytogenetics. WGS identified a cancer-associated event in 51 of 52 patients, including an established subtype defining genetic alterations that were previously missed with standard-of-care (SoC) genetics in 5 of them. Of the 47 true B-other ALL, we identified a recurrent driver in 87% (41). A complex karyotype via cytogenetics emerges as a heterogeneous group, including distinct genetic alterations associated with either favorable ( BCR::ABL1,KMT2A fusion, low hypodiploidy, or complex karyotype to more intensive therapies, such as allogeneic stem cell transplant.,At diagnosis, the genetic classification of adult B-cell precursor acute lymphoblastic leukemia (BCP-ALL) is based on the detection of structural chromosome alterations and altered ploidy states, typically assessed via cytogenetic analysis, fluorescence in situ hybridization (FISH), or reverse transcriptase polymerase chain reaction. These diagnostic strategies inform treatment decisions through the identification of targetable lesions, such as KMT2A-r, low hypodiploid, and complex karyotype and identified JAK-STAT abnormalities as a new high-risk genetic subgroup.UKALL14 is a UK National Cancer Research Institute Adult ALL group study in which patients were stratified based on the Moorman cytogenetic risk classification.Recent comprehensive profiling approaches using whole transcriptome sequencing have been instrumental in the discovery of disease defining alterations in ALL.CRLF2, JAK2, ABL1, ABL2, and PDGFRB) and MLPA .The patients were treated in a randomized trial for adults  with newly diagnosed ALL . Trial participation and correlative research were supported with written informed consent. The study received institutional review board approval. To identify patients with B-other ALL, we performed central review of genetic data collected through SoC testing  as well as research profiling, including FISH (WGS was performed (2× 150 bp) to a target read depth of 60× and 30× for the tumor and healthy samples, respectively. RNA sequencing (RNA-seq) was performed using oligo deoxy-thymidine pulldown for a target coverage of 50 million reads (2 × 75 bp).The WGS and RNA-seq data were aligned to GRCh37d5 using BWA-MEM  and STARhttps://github.com/ThomasBeder/ALLCatchR) classifiers. Sample clustering with the City of Hope B-ALL reference gene expression data set from the MD-ALL R package (https://github.com/gu-lab20/MD-ALL) was used to finalize subtype classification. RNA fusions were detected using FusionCatcherData from 33 RNA-seq cases were analyzed to classify ALL subtypes using the consensus of any 2 of the ALLSorts,IGH locus, we used gGnome (https://github.com/mskilab/gGnome) to construct a graphical representation of the SVs detected in the tumor sample. For each graph, the gGnome “proximity” function was used to “walk” from the Eμ IGH superenhancer locus to genes in GENCODE version 29.,,To detect enhancer hijacking events associated with the DUX4) were manually reviewed using Integrative Genomics Viewer.For DUX4r detection, we used the GRIDSShttps://meme-suite.org/meme/tools/meme.,For single base substitutions (SBSs), mutation signature analysis was performed using the R/Bioconductor MutationalPatterns (version 3.5.6) packageU test was used for between groups comparison of continuous variables using the wilcox.test function.R version 3.6.3 (29 February 2020) was used; Mann-Whitney Comprehensive profiling via WGS characterized arm-level and focal copy number alterations (CNAs), SVs, and acquired somatic and germ line mutations, mapping at least 1 aberrant somatic lesion in 52 of 57 B-other ALL samples in our cohort A. The reArm-level CNAs were the most common class of alteration , followed by focal deletions (<10 Mb) in tumor suppressor genes , and acquired somatic mutations in cancer genes . Most cases  had focal deletions, 73% (38/52) had acquired somatic mutations targeting established cancer genes, 58% (30/52) had recurrent gene rearrangements, and 57% (28/52) harbored arm-level CNAs A.TCF3::PBX1) cases. Cytogenetic analysis of these 5 cases either failed (n = 2) or produced a normal karyotype (n = 3). The median inferred tumor purity for these cases was 46% , compared with 89%  for the remaining patients (n = 47). Of note was the near triploidy case (PD37195), with a germ line TP53  mutation  cases, with an established World Health Organization (WHO) 2016 genetic subtype that was missed by SoC testing : 3 were mutation .DUX4 rearrangements , PAX5alt (n = 7), PAX5 P80R (n = 4), ZNF384 rearrangements , ZEB2/CEBP (n = 4), MEF2D rearrangements , UBTF::ATXN7L3 fusions (n = 2), IDH1/2 mutations (n = 2), and Ph-like JAK-STAT abnormalities (n = 2), along with single cases of IGK::BCL2, IGH::CEBPA, IGH::ID4, and IGH::MIR125B1  cases to be classified into the following subtypes: MIR125B1 . With thMYO18A::FGFR1 fusion [t], which most likely maps to the WHO 2016 myeloid/lymphoid neoplasms with FGFR1 rearrangement classification.PTPN2 p.A108fs∗5 and WT1 p.V371fs∗14, respectively). PD40803 involved a somatic ETV6 p.R399C previously reported as a germ line riskKDM6A (ASXL1 p.R417∗ (PD40826). Lastly, WGS failed to identify a putative driver in 1 case (PD40831), with an estimated tumor purity of 28% (The remaining 6 (13%) B-other cases could not be classified into a recognized B-other subtype . Case PDiskKDM6A , and 1 cy of 28% .To evaluate the diagnostic utility of WGS compared with that of RNA-seq, we compared the diagnostic findings in 31 cases using both WGS and RNA-seq data . WGS anaIGH::DUX4 rearrangements. In a single case with P2RY8::CRLF2 (PD40808), RNA classification was split between Ph-like (ALLCatchR and ALLSorts) and iAMP21 (City of Hope reference data set). In this case, WGS analysis only identified a single chr21 gain rather than a high-level amplification,In 19 of the 31 cases, the underlying lesion contained genomic rearrangement. Evaluation of RNA-seq classification based on gene expression and fusion detection  and 12 cIn 3 cases , additional RNA fusions were not concordant with the consensus RNA classifier subtype , which wZEB2 H1038R, 3 × PAX5 P80R, 2 × PAX5alt, 2 × IDH2 R140Q, 1 × ETV6 R399C, and 1 × WT1 frameshift), RNA classification was assigned to 12 of 13 cases leaving the ETV6 R399C case (PD40803) unclassified in both WGS and RNA-seq. Of the 12 classified cases, 9 were concordant between the WGS and RNA-seq–derived subtypes (IDH2 R140Q mutations (PD40809 and PD40814), and 1 had a frameshift WT1 mutation (PD40836). All 3 cases were classified as PAX5alt by 3 of the 4 RNA classifiers; in comparison, 3 cases with bona fide PAX5 alterations were classified as PAX5alt by 4 of the 4 classifiers  of cases with normal karyotypes had either a cytogenetically cryptic genomic rearrangement (7/16) or an altered ploidy class (2/16).Cytogenetics analysis of the cohort (n = 52) revealed a normal (n = 19), nonspecific abnormal (n = 23), complex (n = 8), or failed karyotype (n = 2). These cytogenetic categories represent highly heterogeneous genetic subgroups A. WGS anIGH::ID4 B. There DUX4-r cases harbored the canonical IGH::DUX4 rearrangement (7/8), but only 1 case had ERG::DUX4 , which has been reported in up to two-thirds of cases with pediatric DUX4-r ALLDUX4-r were female compared with 17 of 44 in the study cohort(39%). Unlike prior observations in both pediatric and adult ALL, suggesting that DUX4-r was associated with favorable outcome,,Most PD40815; . Only a PAX5 abnormalities mapping to PAX5alt (n = 7) and PAX5 P80R (n = 4) subtypes. Notably, 10 of the 11 cases showed evidence of biallelic targeting by either 2 mutations (n = 4) or a single mutation/fusion and concomitant LOH , and FOXP1::PAX5 presented as add(9)(p13). The other 5 PAX5alt contained 3 cases with R38 and R140 biallelic mutations, 1 case harboring an intragenic PAX5 amplification  and AKAP8::ZNF384 (n = 1). We recently reported that patients with ZNF384 are typically younger and have good outcomes.Five cases had a ZEB2/CEBP group: 2 cases with ZEB2 H1038R and concurrent IGH::CEBPB rearrangements and 2 cases with ZEB2 H1038R alone. A fifth ZEB2 H1038R mutation was detected in an IGH::DUX4 case ; however, RNA-seq classified this sample as DUX4-r, and the remaining 4 ZEB2 H1038Rs were all classified as ZEB2/CEBP  and exhibited copy number oscillation involving multiple chromosomes , indicative of chromothripsis (MEF2D::BCL9 (PD40810a) failed cytogenetics and had a relatively stable genome  had a  of CDX2 .40,41 Twutations , a subtyIGH x12 and IGK x1). In addition to the aforementioned DUX4-r (n = 7) and ZEB2/CEBP (n = 2) cases, WGS analysis revealed 4 additional cases of IGK::BCL2, IGH::CEBPA, IGH::ID4, and IGH::MIR125B1. The patient with IGK::BCL2 (PD40797) was classified to be at high-risk  and, although achieving remission after induction, responded poorly to therapy dying shortly after diagnosis (<200 days). The detection of this abnormality at the initial diagnosis might have prompted the re-evaluation of the diagnosis.Lastly, 13 cases involved the hijacking of an immunoglobulin gene enhancer (DUX4 locus were identified (IGH::DUX4 cases (PD37188a and PD40804a). WGS showed evidence that in both cases the 5′ IGH locus translocation mapped to telomeric repeats, whereas the 3′ IGH translocation mapped to the DUX4 loci. Therefore, we can infer that, in these 2 cases, the DUX4 locus along with proximal telomeric repeats was inserted into the IGH locus in an inverted orientation, which confounds detection by RNA fusion calling (ERG::DUX4 (PD40815a) case also showed expression of an alternative exon 6  had indirect evidence from FISH and karyotype, showing a 14q32 deletion correlating to the loss incurred due to an unbalanced translocation to form IGH::ID4  for WGS analysis , with 49 of 69 deletions attributable to RAG activity  enriched in SBS1, a phenotype previously observed in hypermutator cases.,,IGH::CEBPB and 1 IGK::BCL2 case with the highest mutation burden (42%), which we had expected, given the association of this lesion with mature B-cell neoplasms. Lastly, SBS18, a signature dominated by C>A mutations and attributed to reactive oxygen species, was enriched in the MEF2D-r, PAX5, and IDH1/2 subtypes.Analysis of SBS patterns for the established mutation signatures revealed that the majority of SNVs were mapped to the SBS blood signature B-C. SBS The diagnostic work up for adult ALL does not incorporate WGS in most of the centers worldwide. Here, we assessed the utility of WGS profiling in 52 adult ALL cases, that in the absence of informative biomarkers by SoC were classified as B-other.,,ZNF384-r) or poor  outcome.,WGS assigned 88% (46/52) of the cases called B-other to an established genetic subtype of ALL, with ∼20% (10/46) of subtypes being assigned solely via the novel WGS workflows developed in this study. This included 5 cases with WHO 2016 subtypes; 3 of the 5 cases harbored high-risk genetic events that would have changed their UKALL14 risk group and postinduction treatment in the absence of other risk factors. Among the remaining B-other cases, 87% (41/47) were assigned to 1 of the newly described genetic subtypes of ALL.,,DUX4-r) and poor (MEF2D-r and UBTF::ATXN7L3) outcomes. This suggests that a complex karyotype is not a robust classification.Our findings demonstrate that unless cytogenetics reveals an established genetic rearrangement or ploidy subtype, a designation of failed, normal, or complex karyotype frequently misses subtype defining events that can readily be picked up by WGS, as was also demonstrated in our related studies on childhood ALL.IGH::DUX4 case involved a hypermutation caused by homozygous loss of MSH6, a candidate for checkpoint inhibition. A second case involved homozygous loss of CD58 and concurrent LOH of HLA-B, which would likely confer immune escape,,TP53 mutation, which has implications for carrier screening.The WGS also identified events that could alter clinical management. One Integration of RNA-seq for fusion gene detection and gene expression classification enables concomitant validation of WGS based findings using an orthogonal assay. This is of particular importance for transcriptomically defined phenocopy subtypes, such as Ph-like or PAX5alt, for which WGS alone would only be able to evidence previously defined recurrent DNA lesions. RNA-seq can also directly identify fusions caused by complex SVs that are missed in standard WGS fusion calling.Because this was a retrospective study, we used remission samples to source germ line DNA. To avoid tumor contamination, we selected samples with MRD-negative results or MRD <1%. Therefore, the observed genomic subtype frequencies may not reflect the true distribution in adult B-ALL. In this study, we focused on cases that did not harbor informative clinical biomarkers and were classified as B-other. We did not formally assess the performance of WGS and RNA-seq against SoC in patients for whom the ALL subtype was previously determined.For clinical implementation, detailed laboratory analytical and clinical validity studies are warranted to delineate standardization metrics for diagnostic assay deployment and the optimal source of normal DNA to determine assay performance against SoC molecular diagnostic assays across the spectrum of B-ALL subtypes.10.13039/100004325AstraZeneca, GSK, 10.13039/100004319Pfizer, MSD, Bristol Myers Squibb, Amgen, and Millennium Takeda. P.C. is a cofounder, stock holder, and consultant for FL86 Inc. B.P. is on the advisory board for Pfizer. A.V.M. has received honoraria from Amgen. A.K.F. has served as a consultant for Amgen, Pfizer, and Novartis. E.P. is a founder, equity holder, and holds a fiduciary role in Isabl Inc and is an equity holder in TenSixteen Bio. The remaining authors declare no competing financial interests.Conflict-of-interest disclosure: G.G. is a consultant at Isabl Inc. M.F.L. is an employee and equity holder at Isabl Inc. J.S.M.-M. is the founder and equity holder at Isabl Inc. A.A.K. has received honoraria from Kite. L.C.-H. on behalf of the Cancer Research UK and University College London Cancer Trials Centre has received research funding from The current affiliation for S.L. is St George’s University Hospitals NHS Foundation Trust, London, United Kingdom.The current affiliation of K.Z.-A. is The Royal London Hospital, Barts Health NHS Trust, London, United Kingdom.


Sequences flanking the rRNAs are complementary and form long helices known as leader-trailer helices. Here, we employed an orthogonal translation system to interrogate the functional roles of these RNA elements in 30S subunit biogenesis in Escherichia coli. Mutations that disrupt the leader-trailer helix caused complete loss of translation activity, indicating that this helix is absolutely essential for active subunit formation in the cell. Mutations of boxA also reduced translation activity, but by only 2- to 3-fold, suggesting a smaller role for the antitermination complex. Similarly modest drops in activity were seen upon deletion of either or both of two leader helices, termed here hA and hB. Interestingly, subunits formed in the absence of these leader features exhibited defects in translational fidelity. These data suggest that the antitermination complex and precursor RNA elements help to ensure quality control during ribosome biogenesis.Ribosome biogenesis occurs co-transcriptionally and entails rRNA folding, ribosomal protein binding, rRNA processing, and rRNA modification. In most bacteria, the 16S, 23S and 5S rRNAs are co-transcribed, often with one or more tRNAs. Transcription involves a modified RNA polymerase, called the antitermination complex, which forms in response to   Escherichia coli, the large subunit consists of the 23S rRNA [2904 nucleotides (nt)], 5S rRNA (120 nt) and 34 ribosomal (r) proteins, while the small subunit consists of the 16S rRNA (1542 nt) and 21 r proteins. Ribosome assembly occurs co-transcriptionally and entails folding of the rRNA, binding of r proteins, modification of nucleotides and amino acid side chains, and processing of the rRNA by various RNases. Active ribosomal subunits can be reconstituted in vitro by mixing their constituent components  facilitate the process  sites, composed of cis-acting RNA elements boxA, boxB and boxC of the nascent RNA chain, recruit N and host-encoded Nus (N-utilization substance) factors to RNA polymerase (RNAP) , which exhibits enhanced elongation processivity. Antitermination has been most extensively characterized in lambdoid bacteriophages, which use phage-encoded protein N (or Q) to regulate TAC formation. N-utilization (e (RNAP) ,17. Prottem-loop , and Nusop  operons. In this case, modification of RNAP involves NusA, NusB, NusE(S10), NusG, SuhB, and S4, and the resulting antitermination complex is termed rrnTAC —lies at the beginning of the operon, just after promoter P2, and another lies upstream of the 23S rRNA gene . In rrnTAC, the SuhB dimer acts as a structural hub, interacting with NusA, NusE(S10), and NusG. While unrelated and structurally dissimilar to SuhB, N plays an analogous role in the case of N-TAC, interacting with both NusA and NusE(S10). In both complexes, NusA and NusG are stabilized in ways predicted to prevent RNAP pausing and inhibit backtracking, keeping RNAP in an elongation-competent state. The bound antitermination factors probably prevent Rho-dependent and intrinsic termination by blocking Rho and NusA, respectively, from accessing the RNA exit channel.Cryo-EM structures of lambda N-TAC and mination ,40. In brrnB  and qualitative  measurements of the product ribosomes, without worrying about complicating effects on cell physiology. We find that a leader-trailer helix of >17 basepairs (bp) is critical of formation of active 30S subunits in the cell. Other features, including boxA and leader helices hA and hB, are not strictly required. However, ribosomes formed in the absence of these elements are error-prone, suggesting that the antitermination complex and various intergenic RNA features contribute to the process and ensure quality control.In this work, we use an orthologous ribosome system to dissect the key functional elements of the leader and spacer regions. This system enables translation activity to be directly measured, over a >1000-fold range, without affecting cell growth ,45. Varisubunit biogenesis to refer to the entire process of creating translationally-competent particles, including rRNA transcription.Note that throughout this article, we use the term boxC  in the range from 257 nt to 90 nt upstream of the annotated 5′ end of the 16S rRNA and keeping the 3′ most match if multiple matches to boxC are found. The leader is then defined as the sequence from 23 nt upstream of the boxC motif to the 18th nucleotide of the annotated 16S rRNA. Sequences for which the total length of leader and trailer together was below 350 nt were discarded resulting in 1441 total rRNA leader/trailer pairs  or Phusion site-directed mutagenesis (New England Biolabs)  . Plasmid mansoni  were PCR mansoni .antP -SD5′-ATCCC-3′-lacZ). Mutation Δrnc::cat was moved into KLF2513 via P1 transduction, resulting in BRW298. Then, Δ(recA-srl)306 srlR::Tn10 was moved from JC14604 (E. coli Genetic Stock Center) to BRW298 via P1 transduction, resulting in the strain BRW299.Strain KLF2513 is CSH142 carrying λ(l-arabinose (5 mM) and grown for 4 h at 37°C. The cells were washed once in 1 ml Z buffer , and β-galactosidase activity was measured as described (A574)/(OD600·v·t), where A574 is absorbance at 574 nm (characteristic of the product of CPRG cleavage), OD600 is optical density of the cell suspension used, v is the volume of the cell suspension used (in milliliters), and t is time of incubation (in minutes) at room temperature. Strain KLF2674(pBW022) was used as the WT control and KLF2674(pBAD18) was used as the vector-only control.Cells from an overnight culture were diluted 300-fold into 3 ml of fresh Luria broth (LB) containing ampicillin , kanamycin , and escribed . Specifil-arabinose (5 mM) and grown for 4 h at 37°C. Cells were pelleted at 4°C, resuspended in Z buffer, transferred to a 1.5 ml Eppendorf tube, and then re-pelleted. TRIzol (Invitrogen) reagent (1 ml) was added, and the pellet was resuspended by pipetting and vortex mixing (5 min) at room temperature. Chloroform was added, mixed, then the aqueous phase was transferred to a new tube. RNA was precipitated with isopropanol then pelleted. The RNA pellet was washed with 70% ethanol and dissolved in water. Relative amounts of P-16S rRNA were determined using poisoned primer extension, essentially as previously described (Cells from an overnight culture were diluted 300-fold into fresh LB (25 ml) containing Amp (100 μg/ml), Kan (50 μg/ml) and escribed ,60. In arecA-srl)306 srlR::Tn10 into SQZ10  were grown to mid-log phase and subjected to sucrose gradient sedimentation analysis as described . The Escherichia clade structure agrees with the model of Schlessinger. However, our analysis lends no support for a conservation of base pairing between nucleotides −99 to −96 and 1554–1557 of hLT or between nucleotides −1 to −2 and 7–8 of h0 across the Escherichia clade (Young and Steitz 1978) proposed the first secondary structure model of the pre-16S rRNA 78 propos. Ten yeae family , covariae family . During e family . Xenorha0 Figure . A shortia clade .E. coli, we used an orthogonal ribosome system described previously ] resulted in complete loss of activity ] had little if any effect on translation activity ] had a larger effect, but substantial activity (∼30%) was still seen in this strain ] caused no further loss in translation activity compared to ΔboxA alone. Deletion of the leader strand of hLT only, while retaining boxB and boxA , resulted in complete loss of translation activity ] reduced translation activity by ∼30%, and deletion of hB  reduced activity by ∼40% Figure . These d2] reduceboxBA   Figure . In all ) Figure . These drrnTAC, Wahl and coworkers hypothesized that positioning of boxC close to the exit channel of RNAP is important for 30S subunit assembly  resulted in a 50% reduction in translation activity, while the 12-nt insertion (Ins_12) resulted in a 25% reduction  orthogonal ribosomes exhibited ∼15% plasmid-encoded rRNA Figure . StrainsrrnTAC on ribosomal subunit biogenesis, we moved the ΔboxB, ΔboxA and ΔboxBA mutations into the Δ7 prrn strain, which lacks chromosomal rRNA operons and is supported by a single plasmid-borne rrn operon 306 srlR::Tn10 into SQZ10, making a recombination-deficient (Rec−) Δ7 prrn strain, and then replaced the resident plasmid (pHKrrnC-sacB) with each of several rrnB-containing plasmids. In this Rec− background, pBW136 (Δ1575–1598) supported slow growth, and there was no indication of suppressor mutations. This enabled us to measure its doubling time, which was 23 min longer than the control strain   . None oflings/h) . RNA waslings/h) . An RNA assembly ,64,79. TE. coli. The fact that absolutely no functional subunits are detected in the absence of hLT came as a surprise, because mature 16S rRNA supports efficient assembly of subunits in vitro. How can this apparent discrepancy be explained? A growing body of evidence suggests that the free energy landscape of RNA folding in the cell differs from that in the test tube  might interfere with subsequent 16S rRNA processing in our system. Five enzymes have been implicated in 3′ end maturation: an endonuclease, YbeY, and four 3′-to-5′ exonucleases—RNase II, RNase R, RNase PH, and PNPase. These exonucleases act in a redundant fashion to ensure that the 3′ tail of 16S rRNA is fully trimmed . The actIn the cell, there exist pathways to target and degrade defective or damaged ribosomes. These mechanisms, while poorly understood, act on long-lived assembly intermediates and off-pathway (misassembled) particles . In the rpsE-G28E, ΔrpsO), assembly factors , or endonucleases  leads to increased rates of stop codon read-through, frameshifting, and/or spurious initiation . Two of these operons encode the Providencia I leader-trailer structure, characterized by three leader helices of 10–15 bp; whereas, the other operons encode the Providencia II leader-trailer structure, characterized by five leader helices of 3, 6, 8, 10 and >30 bp  similarly have two different leader-trailer structures represented among their rrn operons  leader-trailer helix is observed, well-supported by covariation in multiple clades. Aside from this common feature, considerable structural diversity is evident (Figure  operons . In futuThe custom scripts used during data analysis are available at doi://10.5281/zenodo.7589875.gkad316_Supplemental_FilesClick here for additional data file.


Condensed coacervate phases are now understood to be important features of modern cell biology, as well as valuable protocellular models in origin-of-life studies and synthetic biology. In each of these fields, the development of model systems with varied and tuneable material properties is of great importance for replicating properties of life. Here, we develop a ligase ribozyme system capable of concatenating short RNA fragments into long chains. Our results show that the formation of coacervate microdroplets with the ligase ribozyme and poly(L-lysine) enhances ribozyme rate and yield, which in turn increases the length of the anionic polymer component of the system and imparts specific physical properties to the droplets. Droplets containing active ribozyme sequences resist growth, do not wet or spread on unpassivated surfaces, and exhibit reduced transfer of RNA between droplets when compared to controls containing inactive sequences. These altered behaviours, which stem from RNA sequence and catalytic activity, constitute a specific phenotype and potential fitness advantage, opening the door to selection and evolution experiments based on a genotype–phenotype linkage. Many biological biomolecular condensates are formed from RNA and proteins or peptides , and coaThe ability of coacervate phases to strongly partition a wide range of molecular and macromolecular species , and to 20-spermine coacervates by polynucleotide phosphorylase has been shown to induce transient non-spherical coacervate morphologies , whose RNA ligation activity is based on 5′-triphosphate activated substrates (L) catalyses concatenation of a 31 nt substrate   . The str19-72 into a fixed concentration of RNA  and measuring endpoint activity. The reaction products were separated by urea PAGE and stained using SYBR Gold to visualise all reaction components. The activity of the ribozyme was inhibited in the presence of excess peptide ((Lys)19-72:RNA > 1) 19-72:RNA (5-24) (5-24 ≈ 0.93:1 (Lys)5-24:RNA), corroborating previous observations with poly(L-lysine) and the hairpin ribozyme (HPz) (19-72:RNA and 3:1 (Lys)5-24:RNA, both of which supported coacervation and ribozyme catalysis. Different concentration ratios are required to produce similar droplets for each peptide due to their differing critical coacervation concentrations. Both selected concentration ratios corresponded to points immediately before the plateau in the respective peptide titration turbidity curves and allowed the formation of liquid coacervate droplets without suppression of ribozyme activity. (The phase separation behaviour of the ribozyme system in the presence of poly(L-lysine) was initially investigated by titrating increasing amounts of (Lys)RNA > 1) , but yieRNA > 1) . The rep9-72:RNA . These eA (5-24) , for whime (HPz) . In thisme (HPz) . For furctivity. . Fluoresctivity. .n peptide resulted in an approximately 50-fold increase in the rate of concatenation and led to the formation of substrate chains with an average length approximately 30 nt greater than those produced in solution (k = 1.5 × 10–2 ± 1.0 × 10–3 min–1), and a final average product length (n) of 52.2 ± 0.5 nt was observed , with a final product length of n = 77.8 ± 0.4 nt for (Lys)19-72. Similar values were obtained for the short peptide  suppressed circularisation altogether, whilst the short peptide (3:1 (Lys)5-24:RNA) reduced the formation of circular products.In solution, the E strands . The preL ribozyme is capable of substrate concatenation in both solution and in (Lys)n coacervate droplets, and that its activity is enhanced within the coacervate environment, we aimed to determine whether the elongation of the RNA component would lead to differences in droplet properties, thus altering phenotype. To identify changes due solely to chain concatenation, we developed an inactive mutant of the ribozyme by introducing several point mutations , indicating that the growth was at least, in part, due to coalescence events between the droplets . The coacervate droplets containing the active ribozyme behaved as previously observed, with discrete round morphologies  and litt5-24 and (Lys)19-72 peptides to pre-reacted RNA mixtures. Here, we observed that mixtures containing the active ribozyme and therefore pre-concatenated RNA initially formed non-spherical gel-like condensates with both peptides, which relaxed to form spherical droplets over the course of 24 hr, whilst mixtures with inactive ribozyme yielded spherical droplets from the outset e outset . This suL ribozyme could affect the interactions between different populations of droplets. Using the previously established conditions and concentration ratios, we mixed populations of droplets containing either a Cy5- or FAM-tagged substrate  and monitored mixing and content exchange over the course of 24 hr. These effects can be quantified by the calculation of a Pearson correlation coefficient (PCC), which measures the correlation of pixel intensities between the two fluorescence channels droplets –3. Here,L ribozyme was able to generate RNA strands of >500 nt in length, including circular species, and this activity was enhanced in terms of both rate and yield in (Lys)n coacervates. Although the observation of circular products in concatenation reactions has been previously reported in ribozyme systems 5-24 into a fixed concentration of R3C ribozyme and substrate was similar to that observed for HPz, although in this study the resultant condensed phase appeared liquid rather than gel-like. This variation in condensed phase morphology may be due to the influence of RNA structure, sequence, and hybridisation state on condensation (L ribozyme system. The inhibition of ribozyme activity at excess (Lys)19-72:RNA ratios was comparable to that observed in our previous study  n deviates from the manufacturer’s stated size range, with the (Lys)5-24 used here being predominantly comprised of oligomers between n = 3 to n = 9 in length (We have previously reported that the activity of the hairpin ribozyme (HPz) is greatly enhanced in condensed phases comprised of catalytic RNA and (Lys)peptides . The incensation , with a us study  and is aus study . A previd 19–72) . The pren length . These sThe difference in growth behaviour between active and inactive droplets is a striking demonstration of how the presence of a catalytic species can modulate the physical properties and behaviour of a model protocellular system, in this case by reducing coalescence. The coarsening of liquid coacervate phases is not inevitable: growth can be also be suppressed by active chemical processes hosted within droplets that produce the polymer components , althougn initially form non-spherical gel-like aggregates, which then relax to form spherical droplets over the course of 24 hr.Despite the spherical morphology of the RNA–peptide condensates reported here, the inhibition of growth and wetting may also be due to a phase transition to a gel state. The formation of non-liquid RNA–peptide condensates has been previously reported for poly-rA:rU-peptide mixtures, where RNA base pairing leads to the formation of a kinetically arrested gel-like solid , as wellThe reduction of mixing between populations of droplets containing orthogonally labelled RNA likely results from the changes in material properties and growth behaviour caused by RNA concatenation. The coalescence of the coacervate droplets observed in all inactive systems is likely to be the primary mechanism of mixing in this scenario. Indeed, greater droplet growth was observed in the two-population system than for single-population experiments, which may be due to the necessity of pipetting each droplet population into the sample environment sequentially, thus increasing mechanical agitation and contacts between droplets. All populations at the initial time point contain fusion droplets, which contain separate and unmixed areas of each fluorescent RNA. Notably, fusion droplets in all systems appear evenly mixed after 24 hr, suggesting that significant equilibration occurs before any transition to a highly viscous liquid or solid gel. Mixing may also occur by diffusive transfer of RNA between droplets. As the fluorescently labelled substrate is ligated onto the growing concatemer chain, the diffusion coefficient of the labelled material is expected to decrease over the course of the reaction as its effective length increases, which would in turn slow diffusive transfer in the active system.Taken together, the results reported here describe a range of altered droplet behaviours stemming from ribozyme-driven changes in coacervate physical properties. These physical changes may impact other processes relevant to early compartmentalised replicators and protocells, such as resistance to molecular parasites  and metan condensates 6, 611.77 g/mol; Sigma-Aldrich, St. Louis, MI), formamide , ethylenediaminetetracetic acid disodium salt dihydrate , magnesium chloride hexahydrate ), sodium hydroxide , sodium chloride , urea , hydrochloric acid  (VWR), boric acid , ammonium persulfate 2S2O8, 228.20 g/mol) (VWR), acrylamide , tetramethylethylendiamine , SYBR gold stain (Thermo Fisher Scientific), RNA oligomer length standard (low-range ssRNA ladder) .Trizma base , sodium hexametaphosphate , poly-L-lysine hydrobromide . All RNA and DNA oligomers were obtained from Integrated DNA Technologies  and are listed in 2, 10 mM DTT, 2 mM spermidine, 5 mM of each NTP, 1 U/mL inorganic pyrophosphatase, and 0.5 µM T7 RNA polymerase (purified from a recombinant source in-house). The reaction proceeded for 4–6 hr at 37°C, after which the volume was concentrated by spinning at 15,000 × g, 4°C in Amicon ultrafiltration columns (Merck) with 3 kDa molecular weight cut-off regenerated cellulose filters. Following concentration, the RNA was purified with the Monarch RNA cleanup kit (NEB) following the manufacturer’s instructions, eluted twice in water, and quantified on the NanoDrop OneC (Thermo Fisher Scientific). The resulting ribozymes were gel purified using 12% urea PAGE, whereas the substrate was purified using 20% PAGE. The amount of RNA loaded per well was 5–8 µg mm–2 of the surface area of the bottom of the well. After PAGE, the gel was wrapped in plastic foil and the band of interest was identified by UV shadowing  on an autofluorescent background. The gel slice was excised, crushed, weighed, and soaked in 2 µL of 0.3 M sodium acetate (pH 5.2) per mg of gel at 4°C overnight. Following elution, the gel debris was removed using Costar Spin-X columns  with 0.45 µM cellulose acetate filters. Next, 20 µg RNA -grade Glycogen  and 1.2 volumes of cold isopropanol were added to the solution. The mixture was cooled for 1 hr at –20°C to promote precipitation, and then centrifuged for an additional hour at 21,000 × g, 4°C. The supernatant was removed, and the pellet washed twice with 0.5 volumes of cold 80% ethanol. After washing, the supernatant was completely removed, and the pellet was dried for a few minutes under vacuum and resuspended in ultrapure water. RNA was aliquoted and stored at –80°C.RNA was ordered from IDT or transcribed in-house from DNA templates. DNA sequences containing a T7 promoter upstream of the RNA sequence of interest were ordered from IDT. The DNA templates were prepared by annealing a complementary oligo to the T7 promoter region by heating equimolar mixture of the oligos at 85°C then cooling on ice. The resulting partially double-stranded DNA was used as DNA template for transcriptions. Large-scale in vitro transcription (IVT) reactions were adopted to produce enough RNA for downstream applications. The transcription reaction volume varied from 400 µL to 4 mL and contained the following: 1 µM partially double-stranded DNA template, 30 mM Tris-HCl pH 7.8, 30 mM MgClex = 520 nm for SYBR Gold, 658 nm for Cy5) and analysed with the AzureSpot software . The images were background subtracted using the built-in rolling ball function set to a diameter of 1000.Rotiphorese Gel 40 (19:1) was used to prepare 20% polyacrylamide gel stocks containing 8 M urea and 1× TBE . A 0% gel stock was prepared using the same volumes with water replacing acrylamide. The two stocks were mixed at different ratios to obtain the final desired acrylamide concentration. Polymerisation was initiated by adding 0.01 volumes of 10% APS and 0.001 volumes of TEMED. The gel was cast in an EasyPhor PAGE Maxi Wave (20 cm × 20 cm)  and allowed to polymerise at room temperature for >2 hr, then pre-run at a constant power of 20 W for 45 min. The gel thickness was 2 mm and 1 mm for preparative and analytical gels, respectively. The quenched samples were loaded, and the gel was run for 90 min at 20 W constant power in 1× TBE running buffer. When necessary, the gel was removed from the glass plates and stained with SYBR Gold (Invitrogen) nucleic acid staining dye for 10 min in 1× TBE and 1× SYBR Gold, then washed twice for 5 min in de-ionised water to decrease background fluorescence. Images were acquired using and Azure Sapphire RGB gel scanner . Ratios of RNA to (Lys)n were based on a fixed charge concentration of the RNA , calculated with the following equation:Ribozyme assays were carried out with a 10 µL total reaction volume and the following components: 50 mM Tris-HCl pH 8.6, 10.5 µM ribozyme, 10.5 µM substrate, 10 mM MgClg . PAGE analysis proceeded as described above.Positive charge concentrations were calculated based on the lysine hydrobromide monomer repeat molecular weight (209 g/mol) and mass of poly(L-lysine), considering a single positive charge for each residue. Reactions were set up at room temperature by first adding the all the components except the RNA to allow equilibration of poly(L-lysine) in the buffer. The reaction was then started by adding a mixture of ribozyme and substrate and incubated in a thermocycler at 30°C for 2 hr. Reactions were stopped by adding 1 volume of 5 M NaCl, 1 volume of 1.25 M hexametaphosphate (HMP), and 12 volumes of RNA loading buffer containing 10 mM EDTA, 0.05% bromophenol blue, 95% formamide. The resulting samples were briefly vortexed, denatured for 5 min at 85°C, cooled quickly on ice, and centrifuged for 5 min at 2000 × 2) for 3 hr at 30°C. Varying concentrations of poly(L-lysine) in 1× buffer were then added to the reacted RNA. For every poly(L-lysine) concentration tested, 2 µL of pre-reacted RNA was added to 2 µL of poly(L-lysine) and mixed by pipetting 10 times. The resulting solution was incubated for 5 min, after which absorbance was measured. At least three biological replicates were measured for each concentration. From these data, we selected specific peptide:RNA ratios for both peptides for further experimentation (0.75:1 (Lys)19-72):RNA and (3:1 (Lys)5-24:RNA). The criterion for this selection was the formation of liquid coacervate droplets (determined by turbidity measurements and microscopy) at a peptide:RNA ratio that did not supress ribozyme activity . Both concentrations occur shortly before the respective turbidity maxima in the peptide titration turbidity curve.Measurements were performed using a NanoDrop OneC (Thermo Fisher Scientific) by measuring absorbance at 500 nm as a proxy for coacervate formation. The RNAs were pre-reacted at 2× concentration (21 µM each) in 1× buffer , and either 3 mM (Lys)5-24 or 0.75 mM (Lys)19-72 charge concentration. Aliquoted reactions were quenched by the addition of 2 µL 5 M NaCl, 2 µL 1.25 M HMP, and 34 µL RNA loading buffer  at designated time points . Sample preparation and PAGE were performed as described above. The average substrate length and relative band percentages were calculated as follows:Time-dependent assays were performed using individual aliquots for each time point to mitigate the effects of coacervate adhesion to the PCR tube walls over time. The reaction mixture contained 50 mM Tris-HCl pH 8.6, 10 mM MgClF test for nested models. The simpler first-order model was rejected when p<0.05.The data were fitted in GraphPad Prism using both a first-order (‘one-phase association’) and second-order (‘two-phase association’) kinetic model. First- and second-order kinetics were discriminated between using the extra sum-of-squares 2. The reactions were incubated for 2 hr at 30, 37, and 45°C, then quenched by the addition of nine volumes of RNA loading buffer . They were resolved on an 8% urea PAGE and stained and imaged as described previously.Reactions were carried out with varying ribozyme to substrate ratios, maintaining a total negative charge of 1 mM. Reactions with component ratios of 1:1, 1:2, and 1:4 contained ribozyme:substrate concentrations 10.5:10.5 µM, 8:16 µM, and 5:20 µM, respectively. Buffer conditions were 50 mM Tris-HCl, pH 8.6, and 10 mM MgCl19-72-15 or 3:1 (Lys)5-24 and incubated for 3 hr at 45°C were recovered using a 50 µg Monarch RNA cleanup kit (NEB) and eluted in 12 µL ultrapure water. RNase R  was used to digest a portion of the purified reaction products. The digest reaction mixture (20 µL) contained 1 µg of RNA, 1× RNase R buffer, and 30 U of RNase R, and was incubated for 3.5 hr at 37°C. The digested RNA was recovered with the 10 µg Monarch RNA cleanup kit (NEB), eluted in 6 µL of ultrapure water and mixed with 6 µL of RNA loading buffer. 1.5 µL of the undigested RNA and 5 µL of the digested RNA were resolved on an 8% PAGE, stained, and imaged as described above.The reaction products from 10 µL reactions containing 1:1 ribozyme:substrate with either no poly(L-lysine), 0.75:1 (Lys)2), and were supplemented with either PEG 8000 alone, MgCl2 alone, or both in combination. All reactions comprised a 1:1 ribozyme to substrate ratio and final positive charge concentration of 1 mM . The reactions were set up on ice, started by adding the RNAs and incubated at 30°C for 10 min. After incubation, the reactions containing either PEG 8000 or MgCl2 were quenched with 20 volumes of RNA loading buffer (10 mM EDTA in formamide). For the conditions that combine PEG 8000 and MgCl2 a positive control with added (Lys)n was performed. Consequently, these reactions were quenched by the addition of 1 volume of 5 M NaCl, followed by 1 volume of 1.25 M hexametaphosphate and 18 volumes of RNA loading buffer. All samples were heat denatured for 5 min at 85°C and subsequently resolved on an 8% urea PAGE and imaged as described above.Reactions were carries out in the standard buffer system (50 mM Tris-HCl pH 8.6 and 10 mM MgCl2) with different concentrations of total RNA but maintaining the same 1:1 ratio of ribozyme to substrate. The concentration of each RNA was either 5.25 µM designated as 0.5×, 10.5 µM designated as 1×, or 21 µM designated as 2×. All reactions had 10% Cy5-tagged substrate included. The reactions were set up on ice followed by a 10 min incubation period at 30°C. The reactions were quenched with different volumes of RNA loading buffer such that the final concentration of labelled species is the same in the quenched sample buffer . The 1× reaction was quenched with four volumes of RNA loading buffer. All samples were heat denatured for 5 min at 85°C and subsequently resolved on an 8% urea PAGE and imaged as described above.Reactions were carried out in the standard buffer system . Coacervate droplets were formed by combining a solution of RNA with a solution containing poly(L-lysine), buffer and magnesium chloride and mixing using a pipette in a PCR tube. The mixture was incubated on ice for 5 min, then 5 µL was loaded into the well plate. Individual wells were sealed with a drop of silicon oil to prevent evaporation. After loading, plates were immediately incubated at 30°C. Imaging was performed as soon as the coacervate suspension had settled, typically first at 30 min after mixing. A 3 × 3 image grid, centred at the middle of the well, was captured for each sample at various time points. In most cases multiwell plates were passivated using Pluronic F-68 to prevent droplet wetting and adhesion.Microscopy was performed on a Leica Thunder inverted widefield microscope equipped with a sCMOS camera Leica DFC9000 GTC using a ×63/NA 1.47 objective. Fluorescence channels were λFollowing data collection, grid tiles were inspected, and out-of-focus images were discarded. Only images that remained in focus for all time points in a given sample were carried forward for analysis. Image masks were produced by segmentation in the brightfield channel using the CellPose algorithm  . A rolli Experimental models of simple cell-like compartments can help us to understand how biology operated early in its history. The authors convincingly show how the properties of coacervate droplets can be influenced by the activity of ribozymes inside them. This important result potentially provides a new route for biologists or chemists to establish cell mimics that support the evolution of biomolecules within. public reviews designed to be posted alongside the preprint for the benefit of readers; (ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.Our editorial process produces two outputs: (i) Decision letter after peer review:eLife. Your article has been reviewed by 3 peer reviewers, and the evaluation has been overseen by Timothy Nilsen as the Reviewing Editor and James Manley as the Senior Editor. The reviewers have opted to remain anonymous.Thank you for submitting your article "Ribozyme-phenotype coupling in peptide-based coacervate protocells" for consideration by The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission. (Please note that point 3 should have read: Paragraphs 3-5 of reviewer 2's author recommendations.)Essential revisions:The reviewers were generally quite positive about the work. They have made a number of suggestions that may be useful for future investigation in this area. Of these, some of these are essential as detailed below. We are not asking for any additional experiments, but please discuss the points raised by the reviewers.1. Clarification of the title.2. The results shown in figure 1b, 1d are discordant and this needs to be remedied by either choosing different images or (not as good) explaining in the text why these panels seem to be discordant.3. Paragraphs 3-5 of reviewer 2's author recommendations.Reviewer #1 (Recommendations for the authors):1. As the products of the ligation reaction by the EL R3C ladder ribozyme are mixtures of RNAs with different lengths, the exact requirements  for the phenotypic changes are not clear. Is S2 or S3 enough? Are longer products  more important? Do the elongated RNAs need to remain hybridized with the ribozymes? Droplet formation experiments with S2 or S3  in the presence and absence of the inactive hybridizing ribozyme would clarify these points.2. It is unclear if the droplet property changes observed in this study are advantageous for the contained ribozyme. Is the EL R3C ladder ribozyme still active in the more viscous/solid droplets containing the longer RNAs? A ribozyme activity test started from a substrate long enough to form more viscous/solid droplets might answer this question.Reviewer #2 (Recommendations for the authors):The public review reflects my general view of the paper: it is an impressive achievement to get a ribozyme to control coacervate properties. The authors propose to exploit this by using the droplets to implement selection. There are still questions about the system's suitability for such a task. Readers would benefit from a clearer path forward from the authors, including an experimental assessment of a couple of important points.A direct test of coacervate suitability for such a task would be to carry out a model in vitro selection of ligases based on their activity and influence on coacervate properties, though the system may not yet be at a stage where this is possible. In the meantime some clarity on the following points would help assess the prospects of this:1) Coacervate enhancement of ribozyme activity: The results in Figure 1d and Figure 1b seem discordant. At the 2 h timepoint in Figure 1d, the average product length seems much lower without Lys than with – but the equivalent lanes look the same in 1b with (as far as I can tell) identical conditions. Visual inspection of the gel in 1b also suggests a much higher average product length than calculated in 1d (though perhaps this is a mirage due to staining intensity) and seen in the supporting data. If I'm missing something in my interpretation, perhaps the authors could more clearly explain the differences. This is important for readers to grasp as it underpins the claim that coacervate formation enhances ribozyme activity. I'd recommend showing a gel from the supporting figure clearly showing a condition where ligation is better with Lys. It may also be helpful to quantify the average product length in SYBR Gold stained gels .2) Ribozyme activity: the observed effects of ribozyme activity on droplet properties occur when ~1/4 of the coacervate comprises ribozyme. For selection, there would be much less active ribozyme present. Can small amounts of ribozyme still influence droplet behavior when at lower concentrations (e.g. a mix of active and inactive ribozymes)?3) Droplet fusion: There is convincing resistance of droplets to fusion when both contain active ribozyme. But in a selection setting, where a small fraction of droplets have active ribozyme and the majority do not, the relevant question is whether the droplets with inactive ribozyme will fuse with droplets with active ribozyme, i.e. does ribozyme activity in a droplet make it resistant to fusion or remove its tendency to fuse?Reviewer #3 (Recommendations for the authors):1 – The sentence "The increase in rate observed here is most likely due to the high concentration of ribozyme and substrate that occurs in coacervates directly formed from catalytic RNA and peptides (Ref. 15)" addresses an interesting point: How do we know that the peptide-mediated increase in ribozyme activity is due to coacervate formation as opposed to, for example, an association of freely diffusing peptides with freely diffusing ribozymes? Given that this is not the central point of the paper I would not ask for (probably difficult) experiments but for 1-2 more sentences based on reference 15 that increase the reader's understanding of how the peptide likely mediates through coacervates the increased ribozyme activity.2 – The manuscript is of high interest in the field of origins of life, and needs to discuss in more detail the important implications of coacervates as model systems for early life forms, especially with regard to the effects of ribozyme catalysis on the material exchange between coacervate droplets. Specifically, the coupling of genotype and phenotype, the danger from molecular parasites, and the inflow/outflow of metabolites are likely affected by these changes, and this should be discussed based on the rich literature. I suggest inserting an additional paragraph into the discussion or expanding the last paragraph on these three specific points.3 – While the longer Lys(19-72) peptides do not support ribozyme catalysis (see the first part of the results) they are used as a model system for dye exchange . Please state in the corresponding text the caveat that these longer peptide systems are not model systems for protocells but serve to test the effects of peptide length on dye exchange. Please also discuss whether the less pronounced effect on the exchange rates in coacervates that support ribozyme catalysis would be sufficient for evolutionary importance with respect to protocell function (see (2) above).4 – The discussion mentions that the Lys(5-24) peptides are actually not 5-24 amino acids long but 3 to 9 amino acids in length. To avoid confusing the reader, please correct the length throughout the manuscript, and mention in the materials and methods that "commercially available (Lys)n deviates from the manufacturer's stated size range, with the (Lys)5-24 used here being predominantly comprised of oligomers between n = 3 to n = 9 in length15.". Essential revisions:The reviewers were generally quite positive about the work. They have made a number of suggestions that may be useful for future investigation in this area. Of these, some of these are essential as detailed below. We are not asking for any additional experiments, but please discuss the points raised by the reviewers.1. Clarification of the title.We agree that the previous title was somewhat ambiguous and have therefore incorporated the suggestions of reviewers 1 and 3 to retitle the paper:Ribozyme activity modulates the physical properties of RNA-peptide coacervates2. The results shown in figure 1b, 1d are discordant and this needs to be remedied by either choosing different images or (not as good) explaining in the text why these panels seem to be discordant.The combination of endpoint and kinetic data is potentially confusing, and the effect of phase separation is not obvious from the SYBR Gold stained urea-PAGE gels of endpoint data. However, the use of a stained gel is necessary to visualise all of the components of the system. We have therefore attempted to clarify the data with the following measures:Lane profiles for relevant conditions  have been incorporated into the relevant figures. These profiles illustrate that the intensities of the ligated products are higher in the phase separated system, an effect which is not easily perceived in the SYBR Gold stained gel image.Images of the source urea-PAGE gels for the kinetic experiments  have been converted into a new figure supplement , which clearly shows the difference in rate and activity when monitoring the extension of a fluorescently-tagged substrate strand.We have added some brief explanation to the text to account for the discordance and to draw attention to the newly added features and figures.We hope that these additions are sufficient to remedy the issue and discuss the issues further in our responses to reviewers 1 and 2.3. Paragraphs 3-5 of reviewer 2's author recommendations.Please see author recommendations for detailed responses and revisions.Reviewer #1 (Recommendations for the authors):1. As the products of the ligation reaction by the EL R3C ladder ribozyme are mixtures of RNAs with different lengths, the exact requirements  for the phenotypic changes are not clear. Is S2 or S3 enough? Are longer products  more important? Do the elongated RNAs need to remain hybridized with the ribozymes? Droplet formation experiments with S2 or S3  in the presence and absence of the inactive hybridizing ribozyme would clarify these points.We thank the reviewer for the positive assessment of the work and helpful suggestions. Indeed, the requirements for altering material properties in these experiments are not precisely known. Follow-up studies performed in the manner suggested by the reviewer could elucidate these questions, by determining the physical properties of droplets formed from pre-reacted substrate concatemers with defined lengths or in the presence of a non- or weakly-hybridising ribozyme.et al. 2010) that investigated the effect of polymer length on coacervate physical properties to roughly determine initial and final RNA lengths that were likely to result in an observable change.In designing our experiments, we relied on previous works , we reported that highly viscous or gel-like phases can occur at specific ratios in mixtures of a fragmented hairpin ribozyme and poly(L-lysine), albeit at a much lower overall polymer length. The formation of these phases was associated with increased activity, and both ribozyme activity and strand exchange were maintained, suggesting that high viscosity is not a barrier to ribozyme function in specific cases.In our previous work (Le Vay Reviewer #2 (Recommendations for the authors):The public review reflects my general view of the paper: it is an impressive achievement to get a ribozyme to control coacervate properties. The authors propose to exploit this by using the droplets to implement selection. There are still questions about the system's suitability for such a task. Readers would benefit from a clearer path forward from the authors, including an experimental assessment of a couple of important points.A direct test of coacervate suitability for such a task would be to carry out a model in vitro selection of ligases based on their activity and influence on coacervate properties, though the system may not yet be at a stage where this is possible. In the meantime some clarity on the following points would help assess the prospects of this:1) Coacervate enhancement of ribozyme activity: The results in Figure 1d and Figure 1b seem discordant. At the 2 h timepoint in Figure 1d, the average product length seems much lower without Lys than with – but the equivalent lanes look the same in 1b with (as far as I can tell) identical conditions. Visual inspection of the gel in 1b also suggests a much higher average product length than calculated in 1d (though perhaps this is a mirage due to staining intensity) and seen in the supporting data. If I'm missing something in my interpretation, perhaps the authors could more clearly explain the differences. This is important for readers to grasp as it underpins the claim that coacervate formation enhances ribozyme activity. I'd recommend showing a gel from the supporting figure clearly showing a condition where ligation is better with Lys. It may also be helpful to quantify the average product length in SYBR Gold stained gels .We thank the reviewer for the positive assessment of this work and for highlighting several possible improvements.In response to point 1, we direct the reviewer to our response to Reviewer 1, and to the editorial team in the section “essential revisions”. In brief, the discordance stems from the use of stained gel images to illustrate the overall reaction at its endpoint in Figure 2b, but the use of a fluorescently 5’-labelled substrate strand to determine kinetics in 2d. The use of stained gels allows us to visualise all species, including the ribozyme, substrate and both linear and circular products. However, monitoring the reaction in this way complicates the determination of average substrate length due to problems of band overlap (e.g. ribozyme and S2 product) and the presence of large but low abundance circular products of unknown length, and makes visual assessment of differences in average substrate length at the endpoint of the reaction difficult.We feel that it is important to present a stained gel which displays all reaction components and products, but have aimed to clarify the data by adding lane profiles for certain conditions, which the greater intensity of linear product bands in the presence of pL, adding a new figure supplement based on the fluorescently labelled gels from the source data of the kinetics experiment, and adding some clarification to the text.2) Ribozyme activity: the observed effects of ribozyme activity on droplet properties occur when ~1/4 of the coacervate comprises ribozyme. For selection, there would be much less active ribozyme present. Can small amounts of ribozyme still influence droplet behavior when at lower concentrations (e.g. a mix of active and inactive ribozymes)?This is a valid point that we feel warrants further discussion here and in the manuscript. In a more realistic scenario, it is expected that the concentrations of both functional ribozyme and compatible substrate would be much lower, and that the remaining material would contain non-functional (or at least non-ligating) RNA strands and perhaps other charged polymers.It is therefore necessary to improve the catalytic properties of the ribozyme in order to move towards a more realistic system. In Figure 1 – supplement 1, we show that increasing the amount of substrate relative to the ribozyme actually leads to a reduction in ribozyme activity. We therefore anticipate that optimising the ribozyme for multi-turnover reaction might be required to achieve significant phenotypic change in a system with small amounts of ribozyme but abundant substrate. In our previous work, we demonstrated that the Hairpin ribozyme is capable of strand exchange and therefore multiple substrate ligations in RNA-peptide condensates, perhaps due to the short length of the substrate binding strand. By altering the substrate binding arms of the R3C ribozyme, it might be possible to engineer similar behaviour, perhaps at the cost of reaction rate.sunY ribozyme, which requires only a 5’-G for substrate ligation, would be an interesting candidate for this.In more complex mixtures with a greater variety of RNA strands and therefore a less well-defined substrate pool, a ribozyme with less stringent substrate sequence requirements might be better suited to alter material properties. The We have modified the final paragraph of the discussion to account for some of these shortcomings.3) Droplet fusion: There is convincing resistance of droplets to fusion when both contain active ribozyme. But in a selection setting, where a small fraction of droplets have active ribozyme and the majority do not, the relevant question is whether the droplets with inactive ribozyme will fuse with droplets with active ribozyme, i.e. does ribozyme activity in a droplet make it resistant to fusion or remove its tendency to fuse?This is a highly relevant but experimentally challenging question, which we are currently attempting to address as part of our ongoing projects. The orthogonally labelled substrates transfer between droplet populations by both fusion and diffusion, and as a result all droplets contain measurable amounts of both fluorophores shortly after mixing. This means we are currently unable to investigate the behaviour of each droplet population independently to determine if e.g. an active population resists fusion when mixed with an inactive population. Our preliminary results suggest that in mixtures of active and inactive droplets, the labelled RNA from the active population remains highly localised, whilst the labelled RNA from the inactive population diffuses evenly throughout both populations. Further work is required to investigate fusion in these systems, in particular to establish methods of orthogonally measuring droplet size in the two populations.Reviewer #3 (Recommendations for the authors):1 – The sentence "The increase in rate observed here is most likely due to the high concentration of ribozyme and substrate that occurs in coacervates directly formed from catalytic RNA and peptides (Ref. 15)" addresses an interesting point: How do we know that the peptide-mediated increase in ribozyme activity is due to coacervate formation as opposed to, for example, an association of freely diffusing peptides with freely diffusing ribozymes? Given that this is not the central point of the paper I would not ask for (probably difficult) experiments but for 1-2 more sentences based on reference 15 that increase the reader's understanding of how the peptide likely mediates through coacervates the increased ribozyme activity.2+ and RNA concentrations. These experiments reveal that both high RNA and Mg2+ concentrations are required for enhanced activity. We direct the reviewer to the earlier response to Reviewer 1, and to the new data presented in Figure 1 – supplement 4.We would like to thank the reviewer for this point, and agree that the claim was not fully supported. In response to this and a similar point raised by Reviewer 1, we have performed additional experiments investigating the activity of the ribozyme at varying PEG, Mg2 – The manuscript is of high interest in the field of origins of life, and needs to discuss in more detail the important implications of coacervates as model systems for early life forms, especially with regard to the effects of ribozyme catalysis on the material exchange between coacervate droplets. Specifically, the coupling of genotype and phenotype, the danger from molecular parasites, and the inflow/outflow of metabolites are likely affected by these changes, and this should be discussed based on the rich literature. I suggest inserting an additional paragraph into the discussion or expanding the last paragraph on these three specific points.We agree that the ribozyme induced changes to coacervate properties may impact factors relevant to early compartmentalised replicators such as parasite resistance. However, as the ribozyme system described in this work is not replicative, we would like to limit the discussion of these aspects to avoid lengthening the manuscript further. We have included a sentence in the discussion highlighting these areas as interesting avenues for further study.3 – While the longer Lys(19-72) peptides do not support ribozyme catalysis (see the first part of the results) they are used as a model system for dye exchange . Please state in the corresponding text the caveat that these longer peptide systems are not model systems for protocells but serve to test the effects of peptide length on dye exchange. Please also discuss whether the less pronounced effect on the exchange rates in coacervates that support ribozyme catalysis would be sufficient for evolutionary importance with respect to protocell function (see (2) above).19-72: RNA > 1:1, as determined by the lysine titration PAGE in Figure 1b. In subsequent experiments, we selected a (Lys)19-72: RNA ratio of 0.75:1 at which the compartmentalised ribozymes are highly active and coacervates form in liquid state. We believe that under these conditions the droplets containing longer peptides are an interesting model system. In addition, all experiments are also performed with droplets formed from the shorter peptide and the difference in behaviour between these systems is discussed. This point has been clarified in the second paragraph of the Results section.We thank the reviewer for bringing this miscommunication to light. The longer lysine polymers do indeed inhibit ribozyme activity, but only at ratios when the lysine oligopeptides are in excess over RNA i.e. (Lys)4 – The discussion mentions that the Lys(5-24) peptides are actually not 5-24 amino acids long but 3 to 9 amino acids in length. To avoid confusing the reader, please correct the length throughout the manuscript, and mention in the materials and methods that "commercially available (Lys)n deviates from the manufacturer's stated size range, with the (Lys)5-24 used here being predominantly comprised of oligomers between n = 3 to n = 9 in length15.".19-72: RNA > 1:1, as determined by the lysine titration PAGE in Figure 1b. In subsequent experiments, we selected a (Lys)19-72: RNA ratio of 0.75:1 at which the compartmentalised ribozymes are highly active and coacervates form in liquid state. We believe that under these conditions the droplets containing longer peptides are an interesting model system. In addition, all experiments are also performed with droplets formed from the shorter peptide and the difference in behaviour between these systems is discussed. This point has been clarified in the second paragraph of the Results section.We thank the reviewer for bringing this miscommunication to light. The longer lysine polymers do indeed inhibit ribozyme activity, but only at ratios when the lysine oligopeptides are in excess over RNA i.e. (Lys)


The coronavirus disease (COVID-19) pandemic has resulted in more than six million deaths by October 2022. Vaccines and antivirals for severe acute respiratory syndrome coronavirus 2 are now available; however, more effective antiviral drugs are required for effective treatment. Here, we report that a potent AMP-activated protein kinase (AMPK) inhibitor, compound C/dorsomorphin, inhibits the replication of the human coronavirus OC43 strain (HCoV-OC43). We examined HCoV-OC43 replication in control and AMPK-knockout (KO) cells and found that the virus replication decreased in AMPK-KO cells. Next, we examined the effect of the AMPK inhibitor, compound C on coronavirus replication. Compound C treatment efficiently inhibited the replication and decreased the coronavirus-induced cytotoxicity, further inhibiting autophagy. In addition, treatment with compound C in combination with chloroquine synergistically inhibited coronavirus replication. These results suggest that compound C can be considered as a potential drug candidate for COVID-19. The coronavirus disease COVID-19) pandemic has resulted in millions of deaths owing to the high infectivity and high mortality of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) [9 pandemiin vitro by elevating vesicular pH and regulating the glycosylation of angiotensin converting enzyme 2 [Macroautophagy (autophagy) is an evolutionarily conserved catabolic process that requires lysosomal function, however, its involvement in coronavirus replication is controversial , 3. Coroenzyme 2 , 11, 12.enzyme 2 , 14.Compound C, also known as dorsomorphin, is a potent AMP-activated protein kinase (AMPK) inhibitor widely used to block AMPK-dependent signaling . CompounCoronaviruses are classified into alpha, beta, gamma, and delta coronaviruses, where alpha and beta coronaviruses can infect humans. So far, seven coronaviruses have been known to infect human, including human coronavirus 229E (HCoV-229), HCoV-HKU1, HCoV-NL63, HCoV-OC43, SARS-CoV, MERS-CoV and SARS-CoV-2 . Owing tThe HCoV-OC43 virus was obtained from ATCC , and Rhabdomyosarcoma (RD) cells were obtained from the Korean Cell Line Bank . RD cells were maintained in MEM  containing 10% fetal bovine serum  and 1% penicillin-streptomycin (Welgene). RD cells were infected with HCoV-OC43 as described previously . Cell vi2. Overlay medium was prepared as a mixture of 0.6% agarose in MEM, of which 1 mL was added to each well and the plate was incubated for 5 days. Finally, the cells were fixed with a 10% formaldehyde solution and stained with 1% crystal violet. HCoV-OC43 virus titer in the media was 107 PFU/ml.To enumerate the virus, RD cells were seeded in a 12-well plate one day prior to the assay. The viral samples were diluted 10-fold in MEM with 2% FBS and added to the corresponding wells and incubated for 1 h at 33°C and 5% CO5′- GATGTAGATGCCCGTCTCG -3′ and reverse primer 5′- TGTGGCACACGACTACCTTC -3′. HCoV-OC43 M mRNA was amplified using the forward primer 5′- ACGGTCACAATA ATACGCGGT -3′ and reverse primer 5′- GGGTTGATGGCAGTCGGTAA -3′. The HCoV-OC43 N mRNA was amplified using the forward primer 5′- AGGATGCCACCAAACCTCAG -3′ and reverse primer 5′- TGGGGAACTGTGGGTCACTA -3′. The input RNA was normalized via the amplification of ribosomal protein L4 (RPL4) RNA with the forward primer 5′-GCTCTGGCCAGGGTGCTTTTG-3′ and reverse primer 5′-ATGGCGTATCGTTTTTGGGTTGT-3′.Quantitative RT-PCR was used to measure the level of coronavirus RNA in cells and media, as previously described . BrieflyWestern blotting was used to measure the level of coronavirus protein in cells and media, as described previously , 26. BriTo visualize the cells infected with coronavirus, cells were grown on sterilized glass coverslips and infected with coronavirus. The cells were fixed with 4% paraformaldehyde, blocked with 3% bovine serum albumin in PBS, and stained with anti-OC43 antibody (1:1000 dilution). Finally, the cells were stained with Alexa Fluor 488–conjugated secondary antibody (Thermo Fisher Scientific) and DAPI. Images were captured using a Carl Zeiss LSM710 confocal microscope . Both OC43 positive cells and OC43 negative cells were counted and analyzed to show the percentage of HCoV-OC43 infected cells in each microscopy image.P < 0.05.The results of western blotting, quantitative RT-PCR, and MTT assay were evaluated by a two-tailed Student’s t-test using Excel software . Statistical significance was set as Several studies have suggested that autophagy is involved in coronavirus replication; Thus, we attempted to examine the role of AMPK in coronavirus replication. Previously, we generated AMPK-KO cells and showed that autophagic flux is deregulated in these cells . Here, cTo assess whether AMPK activity and coronavirus infection contribute to autophagy regulation, we examined the level of autophagy marker expression. Coronavirus infection increased the level of LC3-II protein and p62 , which iThe replication of coronavirus was decreased in AMPK-KO cells; therefore, we hypothesized that AMPK inhibition by AMPK inhibitor would also decrease the replication. As predicted, AMPK inhibitor, compound C treatment decreased the level of coronavirus protein in the cell culture media and cell lysates in a dose-dependent manner . We furtWe further examined the effects of compound C treatment on coronavirus infection using immunofluorescence staining. HEK293T and RD cells were infected with HCoV-OC43 virus and incubated with compound C, and viral protein expression was assessed using immunofluorescence staining with anti-OC43 antibody. With an increase in the compound C concentration, the number of coronavirus-positive cells decreased significantly, indicating that compound C treatment inhibited coronavirus infections .Since compound C treatment interferes with coronavirus replication, we expected that the cytotoxicity caused by the infection would be decreased by compound C treatment. In a plaque formation assay, while coronavirus infection induced plaque formation, compound C treatment reduced it . This suFurthermore, examination of cell viability upon coronavirus infection in the presence or absence of compound C showed a significant decrease in cell viability post-infection, which was alleviated with compound C treatment . These rAs AMPK inhibition interferes with autophagy, we hypothesized that an autophagy and AMPK inhibition may synergistically inhibit coronavirus replication. Toward this, we treated cells with chloroquine, a representative inhibitor of autolysosome formation, and compound C . While cWe also examined the expression of autophagy markers in combination with compound C and chloroquine treatment following coronavirus infection . We measIn this study, we examined the effect of compound C/dorsomorphin on the replication of human coronaviruses. Previously, we generated AMPKɑ1-KO cell lines and demonstrated that autophagy initiation and progression were impaired . Here, win vivo. Although compound C is reported to show an inhibitory effect on several kinases other than AMPK, currently, no AMPK-specific inhibitor is available, and the development of more specific inhibitors would be helpful in treating coronavirus-related diseases [Compound C induces apoptosis in cancer cells , and we diseases .in vivo.In addition, we demonstrated that co-treatment with compound C and chloroquine collectively inhibited coronavirus replication . The dosIn this report, we demonstrated that AMPK-KO and an AMPK inhibitor downregulated coronavirus replication. Recent reports have highlighted the requirement of autophagy for efficient replication of coronavirus and that autophagic flux can be blocked by coronavirus protein expression , 30. Intin vitro, and animal experiments should be performed to validate this effect in vivo.HCoV-OC43, which belongs to the beta coronavirus family like SARS-CoV-2, induces a mild disease . TherefoS1 Raw images(PDF)Click here for additional data file.