Methylene blue (MB) is a dye used for histology with clinical importance and intercalates into nucleic acids. After MB staining of formalin fixed paraffin embedded (FFPE) muscle invasive bladder cancer (MIBC) and normal urothelium, specific regions could be microdissected. It is not known if MB influences RNA used for gene expression studies. Therefore, we analyzed MIBC using five different RNA isolation methods comparing patient matched FFPE and fresh frozen (FF) tissues pre-stained with or without MB. We demonstrate a positive impact of MB on RNA integrity with FF tissues using real time PCR with no interference of its chemical properties. FFPE tissues showed no improvement of RNA integrity, which we propose is due to formalin induced nucleotide crosslinks. Using direct multiplex RNA hybridization the best genes for normalization of MIBC and control tissues were identified from 34 reference genes. In addition, 5SrRNA and 5.8SrRNA were distinctive reference genes detecting <200 bp fragments important for mRNA analyses. Using these normalized RNAs from MB stained MIBC and applying multiplex RNA hybridization and mRNA sequencing, a minimal gene expression panel precisely identified luminal and basal MIBC tumor subtypes, important for diagnosis, prognosis and chemotherapy response. Although the molecular distinction between fractionated RNA and DNA was successful in the beginning of the 1900’s , or acidThe methylene blue (MB) dye, a phenothiazine derivate, was synthesized in the middle of the 19th century and produces a blue color when solubilized in water. Due to its small molecular weight, fast diffusion properties and affinity for nucleic acids, MB was initially used to detect tuberculosis in lung tissues  and it iFor decades, tumor tissues archived via formalin fixed and paraffin embedded (FFPE), fresh or flash-frozen (FF) methodologies represented the main pillars for clinical, therapeutic, translational and basic research. Both ways of archiving tumor tissues have pros and cons, but for RNA and DNA sequencing FF tissues are superior regarding RNA quality, although a few laboratories did not find major quality differences compared to FFPE tissues . Researc® FF tissues pre-stained with or without MB. These isolation methods were based upon magnetic beads, columns or organic solvent extraction. MIBC is a grave form of bladder cancer arising from urothelium, where patients have a five-year survival of 40–60% [In the present report, we systematically analyzed muscle invasive bladder cancer (MIBC) tissues using five different RNA isolation methods comparing patient matched FFPE and Tissue-Tekf 40–60% . Over thf 40–60% . Proteinf 40–60% . Furtherf 40–60% . Since c18SrRNA, with the use of 5SrRNA and 5.8SrRNA genes as a new approach for real time qPCR. Considering the recommendation that rRNAs, which constitute 85–90 % of total cellular RNA are highly useful and accurate internal controls and more reliable than other reference genes, a normalization of the isolated RNA to total RNA using especially fluorescent RNA stains is advised [Therefore, our main goal in this investigation was to determine if MB impacts RNA during fractionation from MIBC tissues and influences RNA integrity based upon analyses of ubiquitously expressed reference genes, such as  advised .18SrRNA for our RNA integrity analyses due to its high transcript abundance in total RNA, since it was reported to show increased expression rates and reduced degradation compared to other reference genes [n = 28) and one of the best RNA isolation methods determined in this investigation, multiplex RNA hybridization and mRNA sequencing confirmed luminal and basal MIBC tumor subtypes important for MIBC diagnosis, patient prognosis and chemotherapy [According to multiple publications we chose ce genes . It is vce genes ); we theotherapy . Our resProper handling and chemical processing of tumor tissues as a daily event at Pathology Institutes is absolutely crucial for diagnosis. The retrieval of high quality nucleic acids, such as RNA from archival tissues, is essential for studying the intrinsic gene expression of tumors and can lead to the discovery of new biomarkers as well as profiling tumor subtypes such as MIBC basal and luminal for patient prognosis ,30. PresMIBC tissues were stained with MB and then specific targeted tumor regions, microdissected for RNA isolation. MB staining was similar to the state of the art hematoxylin and eosin (H&E) regarding the histological appearance .For comparison, five different isolation kits were used to extract total RNA from FFPE and FF tissues stained with or without MB . Each me18SrRNA values compared to no MB treatment using qPCR (16H18ClN3S). With this in mind, we asked the question, if other dyes with similar molecular structures or nucleic acid binding properties like MB could also significantly affect 18SrRNA changes using the same tissues. For example, Tolouidine blue (TB), an organic chloride salt with 3-amino-7-(dimethylamino)-2-methylphenothiazin-5-ium as the counterion (C15H16ClN3S), also led to improved 18SrRNA values using qPCR. However, Crystal violet (CV), a hexamethylpararosaniline chloride (C25H30ClN3) led to variable 18SrRNA results . Results confirmed our initial findings with additional FFPE tissues and Method A  with patient matched FF MIBC tissues, a profound statistically significant improvement of 18SrRNA was found for three of four methods , with MB treatment compared to no MB  showed no significant differences of 18SrRNA between FF tissues treated with or without MB. Trizol contains phenol and guanidinium thiocyanate and is recommended for extracting FF tissues, although the process is time consuming and phenol contamination is possible. Importantly, we observed that MB during RNA isolation fractionated into the organic phase, and thus was released from nucleic acids in the aqueous phase, supporting no sustained RNA MB interaction. Although 18SrRNA is a well-known reference gene, importantly, we normalized to the total RNA based on several quantification methods including a fluorescent RNA stain detecting down to 5 ng/mL RNA [18SrRNA to another reference gene (5.8SrRNA) to calculate ΔCts. Analysing ΔCts of the RNAs isolated from the different methods with MB showed that the highly significant changes (A _+MB and C_+MB) did not change in significance  were no longer significant  and 28SrRNA  regions compared to the total area under the curve, including the height of the 28SrRNA peak [http://urx.red/OB4Y) (accessed on 8 October 2021) [In order to determine RNA integrity, we analyzed the RNA integrity number (RIN), which evaluates RNA peak . AnalyziRNA peak . The DV2er 2021) . However5SrRNA  and 5.8SrRNA . Like 18SrRNA, 5.8SrRNA is transcribed by RNA-Polymerase I, whereas 5SrRNA is transcribed by RNA-Polymerase III [5S and 5.8SrRNAs using our primer design shows that amplification for each gene can only occur, if these rRNAs are full length fragments. Therefore, amplification of both rRNAs could reflect a quality qPCR reference for smaller RNAs below 200bp. QPCR results showed that the addition of MB significantly improved 5S and 5.8SrRNA expression similar to 18SrRNA  were generally higher compared to FF tissues (0.94 to 1.23) and human placenta (1.07). Especially for FF tissue isolation with Method C, MB improved the overall ratios .In order to further address the integrity of the different isolated RNAs, we implemented a new reference approach to detect RNA in the amplification range of approximately ~100 bp for qPCRs. Routinely, qPCR primers for reference and target genes are designed to amplify regions between 90 and 150 bp. Uniquely, our designed primers detected the constitutively expressed rase III . In cont 18SrRNA A,B. In a tissues C. Intere18SrRNA, 5SrRNA and 5.8SrRNA values in the presence of MB prior treatment  (COV 61.44%), Ubiquitin C  (COV 61.38%) and TATA-box binding protein  (COV 70.99%). GUSB is involved in the degradation of glycosaminoglycans [TBP) is the DNA-binding subunit of RNA polymerase II transcription factor D, essential for expression of most protein-encoding genes [UBC) is a polyubiquitin precursor, and conjugation of ubiquitin is associated with many cell functions, especially with protein degradation [18SrRNA −3.221 (R2: 0.997), for TBP −3.55 (R2: 0.990) and for UBC −3.69 (R2: 0.979)  COV 61.% and TATng genes . As a meradation . Finally: 0.979) –H. InterKRT5, KRT14, CD44; luminal differentiation: KRT20, GATA3, and FOXA1), 28 tumor samples were each classified as either basal or luminal subtypes . In addition, use of MB also showed a statistically significant benefit to RNA integrity when comparing individual isolation methods for FF tissues   .18SrRNA, 5SrRNA and 5.8SrRNA qPCR were observed for most isolation methods using FFPE tissues with or without MB. On the other hand, we propose that MB RNA intercalation in FF tissues stabilizes and preserves RNA structure from further fragmentation during isolation resulting in a benefit for RNA integrity. In addition to using 5SrRNA and 5.8SrRNA as marker genes for the integrity of smaller RNA fragments below 200 bp, expression of 5SrRNA (via RNA polymerase III) and 5.8SrRNA (via RNA polymerase I) can also represent an advanced quality check of the cellular state. For example, Tetrahymena cells showed 1:1 molar ratios of 5SrRNA: 5.8SrRNA during log growth phase. In contrast, starved Tetrahymena cells synthesized approximately 15% more 5SrRNA over 5.8SrRNA, supporting 5SrRNAs as a biomarker for changes in physiological cellular conditions [5SrRNA:5.8SrRNA gene expression ratios for all RNA isolation methods, FF MIBC tissues demonstrated ratios closer to 1.0, which could reflect more stable conditions with less cellular stress following loss of blood supply prior to fixation for these six bladder tumors  identification in body fluids [Since RNA fragmentation is ongoing in tissues prior to fixation and upon formalin treatment when nucleic acids cross link, specifically RNA, this leads to hydrolysis ,47,48. Tnditions . Examinir tumors C. Alterny fluids .Another important advantage of MB is the similar staining of tissues compared to H&E . The relTBP as most stable genes using qPCR . All the procedures were performed in accordance with the ethical standards established in the 1964 Declaration of Helsinki and later amendments. All patients gave written consent for the use of their tumor or normal tissue material.® embedded fresh-frozen (FF) archived tissues (2008–2019) included in this study were: (1) FFPE MIBC (n = 6) and matched FF MIBC samples (n = 6); FFPE MIBC samples (n = 23); (2) FFPE normal control urothelium (n = 5) from non-cancer patients. In addition, five control placental tissues (3rd trimester) were flash-frozen into liquid nitrogen.Formalin-fixed, paraffin-embedded (FFPE) and Tissue-TekTissue de-paraffinization and re-hydration: All tissues were processed according to our standard methodology: FFPE 10 µm tissue cuts were first treated at 70 °C for 1 h in a dry oven to facilitate paraffin melting. Tissue cuts were then incubated for 10 min at RT in a sequential order with the following solutions for further de-paraffinization and rehydration: (1) 3 × Roti-Histol ; (2) 100% Ethanol ; (3) 96% Ethanol; (4) 70% Ethanol and (5) dH2O with 0.1% DEPC. Matched FF 10 µm tumor tissues were incubated 2 times for 5 min with 70% Ethanol and dH2O to remove Tissue-Tek®.MB tissue staining: Following tissue de-paraffinization and re-hydration, tissue cuts were incubated with or without MB  for: (1) FFPE and FF 10 µm tissue sections on glass slides to demonstrate specific histological staining and tumor quantity  and washed 2 times each 10 s with sterile dH2O . Then samples were centrifuged  to remove the dH2O, and the first solution of the corresponding RNA method was added.quantity ; or (2) Hematoxylin/Eosin (H&E) tissue staining: A modified shortened protocol for H&E histological staining of tissue cuts was used: 1 mL hematoxylin  was added to the slide and incubated for 3 min at RT. The slides were washed 2 times with dH2O (each 1 min) and 1 mL of bluing buffer (Dako #CS702) for 1 min was added. After 1 time washing with dH2O (1 min), 1 mL eosin was added for 1 min at RT, again washed with dH2O . Lastly, all H&E and MB stained tissue slides for histological analyses were cover slipped with glass slides and a mounting buffer .n = 6), each 10 µm thick, were stained either with or without MB as above and then immediately processed using different RNA isolation methods to determine RNA integrity. Regarding all methodologies below, the Proteinase K digestion step was extended to overnight for all FF tissues, if applicable. The four RNA isolation methods are shown in Six MIBC FFPE and matched FF tumor tissues ((1) Maxwell RSC DNA Blood kit  (Method A). For RNA tissue extraction a purchased incubation buffer  was added replacing the DNA extraction buffer. Briefly, samples were heated to 80 °C for 10 min, then Proteinase K and Protector RNase Inhibitor  were added, followed by incubation at 56 °C for 1 h while shaking. Afterwards, an RNA lysis buffer  was added and the samples were pipetted into the cartridges (supplied by the manufacturer) and processed by a Maxwell RSC machine. Since this isolation method does not involve a DNase I step, 20 U RNAse-free DNase I  was added for 1 h at 37 °C. (2) Maxwell RSC RNA FFPE kit  (Method B). Samples were processed according to manufacturer’s instructions, starting at the Proteinase K incubation along with the addition of an RNA Protector RNase Inhibitor. (3) High Pure FFPE RNA Isolation Kit  (Method C). Samples were processed according to manufacturer’s instructions, starting at the Proteinase K incubation along with the addition of an RNA Protector RNase Inhibitor. (4) XTRAKT FFPE kit  (Method D). Samples were processed according to manufacturer’s protocol. (5) Trizol  (Method E). Only FF MIBC tissues and FF normal placental tissues  were isolated for RNA according to manufacturer’s instructions with some modifications. Briefly, Isopropanol was added to the aqueous phase  in the presence of 20 µg Glycogen  at −20 °C overnight, the samples were centrifuged  and the supernatant removed. RNA was washed with 80% ethanol, air dried, solubilized in DEPC treated dH2O, DNAse I treated as above, precipitated with 0.6 M sodium acetate in the presence of ethanol and incubated overnight at −20 °C. Samples were centrifuged, washed with 80% ethanol, air dried and solubilized with DEPC treated dH2O.The following total RNA isolation methodologies were used for comparison: g, 1 h, 4 °C) and the supernatant removed. RNAs were washed with 80% ethanol, air dried and solubilized in DEPC treated dH2O and then quantified using the QuantiFluor® RNA System  and Nanodrop 2000 (Thermo Scientific). RNA quality, like the RNA Integrity Number (RIN) and the DV200 was assessed using the Bioanalyzer 2100 with the corresponding 2100 Expert Software from Agilent.After RNA isolation was performed using the different methods described above, all RNAs except for Trizol isolated, were further purified using an equal volume of chloroform , centrifuged , a back-extraction with TE buffer  to ensure no loss of RNA during extraction and then the aqueous phases containing RNA were precipitated with 100% ethanol in the presence of Glycogen and 0.6 M sodium acetate and incubated at −20 °C overnight. RNA samples were centrifuged . All primer nucleotide sequences for qPCR are shown in For cDNA a total of 1 µg RNA per sample was reverse transcribed to obtain a concentration of 20 ng/µL cDNA using Applied Biosystems High Capacity cDNA Reverse Transcription Kit . For qPCR, 40 ng cDNA, SYBR Select Master Mix , 200 nM of each primer were analyzed using the StepOnePlus Real-Time PCR Systems (Applied BiosystemsThirty-four reference genes and six MIBC subtyping target genes with NCBI Accession numbers  were anaTM 3′ mRNA-Seq Kit FWD (Lexogen GmbH) which allows for the generation of Illumina-compatible libraries from poly-adenylated RNA. 250 ng of purified total RNA were used in input for library preparation which was carried out according to manufacturer’s instructions. The QuantSeq protocol creates libraries close to the 3′ end of the transcripts generating only one fragment per transcript. Sequencing of the QuantSeq libraries was performed on the Illumina NovaSeq 6000 platform  yielding at least 20 M clusters per sample. Pre-processing of demultiplexed raw data (FASTQ files) was performed via the nf-core RNA-seq pipeline v.3.3 [mRNA sequencing was performed using the Lexogen QuantSeqne v.3.3 . In partne v.3.3  using STne v.3.3 . Read cone v.3.3  within Rne v.3.3 .https://github.com/cit-bioinfo/BLCAsubtyping) [Twenty eight MIBC samples were classified into luminal and basal subtypes based on three different approaches: (1) Protein immunohistochemistry (IHC) determination of luminal and basal subtypes adapted from a previous report . (2) RNAbtyping) .2) as well as all graphs, were performed using GraphPad Prism 9. A p-value < 0.05 was considered statistically significant. Unsupervised hierarchical clustering was performed with JMP SAS 13.2.All statistical analyses such as two-tailed Mann––Whitney U test, coefficient of variation (COV), ANOVA and Pearson correlation coefficients (R18SrRNA and 5SrRNA and 5.8SrRNA. When isolating RNA from more commonly available FFPE archival tissues, implementing MB becomes a choice depending on the amount of targeted tissue within a sample for RNA isolation and analyses. This becomes important since most methods extracting RNA from FFPE tissues did not show any differences in RNA integrity with or without MB. However, when using microdissection, MB helps to identify specific tissue regions leading to enrichment of an RNA population. As a recommendation, staining with MB and implementing Methods B or C results in excellent 18SrRNA using qPCR, which also demonstrated 100% congruence of MIBC tumor subtyping gene expression using direct multiplex RNA hybridization compared with IHC and mRNA sequencing. Our above recommendations could also be important for spatial sequencing and laser dissection.In conclusion, we outline several messages when isolating RNA from archival tissues. MB can easily be used for staining tissues of interest for microdissection and RNA isolation resulting in no artifacts due to its chemical properties. We recommend employing MB when extracting RNA from FF tissues especially using Methods B and C, which leads to improved RNA integrity and reference gene expression, like


Escherichia coli. Specifically, de-novo-designed riboregulators, known as toehold switches, were concatenated to enhance the functionality of an OR gate, and a previously utilized antisense RNA strategy was further optimized to construct orthogonal NIMPLY gates. These optimized synthetic logic gates were able to be seamlessly integrated to achieve final arithmetic operations on small molecule inputs in cells. Toehold-switch-based ribocomputing devices may provide a fundamental basis for synthetic RNA-based arithmetic logic units or higher-order systems in cells.The development of computational logic that carries programmable and predictable features is one of the key requirements for next-generation synthetic biological devices. Despite considerable progress, the construction of synthetic biological arithmetic logic units presents numerous challenges. In this paper, utilizing the unique advantages of RNA molecules in building complex logic circuits in the cellular environment, we demonstrate the RNA-only bitwise logical operation of XOR gates and basic arithmetic operations, including a half adder, a half subtractor, and a Feynman gate, in  Synthetic biology aims to create technologies for designing and building biological systems with programmable and predictable dynamics . Since tNucleic-acid-based genetic devices have made remarkable progress in molecular computing and may provide the required platform for scalable synthetic biological systems. Complex logic circuits and advanced computing systems have been implemented using toehold-mediated strand displacement, including a bistable circuit , a squarBuilding on the success of synthetic RNA regulatory parts and inspired by natural RNA regulators , severalE. coli using a de-novo-designed toehold switch and antisense RNAs. Synthetic RNA-based ALUs could lay the foundation for making sophisticated molecular devices with neural-network-like capabilities for biomedical applications.In particular, toehold switches may form the basis for constructing an arithmetic logic unit (ALU) in vivo. A generalized toehold switch architecture, termed ribocomputing devices, concatenated several toehold switch sensor domains and utilized the self-assembly of RNA species to compute multi-input AND/OR/NOT operations . The desThe NIMPLY gate often used in synthetic biology and genetic circuits ,70 was pE. coli, the three circuit components—switch RNA, trigger RNA, and antisense RNA—should be selected from the existing library with appropriate modifications . GFP was used to characterize the switch output performance via flow cytometry. First, the OR gate functionality was verified in the absence of antisense RNAs, where the GFP fluorescence was increased at least 100-fold in the presence of either trigger RNAs  dissociating the trigger from the switch or (2) capturing the trigger freely floating in the cytoplasm . Single Encouraged by the optimized NIMPLY gates, we then aimed to construct an XOR gate with two chemical inducers as inputs: IPTG and anhydrotetracycline (aTc). An XOR gate provides a true output with an odd number of true inputs a. In theE. coli BL21 AI strain, where genomically encoded T7 RNA polymerase was induced by arabinose. A number of basic molecular interactions were verified for the XOR gate: the crosstalk between the switch and antisense RNAs was negligible  changes from BUFFER gate to NOT gate in response to the input signal a. At theE. coli using de-novo-designed toehold switches and antisense RNAs. A systematic approach was taken where the basic building block, a NIMPLY gate, was optimized and then subsequently used for an XOR gate, which in turn could be used for basic ALUs. While the NIMPLY gate design was previously demonstrated [In this study, we present the binary operation of XOR gates, cellular arithmetic operation including a half adder and a half subtractor, and a Feynman gate in single-cell nstrated , it was nstrated ,80, we tNotably, the antisense RNA designs can be extended to other related synthetic RNA regulators. As an example, a recently reported 3-way junction (3WJ) repressor  can be aThe successful demonstration of a synthetic XOR gate can be seen as a benchmark for systematic synthetic gene circuit construction. Previously, several lines of work demonstrated RNA-based XOR gates, including sRNA , miRNA , and gRNE. coli. Second, the ALUs can be designed with almost no sequence constraints with in silico screening and optimization. Third, a large library of orthogonal toehold switches provides the required parts for building complex systems. Lastly, the system inherits the general advantages of RNA-based operations, including a fast response time, reduced resource usage, and multiplexing [Herein, we provided a framework for constructing several synthetic RNA-only basic ALUs with de-novo-designed toehold switches at the single-cell level. This design paradigm offers excellent programmability with simple structural specifications rather than sequence constraints. First, the concatenation of switch RNAs can effectively reduce the encoding space of genetic programs and, therefore, enable the operation of complex systems in iplexing ,91,92. Riplexing . Notablyiplexing  showcaseE. coli strains were used in this study: BL21 DE3  gal dcm), BL21 AI  gal dcm araB::T7RNAP-tetA), and DH5α  λ−).The following E. coli DH5α and purified using the EZ-PureTM plasmid Prep Kit. Ver. 2 (Enzynomics). Plasmid sequences were confirmed by DNA sequencing after every cloning step. Plasmids were transformed through chemical transformation [The backbones for the plasmids used in this research were taken from the commercial vectors pET15b, pCDFDuet, pCOLADuet, and pACYCDuet (EMD Millipore). The switch RNA of the NIMPLY complex was constructed using ACTS Type II N3 and ACTS Type II N7 from previous research  and was ormation .E. coli BL21 DE3 and AI strains were used; they contain chromosomally integrated T7 RNA polymerase under the control of IPTG-inducible lacUV5 promoter and arabinose-inducible PBAD promoter, respectively. For the in vivo characterization of the NIMPLY complex in E. coli BL21 DE3 cells were cultured on LB agar plates (1.5% agar) with appropriate antibiotics: pACYCDuet (25 μg/mL Chloramphenicol), pCDFDuet (50 μg/mL Spectinomycin), and pET15b (100 μg/mL Ampicillin). Single colonies were grown overnight (~16 h) in 96-well plates with shaking at 800 rpm, 37 °C. Overnight cultures were diluted 1/100-fold into fresh media and returned to shaking . After 80 min, cell cultures were induced with 1 mM IPTG (Promega) and returned to the shaker  until fluorescence measurement after 3 h 30 min. For the experiments on the toehold-switch-based XOR gates, a half adder, a half subtractor, and a Feynman gate, chemically transformed E. coli BL21 AI cells were cultured on LB agar plates  with appropriate antibiotics. All antibiotics were purchased from Gold biotechnology: pACYCDuet (25 μg/mL Chloramphenicol), pCOLADuet (50 μg/mL Kanamycin), pCDFDuet (50 μg/mL Spectinomycin), and pET15b (100 μg/mL Ampicillin). Single colonies were grown overnight (~16 h) in 96-well plates with shaking at 800 rpm, 37 °C. Overnight cultures were diluted 1/100-fold into fresh media and returned to shaking . After 80 min, cell cultures were induced with 1% (w/w) arabinose (Gold biotechnology) to produce T7 RNA polymerase for 1 h with shaking . Then the cell cultures were induced with 0.5 mM IPTG, 100 ng/mL aTc (Takara) for the XOR gates and 1 mM IPTG, 200 ng/mL aTc for a half adder, a half subtractor, and a Feynman gate, and returned to the shaker  until fluorescence measurement after 3 h 30 min.For in vivo experiments, For the experiment on the XOR gate with a gradient concentration of chemical inducers d, 200 µLw/v) para-formaldehyde solution (Sigma Aldrich) and fixed for 15 min at room temperature. After fixation, samples were washed twice using 1X phosphate-buffered saline . Fixed cells were diluted by a factor of ~5 into 1X PBS. Cells were detected using a forward scatter (FSC) trigger, and at least 100,000 events were recorded for each measurement. Cell population was gated according to the FSC and side scatter (SSC) distributions as described previously [GFP fluorescence was measured using flow cytometry  after fixation. The cell pellet was resuspended with 2% (eviously . To evalE. coli. In conclusion, toehold-switch-based ribocomputing devices can provide a platform for synthetic RNA-based higher-order circuits in cells.Expanding the pool of programmable and predictable logic gates is one of the important goals of synthetic biology. Here, we aimed to demonstrate several RNA-only logic gates using toehold switches and antisense RNAs. RNA-only XOR gates, serving as the basic building blocks of arithmetic logic circuits, were constructed using orthogonal NIMPLY gates. Subsequently, the optimized synthetic logic gates were incorporated into arithmetic operations and reversible logic gates via a bottom-up approach in single-cell


We systematically investigated the influence of locked nucleic acid (LNA), unlock nucleic acid (UNA), and 2’-O-methyl-RNA (2’-O-Me-RNA) residues on the thermal stability, structure folding topology, biological activity and enzymatic resistance of three sequence-related DNA G-quadruplexes. In order to better understand the mechanism of action of the studied modifications, a single-position substitution in the loops or G-tetrads was performed and their influence was analyzed for a total of twenty-seven modified G-quadruplex variants. The studies show that the influence of each modification on the physicochemical properties of G-quadruplexes is position-dependent, due to mutual interactions between G-tetrads, loops, and additional guanosine at 5’ or 3’ end. Nevertheless, the anticancer activity of the modified G-quadruplexes is determined by their structure, thus also by the local changes of chemical character of sugar moieties, what might influence the specific interactions with therapeutic targets. In general, UNA modifications are efficient modulators of the G-quadruplex thermodynamic stability, however they are poor tools to improve the anticancer properties. In contrast, LNA and 2’-O-Me-RNA modified G-quadruplexes demonstrated certain antiproliferative potential and might be used as molecular tools for designing novel G-quadruplex-based therapeutics. They G-quadruplex forming sequences are present in the human genome and play important roles in pivotal biological processes within cells such as replication, transcription, and translation , 5. Theyanti conformers of guanosine [syn-oriented guanosine within TBA G-tetrad by 2’-O-Me-RNA-G caused structure destabilization, whereas the presence of this modification at anti-position preserved G-quadruplex structure in K+ environment [Among different chemical modifications of sugar moiety, the unlock nucleic acid (UNA), locked nucleic acid (LNA), and 2’-O-methyl-RNA (2’-O-Me-RNA) have attracted great attention and their influence on the G-quadruplex structure and thermodynamic stability has been extensively studied , 14, 15.uanosine , 21, 22.uanosine . Similaruanosine , 25. SinHerein we present a systematic study on the influence of UNA, LNA, and 2’-O-Me-RNA on thermodynamic stability, biological activity and enzymatic resistance of three sequence-related DNA G-quadruplexes which show considerable antiproliferative properties against HeLa cancer cells . The strThe synthesis of the oligonucleotides listed in −4 to 10−6 M. A specific amount of each oligonucleotide was evaporated to dryness and dissolved in buffer containing 100 mM potassium chloride (KCl), 20 mM sodium cacodylate and 0.5 mM Na2EDTA (pH 7.0). The concentrations of single‐stranded oligonucleotides were firstly calculated based on the stock solutions absorbance at 85°C. The extinction coefficients were calculated using the OligoAnalyzer tool (Integrated DNA Technologies). Absorbance versus temperature curves were acquired using the UV melting method at 295 nm with the temperature range of 95°C to 3°C and a temperature decrease of 0.2°C/min using a JASCO V‐650 (Cremella (LC) Italy) spectrophotometer equipped with a thermoprogrammer. The thermodynamic parameters were analyzed and determined using MeltWin 3.5 software. The melting temperatures calculated for the 10−4 M concentration of the oligonucleotide are denoted as TM.UV melting analysis was accomplished for nine different concentrations of each oligonucleotide in the range of 102EDTA (pH 7.0) to reach a sample concentration of 3.0 μM. The G‐quadruplex samples were denatured at 90°C for 3 min and then cooled to room temperature overnight, followed by data collection. The spectra were recorded in triplicate in the 210–300 nm wavelength range, at 37°C and the buffer spectrum was subtracted from the sample spectra. Data analysis was made in the Origin v8.5 software.The CD spectra were collected using the JASCO J‐815 (Cremella (LC) Italy) spectropolarimeter. Each oligonucleotide was evaporated to dryness and dissolved in 1ml buffer containing 100 mM KCl, 20 mM sodium cacodylate and 0.5 mM NaHeLa) cell line  was cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) , 1% Antibiotic–Antimycotic solution  and 1% MEM vitamin solution . The cells were grown in an incubator at 37°C with 5% CO2 and with humidity of 95%.The human cervical adenocarcinoma  in RPMI 1640 without phenol red media, was added to the wells. The cells were incubated at 37°C with 5% CO2 and 95% humidity for 4h. Following, the medium was removed and replaced with 100 μl/well solution of 70% isopropanol and 40 mM HCl to liquefy the blue‐purple crystals of formazan. The plates were shaken at 300 rpm at room temperature for 30 min. For the quantification of the free formazan a microplate reader xMark  was used and the absorbance was measured at 570 nm. Data analysis was made using Microsoft Excel 2016 software. Cell viability was calculated according to the equation: % Viability = (mean OD of the cells with oligo/ mean OD of the control cells) x 100. Each experiment was repeated in triplicate, and the results were expressed as the means ± SD.The G‐quadruplexes antiproliferative properties were measured by using the MTT assay. Each oligonucleotide had a final concentration of 10 μM and was dissolved in 1× PBS buffer with 100 mM potassium chloride (KCl), followed by denaturation at 90°C for 5 min with subsequent cooling to room temperature overnight. The experiments were conducted on cervical HeLa cell line, being seeded in 96‐well plates at a density of 500 cells/well in 100 μl of RPMI 1640 medium  supplemented with 10% FBS  and MEM 1% vitamin solution . The 96‐well plates were incubated at 37°C, with 5% COIn order to analyze the stability tendency of the G-quadruplex, a nuclease stability assay was conducted by CD spectroscopy method. Approximately 7 nmol of stock solution of each G-quadruplex was evaporated to dryness, dissolved in 1xPBS with 100mM potassium chloride (KCl) buffer, denatured 5min at 90°C and then cooled to room temperature overnight. The degradation patterns were analyzed by monitoring the CD signal decrease of each sample in 400 μL of RPMI 1640 medium  with 10% fetal bovine serum (FBS)  and incubated at 37°C. The CD spectra were recorded at 4°C using JASCO J‐815 (Cremella (LC) Italy) spectropolarimeter equipped with a Peltier temperature control system after 0h and 24h of incubation. The CD spectra were recorded in duplicate in the 235–330 nm wavelength range. Each spectrum presented was corrected for the spectrum of the reaction medium (RPMI with 10%FBS). Data analysis was performed using Origin v8.5 software where the maximum CD signal was obtained for the 0 and 24h. The stability percentage of the G-quadruplexes was calculated in Excel, where the signal intensity at 0 time corresponded to 100% of oligonucleotide viability.The results are reported as the means ± standard deviation, and at least 3 independent biological replicates were performed for the MTT assay. Data analysis was made using Sigma Plot software , and the statistical significance treatment in cells between the native sequence and modified sequences was tested by one‐way ANOVA. Normality was tested by the Shapiro–Wilk test. The differences were considered statistically significant for p < 0.001.i.e. loops composition and number of G-tetrads, and according to the considerable inhibitory effect on the HeLa cancer cells growth. Moreover, all of the oligonucleotides showed moderate stability in human serum, making them good candidates for improving their therapeutic potential, most probably due to the synergistic toxicity of G-quadruplex degradation products [i.e. UNA, LNA and 2’-O-Me-RNA which were reported previously as modulators of the TBA G-quadruplex physicochemical and biological properties. Importantly, the studies based on isosequential G-rich oligonucleotides with minor structural variations give an unprecedented opportunity to understand more globally the influence of chemical modifications on G-quadruplex structure and biological activity.Diverse nucleic acid chemistries, as well as various G-quadruplex architectures, make designing of an optimal G-quadruplex therapeutic agent relatively challenging. In this article, we selected three G-quadruplex forming sequences  from a pproducts . For fur3T4G3)2 (ON1) forms an antiparallel, dimeric G‐quadruplex with three G‐tetrads and diagonal loops 2 2  from one of the strands are positioned outside the G-quadruplex core [According to previously published NMR studies, oligonucleotide d2 ON2,  in Na+ s4)2 ON3,  folds inG-quadruplex formation of modified oligonucleotides was evaluated by UV melting analysis, providing comprehensive thermodynamic parameters of the twenty-seven modified variants and information about stoichiometry of the folding process. In order to study the effect of the chemical modifications on the G-quadruplex topology, the circular dichroism (CD) spectroscopy was also performed. The biological studies were performed by MTT assay in HeLa cancer cell line to investigate the antiproliferative activity of the modified variants. The nuclease stability was monitored by CD measurements to evaluate oligonucleotides viability tendency after 24h incubation in RPMI medium supplemented with 10% FBS. The extensive variety of analyses permitted us to analyze the influence of the UNA, LNA and 2’-O-methyl-RNA modifications on the G-quadruplex structure, topology, anticancer potential, and enzymatic viability.i.e. LNA, UNA and 2’-O-Me-RNA were placed in three different positions of ON1, ON2 and ON3 G-quadruplexes  in comparison with ON2 and ON3  due to the lack of additional guanosine residues at the terminal positions  and ON2–L6 (position T6) is slightly disadvantageous for the G-quadruplex thermal stability leading to decrease of TM value by 2.6°C and 3.7°C, respectively. However, the presence of LNA-T at position T5 of ON3–L5 increases G-quadruplex thermal stability (ΔTM = +3.4°C). The replacement of thymidine by LNA-T in the loop near the guanosine residue  was favorable for the G-quadruplex thermal stability increasing TM value by 1.7°C for ON1–L7 and 1.2°C for ON2–L8. In contrast, a decrease in melting temperature by 1.4°C was caused by the presence of LNA-T at position T7 of ON3–L7. The unmodified ON1–ON3 G-quadruplex structures are characterized by a number of interactions between the thymidine residues in the loops i.e. stacking interactions and H-bonding between each other or with G-quartet residues [ON3 in reference to ON1 and ON2 might be due to more favorable orientation of LNA–T for the interactions within ON3 or the fact that T5 from both strands are not involved in the additional interactions with G-tetrad resulting e.g. in less significant disruption of the structure. In contrast, in ON1 and presumably also in ON2 the thymidine residues at T5 and T6 positions of both strands are involved in various stacking interactions and H-bonding, whereas the effect is opposite for the T7 and T8 positions. In this case, the thymidine residues of all studied G-quadruplexes in both strands are involved in the variety of interactions with the remaining residues within the structure. Therefore, the final effect of the LNA–T substitution might be dependent on the character of interactions and on the favorable or unfavorable orientation of the LNA–T residue forced by locked nature of the modification or the topology change induced by LNAs (see next section).The replacement of guanosine with LNA-G in the G-tetrad (G2 position) caused stabilization of the three G-quadruplex variants leading to an increase in Tectively , Fig 2. residues , 21, 22.ion only , 20, 24.ructures . Differeresidues –31. The In general, the presence of LNA-T modification within a loop, results more often in a slight enhancement of the thermal stability of G-quadruplex structures when the modification is positioned near the G-tetrad. However, the most significant improvement of the G-quadruplex thermal stability occurred when the LNA was placed directly within G-tetrad.ON1, ON2 and ON3 G-quadruplexes , whereas the decrease of ON2–U2 melting point in reference to ON2 was -13.5°C. This could be due to the presence of specific G-quadruplex structure of ON2–U2, where two guanosine residues are aligned at the same side of the G-tetrad, mimicking almost half of the G—tetrad and providing additional stabilizing element which neutralizes unfavorable consequences of the presence of UNAs. However, the influence of folding topology changes revealed by CD spectra analysis described below should be also considered. Agarwal et al. reported that substitution of single UNA modification in the G-tetrad results in considerable destabilization of the structure with significant decrease in melting temperature [ON1 and ON3 or T6 of ON2) demonstrated to be thermally advantageous for all studied G-quadruplexes . In contrast, the substitution of thymidine residue at position T7 (ON1 and ON3) or T8 (ON2) resulted in decrease of thermal stability of ON1 and ON3 by 1.9°C (ON1–U7) and 5.3°C (ON3–U7), whereas destabilization of ON2 was less significant . Previously published data indicated that UNA modification in the loop stabilizes G-quadruplex structure and destabilizes when present in the core [ON1–ON3 G-quadruplex structural elements are opposing i.e. UNA stabilizes the structure at the positions at which LNA is unfavorable and vice versa. This clearly suggests that flexibility or stiffness of sugar moiety is one of the pivotal factors for G-quadruplex structure stability.As expected, the replacement of guanosine residue in the G-tetrad by UNA-G at G2 position was highly disadvantageous for thermal stability of ruplexes , Fig 2. perature . Thus, tthe core , 17. OurON1 and ON3 (M = -0.1°C for ON1–M2 and -1.8°C for ON3–M2). In contrast, the presence of 2’-O-Me-RNA-G at G2 position of ON2 increased the thermal stability by 5.6°C. This diverse behavior might be due to the differences in G-quadruplex structure of ON1–ON3, what stays in accordance with previous investigations indicating that 2′-O-methyl-RNA is able to variously modulate G-quadruplex stability depending on the type of the G-quadruplex folding [ON2–M2 can be also the contributor to various effects observed for the variants modified within the G-quadruplex core.Replacement of one of the guanosine residues in the G-tetrad by 2’-O-Me-RNA-G at G2 position caused a minor decrease in the thermal stability of  folding . HoweverON1 (ON1–M5) and ON3 (ON3–M5) demonstrated only a slight increase in thermal stability of ON1 and ON3 . In contrast, the presence of 2’-O-Me-RNA at position T6 of ON2 (ON2–M6) caused a decrease in the thermal stability (ΔTM = -2.4°C). Replacement of thymidine by 2’-O-Me-RNA in the loop near the guanosine residue at position T7 of ON1 and ON3 and at position T8 of ON2 resulted in decrease of the G-quadruplex thermal stability by 1.1°C (ON1–M7), 1.9°C (ON2–M8) and 0.7°C (ON3–M7).The thymidine replacement within the loops by 2’-O-Me-RNA residues at position T5 of C3’endo sugar puckering, and both were reported to increase thermal stability of G-quadruplexes when they replace guanosine residue having anti conformation [In general, the 2’-O-Me-RNA modification within G-tetrads induced mild changes in the thermal stability of studied G-quadruplexes, in comparison to LNA and UNA modifications. According to already published data, LNA and 2’-O-Me-RNA modifications adopt ormation , 25. Theormation .ON2 G-quadruplex demonstrated to be more tolerant for chemical modifications in the G-tetrad, showing the most restrained thermal stability changes among all three types of studied G-quadruplexes. This might be due to the extra guanosine residue at the 5’ terminal position of oligonucleotide, which results in formation of G-quadruplex core with two guanosine residues aligned at the same side of the G-tetrad mimicking almost half of a G-tetrad and providing more stability to the G-quadruplex structure. Even though ON3 includes the extra guanosine at the 3’ end, the positioning of two guanosine residues within G-quadruplex structure is at opposite sides of the core, not leading to extra stability as it is observed in ON2.Overall, the Circular dichroism spectroscopy is a primary technique for the characterization of G-quadruplex topology and is based on changed absorbance of circularly polarized light, providing different CD spectral characteristics , 34. Var3T4G3)2 (ON1) and d(G4T4G3)2 (ON2) show reshaping of CD pattern which is typical for an antiparallel G-quadruplex topology with changed glycoside bond angle arrangement of guanosines in the G-tetrads or for structural polymorphism with a predominance of antiparallel folding topology. Our findings stay in accordance to previous reports [3T4G4)2 (ON3) demonstrated a hybrid topology. The CD spectra of ON1, ON2 and ON3 variants containing LNA, UNA or 2’-O-Me-RNA residues were analyzed to determine the influence of a particular modification on the G-quadruplex topology.The CD spectra of d. The ON2–L2 demonstrated a shift of the positive signal at 260 nm to around 250 nm, maintaining the positive signal at 295 nm , though the LNA modifications in the loops did not change CD pattern of ON3. Previously reported studies indicate that LNA single substitution within G-tetrad leads to a change of G-quadruplex topology from antiparallel to parallel, being partially in accordance with our findings [topology . Surprist 295 nm . The varwith LNA  revealedfindings , 24.ON1  being unfolded at the physiological temperature of 37°C. The CD spectra of ON1–U5 clearly showed a shift of CD pattern presenting two maxima near 260 nm and 290 nm and a minor negative band near 240 nm. The above change might indicate a hybrid topology of ON1–U5 G-quadruplex. In contrast, ON1–U7 demonstrated a shift of topology from antiparallel to parallel. A significant reduction of CD bands was also observed for ON2–U2 containing UNA residue within G-tetrad and showing low stability of G-quadruplex structure  induced only a shift of G-quadruplex topology from hybrid to parallel, with no significant signal reduction . The CD spectra of ON1–M5 and ON1–M7 are also characterized by a minor shift of CD bands in reference to ON1, with two positive signals around 265 nm and 285 nm, however their topology is unclear. The CD spectra of ON2 variants, i.e. ON2–M2 and ON2–M8  caused only minor, 10–30% inhibition of cancer cells growth. These LNA-modified G-quadruplexes were characterized by substantial improvement of the thermal stability, clearly demonstrating that the antiproliferative effect is not directly dependent on the thermal stability of the structure.The antiproliferative activity of activity . The majHela cells viability in comparison to the impact of parental sequences. In general, the presence of UNA within majority of studied G-quadruplexes caused a significant increase in the number of viable cells from 43.9, 39.0 and 33.3% for the unmodified ON1, ON2 and ON3 G-quadruplexes, respectively, to 70–90% for the variously modified variants. The only exception was ON2–U2, which inhibited cells growth by 47.3% (52.7% of viable cells). The above results clearly show that UNA modifications are unfavorable for the antiproliferative properties of the G-quadruplexes, being in accordance with the previous studies reported by Kotkowiak et al. indicating very moderate ability of UNAs to influence anticancer properties of TBA [The treatment of cells with UNA-modified G-quadruplex variants resulted in increased s of TBA .ON1 (ON1–M2) resulted in a minor loss of ON1 antiproliferative activity, decreasing HeLa cell viability up to 60.5%. In contrast, the presence of 2’-O-Me-RNA modification within ON1 loops maintains the inhibitory potential of the G-quadruplex leading to cell viability at the level of 53.8% for ON1–M5 and 50.6% for ON1–M7. For the ON2 variants modified with 2’-O-Me-RNA we could observe that none of them maintain similar antiproliferative potential when compared with ON2. Treatment of cells with ON3 variants modified with 2’-O-Me-RNA in the G-tetrad (ON3–M2) or in the middle of the loop (ON3–M5) maintains the growth inhibition capacity of ON3 with no statistically significant difference when compared with the parental sequence. The presence of 2’-O-Me-RNA residue at position T7 of the loop (ON3–M7) demonstrated also certain antiproliferative potential, however the level of viable cells was slightly higher than for ON3 (51.9% of viable cells).The presence of 2’-O-Me-RNA residue within G-tetrad of ON1 and ON3 demonstrated certain antiproliferative effect, whereas ON2 variants showed substantially decreased inhibitory activity. Importantly, the presence of UNA residues within G-quadruplex structures appeared detrimental for inhibitory activity of the majority of ON1–ON3 variants. The increased flexibility of G-quadruplex structures induced by the presence of UNA modifications might interfere with the favorable interactions between the G-quadruplexes and a target, being the reason for the decreased inhibitory effect observed for UNA-modified ON1–ON3 variants.The above data indicates that the LNA chemical modifications can be introduced in different loop positions without a significant influence on the antiproliferative potential of majority of the G-quadruplex variants. However, the presence of LNA residues is detrimental for the biological activity when modifications are placed in G-tetrads. All 2’-O-Me-RNA-modified variants of The biological stability of G-quadruplexes is a key feature of therapeutic oligonucleotides. However, the degradation by exonucleases under physiological conditions is one of the main problems in applications of therapeutic oligonucleotides , 8, 11. ON1, ON2 and ON3, respectively  did not change the oligonucleotide enzymatic resistance. However, a decrease of stability in the 8–11% range was observed for ON2–L2 and ON2–L6. The presence of LNA within ON3 decreased nuclease stability of parental G-quadruplex by 14.9% (ON3–L2), 24.1% (ON3–L5), and 9.5% (ON3–L7). In general, LNA modification did not demonstrated universal tendency for improvement of the nuclease stability of studied G-quadruplexes. Despite the above results, LNA rarely might be also a modification of choice for modulation of G-quadruplex properties, as presented for ON1–L5 which was characterized by moderately enhanced enzymatic resistance without major influence on the antiproliferative potential of G-quadruplex.The substitution of LNA modifications within M [ON1–U2 and ON3–U2 demonstrated a significantly elevated levels of undegraded oligonucleotide after 24h in reference to unmodified parental compounds , whereas a similar stability tendency in comparison to unmodified ON1 was observed for ON1–M7. The ON2 variants modified with 2’-O-Me-RNA were characterized by 16.1% and 4.3% increase in the nuclease stability for ON2–M2 and ON2–M6, respectively. In contrast, ON2–M6 demonstrated a decrease of undegraded oligonucleotide level by 12.3%. The presence of 2’-O-Me-RNA residue within ON3 G-tetrad (ON3–M2) induced minor, 5.4% increase of the G-quadruplex nuclease stability. However, the modification of the loops by 2’-O-Me-RNA led to a decrease in enzymatic stability by 2.2% and 7.5% for ON3–M5 and ON3–M7, respectively.The 2’-O-Me-RNA-modified The nuclease stability of the analyzed G-quadruplexes is neither directly proportional to their thermodynamic stability nor to their antiproliferative properties. The 2’-O-Me-RNA revealed to be the most neutral modification which enhances or maintains the G-quadruplex enzymatic resistance without significant depletion of the G-quadruplexes antiproliferative potential. Surprisingly, the presence of UNA within G-tetrads of two studied types of G-quadruplexes appeared favorable for the biological stability of these oligonucleotides despite a large destabilization of G-quadruplex structure and unfavorable changes of antiproliferative potential.in vitro, without significant changes in the thermal stability, structure folding topology and with maintaining certain enzymatic resistance. Moreover, G-quadruplexes modified by LNAs within the loops demonstrated to have similar antiproliferative potential to the native sequences. Both modifications were confirmed to be modifications of choice for designing new potential G-quadruplex-based therapeutic candidates for future pre-clinical investigations.Chemical modifications play important role in the G-quadruplexes design, since they can modulate their properties. In this study, the structural and biological properties of twenty-seven G-quadruplex variants containing LNA, UNA or 2’-O-Me-RNA modifications in the loop or in the G-tetrad were examined. As this was a preliminary evaluation of these novel G-quadruplexes, a single 10 μM concentration of oligonucleotides was used to provide some general conclusions to foster more detailed investigations. The above studies suggest that the G-quadruplex structural elements cannot be considered separately without consideration of their involvement in the interactions with other parts of the structure, therefore the structural prediction still represents a great challenge. The influence of each modification on the physicochemical properties of G-quadruplexes can be different, depending on mutual interactions between G-tetrads, loops, and in some cases also additional guanosine at 5’ or 3’ end. Moreover, the inhibitory activity of the G-quadruplexes is strongly dependent not only on the structure but also on local changes of residues chemical characteristics and on the specific interactions with the binding target. To summarize, UNA modifications can be modulators of G-quadruplex thermodynamic stability but are rather of little use for improvement of anticancer potential of studied G-quadruplexes. In contrast, 2’-O-Me-RNA modified G-quadruplexes were found to be effective in inhibition of HeLa cancer cells proliferation S1 File(DOCX)Click here for additional data file.


Bunyaviruses are negative sense, single-strand RNA viruses that infect a wide range of vertebrate, invertebrate and plant hosts. WHO lists three bunyavirus diseases as priority diseases requiring urgent development of medical countermeasures highlighting their high epidemic potential. While the viral large (L) protein containing the RNA-dependent RNA polymerase is a key enzyme in the viral replication cycle and therefore a suitable drug target, our knowledge on the structure and activities of this multifunctional protein has, until recently, been very limited. However, in the last few years, facilitated by the technical advances in the field of cryogenic electron microscopy, many structures of bunyavirus L proteins have been solved. These structures significantly enhance our mechanistic understanding of bunyavirus genome replication and transcription processes and highlight differences and commonalities between the L proteins of different bunyavirus families. Here, we provide a review of our current understanding of genome replication and transcription in bunyaviruses with a focus on the viral L protein. Further, we compare within bunyaviruses and with the related influenza virus polymerase complex and highlight open questions. Bunyavirales constitutes a diverse group of predominantly insect or rodent-borne viruses with a segmented single-stranded RNA genome of negative polarity. In 2017, the formerly separated families of Bunyaviridae and Arenaviridae were combined to form the order of Bunyavirales [https://talk.ictvonline.org/taxonomy/). Some bunyaviruses such as Lassa virus (LASV), Rift Valley fever virus (RVFV), and Crimean-Congo hemorrhagic fever virus (CCHFV) are of general public health concern as emphasized by their inclusion in the WHO R&D Blueprint list of priority diseases [The order diseases  because diseases . A comprde novo typically by a prime-and-realign mechanism and proceeds through a complementary positive-sense intermediate of the viral genomic RNA (cRNA) [cis-acting elements essential for genome replication and transcription processes.After attachment and entry of bunyaviruses into host cells, the viral RNA (vRNA) genome segments associated with the viral L protein and the nucleoprotein (NP) are released into the cytoplasm where viral transcription and genome replication take place. The 3′ and 5′ termini of each genome segment are highly conserved within each bunyavirus species. They are also highly complementary and thus capable of forming a double-stranded RNA (dsRNA) stem, allowing each genome segment to potentially form a pseudo-circularized panhandle . HoweverA (cRNA) . Some buBunyavirales order were long awaited in the field and their appearance is summarized in Peribunyaviridae family, using a construct with a truncated C-terminus [apo form by low-resolution cryogenic electron microscopy (cryo-EM). A high-resolution cryo-EM structure of full-length LACV L was subsequently determined in 2020, containing the C-terminal region (CTER) that was missing in earlier structures [(Phenuiviridae) [Arenaviridae) [Arenaviridae polymerase activity [in vitro and cell-based assays [Although several bunyavirus NP structures have been published since 2010 –37, struterminus . It was ructures . Concomiviridae) –44, as wviridae) . Howeverviridae) ,47, snapviridae) –50. LACVviridae) . SFTSV aviridae) ,50  an endonuclease domain (ENDO) that undergoes large, rigid-body displacements during L protein activity; (ii) an elongated linker-region; and (iii) a PA-C-like domain that is defined as core lobe and vRNA-binding lobe (vRBL) in proteins . Both thThe globular L protein CORE is composed of the elongated PA-like linker, the PB1-like region, and the PB2-like lid. It contains a large central cavity that encompasses the RdRp active site including the canonical motifs A to F that are essential for RdRp activity. These motifs (i) select and stabilize the NTP that is complementary to the RNA template; (ii) coordinate two metal ions that are necessary for a nucleophilic attack on the α-phosphate of the fixed nucleotide; (iii) translocate the template and the nascent product after nucleotide incorporation; and (iv) reinitiate the catalytic cycle. Motifs A to E are localized in the palm whereas motif F is part of the fingers. Additional specific motifs of segmented negative-strand viruses (sNSV) include motif G in the thumb and H in the fingers domain ,40,48,66Bunyavirales and also in comparison to the PB2 protein of Orthomyxoviridae [The CTER displays the highest sequence divergence within the oviridae ,67. It coviridae ,49,60,61oviridae ,49,60,61oviridae ,60. The oviridae , whereasoviridae . The SFToviridae ,42. Inteoviridae . Both thoviridae .Structures of LASV, SFTSV, and LACV L proteins in pre-initiation and early elongation conformations show binding of the conserved genomic 3′ and 5′ RNA termini, which constitute the usually highly complementary and conserved viral promoter, with a length of up to 19 nucleotides ,48,50. Tin vitro polymerase activity could be observed in the absence of the 5′ RNA, mutations in this binding site resulted in a complete RNA synthesis defect of phenuivirus and arenavirus L proteins in cell-based mini-replicon assays [In LACV L, 5′ hook binding has been shown to result in the ordering of the fingertips region of the RdRp that corresponds to motif F and stabilization of residues surrounding the NTP entrance tunnel . For SFTn assays ,50, whicn assays . The pren assays –50,70,71Peribunyaviridae) and LASV where the complementarity of nucleotides within the distal duplex was shown to be critical for genome replication and transcription [The 5′ RNA region upstream of nucleotide 10 forms a duplex with the complementary 3′ RNA, denoted the distal duplex . Structucription –74.in vitro protein–RNA interaction studies led to the conclusion that nucleotides 2 to 5 of the 3′ terminus were essential for template binding, although the existence of a secondary binding site was unknown at that time [In addition to the expected position of the 3′ RNA towards the active site, a secondary binding site has been observed for LACV, SFTSV, and LASV L . It is fhat time . Togethehat time ,73. Bindhat time ,50. The Promoter-bound pre-initiation structures were important to better understand the organization of the L protein. In combination with further structures of functionally relevant conformations, they foster understanding of the molecular mechanisms underlying L protein catalytic activity.in vitro and in cells. For both LASV and LACV, mini-replicon assays revealed that the tag necessary for protein purification should not be added at the N or C termini as this significantly reduced L protein activity [in vitro [in vitro activity [For LACV, SFTSV, and LASV, stalling the L protein in active states for structural determination was linked to the availability or development of functional assays both activity ,59. Instactivity  or at thin vitro ,50. In tin vitro . Additioactivity .de novo formation of a 2 to 3 nucleotide primer, which is also the rate-limiting step in the genome replication process, a realignment of the primer to the template terminus takes place. During this realignment step, the template moves backwards in the active site so that the newly aligned primer can be subsequently extended by the polymerase during elongation  [For some RdRps that perform terminal initiation of coming”) ,87–89. Fcoming”) , consistcoming”) –45,48–50coming”) . The seqcoming”) . Howevercoming”) .The arenavirus genome 5′ end contains an additional triphosphorylated G . It was In hantaviruses, the product of genome replication is monophosphorylated at the 5′ end. It has been suggested that initiation starts internally at nucleotide +3 of the template with an initial GTP generating a short primer . This shFor phenuiviruses, particularly SFTSV, both terminal initiation and internal initiation at position +3 with subsequent realignment of a dinucleotide could be equally well explained by biochemical data . In bothOverall, structural data of genome replication at the initiation stage are still rare, likely due to the transient nature of these conformations. Therefore, although there have been many hypotheses about the presence and location of priming loops within the L proteins of LASV, JUNV, SFTSV, and RVFV, all of these remain highly speculative for now ,44,45.Structures of L proteins stalled at elongation have been determined for LASV, LACV, and SFTSV ,48,50. Ltrans, the 5′ end must be released from its dedicated binding site to pass the active site and allow for production of a full-length cRNA or vRNA. After product disassociation, one can speculate that the template backtracks so that the 5′ end rebinds to the 5′ binding site in the same L molecule, but there are no structural data published supporting this hypothesis yet. Notably, in case of arenaviruses, the nontemplated extra G nucleotide added to the 5′ terminus during prime-and-realign is not replicated and therefore does not lead to the addition of an extra C at the 3′ end. RNA synthesis is therefore terminated in a way that retains the original 3′ sequence. The genome segments packaged into nascent virions, therefore, contain only a single-nucleotide G overhang at the 5′ end [To date, there are no structural data visualizing chain termination in bunyavirus genome replication. If it is not provided in e 5′ end .in vitro, although their essential role in viral transcription was detected in cell-based studies [Viral transcription is initiated by a primer, a capped host RNA fragment obtained by cap-snatching. In the case of influenza virus, cap-snatching occurs in the nucleus and is rather well understood, while for the cytoplasmically replicating bunyaviruses, many aspects, such as the nature, source, and location of the target capped RNA, remain obscure as reviewed previously . However studies ,102–106.in vitro possess a catalytically important histidine residue upstream of the PD(E/D)K motif, which is implicated in first cation coordination, whereas the ENDOs with very low or absent in vitro activity contain a glutamate or aspartate residue instead. Structural knowledge was initially obtained via X-ray crystallography studies of isolated ENDO domains from LACV (Peribunyaviridae), Lymphocytic choriomeningitis virus, LASV, Pichinde virus (PDB-ID: 4i1t), California Academy of Sciences virus (CASV) (Arenaviridae), Hantaan virus, Andes virus (Hantaviridae) as well as Toscana virus and SFTSV (Phenuiviridae) [Structurally, all ENDOs have a similar two-lobed, kidney-shaped architecture, harbor a PD(E/D)K active site motif, and coordinate two divalent metal ions essential for catalytic activity ,103,104.viridae) ,103,104.viridae) . In the viridae) .7GTP for SFTSV and RVFV [The global fold of the CBD and CBD-like domains (determined either isolated or in the context of the full-length L) is conserved. It is composed of a β-sheet that packs against a conserved α-helix, and additional elements that vary between families. The cap-binding residues are located on the first β-strand of the central β-sheet and either in a β-hairpin  or a loop (for LACV) connecting two strands of the β-sheet. The CBD has been solved in complex with capped RNA for LACV, and with mand RVFV ,49,61. Cand RVFV ,60. The and RVFV ,97. It tapo structure of SFTSV L protein, the cap-binding site was found to be blocked by an arginine sidechain stacking between the two aromatic sidechains responsible for cap-binding revealing a potential regulatory mechanism of cap-snatching [Despite the global similarities, the structure of LACV CBD was somewhat surprising as it does not bind the cap mainly via aromatic stacking as observed in many other cap-binding proteins ,107–109.natching –43.The structure of LACV L has been determined in three different conformations related to viral transcription, respectively called “Capped primer cleavage,” “initiation,” and “early elongation”  Fig 6)..Fig 6).The capped primer cleavage conformation is suggested to be compatible with cleavage of the capped RNA by the ENDO during cap-snatching. Compared to the pre-initiation conformation, the capped primer cleavage structure reveals a large conformational change of the CTER and the ENDO together with a 20° rotation of the ZBD towards the CORE generating a closed conformation of the entire LACV L protein. A concerted movement of the CBD brings the cap-binding site, which was exposed to the solvent in the pre-initiation state, to a more buried conformation, where it interacts with the CORE. Although only the first two residues of the capped RNA are resolved in this structure, it is conceivable that the RNA projects towards the ENDO active site as the path is open. So far, no structure of LACV L with RNA bound to the ENDO has been published. Although this has been achieved for SFTSV L, transcription state structures of the phenuivirus L are still missing .Following cleavage, the capped RNA primer needs to be reoriented towards the RdRp active site. It was suggested that for the LACV L, primer RNA entry into the RdRp active site cavity results from the electrostatic repulsion of the ENDO, which reorientates between the cleavage and initiation conformation . In the Prime-and-realign has also been observed in transcription of sNSVs –116. Whein vitro [In the transcription initiation structure of LACV L protein, capped RNA entry into the active site cavity is correlated with the retraction of the PR-loop that stabilizes the 3′ template in the cleavage conformation . During in vitro .Following initiation, the L protein proceeds to elongation. In the LACV L transcription elongation structure, this is correlated with movements of the CBD and the ENDO back to the position they had at pre-initiation stage  compare and 6. TDifferent to cellular mRNA and influenza virus transcription, bunyavirus transcription products are usually not polyadenylated, the only exception being hantaviruses, in which mRNAs transcribed from the M genome segment terminate in a poly-U stretch and therefore contain a poly-A sequence . Most RNin vitro and reduces RdRp activity in a cell-based minigenome system [Given that the L protein produces different products (mRNA and cRNA / mRNA and vRNA) from the same template, there must be some kinds of regulatory mechanisms in place to control the generation of these different RNA species. Notably, many bunyaviruses use the vRNA as a template for viral genome replication and transcription but prefer genome replication in presence of the cRNA as a template. For BUNV, it has been reported that the sequence of the 3′ RNA terminus is crucial for this preference but not the only determinant . On the e system . Howevere system ,52,127.in vitro and cell-based assays [Arenaviridae family, which was found to not contain any Z protein or similar. It is unclear which protein drives budding and RNP recruitment in this case [Another regulatory switch occurs when arenavirus Z protein binds to the L protein. Z has been shown to inhibit the L protein RdRp activity in both d assays –59,132. d assays ,133 and d assays  in the pd assays –53. The d assays ,51–53. Ad assays . Consided assays ,135. Thed assays , a late d assays ,138, andd assays ,140. Thehis case .Structurally, the bunyaviral RNP, the functional unit of genome transcription and replication, is a complex of vRNA or cRNA encapsidated by the NP and associated with L. The lack of structural data exploring how these RNPs, firstly, scaffold the processes of genome replication and transcription and, secondly, are coreplicationally assembled is one of the major outstanding problems for bunyavirus research. However, although at low resolution, recent studies looking at isolated RNPs of BUNV and MACV have provided us with important new insights into these complexes ,143. ForIt is clear that L and NP need to interact during genome replication and transcription. Firstly, for both processes, NP needs to be stripped from the template as it enters the polymerase active site and the NP needs to be rebound to the outgoing template to preserve RNP integrity. Secondly, for replication only, the nascent vRNA and cRNA are packaged by newly synthesized NP, whereas transcripts are not packaged. It is plausible, therefore, that NP plays a regulatory role in balancing viral transcription and genome replication , althougDue to its perennial threat to global public health through seasonal epidemics and periodic pandemics, the influenza orthomyxovirus has received considerably more attention than other segmented RNA viruses such as bunyaviruses, which tend to cause much more localized outbreaks. This also extends to detailed understanding of the transcription and replication mechanisms involving the polymerase. It is therefore interesting to compare the heterotrimeric orthomyxovirus polymerase complex to the monomeric bunyavirus L proteins and identify common and distinct features. Although these viruses are closely related, the location of genome replication and transcription inside the infected cell is distinct with the orthomyxovirus polymerase complex localizing to the nucleus, whereas bunyavirus L protein remains in the cytoplasm. However, it is now well established that the overall architecture of the monomeric L protein corresponds to a concatenation of the three influenza virus polymerase subunits in the order PA-PB1-PB2 ,93 comp.In all segmented RNA viral polymerases, the mobile ENDO and CBD coordinate cap-snatching. Unlike for bunyaviruses, the source of, and mechanism of access to, the capped primers required for transcription is known for influenza virus. In the nucleus, cap-snatching occurs through the binding of the influenza virus polymerase to the Serine 5 phosphorylated CTD of initiating cellular Pol II giving access to nascent capped transcripts –155. Conde novo initiation happens terminally for vRNA to cRNA replication, and internally for cRNA to vRNA replication [Significant structural differences between influenza virus and bunyavirus polymerases mainly lie in the idiosyncratic domains in the CTER beyond the mid-link and CBD. In influenza virus polymerase, these are the 627 and NLS  domains. The 627 domain is critical for mediating host factor ANP32-dependent asymmetric dimerisation of the polymerase during genome replication, and the NLS enables nuclear import of the PB2 subunit through interaction with importin-α. Since they interact with host proteins, both these domains contain important host-specific amino acid variations . In LASV, there is also a 627-like domain but with a distinct extreme CTER. In LACV and SFTSV, this region contains unique structures, respectively, a ZBD with a protruding β-hairpin strut and a loop-like lariat, both of which bridge to the CORE, stabilising the whole CTER in certain functional states. Unfortunately, to date, there is no knowledge about how any host factor interacts with L proteins. Another important difference stems from the fact that the two single-stranded ends of the promoter are less complementary and crucially of different lengths in influenza virus when compared to bunyaviruses. A consequence of this is that for influenza virus polymerase, lication , since tlication . No obviapo structure, the ENDO and CBD were visible and their position opposite compared to the influenza virus polymerase [Another difference between influenza viruses and bunyaviruses is the relative position of the CORE, ENDO, and CBD at transcription. In the influenza virus polymerase complex, the CBD undergoes a large rotation to first bring the capped RNA towards the ENDO for cleavage and subsequently insert it into the RdRp active site for transcription priming . This dilymerase , the traIn some bunyaviruses, the ENDO and CBD have been observed to be self-inhibited, which is not the case for influenza virus . For LASV, two different examples of self-inhibition of the ENDO by other peptides of the L protein have been observed, and for SFTSV, the CBD is self-inhibited by an arginine (R1843) from an adjacent L protein region, necessitating a conformational change to allow capped primer binding. Finally, there are key functional differences between mechanisms of termination of influenza virus polymerase and bunyavirus L proteins. Whereas during transcription termination, influenza virus polymerase polyadenylates the viral mRNA by stuttering, the mechanism of which is now rather well understood , for mosIn recent years, tremendous progress has been made in characterizing the structure and function of the L protein, which, in turn, has given new insight into the detailed mechanisms of bunyavirus genome replication and transcription. These new structural data open a new era in bunyavirus research where functional studies on genome replication and transcription can be supported by structural data or structure predictions. However, many gaps need to be filled to achieve a complete picture of these processes and their regulation. What is the role of L protein dimers? What determines the different modes of termination during transcription and replication? How is the 5′ RNA released from its dedicated binding pocked during genome replication? How do the different primer lengths detected in bunyavirus families  relate tAlthough bunyaviruses have a relatively simple composition, their proteins and RNA are multifunctional, multifaceted, and depend not just on each other but also on host factors. Dissecting the exact and potentially multiple roles of each functional element within the RNA and proteins will allow for comparisons to be made and commonalities to be identified between bunyavirus families. Although this will be challenging, it is essential and will allow for the rational design of antiviral strategies. Looking at the progress made on related viruses, we expect the next major step to be the determination of the structure and dynamics of functional RNPs and modelling of their assembly and structural changes during genome replication and transcription processes. Discovery of host factors involved in these processes will be instrumental to obtain a full picture. Maybe electron tomography will allow us to visualize and understand these processes in the context of the cell. We therefore look forward to even more exciting research on these highly relevant viruses.


RNA editing processes are strikingly different in animals and plants. Up to thousands of specific cytidines are converted into uridines in plant chloroplasts and mitochondria whereas up to millions of adenosines are converted into inosines in animal nucleo-cytosolic RNAs. It is unknown whether these two different RNA editing machineries are mutually incompatible. RNA-binding pentatricopeptide repeat (PPR) proteins are the key factors of plant organelle cytidine-to-uridine RNA editing. The complete absence of PPR mediated editing of cytosolic RNAs might be due to a yet unknown barrier that prevents its activity in the cytosol. Here, we transferred two plant mitochondrial PPR-type editing factors into human cell lines to explore whether they could operate in the nucleo-cytosolic environment. PPR56 and PPR65 not only faithfully edited their native, co-transcribed targets but also different sets of off-targets in the human background transcriptome. More than 900 of such off-targets with editing efficiencies up to 91%, largely explained by known PPR-RNA binding properties, were identified for PPR56. Engineering two crucial amino acid positions in its PPR array led to predictable shifts in target recognition. We conclude that plant PPR editing factors can operate in the entirely different genetic environment of the human nucleo-cytosol and can be intentionally re-engineered towards new targets.  The term RNA editing had originally been coined for the seminal discovery of post-transcriptional uridine insertions into mitochondrial pre-mRNAs of trypanosomes, reconstituting proper reading frames . NumerouAfter the original discovery of cytidine-to-uridine (C-to-U) RNA editing in plant mitochondria more than three decades ago  it quickAt around the same time when plant organelle C-to-U editing was discovered, two different types of RNA editing were also reported in the animal kingdom. The discovery of a cytidine-to-uridine exchange introducing an early stop codon in the apolipoprotein B mRNA in the intestine even antedated the reports on plant organellar RNA editing ,15. It iPhyscomitrium patens are of this type, while much more diverse and complex editosomes requiring assembly of multiple interacting proteins have evolved in flowering plants , the constructs were introduced into the dephosphorylated  eukaryotic expression vectors pEYFP-C1 and pCMV-HA (Clontech TaKaRa), respectively, to create the final fusion protein coding sequences. Plasmid DNA for all constructs was isolated and purified using the NucleoBond® Xtra Midi kit (Macherey Nagel). Construct sequences were verified by Sanger Sequencing (Macrogen Europe). To generate constructs with coding sequence mutations, rolling-circle  following the PEI MAX user manual. Cells were incubated at 37°C and 5% CO2 until harvest. Expression of EYFP constructs was verified via fluorescence microscopy. Cells were harvested by trypsinization  after 14, 20 or 40 h. Samples were pelleted at 300 g for 5 min at 4°C, washed with cold PBS, frozen in liquid nitrogen and stored at −80°C.HeLa, HEK-293 and MCF-7 cells were grown in DMEM and IMR-90 cells in MEM (Pan Biotechnologies) media, supplemented with 10% fetal calf serum and 1% Penicillin/Streptomycin, respectively, and incubated at 37°C and 5% COTo verify the expression of recombinant proteins in human cells, cells were seeded on 12 mm cover slips. After transfection, cells were fixed in 4% paraformaldehyde (PFA) in PBS, pH 7.4 at room temperature for 15 min. Next, cells were permeabilized with 0.5% Triton in PBS for 5 min and blocked in Roti Immunoblock (Roth) at 4°C overnight. To stain HA (Hemagglutinin peptide)-tagged or EYFP (Enhanced Yellow Fluorescent Protein)-tagged proteins, respectively, the fixed cells were incubated for 1 h in a 1:500 diluted solution of primary antibody , washed in PBS and afterwards incubated for 1 h in a 1:1000 diluted Alexa594 labelled secondary antibody (Thermo Fisher) solution and DAPI . After final washing, the cells were mounted on microscope slides using Fluoromont-G mounting medium (Southernbiotech). The localization of EYFP- and HA-tagged PPR proteins was examined on a Nikon Eclipse Ti2 system, equipped with a PlanFluor 40× Oil objective (NA 1.3) using the NIS-Elements AR software and ImageJ/Fiji version 1.53c for Windows. Transfection rates were calculated using Fiji as outlined in Cell transfection was executed as described above. After harvesting, half of the cells were pelleted and frozen. The pellets were resuspended in 50 μl 4× sample buffer , vortexed, heated for 10 min at 98°C under continuous shaking and centrifuged for 10 minutes at 11 000 g. 5 μl were used for SDS polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were transferred to a nitrocellulose membrane via wet electroblotting. After test-staining of total protein with Ponceau, the membrane was blocked with 1:10 RotiBlock for 60 min. For HA-tagged constructs or EYFP-tagged constructs, membranes were incubated with primary α-HA antibody  or monoclonal α-GFP mouse (Roth) antibody, respectively, diluted 1:2000 in 1:10 RotiBlock, at 4°C overnight or at room temperature for 1 h under careful shaking. After washing with TBS-T  for 5 min three times, membranes were incubated with the respective peroxidase conjugated secondary antibody (Jackson ImmunoResearch), diluted 1:10 000 in PBS, for 30–60 min at room temperature under careful shaking. Washing was repeated. The immunoblots were overlayed with a 1:1 Luminol mixture (Solution A and B Amersham ECL start Western blotting detection reagent) and chemiluminescence was imaged via ImageQuant LAS 4000 mini and the AxioVision software. Subsequently, the membranes were incubated at room temperature for one hour with a primary α-tubulin rabbit  antibody, diluted 1:1000 in 1:10 RotiBlock, washed three times for 5 min in TBS-T and further incubated for 30 min with a peroxidase conjugated secondary antibody (Jackson ImmunoResearch), diluted 1:10 000 in PBS. Initiation and imaging of chemiluminescence was repeated.18 primers  were used. A reverse primer binding downstream of the target and a forward primer binding in the PPR protein coding region were used for RT-PCR amplification . A DNase I (Thermo Fisher Scientific) treatment was executed to prevent DNA carryover. For reverse transcription , Oligo dT6-MBP-PPR65 in the IMR-90 transcriptome, 1.2 × 106 IMR-90 cells were seeded on 10 cm culture dishes. The next day, media was replaced  and cells were transfected with 18 μg of plasmid DNA using 72 μl of PEI MAX reagent (Polysciences) per plate. An EYFP expression plasmid was co-transfected with the HA-His6-MBP-PPR65 expression plasmid in a 2:8 p(EYFP):p(HA-His6-MBP-PPR65) ratio to allow Fluorescence Activated Cell Sorting (FACS) of transfected cells. As control, cells were transfected with plasmids, encoding only the EYFP or EYFP and HA-His6-MBP protein tags, respectively. The media was exchanged after 6 h. 20 h after transfection, the cells were trypsinized, pelleted and taken up in cold PBS. 100 μl aliquots of the cell suspensions were retrieved prior to cell sorting, to confirm the editing efficiencies as described above. EYFP-positive cells were FACS-sorted at the Flow Cytometry Core Facility of the University of Bonn. The EYFP positive cells were pelleted, frozen in liquid nitrogen and stored at -80°C. RNA isolation was carried out using an RNA purification kit (Macherey Nagel) and subsequent DNase I treatment was executed as described above. Library preparation (polyA enrichment) and Illumina sequencing (150 bp paired-end) were performed by Novogene. Total RNA was sequenced for a minimum of three independent replicates per functional PPR56 and PPR65 construct, respectively. A total list of RNASeq samples is given in To investigate off-targets of EYFP-PPR56 and HA-Hiswww.bioinformatics.babraham.ac.uk/projects/fastqc). Adaptors and low-quality sequences were trimmed, using Trimmomatic 0.39  were sequenced. For that purpose, the control RNA reads were mapped to the NCBI human GRCH38 RefSeq Transcripts  via BBMap with default settings and options outu/outm, maxindel = 200k, mdtag = true, sam = 1.4 and pairedonly = true. The SAM output file was converted into BAM format via samtools (v1.13) view (www.R-project.org) and RStudio v1.2.5033  to be used as customized transcriptome in subsequent analyses.To generate specific transcriptome references for the IMR-90 cell lines, control samples expressing EYFP (PPR56 reference) or EYFP and HA-His13) view . The abu13) view . All traThe trimmed reads of each sample were mapped to the generated customized transcriptome individually using bbmap.sh  with default settings and the options outu/outm, maxindel = 200k, mdtag = true, sam = 1.4, subfilter = 3 and pairedonly = true. The output SAM files were converted into BAM format (samtools view), deduplicated , coordinate-sorted (samtools sort) and indexed (samtools index) via Samtools 1.10.www.R-project.org) and RStudio v1.2.5033 .SNPs between the sample RNAs and the two customized reference transcriptomes were called through JACUSA v2.0.2 ,46. The To scan the customized transcriptome based on NCBI human GRCH38 RefSeq Transcripts for putative editing targets of PPR56, the TargetScan tool of PREPACT  was emplPhyscomitrium patens (E. coli (Pentatricopeptide repeat (PPR) proteins PPR56 and PPR65 are C-to-U RNA editing factors functionally characterized in the model moss m patens ,49. Bothm patens . Importa, we first wished to test different arrangements of N-terminal tags for expression in human cell lines  that proved successful for expression in E. coli and EYFP, the Enhanced Yellow Fluorescent Protein .All PPR56 and PPR65 construct variants Figure  were ini2+-binding centers in the DYW domains and thus abolish cytidine deaminase activity  that were secondarily targeted to the introduced artificial targets by promiscuous protein-protein (a/o protein-RNA) interactions. To explore this possibility, we introduced two single amino acid mutations each into the DYW domains of both RNA editing factors that could be expected to affect the known Zny Figure . Changiny Figure . Aside fy system .4 in the standard culture media (a priori containing approximately 4 μM zinc) could not enhance RNA editing. It rather yielded the opposite effect that became visible with the presence of 100 μM Zn2+ at extended incubation times of 40 h, likely reflecting an overall zinc toxicity at this concentration after extended incubation times  and the addition of zinc supplements  to the HeLa cell culture media  showing different frequencies of RNA editing in the initially tested HeLa cells were alternatively transfected into IMR-90 (non-transformed human lung fibroblast cell line), HEK-293 (human embryogenic kidney cells) and MCF-7  cells .To check whether plant-type RNA editing could similarly be recapitulated in other human cell lines, four selected constructs  for A or U in positions −17, −16 and −15, likewise fit the native targets of PPR56  amino acids, whose identities are known to distinguish between keto and amino nucleotides, in two selected PPRs in PPR56 towards changing their preference for purine nucleotides. PPR S-7TD was changed to S-7TN changing its conceptual preference from G to A and, conversely, PPR S-4TN was altered to S-4TD changing its predicted preference from A to G  to being the dominant nucleotide in the S-7TN mutant off-target collection. Similarly, although less dramatically, also in the case of the S-4TD mutant off-target collection we found a shift in the conservation profile for the nucleotide directly upstream of the one targeted by the mutation introduced into the PPR. Finally, we found that the moderate, and non-canonical selectivity for G exerted by P-6ND is significantly more pronounced in case of the S-7TN mutant.The successful functional redirection of the RNA editing activity of PPR56 by the single amino acid exchanges in selected PPRs Figure  led us tPrime examples for off-targets efficiently edited by the S-7TN mutant protein Figure  or by thQuite expectedly, the changes in the off-target profiles are not only of a simple yes-or-no quality but also expressed in the different frequencies of RNA editing at some off-targets shared between the three data sets Figure . An editA significant increase to an editing efficiency of 78% is found for an editing site in the 3’-UTR of the PATL1 mRNA for the S-4TD variant of PPR56 Figure  while thA somewhat surprising collateral result of our extensive off-target analyses are the overall numbers of off-targets, which are strongly decreased to less than 350 for the S7-TN mutant but significantly increased to >2200 for the S-4TD mutant .To identify differential contributions by the nine PPRs of PPR56 that likely participate in target selection following the established PPR-RNA code, we scored matches, transitions and transversion mismatches separately for the three off-target data sets of the native protein and the two mutants investigated here . CharactFinally, we analyzed the correlation of RNA editing efficiencies and the respective matching scores as defined for the conceptual fits between PPRs at the off-target sequences. A general trend for higher RNA editing efficiencies with improved matches can expectedly be seen, but the overall correlation is only moderate . NotablyAs of today, and despite ca. 100 meantime functionally characterized plant C-to-U RNA editing factors ,56, therE. coli  in E. coli (Their capacity for site-specific RNA binding has raised speculations to use PPR proteins for specific manipulation of RNA targets . These s E. coli .An important issue to be considered, however, may be the variable RNA editing efficiencies observed in the different cell lines that we have explored here. The evidently different degrees of RNA editing in the four human cell lines Figure  may indiin planta (Along the same lines, the strength of expression of the respective editing tool may play an important role. Notably, more than 4,000 off-targets have been found upon APOBEC3A overexpression , which in planta ,91.The prime alternative to PPR-based approaches for targeted transcriptome editing uses CRISPR-Cas13 variants with engineered nucleobase deaminases specifically targeted to RNAs by guiding RNAs . An impoAny approach targeting specific RNAs or transcriptomes for editing should take possible collateral effects also on the genome into account. The capacity of binding not only to RNA but also to single-stranded DNA is evident for designer P-type PPR proteins . Hence, Whatever biotechnological developments may prove to be the most efficient and/or most specific approach, extensive and careful off-target analysis will be mandatory. The DYW domain cytidine deaminase could be promising also for fusion to Cas proteins in CRISPR-based approaches. Controlled cytidine deamination by the DYW domain might be enabled by the just recently identified autoinhibited ground state of the DYW domain caused by a conserved gating domain, which regulates the active site .E. coli assay setup. One notable case in point are characteristic differences found for the eukaryotic versus the prokaryotic setup for PPR56 upon mutation of key PPR residues (Figure E. coli may even prove to be generally superior in eukaryotic systems. In any case, the much larger endogenous background transcriptome of the eukaryotic cells will allow for much larger off-target data sets that are helpful to understand RNA binding properties and editing efficiencies. Our observations of collateral changes in the off-target profiles also for positions next to the ones directly juxtaposed with specifically mutated PPRs (Figure The successful functional expression of plant C-to-U RNA editing factors in human cells reported here significantly complements and extends the insights gained from the previously established s Figure . It remas Figure  are primwww.bioinformatics.babraham.ac.uk/projects/fastqc. BBTools suite is an open-source initiative and accessible via sourceforge.net/projects/bbmap. NCBI human GRCH38 RefSeq Transcripts can be downloaded via www.ncbi.nlm.nih.gov/genome/guide/human. R v4.1.1 is available at https://www.r-project.org and RStudio v1.2.5033 at www.rstudio.com. The TargetScan tool of PREPACT is an online tool accessible via http://www.prepact.de/prepact-main.php. The RNAseq data have been deposited in the SRA archive as BioProject PRJNA832818.FastQC analyses is available via gkac752_Supplemental_FileClick here for additional data file.