C. elegans genome engineering. Following the discovery that the Drosophila mariner element Mos1 could mobilize in the C. elegans germline , tools for efficient Mos1-mediated transgene insertion have been developed and widely adopted (Robert and Bessereau 2010). In parallel, the establishment of effective antibiotic selection in C. elegans offered increased versatility for genetic crosses and the advantage of injecting into phenotypically wild-type animals .Insertion of single-copy transgenes is an important tool in Mos1-mediated single-copy insertion (mosSCI) system is a toolkit of choice for this approach . The ttTi5605(mos1)II insertion site in particular has been used for stable germline expression, and plasmids targeting ttTi5605 are also compatible with the ‘universal’ mosSCI insertion sites .Reliable, site-specific, single-copy transgene insertion remains a key technique for applications such as synthetic reporters and rapid testing of genetic rescue constructs, especially when germline expression is required. The highly developed ttTi5605, we constructed two modified mosSCI plasmids , however existing ttTi5605-mosSCI plasmids use unc-119(+) selection. Our plasmid allows use of the ttTi5605 site without the need for injection into unc-119(ed3) mutants, which are more challenging to grow than wild-type animals and do not have optimal germline health on standard E. coli OP50 as a food source.To add to the suite of tools for targeting plasmids . First, unc-119 selection-based ttTi5605-mosSCI plasmid (pCFJ151) by adding loxP recombination sites flanking the unc-119(+) selection cassette (unc-119(+) selection cassette using an existing Cre recombinase protocol . Since unc-119(+) is a frequently used selection marker, this new feature will add flexibility for construction of strains with multiple transgenes.Second, we modified the standard cassette . This chet al. 2009). We performed three independent test injections using different transgenes and found both of our plasmids to be effective for mosSCI (unc-119(+) cassette (unc-119(+) excision, which can be easily screened for using a visible phenotype, excision of NeoR would require screening by PCR or by loss of drug resistance. Overall, we hope that these excisable unc-119(+) and NeoR mosSCI plasmids will be useful companions to the extensive mosSCI plasmid and strain toolkit .For increased versatility, both of the plasmids described here are minimal and do not encode additional tags or fluorescent proteins. Multiple-fragment transgenes can be seamlessly fused and cloned into these plasmids using the Gibson isothermal assembly technique by designing PCR amplicons, synthetic dsDNAs, restriction fragments or oligos with overlapping homologous ends  was constructed in two steps from pCFJ151 (Addgene # 19330). Briefly, the C. briggsae unc-119(+) selection cassette was removed, leaving a modified minimal multiple cloning site (MCS) between the ttTi5605 insertion homology arms. A PCR amplicon containing C. br. unc-119(+) flanked by loxP sites was cloned into the BssHII(PteI)/BglII sites. To construct plasmid pALS297 , the loxP::C. br. unc-119(+)::loxP selection cassette was excised from pALS186 by BssHII(PteI)/BglII digestion and replaced by an MluI/BglII-digested PCR amplicon containing the NeoR selection cassette flanked by loxP sites. The NeoR selection cassette (Prps-27::NeoR::unc-54-3ʹutr) was amplified from pCFJ910 (Addgene # 44481).unc-119(ed3) allele. Segregation of unc-119(ed3) was followed by the unc phenotype. The ttTi5605(mos1) insertion site was followed using a three-oligo PCR  modified from the Wormbuilder site (http://www.wormbuilder.org/) to simultaneously amplify the ~720 bp product from the mos1 insertion allele and the 455 bp product from the wild-type allele. PCRs were performed with DreamTaq Green 2X PCR Mastermix (Thermo #K1081) using 1 uL of single worm lysate in a 20 uL reaction at an annealing temperature of 57°C.To create strain ALS344, strain EG6699  was backcrossed 4X to wild-type N2 animals to remove the Cloning transgenes into mosSCI plasmids. Transgenes of interest were assembled from multiple PCR amplicons with ~20 nucleotide overlapping ends into mosSCI plasmids by Gibson Isothermal assembly  using a home-made enzyme mix or a kit (NEB #E2621S). For assembly into AvrII(XmaJI)/XhoI-digested pALS297 or pALS186, the first fragment of the transgene is amplified using the forward primer op297-p186AvrIIF (Table 1) and a transgene-specific reverse primer. The last fragment of the transgene is amplified using a transgene-specific forward primer and the reverse primer op297XhoIR or op186XhoIR (Table 1) for assembly into pALS297 or pALS186, respectively.Microinjection and screening. Worm mosSCI transgenesis was performed according to the direct injection protocol  as described in detail on the Wormbuilder site (http://www.wormbuilder.org/). Transgene-containing mosSCI plasmids were purified by ZymoPURE plasmid miniprep (Zymo #D4209). Co-injected plasmids were purified by HiSpeed Plasmid Midi kit (Qiagen #12643). For unc-119(+)selection, plasmids were injected into strain EG6699 , which was grown on E. coli HB101. For NeoRselection, plasmids were injected into strain ALS344 (Table 1) and G418 was added to plates the day after injection as described  (125 uL of 25 mg/mL G418 per 35 mm plate).Following heat-shock and counter-selection for co-injection markers, inserts were verified by PCR using pairs of primers where one primer hybridized to genomic DNA flanking the insertion site and outside of the vector homology arms  and the second primer hybridized either within the transgene or selection cassette (Table 1). Amplification was carried out using either DreamTaq Green 2X PCR Mastermix  or Phusion polymerase , respectively, according to the manufacturer’s instructions in 20 uL reactions containing 1 uL of a 6uL single worm lysate as the template.Excision of the unc-119(+) selection cassette. The C. br. unc-119(+) selection cassette was excised using Cre recombinase as described  using pDD104 . Briefly, 15-20 animals were injected and moved onto 5-6 plates (3 P0 worms/plate). Approximately 15 F1 animals expressing the mCherry co-injection marker were singled. From the progeny of these animals, unc F2 animals were singled to establish excised lines. At least two independent lines were recovered from each of two independent trials. Cre-mediated excision of the NeoR selection cassette has not been tested.Table 1: List of plasmids, strains and oligonucleotides. Oligonucleotides with NM or JL designations were described by or adapted from Dr. M. Nonet (Washington University School of Medicine in St. Louis) or Dr. J. L. Bessereau (Universitéde Lyon), respectively, based on protocols referenced on the Wormbuilder site (http://www.wormbuilder.org/). *, N20-22  represents 20-22 transgene-specific nucleotides.


Vitis vinifera L. Cv. “verdejo” and “palomino”) were studied as natural preservatives in deer burgers compared with the traditional additive sodium ascorbate. Burgers packed in modified atmosphere packaging and stored in refrigeration were analyzed at 0, 4, 8, and 12 days. The addition of lees (2.5% and 5%) produced a reduction of pH and variations in color (L* and a*), higher antioxidant capacity and phenolic content, lower lipid and protein oxidation, and the inhibition of psychotrophic aerobic bacteria and enterobacteria during the storage time. Likewise, burgers with lees kept the aldehydes concentration (volatile compounds indicators of lipid oxidation) over storage time, while esters, acids, and other compounds, previously present in lees, increased. These changes provided new odor and taste attributes like wine, bakery, and raisin notes. Therefore, the addition of wine lees had an antioxidant and antimicrobial effect and produced new sensory attributes in deer burgers.Wine lees from two grape varieties ( Meat and meat products are widely considered as a main part of the human diet since they are a resource of bioavailable amino acids, proteins, lipids, minerals, and vitamins. Consumers demand meat products because they are easy to store and cook. In general, they are elaborated mainly with livestock animals like pork, beef, or poultry, but today, consumers look for other unusual species. In this sense, venison meat from wild red deer is a viable alternative to traditional meats in an increasingly demanding market. It is considered a “natural” meat, obtained from free-range animals and free of hormone, antibiotics, and other products. Also, its nutritional quality allows consumers to follow a healthy diet due to its low fat and saturated fatty acid content and high protein and trace mineral content . DespiteAlthough the food industry has commonly employed synthetic additives to maintain the quality of meat products, consumers prefer the use of natural preservatives. Essential oils, aqueous extracts, powders, and other plant products have been proposed as natural additives due to their phenolic content being responsible for the antioxidant and antimicrobial capacities . HoweverIn particular, the wine industry produces 2–3 million tons of waste products per year only in Spain, which need to be processed . ConsequSaccharomyces cerevisiae have been proposed in the food industry as natural antioxidants [Inactive dry yeasts (IDY) from oxidants , but fewoxidants ,12. Neveoxidants ,14,15.Taking into account the functional properties of wine lees, this research has been carried out to find an alternative to traditional and synthetic additives in the meat industry. With this aim, the antioxidant and antimicrobial efficiency of wine lees added to deer burgers packed in MAP to enhance their self-life was checked. Also, changes in the chemical and sensorial properties of burgers were studied.Cervus elaphus) and pork fat. Boneless legs of wild stags hunted in “montería”, following Spanish Law 2/1993, were used. Fat from female pigs of the same crossbreed were used. These raw materials were obtained from a local supplier . Other ingredients used in the formulation were mineral water, sodium chloride, and sodium ascorbate . Lyophilized lees from the fermentation by Saccharomyces cerevisiae of must from “verdejo” (Lv) and “palomino” (Lp) grape varieties were employed. Both types of wine lees were supplied by the Department of Analytical Chemistry of University of Cádiz (Spain).Burgers were prepared employing lean from wild red deer (w/w) of ingredients: 70% deer lean, 15% pork fat, 14% mineral water, 1% sodium chloride, and 400 ppm of sodium ascorbate. Deer lean and pork fat were previously minced separately in an Unger W-98 table-top mincer  through an 8 mm plate. Sodium chloride and sodium ascorbate were dissolved in water, added to the ground raw materials and homogenized manually for 3 min. Burgers with wine lees were made using the base formulation of C but adding “verdejo” and “palomino” lees in two proportions (w/w), 2.5%  and 5% , instead of sodium ascorbate. Deer burgers (100 g) were formed employing a conventional burger-maker. Burgers were individually packed in thermoformed polypropylene plastic trays  and heat sealed by a transparent top film (polyamide 15 and polypropylene 50). High-oxygen atmosphere composed of 80% O2 and 20% CO2  was applied using an Orved packing machine (Andher). In order to simulate the supermarket conditions, samples were kept in a refrigerated display with transparent glass doors and white light lamps of 150 lumens  for 12 days. Storage was carried out at 4 °C and 12 h/day of light exposure. Three burgers of each treatment were taken at 0, 4, 8, and 12 days of chilled storage for the microbial and physicochemical analyses. Volatile compound analysis was performed at days 0 and 12. Sensorial analysis was made only before storage.Different types of burgers were prepared. Control burgers (C) were obtained by mixing the following amounts  equipped with a glass electrode probe . Moisture was measured in each sample by oven-drying in duplicate . Color parameters L* (lightness), a* (redness), and b* (yellowness) were evaluated in three different points after 30 min blooming time using a Minolta CM-2600d portable colorimeter . Illuminant D65 and 10° standard observer were fitted. To determine the total phenolic content (TPC) and radical scavenging activity, a previously sample extraction in duplicate was carried out, and then, for the consequent analysis by Folin index, DPPH and ABTS methods was applied . Lipid oEnterobacteriaceae were enumerated on Violet Red Bile Glucose Agar  by anaerobic incubation, getting it by anaerobic gas generating sachets , at 37 °C for 1 day. Lactic acid bacteria (LAB) were analyzed on de-Man Rugosa Sharpe agar  at pH 5.7, incubated under anaerobic conditions  at 30 °C for two days. Yeasts were incubated in Rose Bangal agar (Pronadisa) containing chloramphenicol (0.1 g/L) at 30 °C for two days. Microbial analysis was carried out in duplicate in each sample.Microbiological quantification was carried out by bacterial counts on selective media and conditions. Total viable counts (TVC) and psychotrophic aerobic counts were performed using plate count agar , being incubated at 30 °C for three days and at 4 °C for six days, respectively. Volatile compounds of samples were analyzed by headspace/solid phase microextraction (HS-SPME) and gas chromatography coupled to mass spectrometry (GC-MS) following the method described by Soriano et al., 2018 . ExtractDM [Identification of volatile components was performed compared to the authentic standards from Sigma-Aldrich . The tentative identification of compounds for which it was not possible to find reference volatiles was carried out by comparison of their mass spectra with spectral data from Wiley G 1035 A and NBS75K libraries and by the linear retention index (LRI) comparison. Semiquantitative analysis of the compounds was performed assuming that component response factors were the same as the response factor for the internal standard. The concentration of each volatile compound was expressed in ng/gDM .A quantitative descriptive analysis (QDA) was carried out in samples according to UNE-EN ISO 13299:2016. Burgers at day 0 were analyzed before and after cooking. Analysis was performed by a 10-member trained panel, composed of seven women and three men aged between 22 and 60 years of age and staff of the Food Technology Area of the University of Castilla-La Mancha. The panel had previous experience in meat product analysis, including burgers. One training session was held, employing commercial deer burgers from a local supplier . The analysis was executed in a tasting room designed and equipped in accordance with UNE-EN ISO 8589:2010. Prior to QDA, a session was developed with lees at work concentrations (2.5% and 5% in water) in order to generate sensory attributes. The panel evaluated appearance and odor attributes in raw burgers and appearance, odor, taste, and texture attributes in cooked burgers. Attributes were assessed using 10-cm non-structured line scales with two anchors at the ends. Burgers were cooked on a griddle until reaching an internal temperature of pasteurization (72–73 °C). Raw samples were presented in dishes at 20–22 °C, and cooked samples in individual pieces covered with aluminum foil at 50 °C. All samples were coded labelled (number-letter-number). Sensorial analysis was carried out in a single session.p < 0.05. Principal component analysis (PCA) was applied to find the relationships among different type of sample and times of storage regarding volatile composition. Statistical analysis was carried out using the IBM SPSS 24.0 software for windows statistical package .In order to determine the influence of wine lees addition or storage time on the different variables a one-way analysis of variance (ANOVA) and a subsequent post hoc Student–Newman–Keuls test were carried out. In the case of volatile and phenolic composition of lees, a t Student test was executed to observe the significant differences between type of lees and in volatile compounds between day 0 and 12 in burgers. A significant difference was considered when 2 of MAP and the water of the samples. Likewise, lees addition in burgers caused a decrease of the pH, probably due to the acidity of lees extracts.The moisture content was not significantly affected during storage. On the other hand, burgers with lees showed lower moisture values than control, mainly those with the highest concentration of lees (Lvb and Lpb).Regarding color parameters, lees addition caused few variations in the component L*, although at the end of storage, control burgers presented the highest values of L*, indicating the surface discoloration of meat during chilled storage . ComponeThe microbiological counts of burgers packed in MAP during chilled storage are shown in p < 0.001) was observed during storage in all samples. In general, wine lees addition did not affect TVC (p > 0.05). In this sense, other natural extracts (chestnut and seaweed) did not influenced TVC throughout storage, while tea and grape extracts and tomato powder led to a decrease in pork burgers [p < 0.001) in psychotrophic aerobic bacteria counts, especially in control burgers, reaching values of 8 log CFU/g, while burgers with lees kept the initial counts during the first eight days of storage (5.7–6.5 log CFU/g). Enterobacteriaceae counts exhibited a significant increase (p < 0.001) over time in control burgers reaching 3.3 log CFU/g at the end of storage. Burgers with 2.5% wine lees (Lva and Lpa) showed this growth pattern but lower counts (2.6–2.9 log CFU/g). No growth was observed in burgers with 5% lees (Lvb and Lpb) maintaining 2.4–2.6 log CFU/g during storage.Freshly made burgers showed a TVC of 4.8–5.2 log CFU/g, but a significant increase  in LAB counts was observed during chilled storage in all samples, however no significant effect of wine lees addition was observed after 12 days of storage.It is known that LAB constitute a wide part of the natural microbiota of meat packed in MAP . A signip < 0.001) than control.Similarly, yeast counts increased with storage time, although, in the case of burgers with 2.5% lees (Lva and Lpa), the final yeast counts were significantly lower (DM) than burgers with sodium ascorbate  showed the highest values of radical scavenging activity, tested by both methods, but ABTS was more efficient to highlight the differences. Other authors have also observed a greater antioxidant activity using natural preservatives instead of ascorbic acid .Lipid oxidation was measured by thiobarbituric acid-reactive substances (TBARs) analysis, which determines the content of secondary lipid oxidation products responsible of the meat off-flavors. TBARs values increased over the storage time in all burger types .This rise was also observed in other studies with pork and beef burgers ,30, and Carbonyls are one of the main products of oxidized proteins and they have been widely used as indicators of protein oxidation in food and biological systems . Table 3No effect was observed due to the concentration or type of lees used, despite the higher content of phenolic compounds in “palomino” lees .Lipid oxidation produces different volatile organic compounds (VOCs), which give rancid and unpleasant flavors and decrease the sensorial quality of meat products . MoreoveTo the best of our knowledge, the volatile profile of wild red deer meat has been little studied, only being reported in smoked dried meat  and raw Aldehydes are indicators of raw and cooked meat rancid odors due to their high concentration and low odor threshold  and showOther compounds also originated by lipid oxidation such as hydrocarbons and ketones showed less changes because of the storage and lees addition . ControlHydrocarbons are considered the main volatile compounds formed via lipid oxidation in deer cecina (Spanish smoked dry-cured meat product) , but theAliphatic alcohols suffered a considerable increase at the end of storage, especially in the case of 1-hexanol, which was the predominant alcohol in deer burgers after 12 days of chilled storage, followed by 1-octen-3-ol. It is remarkable that at the end of storage, many alcohols like 1-pentanol, 4-heptanol, 2-ethyl-1-hexanol, and 1-octanol showed significantly lower concentrations in burgers with wine lees than in control samples.Aliphatic acids detected in deer burgers were produced mainly by the degradation of triglycerides and phospholipids, while esters could be generated from the esterification of several alcohols and carboxylic acids in meat products during storage ,47.Esters provide fruity notes, mainly those derived from short-chain acids, and slight fatty odor from long-chain acids. The addition of lees caused a high increase in the total acids and esters of burgers since these compounds are formed during wine fermentation and they are present in wine lees . Among tStorage led to an increase in the total of benzene compounds in the control and lees samples. Some of them, such as benzaldehyde and phenylethyl alcohol, derived from wine fermentation, increased their concentrations due to the use of lees. These compounds could improve the aroma of deer burgers with characteristic notes, while others, such as p-cymene or trimethyl benzene, with high odor thresholds, may not significantly affect the aroma of meat products .Sulfur compounds can arise from the catabolism of sulfur-containing amino acids or generated by microorganisms ,48. TheyFurthermore, some furanic compounds were identified, 2-pentyl furan being the most abundant, although small variations were observed with respect to control samples during storage.In order to highlight the differences in deer burgers based on their volatile composition, PCA was applied to the completely volatile data set . Table 5Extracts addition could have a negative effect on the appearance, aroma, and taste, especially when their concentrations are high . To deteRegarding cooked burgers B,C, contEnterobacteriaceae counts, from the moment of their addition. On the other hand, lees provide a higher antioxidant capacity and phenolic content than sodium ascorbate, mainly at the highest concentrations. This fact results in a higher protection against lipid and protein oxidation of burgers during the storage in MAP. Although most of the volatile compounds increased at the end of storage in all samples, aldehydes that are markers of lipid oxidation, maintained their concentration in burgers with lees. However, some compounds present in lees such as esters, acids, and benzenic compounds greatly contribute to increase the volatile fraction of the experimental burgers, providing them new odor and taste attributes.The replacement of sodium ascorbate by lees (2.5% and 5%) causes a reduction of the pH and modifies color parameters (increase of L* and reduction of a*), protecting the discoloration of deer burgers during a storage time. Moreover, lees reduce the psychotrophic aerobic bacteria counts during the first eight days of storage in MAP, as well as the Therefore, wine lees from the oenological industry could be a good alternative as natural preservatives in meat products. A widespread use would contribute to the revalorization of this byproduct.


Intraluminal vesicles (ILVs) are released into the extracellular space as exosomes after the fusion of multivesicular bodies (MVBs) with the plasma membrane. miRNAs are delivered to the raft-like region of MVB by RNA-binding proteins (RBPs). RNA loading into exosomes can be either through direct interaction between RNA and the raft-like region of the MVB membrane, or through interaction between an RBP–RNA complex with this raft-like region. Selection of RNA aptamers that bind to lipid raft region of liposomal membranes was performed using the selection-amplification (SELEX) method. The pool of RNA aptamers was isolated, and the binding of this pool to lipid-raft regions was demonstrated. Sequencing of clones from rafted liposome-eluted RNAs showed sequences apparently of independent origin. Bioinformatics analysis revealed the most frequent raft-motifs present within these sequences. Four raft RNA motifs, one of them an EXO motif, have been identified. These motifs appear to be most frequent both in the case of raft RNA aptamers and in the case of exosomal pro-tumoral miRNAs transferred from cancer cells to macrophages, natural killer cells and dendritic cells, thus suggesting that the selection for incorporation of these miRNAs into ILVs is based on their affinity to the raft-like region of the MVB membrane. Exosomes are a class of extracellular vesicle (EV), 50–120 nm in diameter, originating from endosomes . They arCellular membranes coexist, at physiological temperatures, as a mixture of fluid and liquid-ordered domains influenced by membrane proteins . The proSec [Structure-dependent RNA binding was demonstrated for liquid-ordered membrane lipid rafts . BindingSec . This RNSec ,17.RNA loading into exosomes in mammalian cells appears to be independent of ESCRT and dependent on ceramide . RNAs caThe tumor microenvironment consists of various components such as endothelial cells, cancer associated fibroblasts (CAFs), innate immune cells, adaptive immune cells , and the extracellular matrix. The innate immune cells include dendritic cells (DC), macrophages, neutrophils, myeloid-derived suppressor cells (MDSC), mesenchymal stem cells (MSCs), and natural killer cells (NK) . Of thesThe new concept, presented in this study, is to search for membrane RNA aptamers specific for membrane lipid rafts. The RNA aptamers are selected for lipid raft region formed within the lipid bilayer during liposome formation. We also present a correlation analysis of raft motifs within both the raft RNA aptamer sequences and the sequences of exosomal pro-tumoral miRNAs transferred from cancer cells to macrophages, natural killer cells and dendritic cells.We performed the selection-amplification procedure in order to select a pool of RNA aptamers specific to lipid rafts formed within a lipid bilayer. The analysis of these rafted liposomes using a Zeta-sizer, revealed a homogenous pool of particles of ca. 100 nm in diameter .32P RNA content by scintillation counting, and for liposomes by A400 turbidity. There is an increase in the percent of RNA bound to liposomes as a function of the cycle number. Before cycle one, there is no binding of the pool of RNA 70N/50N to the liposomal membranes containing lipid rafts.The progress of selection for RNA aptamers that can bind liquid-ordered domains in a lipid bilayer membrane is shown in After 7 cycles, the pool of selected RNA aptamers is tested for binding to lipid raft-containing liposomal membranes, and RNA binding is 14%. We applied FRET microscopy of giant lipid vesicles (GVs) to confirm binding of raft RNA aptamers to liquid-ordered domains (lipid raft regions) within lipid bilayers A,B.In this series of micrographs , liquid-We used fluorescence and FRET microscopy techniques applied to neuroblastoma cells. Raft RNA aptamers were labeled with green YOYO-1, which undergoes a large fluorescent enhancement upon intercalation into RNA structures , and memWe have identified, from published research  method and the gel filtration technique on a column that voids liposomes but includes RNAs of this size. RNA aptamers are single-stranded oligonucleotides usually composed of ca. 20 to 100 nucleotides . Their uRNA aptamers specific for lipid rafts  have been identified , and bifmiRNAs can be delivered to the outer (cytoplasmic) surface of the MVB limiting membrane by RBPs ,19,20,21When cancer cell-derived exosomes reach the recipient immune cell, exosomal miRNAs can act on the different immune cell types through three main mechanisms : functioThere are also other routes of tumor-derived extracellular miRNA transfer, which are independent of exosomes, but incorporate other extracellular vesicles , RNA-binding proteins (RBP), or high/low-density lipoproteins (HDL/LDL) .In conclusion, our selection of raft RNA aptamers and subsequent correlation analysis of 4-nts motifs from two sets of RNAs: exosomal pro-tumoral miRNAs and raft RNA aptamers, indicates that the raft-motif present within the miRNA sequence is an important factor for miRNA selective loading into cancer cell-derived exosomes.N-stearoyl-d-erythro-sphingosylphosphorylcholine . T7 RNA polymerase (used for in vitro transcription) was obtained from Epicentre . Fluorescent probes: YOYO-1, cholera toxin subunit B (recombinant) Alexa Fluor 555 conjugate (CTB555) were purchased from ThermoFisher Scientific . IMR-32 neuroblastoma cell line was purchased from the European Collection of Authenticated Cell Cultures (ECACC), a Culture Collection of Public Health England, UK, through Sigma-Aldrich .Chemicals were analytical-reagent grade whenever available. Sephacryl S-1000 was obtained from Pharmacia. The following were purchased from Avanti Polar Lipids : 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC); cholesterol (CHOL); 2 and 2 mM CaCl2, pH 7.0) [The appropriate lipids  were dissolved in chloroform or chloroform/methanol (2/1), solvents were evaporated under a stream of nitrogen gas, the pellet was desiccated under vacuum for at least 2 h, resuspended in HEPES buffer  . Followi32P-labeled RNA  with liposomes  was performed in HEPES buffer at room temperature for 5 min, followed by gel filtration on a Sephacryl S-1000 1 mL column. The initial RNA pool with 50-mer random region (50N) and 70-mer random region (70N) was generated by T7 polymerase by in vitro transcription of a DNA template strand of the sequence: 5′-TGG TCA TGT GAT CGG CGT ATG—50N (or 70N)—TAT CGT GTC ATC GTC GTC CCT ATA GTG AGT CGT ATT A-3′ (the underline sequence is the T7-promoter region). Approximately 1015 molecules of RNA transcribed from independently synthesized DNA templates were heated in water at 65 °C for 5 min, 10xHEPES buffer was added, and solution was cooled to room temperature over 10 min to allow for RNA folding.The selection was performed as described . Briefly15 sequences. In step B, ssRNA is first incubated (counterselection) alternatively with liposomes without lipid rafts  or with buffer only (to remove non-specific RNA aggregates), then ssRNA is incubated with raft-liposomes, and next the liposome/RNA complexes are separated from unbound species by gel chromatography using Sephacryl S-1000. The eluted fractions were analyzed for 32P RNA content by scintillation counting and for liposomes by A320 turbidity . Co-elution of RNA and liposomes indicates binding. RNAraft are these RNA sequences that can bind to membrane lipid rafts. In step C, RNAraft sequences were extracted from the liposome suspension by precipitation. In step D, the ssRNAraft sequences were reverse transcribed to give a cDNA copy of the winning  sequences. In step E, pcr amplification completes the selection cycle and provides a dsDNA template enriched in the winning sequences for the next cycle.Selection-amplification (SELEX) procedure  starts w32P-labeled RNA in water was denatured for 3 min at 65 °C. Buffer A was added and RNA was folded by slow cooling to room temperature. LUV (5 mg of lipids/mL) were exposed to folded radiolabeled RNA (0.25 μM) for 5 min. Gel filtration of RNA/liposome mixture was performed using Sephacryl S-1000 Superfine. The eluted fractions were analyzed for 32P RNA content by scintillation counting and for liposomes by A320 turbidity. Co-elution of RNA and liposomes indicates binding. Recovery of both vesicles and RNA was typically complete (>95%).The RNA—liposome binding assay was performed as described . BrieflyFor fluorescence microscopy, raft-containing GVs were prepared . BrieflyThe fluorescence dye YOYO-1 iodide  was mixed with the pool of RNA aptamers from the seventh cycle in the HEPES buffer. There was no observable fluorescence of YOYO-1 in the presence of lipid vesicles alone ,31. YOYOIMR-32 cells, a human neuroblastoma cell line, were cultured in Eagle’s Minimum Essential Medium (Sigma) supplemented with 1% non-essential amino acids (NEAA), 2 mM glutamine, 0.5 mg/mL of streptomycin sulfate, 100 units/mL of penicillin G, and 10% fetal bovine serum (FBS) in a 5% CO2 and 95% air humidified atmosphere at 37 °C.IMR-32 cells were grown on slides with the 8 well Thermo Scientific Nunc Lab-Tek II Chamber Slide System. After fixation with 2% paraformaldehyde for 15 min at 37 °C, and blocking (30 min room temperature) with 2% (w/v) BSA at 4 °C, the cells were incubated for 2 h at room temperature with CTB555. CTB555 was used to visualize cholesterol-enriched lipid rafts in the plasma membrane . The flu


We identified two novel fusion transcripts in GBM tissue and five novel fusions in the matched plasma of GBM patients. The fusion transcripts FGFR3-TACC3 and VTI1A-TCF7L2 were detected in both tissue and matched plasma. A longitudinal follow-up of a GBM patient with a FGFR3-TACC3 positive glioma revealed the potential of monitoring RNA fusions in plasma. In summary, we report a sensitive RNA-seq-based liquid biopsy strategy to detect RNA level fusion status in the plasma of GBM patients.Sequencing studies have provided novel insights into the heterogeneous molecular landscape of glioblastoma (GBM), unveiling a subset of patients with gene fusions. Tissue biopsy is highly invasive, limited by sampling frequency and incompletely representative of intra-tumor heterogeneity. Extracellular vesicle-based liquid biopsy provides a minimally invasive alternative to diagnose and monitor tumor-specific molecular aberrations in patient biofluids. Here, we used targeted RNA sequencing to screen GBM tissue and the matched plasma of patients ( Glioblastoma Multiforme (GBM) is the most common malignant primary central nervous system tumor. It is highly aggressive, with a median overall survival of 15–23 months despite aggressive treatment .Emerging studies in cancers have demonstrated the role of oncogenic fusions, such as EML4-ALK in non-small-cell lung cancer , BCR-ABLConventionally, tumor tissue biopsy is used for histological and molecular analysis, including fusion identification . In the Novel sequencing technologies have been used to identify tumor-specific molecular signatures in the EV-derived genetic cargo ,19,20,21n = 9) and healthy controls (n = 10) were included in the RNA-seq exploratory fusion discovery study. Our GBM patient population included n = 3 patients with gene fusions (P1–P3) and n = 6 (P4–P9) patients with no gene fusions, as reported by Massachusetts General Hospital (MGH) Solid Fusion Assay. The demographic, histologic and molecular characteristics of the GBM patient cohort and the demographics of healthy controls are summarized in Table 1. The gene fusions analyzed by the MGH Solid Fusion Assay and RNA-seq are listed in A cohort of adult GBM patients with prior molecular characterization  with gene fusions reported in the MGH Solid Fusion Assay revealed fusions in P2 . No fusion transcripts were detected in P1 and P3. FGFR3-TACC3, VTI1A-TCF7L2 fusion transcripts were detected in both the tissue and matched plasma of patient P2 A. The anEight novel fusion transcripts were detected by an RNA-seq analysis in the healthy controls H1 , H3 (FIP1L1-SCFD2), H5 (ELL-TAL1), H7 (FIP1L1-ATP8A1) and H8 . TMEM91-TAL1 was the only fusion transcript commonly reported in both the patient and healthy control plasma B. The su1) and post-surgery (t2) and identified no fusions on RNA-seq analysis. There was no sufficient tumor tissue available from this time point for sequencing analysis.Patient 2 presented with seizures secondary to a left frontal mass , with im3) also revealed a VTI1A-TCF7L2 fusion. Additionally, fusions UBE2L3-VPS39 and FGFR3-TACC3 were also detected. A plasma RNA-seq one month later revealed no fusions. We hypothesized that the FGFR3-TACC3 fusion identified in the plasma at t3 was derived from the initial FGFR3-TACC3-positive diffuse astrocytoma. Approximately 1.5 years following initial diagnosis, the patient developed a new left occipital lesion, unrelated to the previous tumor. MRI revealed an independent new left occipital mass in addition to the residual frontal diffuse astrocytoma. The patient underwent a subtotal resection with pathology revealing a new isocitrate dehydrogenase (IDH) wild-type GBM negative for FGFR3-TACC3 by the MGH Solid Fusion Assay . An RNA-RNA fusion transcripts are major drivers of cancer, and their accurate detection and monitoring is critical to advancing clinical care. Gene fusions occur in approximately 30–50% of patients with GBM ,24,25. CHere we used a targeted RNA-seq panel (QIAseq Targeted RNAscan Human Oncology Panel) to screen the GBM tissue and matched plasma samples of nine GBM patients for both previously known and novel RNA fusions, demonstrating the feasibility of using an RNA-seq-based liquid biopsy to profile the transcriptome level gene fusions in EVs derived from the plasma of patients with GBM. There was a 100% concordance between the common gene fusions detected by MGH Solid Fusion Assay and RNA-seq, which was both reassuring and indicative of the specificity of the RNA-seq assay. We observed a clustering of the novel gene fusions VTI1A-TCF7L2 and SND1-TMEM178B along with the commonly reported FGFR3-TACC3 and AGK-BRAF fusions in the GBM tissue of patients P1-P3, who were fusion-positive by the MGH Solid Fusion Assay. Only one fusion, VTI1A-TCF7L2, was identified in the GBM tissue of patients P4–P9, who were fusion-negative by the MGH Solid Fusion Assay. This indicates the increased propensity of certain GBM tumors to harbor multiple fusions in their tissue due to genomic instability. Five novel fusion transcripts TMEM91-TAL1, CRTC1-ABHD12, VTI1A-TCF7L2, UBE2L3-VPS39 and RAB7A-FOXP1 were identified in the plasma of GBM patients. Eight novel fusion transcripts TMEM91-TAL1, CDCA7L-MLLT3, UBA5-FOXP1, FIP1L1-SCFD2, ELL-TAL1, FIP1L1-ATP8A1, GLB1-TAL1 and RASA3-TAL1 were identified in healthy plasma. Interestingly, TMEM91-TAL1 was the only fusion common in both plasma cohorts.The detection of the FGFR3-TACC3 fusion transcript in the plasma is of important clinical value. This fusion is formed by the confluence of its fusion partners that code for fibroblast growth factor receptor 3 (FGFR3) and transforming acidic coiled-coil (TACC3). This chimeric protein localizes to mitotic spindles, resulting in chromosomal segregation defects and the induction of aneuploidy, leading to cancer . This onThe VTI1A-TCF7L2 fusion transcript was identified in the GBM tissue of patients P2 and P3; additionally, this fusion was also identified in the plasma of patient P2 in a longitudinal setting. Although the exact function of the fusion protein has not been described, VTI1A-TCF7L2 has been implicated in several cancers, including lung, colorectal cancer (CRC), breast cancer and glioma ,35. The Shah et al. analyzed the transcriptome of 185 GBM tumors and identified two major genomic hotspots at the chromosomal locations 7p and 12q. Additional regions, including chromosomes 1, 4, 6 and 19, also revealed a high frequency of fusions. Furthermore, they showed that nearly 20% of GBMs have a propensity to harbor more than one fusion, and the majority of the detected fusions were private fusions occurring only in one patient . We deteWe also detected four novel fusions involving the gene domain TAL1, including TMEM91-TAL1, ELL-TAL1, GLB1-TAL1 and RASA3-TAL1. Interestingly, all of them were detected in the plasma of healthy controls, while TMEM91-TAL1 was detected in the plasma of two patients with GBM and a healthy control. The TAL1 gene is an essential transcriptional regulator of hematopoiesis, in particular for the specification of hematopoietic lineages . TAL1 isIn addition, we report several novel gene combinations , of which individual fusion partners have been previously reported in the context of several cancers, including MLLT3 in leukemia , SND1 , which might undermine the sensitivity of the assay, as larger fragments are more favorable for fusion detection. Moreover, the low RNA yield and heterogeneity of the RNA isolated from plasma EVs limits fusion discovery and requires further optimization of the fusion discovery platforms to identify genetic alterations from the low-input EV RNA. This heterogeneity of the EV RNA between biological replicates (multiple plasma samples from the same patient) challenges the reproducibility in fusion discovery. However, this is a common limitation for any EV-based biomarker discovery study . FurtherThis proof-of-principle study demonstrates the feasibility of utilizing targeted RNA-seq for the fusion transcript profiling of EV RNA derived from the plasma of patients with GBM. The detection of fusion transcripts in biofluids can open avenues to aid in the management of patients with GBM in the contexts of diagnosis, prognosis, monitoring as well as patient stratification for novel clinical trials. Further studies with more sensitive sequencing technologies are required to explore the clinical implications of fusion transcript detection in plasma.n = 9) consisted of pathology-confirmed IDH wild-type GBM (WHO grade IV) patients ranging from 41 to 74 years who underwent surgery at Massachusetts General Hospital (MGH). Tumor tissue and matched plasma samples were collected. The patient demographics, pathological diagnosis and molecular characteristics are reported in n = 3) as well as unrelated healthy volunteers (n = 7) were also collected. The demographics of the healthy controls are reported in The study population  and stored at −80 ℃ before use. The frozen tumor tissue was thawed at room temperature and the RNA was extracted using an RNeasy Mini kit according to the manufacturer’s instructions, with 30 min of on-column DNase digestion using the RNase-Free DNase kit  to eliminate the genomic DNA (gDNA). Each tumor RNA sample was eluted in 40 μL nuclease-free water  and stored at −80 ℃ before use.g for 10 min and filtered using 0.8 μm filters prior to being aliquoted into cryovials . All the aliquoted samples were stored at −80 °C.Whole blood samples were collected using BD Vacutainer® blood collection tubes . Within 2 h of collection, the samples were centrifuged at 1100× The RNA was isolated from 2 mL of plasma from the patients as well as healthy controls using the ExoRNeasy Maxi kit (Qiagen) according to the manufacturer’s instructions. The EV RNA was eluted in 14 μL of nuclease-free water and stored at −80 ℃ before use.The concentration of the tumor RNA samples was determined using the Qubit RNA HS Assay Kit on a Qubit 4 fluorometer (both Thermo Fisher Scientific). The RNA was assessed for quality and integrity using the profiles generated by an Agilent Bioanalyzer. For the tumor tissue, the RNA was diluted to meet the recommended range and assessed using an Agilent RNA Nano Kit; the plasma RNA was directly assessed using an Agilent RNA Pico Kit according to the manufacturer’s instructions .The tumor RNA was also assessed for gDNA contamination by a GAPDH RT-qPCR (cutoff Ct 30) using a 100 ng tumor RNA template, 2× Power SYBR™ Green PCR Master Mix  and 4µM of forward and reverse primers  in a total reaction mix of 25 µL. The thermocycling conditions were as follows: 95 °C for 10 min, 40 cycles of 95 °C for 15 s and 60 °C for 1 min with data collection. The cycling was performed on a StepOnePlus Real-Time PCR System (Applied Biosystems). The Ct values were analyzed using the StepOne Software (v2.3) with auto settings.A QIAseq Targeted RNAscan Human Oncology Panel and QIAseq 12-Index (both Qiagen) were used for the library prep and sample indexing. An amount of 100 ng of the tumor RNA and 5 μL of the EV RNA were used as an input for the library preparation, as per the manufacturer’s instructions. The sample libraries were diluted to meet the recommended concentration range and assessed using a DNA High Sensitivity Chip (Agilent Technologies) on an Agilent Bioanalyzer to determine the size distribution, and were quantified on a Qubit 4 fluorometer using a Qubit dsDNA HS Assay kit (Thermo Fisher Scientific). The molarity of the sample libraries was calculated based on the concentration and median library size. The quantified libraries were normalized to 4 nM and pooled at equimolar ratios. The sequencing libraries were finally denatured and diluted to 6 pM according to the guidelines . Paired end sequencing was performed on a MiSeq Sequencer to generate FASTQ files only, using a MiSeq Reagent Kit V2, 300 cycles (Illumina), with 231 cycles for read 1 and 71 cycles for read 2, as per the panel instructions. Homo sapiens, GRCh38.p10) using STAR software (version 2.5.2a). The R1 reads containing gene-specific primers were first mapped with downstream endogenous bases; the unmapped segments were mapped in the 2nd pass. Quality metrics, including mapping status, RNA control primers, mapped read length and splice distance statistics were generated for a data evaluation. An integrated Qiagen panel-specific data analysis pipeline was adopted here for data processing . The rawThe GeneGlobe data analysis pipeline integrates a list of predefined filters to avoid reporting artifacts caused by sequence homology, read-though RNA or signal noise. For a fusion to be called by the GeneGlobe data analysis pipeline, the fusions must (1) contain exons from different genes within one read or read-pair; (2) be at least 12 base pairs long; (3) pass all filters or be flagged with at most one filter; (4) be curated fusions, which are extensively described in the literature (and will be reported even with limited evidence). Our final reporting strategy includes (1) curated fusions and (2) high confidence fusions, which passed all filters defined by GeneGlobe. Low confidence fusions, which have not passed all the GeneGlobe defined filters or are not curated are not reported as true fusions. Fusions COL1A1-COL1A2 and HACL1-COLQ are reported by GeneGlobe as a result of the bioinformatics artifact and are thus excluded from our analysis . The sumWe validated all the positive fusions reported by RNA-seq using a droplet digital PCR (ddPCR) assay. The primers (Invitrogen) were designed to target the fusion sequence using Primer-Blast (NCBI) and were shown in In this study, we used targeted RNA-seq to screen tumor tissue and matched plasma samples from GBM patients for the presence of known and novel fusion transcripts. We successfully detected a range of previously reported oncogenic fusions  as well as novel fusions, including VTI1A-TCF7L2 and SND1-TMEM178B. The fusion transcripts FGFR3-TACC3 and VTI1A-TCF7L2 were detected in both the tissue and matched plasma. Furthermore, we identified an FGFR3-TACC3 fusion transcript in the patient plasma in a longitudinal setting. Our study demonstrates the feasibility of RNA-seq-based fusion transcript detection and paves a path for liquid biopsy-based fusion transcript profiling for diagnosis and longitudinal monitoring.


RNA editing occurs in all kingdoms of life and in various RNA species. The editing of nuclear protein-coding transcripts has long been known in metazoans, but was only recently detected in fungi. In contrast to many metazoan species, fungal editing sites occur mostly in coding regions, and therefore, fungal editing can change protein sequences and lead to modified or new functions of proteins. Indeed, mRNA editing is thought to be generally adaptive on fungi. Although RNA editing has been detected in both, Ascomycota and Basidiomycota, there seem to be considerable differences between these two classes of fungi concerning the types, the timing, and the purpose of editing. This review summarizes the characteristics of RNA editing in fungi and compares them to metazoan species and bacteria. In particular, it will review cellular processes affected by editing and speculate on the purpose of editing for fungal biology with a focus on the filamentous ascomycetes.Fungi show various types of mRNA editing in nuclear transcripts.• Fungal editing leads to proteome diversification.• Filamentous ascomycetes may require editing for sexual sporulation.• Wood-degrading basidiomycetes may use editing for adaptation to different substrates.•  The editing of RNA is conserved through all kingdoms of life. Editing occurs in different RNA species, including mRNA, rRNA, and tRNA (reviewed in Knoop apoB) transcript by APOBEC-1 , listing editing events from the six ascomycetes  editing, Table Tens of thousands of editing sites were then identified in S. macrospora , and meiotic silencing by unpaired DNA (MSUD) (Gladyshev atg) have been shown to be required for sexual fruiting body formation in filamentous ascomycetes  and regulators need to be identified. It has been shown that editing indeed changes protein sequences, but the effect of editing on protein functions is not fully understood and especially in the basidiomycetes, diversified protein functions have yet to be tested. Taken together, more research is needed to understand the mechanism(s) and the role(s) of mRNA editing for the kingdom fungi.


The introduction of fluorophores into RNA for both in vitro and in cellulo studies of RNA function and cellular distribution is a subject of great current interest. Here I briefly review methods, some well-established and others newly developed, which have been successfully exploited to site-specifically fluorescently label interior positions of RNAs, as a guide to investigators seeking to apply this approach to their studies. Most of these methods can be applied directly to intact RNAs, including (1) the exploitation of natural posttranslational modifications, (2) the repurposing of enzymatic transferase reactions, and (3) the nucleic acid-assisted labeling of intact RNAs. In addition, several methods are described in which specifically labeled RNAs are prepared de novo. The introduction of fluorophores into RNA for both in vitro and in cellulo studies of RNA function and cellular distribution is a subject of great current interest. Excellent comprehensive reviews have recently been published on the synthesis of fluorescent analogs of nucleosides ,2,3,4,5,3U, and 4-thiouridine, s4U can be derivatized with appropriately reactive fluorophores, as described below.There are more than 100 known posttranscriptional modifications of tRNAs [Phes [Phe is environmentally sensitive [Phe binding to various ligands [310–315 ~ 4000) [The fluorescent nucleoside wybutosine, or its derivative hydroxy-wybutosine , is founPhes ,12. Wybuensitive ,14. This ligands ,16 and i ligands ,18. Howe ~ 4000) ,20 and l ~ 4000)  limit itd-loop of the overwhelming majority of tRNAs, located at positions 16, 17, 17a 20, or 20a [Tyrs [DHU is formed by post-transcriptional reduction of uridine and is the most widespread modified base which can be straightforwardly labeled with fluorescent groups. Four dihydrouridine synthases (Dus1–Dus4) have been identified, which use NADPH as the external reductant that is coupled with an enzyme-bound FMN cofactor. Collectively the Dus enzymes are responsible for the presence of one or more DHUs in the , or 20a , as well0a [Tyrs . One or 0a [Tyrs . A pract0a [Tyrs ,24, but 4 reduction in DHU residues allowed covalent introduction of weak base NH2-containing fluorophore nucleophiles into tRNAs with substantial retention by the labeled tRNAs of protein synthesis activity. Their work made extensive use of proflavin derivatives of tRNA [Zachau and Wintermeyer and their co-workers demonstrated that NaBH of tRNA . More re of tRNA ,23 and h of tRNA ,27 and A of tRNA  have bee of tRNA . The sectRNAs fluorescently labeled via DHU chemistry have been used widely in in vitro ensemble ,31,32 anE. coli tRNAs, each at position 47 within the variable loop, by transfer of acp from S-adenosylmethionine (SAM) [1acp3 Ψ [N-hydroxysuccinimide esters of Cy dyes [A recently identified enzyme, tRNA U47 acp transferase A, abbreviated as TuaA, is responsible for modifying eight ne (SAM) , and Tsr[1acp3 Ψ . The ami Cy dyes . The res Cy dyes ,53,54,554U, in bacteria and archaea, using cysteine as the ultimate sulfur donor, via a complex set of reactions. The s4U nucleoside is almost always found at position 8 of most bacterial and archaeal tRNAs. As TtuI modifies U8 of all bacterial tRNAs, there is no RNA sequence motif determining the specificity for U8 thiolation. Lauhon et al. [E. coli tRNA could serve as a minimal substrate for s4U8 synthesis   catalyzen et al.  determinynthesis . A subse4U, and the resulting fl-tRNAs have been used in single molecule studies of protein synthesis [4U at much higher efficiency with exquisite specificity for s4U utilizes a two-step scheme in which the thione is first oxidized by periodate to the sulfonate, which is subsequently displaced by fluoresceinamine (FAM-NH2) [Cy-maleimides have been used to derivatize synthesis . HoweverFAM-NH2) .3U, and s4U chemistry would be restricted to tRNAs. The recent demonstration that DHU is also found in many mRNA sites [3U and s4U modifications are also present in RNAs other than tRNA, but also whether directed evolution of the enzymes involved in DHU, acp3U, and s4U formation would be successful in broadening substrate specificities [It has generally been assumed that fluorescent labeling via DHU, acpNA sites  raises tNA sites  and/or sficities .Two recent reviews provide excellent overviews of the repurposing of enzymes to site-specifically label RNAs, at internal positions as well as at the 5′-mRNA cap and 3′-terminus ,8. Here S. cerevisiae tRNAPhe transcript by click chemistry, using the enzyme Trm1 that is specific for methylation at this position, and replacing S-Me with S-pent-2-en-4-ynyl [6A formation, METTL3-14 and METTL16, which target the underlined A in the sequences GGACU and UACAGAGAA, respectively. Replacing the S-Me group in SAM with Se-propargyl affords relatively high yields of N6-propargylA (70–80% relative to S-Me) at targeted A residues within model RNAs 20–61 nts long, which are subsequently labeled by click chemistry with Cy5-azide. For METTL3-14, a similar strategy can be used to introduce photocaging groups  at the N6 position. An important limitation of this approach is that these two SAM-Mtases can methylate sequences in addition to the most preferred sequences shown above. Thus, for example, METTL3-14 has broad specificity for DRACH sequences , making it likely that, in general, several sites in long RNAs containing >150 nts will be targeted by this enzyme.Several SAM-Mtases catalyze transfer reactions in which the sulfonium-bound methyl group is replaced with an extended side chain containing a functionality  which can be straightforwardly derivatized with commercially available fluorophore-containing reagents. This approach was first used to site-specifically place an AlexaFluor594 dye at the N2 of G26 in an n-4-ynyl . An apprn-4-ynyl  demonstrE. coli tRNAPhe, but is a good catalyst for methylating a variant substrate with altered residues in the loop region (nts 54-60). Given the ability of SAM-Mtases to tolerate replacement of the S-Me group with other alkylating agents, as described above, this alteration of specificity might have future utility for introducing fluorophores at selected RNA locations.The enzyme, TrmA, methylates uridine-54 of the T-arm of tRNA, a 17-nucleotide stem loop nts 49-65) which is structurally conserved and is the minimal substrate for the enzyme . By muta which isBacterial TGT has also been used to site- specifically label RNAs . As show3), giving an acylated product suitable for further reaction via click chemistry with a suitable fluorophore partner. As a demonstration of the utility of the approach, a 65 nt small nucleolar RNA, SNORD78, was labeled successively with fluorophores at two different positions, Alexa488 at G14 and TAMRA at A49, generating a strong FRET signal. In this approach the sizes of the gaps can be varied, but the most site-specific modifications occur with a loop or gap size of one. Even here, however, small amounts of secondary acylation (20–30%) are found at the position adjacent to the targeted site. Further development of this approach will be needed to suppress such secondary acylation. Xiao et al.  have int1, N6–ethenoadenosine (step iv) which can be labeled by click chemistry with a fluorophor. Using this approach, two different A residues targeted within a 275 nt long riboswitch were labeled with yields of each dye derivatized site of between 14–17%. These values are markedly lower than an average value of ~65% which had been previously found for site-specific labeling of single-stranded RNA regions by the same methodology [Zhao et al. ,69 have hodology , an indihodology  and doubhodology ,69.cis, the recognition sequences can be altered to target any internal RNA nucleotide in trans. The attached reactive groups, amine, ethyne, or azide can be readily derivatized with fluorophores. This procedure has been applied to simple RNA sequences, giving yields of incorporated fluorophores of 50–80%. Substitution of tenofovir diphosphate in place of ATP resulted in formation of a more stable reaction product [Ghaem Maghami et al.  have rec product . More coPLOR incorporates labels at specific positions of RNAs by combining solid-phase chemical synthesis with highly processive DNA-dependent RNA polymerases (RNAPs) to generate specifically labeled RNAs . PLOR prIn one application PLOR has been used to incorporate Cy3 and Cy5 into positions 24 and 55, respectively, of a 71nt adenine aptamer, permitting monitoring of the increase in FRET efficiency on adenine addition. The PLOR approach continues to be developed, most recently by optimizing linker sizes of DNA templates, the lengths and base composition of the RNA synthesized in the initiation stage, and methods for coupling fluorophores to modified nucleotides ,78.thG, shown in thG and native nucleoside triphosphates, which enforces initiation with the unnatural analogue. The resulting 5′-end modified transcripts are then mono-phosphorylated and ligated to provide longer site-specifically modified RNA constructs  ribozyme, with two of the four yielding active ribozymes, and with overall ligation yields of 20–40%. It will be interesting to see whether this method can be applied to other substituted guanosines, or to fluorescent analogues of other nucleosides.Li et al.  have desnstructs . This apThe expansion of the genetic alphabet via the creation of a wide variety unnatural base pairs (UBPs) has been the object of a number of laboratories in recent years, as recently reviewed ,80. SitethG method (As this review makes clear, there are many potential methods available for site-specific fluorescent labeling of RNA interior positions, so that the choice of which method to employ will typically depend on the size, sequence and structure of the targeted RNA. That said, it is worth considering whether any of the methods considered could become general, in the sense that it could be readily employed by most laboratories to any RNA of choice. At present, three methods come closest to this goal. Two of these, which label intact RNAs, are RNA acylation at DNA-induced loops or gaps  and evolG method  has the G method  each emp


Catalysis by nucleic acids is indispensable for extant cellular life, and it is widely accepted that nucleic acid enzymes were crucial for the emergence of primitive life 3.5‐4 billion years ago. However, geochemical conditions on early Earth must have differed greatly from the constant internal milieus of today's cells. In order to explore plausible scenarios for early molecular evolution, it is therefore essential to understand how different physicochemical parameters, such as temperature, pH, and ionic composition, influence nucleic acid catalysis and to explore to what extent nucleic acid enzymes can adapt to non‐physiological conditions. In this article, we give an overview of the research on catalysis of nucleic acids, in particular catalytic RNAs (ribozymes) and DNAs (deoxyribozymes), under extreme and/or unusual conditions that may relate to prebiotic environments. Many different physicochemical parameters influence nucleic acid catalysis, including temperature, pH, pressure, dehydration and metal ion composition. In order to explore plausible scenarios for early molecular evolution and investigate to what extent nucleic acid enzymes can adapt to non‐physiological conditions, it is essential to understand how different environmental conditions both inhibit and potentiate nucleic acid catalysis. This review demonstrates that nucleic acid catalysts can tolerate a wide range of challenging environments, and in many cases are enhanced by environmental factors. For RNA (ribozymes), the most iconic example is the ribosome,While the main function of DNA in biology is the storage of genetic information, a large number of artificial DNA catalysts have also been isolated by in vitro selection. These deoxyribozymes, or DNAzymes, catalyze a range of bond forming reactions, including the Diels–Alder reaction,Finally, synthetic nucleic acids are also capable of catalysis. In particular, Taylor et al. selected artificial endonuclease and ligase enzymes from random pools of arabino nucleic acid (ANA), 2′‐fluoroarabino nucleic acid (FANA), hexitol nucleic acid (HNA) and cyclohexene nucleic acid (CeNA).While these studies convincingly demonstrate the broad catalytic potential of polynucleotides, they leave open the question of whether some of these reactions could have contributed to early biocatalysis, and whether they are compatible with the environmental conditions on early Earth.Since the beginning of the Origin of Life field, great efforts have been made to determine, or at least constrain, the conditions under which life originated. Definitive answers have been elusive, due to the extreme timescales under consideration and the combined uncertainties of when, where and how the first primitive forms of life emerged. The lack of fossil evidence of early life, the large number of possible geochemical environments and the difficulty in determining conditions on early Earth make this an almost intractable problem for origin of life researchers, amongst whom there is little consensus on these questions.2+, but low in concentrations of free transition metal and group 12–16 ions due to the formation of insoluble sulfide compounds.2+ as a cofactor, until the advent of aerobic conditions caused the oxidation of Fe2+ to Fe3+, necessitating its replacement by Mg2+ or other metal ions.2 concentrations, likely rose monotonically from pH 6.6 in the Hadean era to pH 7.9 by the Cambrian era.The many studies that aim to constrain the global climate and conditions on early Earth allow some experimental boundaries to be set: As today, divalent magnesium and calcium were abundant in the oceans of early Earth. Historical ocean solute composition is dependent on both pH and reducing potential. Assuming an acidic ocean pH around 4 Ga, hydrogen sulfide present in seawater would have created a reducing environment rich in FeEstimates of temperature are more variable, spanning climates ranging from frozen to near boiling. Oxygen, iron and silicon isotope studies suggest temperatures of 70 °C up until as late as 3.3 Ga, a theory additionally supported by evidence of a low viscosity Archean ocean.Although these studies provide some useful constraints on the conditions at the Origin of Life, a broad range of conditions remain feasible. The exact microenvironment in which the first replicators emerged was likely more significant than the global conditions at the time. For example, ‘warm little ponds’ on land would be subject to temperature, composition and concentration fluctuations due to evaporation and condensation driven by day–night cycles,In this focus review, we will explore the range of conditions under which nucleic acid catalysis is possible, highlighting how nucleic acids can adapt to extreme conditions, and how these conditions can both support and potentiate function. In order to understand the emergence of life, we must understand the environmental factors that would have acted upon the first functional nucleic acids, for example, in an RNA, proto‐RNA or mixed nucleic acid world scenario. In addition, many nucleic acid enzymes catalyze industrially relevant processes and, as such, challenging conditions may be required to increase reaction rates, shift reaction equilibria or improve substrate or product solubility. In both cases, reaction conditions may deviate strongly from in vivo or typical in vitro environments.22.1The range of conditions in which catalytic nucleic acids are functional is largely determined by the mechanism by which nucleic acids can fold into catalytically active three‐dimensional structures. Nucleic acid folding differs to that of proteins, which in many cases tend to fold via rapid, cooperative two‐state thermodynamic transitions, with no detectable intermediate structures.2.22+ and K+, both of which interact with RNA predominantly through electrostatic forces.2+ ions enable the formation of complex folds that allow nucleic acids to stabilize specific structures, recognize binding partners and mediate catalytic processes.2+ can be divided into two populations  in sea water after Na+, it is conceivable that similar Mg2+ concentrations were present in an Archean ocean,2+, Ca2+, Fe2+, Sr2+, Ba2+, Na+ and polyamines.Tetrahymena group I ribozyme, which was derived from a self‐splicing Tetrahymena preribosomal RNA and catalyzes a reaction mimicking the first step of splicing,Tetrahymena group I ribozyme folds into a native‐like state in the presence of various counterions, folding of the catalytically active state requires site‐specific binding of Mg2+ or Mn2+.Given that magnesium is the seventh most abundant element in the Earth's crust, and that the Mg2+.2+ concentrations of approximately 1 mm.2+ in vivo is alleviated by the presence of RNA chaperone proteins, which promote RNA folding and annealing.m MgCl2.2+ concentrations and near‐optimal activity occurs between 0.1 to 100 mm in vitro.All of the larger natural RNA enzymes, such as RNAsePLike ribozymes, DNAzymes use diffuse electrostatic and specific metal ion interactions for activity and folding. Notably, the high stability, cost‐effective production, and easy chemical modification of DNA has enabled the systematic selection of a large number of DNAzymes and aptamers capable of selective metal ion detection. These DNAs can bind to and distinguish between an impressive range of species, including alkali metal ions, alkaline earth metal ions, transition metals, noble metals, post‐transition metal ions and lanthanide and actinide ions for catalysis.2 concentration for RNAseP RNA folding and activity is reduced from 60 mm to 10 mm in the presence of 1 mm spermidine.It should be mentioned that non‐metallic ions can also support folding of nucleic acids into active conformations. For example, polyamines can aid RNA folding; the required MgCl3+) are also of interest, as their interactions with nucleic acids are very different from typical divalent metal ions due to their unusual coordination chemistry. In particular, the absence of a strong ligand field allows for a high degree of structural diversity in lanthanide complexes, as ligands alone dictate the symmetry and coordination of complexes.m concentrations are required for binding), but they can also directly interact with the nucleobase moieties.3+ ions can accelerate a small Pb2+‐dependent ribozyme called the leadzyme,3+‐dependent RNA‐cleaving DNAzymes were discovered by in vitro selection experiments,3+‐specific ribozymes have not yet been described in literature, and at a first glance rare earth metals have little relevance for origin of life scenarios due to their low aqueous solubility. However, low concentrations of lanthanides are available, for example, under hot acidic conditions in volcanic mudpots, and Ln3+ ions are essential under these conditions for some acidophilic microbes that use methane as an energy source.Lanthanides Ln+ are als2.43+, La3+ and Tb3+, Pb2+, and Zn2+ catalyze rapid RNA cleavage in aqueous solutions.2+ is only about 4 % as active as Pb2+, and other metal ions such as Cd2+, Mn2+, Cu2+ or Mg2+ catalyze degradation one to two orders of magnitude slower than Zn2+.2+) can reduce RNA half‐lives down to minutes.2+ and high temperatures, such as hydrothermal vents, are unsuitable settings for RNA‐based scenarios of molecular evolution. Likewise, free Ln3+ ions are highly nucleolytic under basic conditions, as their ions form multinuclear complexes and cleave RNA nonspecifically at low mm concentrations with a rate acceleration as large as 108–1012‐fold.IV to accelerate DNA hydrolysis up to 1011‐fold under neutral conditions, reducing the half‐life of the phosphodiester linkage in DNA from millions of years down to a few hours.While metal ions assist nucleic acid folding and catalysis in many cases, they are often also a threat to the chemical integrity of RNA Figure ;97 heavyPossible modes of metal ion‐catalyzed nucleic acid hydrolysis include Lewis acid catalysis, Brønsted base catalysis, nucleophilic catalysis by metal‐bound hydroxides and simple electrostatic stabilization of transition states by positively charged metal ions Figure . The ind+‐dependent DNAzymes were isolated by targeted in vitro selection.Facing the threat of degradation by metal ions, in particular in the case of RNA, it is interesting from a prebiotic perspective that a number of nucleic acids are capable of efficient catalysis without divalent metal ions. In particular, several families of small nucleolytic ribozymes reversibly catalyze metal‐independent and site‐specific cleavage/ ligation of the RNA backbone, and can accelerate this reaction by approximately a million‐fold using general acid base catalysis.2.52+ during nucleic acid folding and catalysis, Fe2+ is of great prebiotic interest as it was likely to be highly abundant on Earth before the advent of photosynthesis.2+ was speculated to be present in micro‐ to low millimolar quantities during early Archean Earth.2+ during RNA cleavage catalyzed by several DNAzymes.2+ was used during pH‐dependent selection for RNA‐cleaving ribozymes, where it enabled the discovery of novel catalytic motifs that are absent in typical selections using Mg2+.2+ with Fe2+ in an anoxic environment enabled various natural RNAs, such as tRNA or ribosomal RNA, to catalyze single‐electron transfer reactions, which are typically limited to cofactor‐dependent protein enzymes.Of the various ions that can replace Mg2+ has also been proposed as a key divalent transition metal ion in prebiotic chemistry.2 reduction and biomolecular polymerization occurred, driven by UV light. Indeed, Zn2+ can substitute Mg2+ as the only divalent metal ion during RNAseP catalysis, but only in the presence of high concentrations of ammonium salts.2+ was also shown to be strongly beneficial for DNA‐catalyzed DNA cleavage. The artificial deoxyribozyme 10MD5 is a bimetallic metalloenzyme  that catalyzes the Mn2+/Zn2+‐dependent DNA phosphodiester bond hydrolysis with at least a 1012‐fold rate enhancement.2+‐dependent monometallic DNAzyme.2+ alone for catalysis were identified by in vitro selection.Zn2+ ions in particular extended the catalytic properties of ribozymes under the anoxic conditions of the late Hadean and early Archean. Further research in this field could uncover new, unexpected catalytic nucleic acids that increase the plausibility of an early metabolism mediated by nucleic acids.In summary, the availability of metal ions such as magnesium was most likely not a critical factor for early nucleic acid enzymes . However, it is possible that Fe33.1Another crucial physicochemical parameter for early nucleic acid catalysis and stability is pH. Estimates of environmental pH on early Earth are largely hypothetical (see introduction), but most evidence suggests that oceanic pH was initially acidic pH 6.6,, 50.The 2 levels in the Hadean era may have led to a variety of acidic aqueous environments,DNA is less stable than RNA under acidic conditions due to increased depurination below pH 3,3.2aK in the neutral range.aK values far from neutrality and are therefore suboptimal for general acid‐base catalysis.aK of both general acid and base towards neutrality, a similar effect to that found in some proteins.aK of N1 in the catalytically active guanosine base.The direct effect of pH on catalysis is inherently dependent on the type and mechanism of the reaction. Catalysis by nucleic acids can occur via transition state stabilization (e.g. by hydrogen bonding or electrostatic stabilization), general acid and/or base catalysis (i.e. by enhancing the nucleophilicity of attacking groups by deprotonation or by stabilizing leaving groups by protonation), or by facilitating active conformational states such as the formation of an in‐line transition state during nucleophilic attack.5 to 106 ribozymes for the HP ribozyme at neutral pHkcat due to the low reactivity of the neutral base moieties.Generally, the acid‐base mechanism employed by small ribozymes makes them robust towards changes in pH and enables significant cleavage and ligation activity at pH >6. However, the rate enhancement is limited by the small fraction of ribozymes that, on average, have the correct ionization state for general acid‐base catalyzed cleavage  due to the protonation of G‐C base pairs and resultant Hoogsteen base pair formation.3.32+ or Fe2+ was included as the divalent metal ion during selection, different sequences and secondary structure motifs were isolated. Neutral pH in the presence of Fe2+ led to the selection of hammerhead (HH)‐like motifs, whilst at pH 5 a variety of previously unknown motifs were discovered and the abundance of HH motifs dropped to less than 0.1 %. Thus, both pH and substitutions between Fe2+ and Mg2+ strongly influence the relative fitness of different motifs.Indeed, in vitro selection experiments have shown that nucleic acids can be readily evolved towards improved catalysis at lower pH where the chemical stability of the RNA backbone is strongly increased. For example, a de novo selection of self‐cleaving ribozymes at low pH resulted in a variant that showed pH‐dependent kinetics with an optimum of around pH 4.2+, demonstrating that low pH can facilitate the Mg2+‐free cleavage of RNA by a DNAzyme.Short RNA‐cleaving DNAzymes have also been evolved to function in trans at low pH. The reaction proceeds optimally at pH of 4–4.5 in the absence of MgLigation reactions represent an important catalytic function, for example, for nucleic acid self‐replication.Early peptide synthesis and even translation may have also occurred over a broad pH range. The peptidyltransfer reaction that takes place at the heart of the ribosome does not involve acid‐base catalysis and so is relatively pH‐insensitive.4mT) of nucleic acids is sufficiently low to reduce the catalytic potential at even slightly elevated temperatures. In addition, the faster reaction kinetics at elevated temperatures are offset by the increasing rate of phosphodiester hydrolysis, especially in the presence of divalent metal cations such as magnesium as discussed above, which prevents sustained catalysis.Temperature is a further critical parameter in nucleic acid catalysis and stability Figure . As for 4.1Temperature estimations of the early Earth are a matter of debate. Several lines of evidence exist that support a hot climate during the Archean eon, 4 to 2.5 billion years ago, by which point the Earth's crust is thought to have cooled sufficiently to allow for the dawn of life. Based on oxygen and silicon isotope analyses in sedimentary rocks,Despite the temperature sensitivity of RNA, living systems have adapted to survive at extreme temperatures. Comparison of homologous ribozymes in mesophilic and thermophilic organisms reveals how sequence adaptations can lead to higher temperature stability. A study on RNase P homologs in mesophilic and thermophilic bacteria by Pan et al. observed that folding was more cooperative for thermophilic RNA, and the folding pathway proceeded via a different set of intermediate structures despite the high similarity of the final states.4.2Tetrahymena ribozyme.Several reports focusing on heat adaptation of nucleic acid enzymes to higher temperatures have been published. Guo et al. used directed evolution to select for thermally stable variants of the Schistosoma mansoni HH ribozyme is most active at ≈70 °C in vitro without additional sequence optimizations.Thermus thermophilus where it catalyzes efficient cis‐ and trans‐ cleavage of mRNA in vivo at temperatures up to 80 °C. In this case, temperature modulates the rate limiting steps of the reaction: at 37 °C, catalysis is limited by substrate dissociation, whereas at high temperature RNA degradation, ribozyme‐substrate association, and secondary structure denaturation limit activity.Saksmerprome et al. discovered highly thermostable variants of the HH ribozyme.2+‐dependent RNA‐cleaving DNAzymes with activity at 90 °C.2+ and maintaining catalysis with minimal secondary structure.DNAzymes capable of high temperature catalysis have also been obtained by in vitro selection. Nelson et al. selected a range of Zn2+ dependency of nucleic acid folding and catalysis, which, in the case of RNA, helps to reduce degradation. More work investigating the stabilization of more primitive, short ribozyme systems is required to examine the range of temperatures that permit the emergence or even self‐replication of functional RNAs at increased temperatures. DNA is more resistant to degradation than RNA, so selected DNAzymes can operate at up to 90 °C by relying on metal cofactor binding rather than the maintenance of a well‐folded active site. It is as yet unknown whether such systems are limited to simple reactions such as substrate cleavage.These studies demonstrate that the catalysis of nucleic acids can be retained at elevated temperatures. Temperature adaptation in ribozymes is generally achieved through additional RNA‐RNA interactions stabilizing both the catalytically active conformation and RNA‐substrate interactions, allowing activity to be sustained up to 80 °C. These adaptive mechanisms may generally also decrease the M5−1), whilst the DNA double helix dissociation has a positive ΔV of 1–5 mL mol−1.mT, with duplexes that melt below 50 °C being destabilized by pressure and those melt above 50 °C being stabilized.Vs and melt under pressure.In addition to temperature and pH, hydrostatic pressure is also a potentially important environmental factor when considering oceanic or subterranean origins of life. High‐pressure conditions are typically defined as 10 MPa or greater, corresponding to a water depth of 1000 m or more. 88 % of the volume of modern oceans may be considered high pressure, with an average pressure of 38 MPa and a maximum on the abyssal plane of 110 MPa.Phe up to 1 GPa.obsk) and overall equilibrium constant of HP ribozyme self‐cleavage decreases with increasing pressure.2+ typically required for cleavage under ambient pressure.V is associated with the cleavage reaction itself.RNA structures are also remarkably stable under high hydrostatic pressure: few structural changes are observed in tRNA2+‐catalyzed degradation of RNA. When considering undersea environments, the resistance of nucleic acid to pressure‐induced denaturation lends support to a nucleic acid‐based origin of life, especially when considering the drastic effect of such conditions on protein folding.These studies demonstrate that hydrostatic pressure can promote nucleic folding and compensate for a lack of magnesium in certain nucleic acid catalysts. The increase in melting temperature associated with pressurization could permit increased reaction temperatures for weakly folding systems, and be used to avoid Mg6Apart from the typical physicochemical parameters such as pressure, ionic conditions, pH and temperature described above, more exotic environmental conditions can strongly influence nucleic acid catalysis. A notable example is the extraordinary effect of dehydrating conditions on ribozyme and deoxyribozyme catalysis induced by freezing, evaporation, or the presence of organic solvents.6.12+, which are usually indispensable for catalysis in low‐salt conditions.2+‐independent RNA ligation by HP ribozymes in both trans and in cis, while disfavoring the reverse cleavage reaction.The discovery that freezing or evaporation can enhance or even trigger ribozyme catalysis was serendipitous. The first reports of (undesired) HH ribozyme activity at sub‐zero temperatures came from investigations of the autocatalytic processing of dimeric tobacco ringspot virus satellite RNA (STobRV RNA) by Prody et al.2+ can be compensated by high concentrations (>1.5 m) of monovalent cations.2+ ions in dilute aqueous solution are still vital for tertiary RNA folding and stabilization of the active conformation.A first conjecture as to why monovalent salts are important for HP ribozyme catalysis under frozen conditions is provided by previous studies, which have shown that the absence of M2+‐independent ribozyme catalysis in presence of primary alcohols or poly(ethlyene glycol) (PEG) might, as in freezing, be at least partially due to the enhanced RNA‐Na+ interactions that can compensate for the missing divalent metal ions.2+‐dependent activity of ribozymes and mitigates the effects of destabilizing mutations, although higher levels of ethanol in the presence of Mg2+ diminishes this activity, presumably due to RNA aggregation.The activation of the HP ribozyme by the high salt concentration in eutectic brine does not at first seem to explain the alcohol‐induced activation of catalysis, since the typical alcohol concentrations used to trigger ribozyme catalysis are not sufficient to co‐concentrate or precipitate monovalent counter‐ions.Kazakov et al. also reported that HP ribozyme‐catalyzed ligation during evaporation is considerably improved by the presence of PEG, which had no impact on ligation under aqueous or frozen conditions or ethanol‐induced ligation. The authors concluded that PEG might decrease the rate of evaporation, thereby extending the windows of partial dehydration where the water activity is still sufficient to allow hairpin ribozyme catalysis.The formation of active ribozyme conformations in the absence of divalent metal ions can be induced by conditions that promote electrostatic shielding and RNA compaction, such as partial dehydration, up‐concentration of monovalent cations, or reduced dielectric constant. Furthermore, the effective increase in RNA concentration during freezing facilitates RNA‐RNA association, even from very stable monomeric structures,m−1 under aqueous conditions  thereby providing unproductive complexes a “second chance” at catalysis.Both HP and HH ribozyme ligation yields strongly benefit from repeated freeze‐thaw (FT) cycling.2+‐dependent ribozymes such as the R18 RNA polymerase, which catalyzes templated primer extension using nucleoside triphosphates.2+ salts. The authors also investigated the impacts of different negative counter‐ions to Mg2+, and found that they markedly influence activity, presumably due to their influence on the eutectic freezing point, which dictates the concentrating effect of the eutectic brine. The ice microstructure has been shown to provide a quasi‐cellular compartmentalization enabling robust phenotype‐genotype linkage, which is one of the key requirements for Darwinian Evolution.Attwater et al. demonstrated the beneficial effects of a frozen environment on strictly M6.2+.m NaCl, and is completely independent from divalent metal ions. The EtNa RNA cleavage rate can be directly used as a biosensor for the precise measurement of alcohol levels in spirits such as whisky or vodka. Interestingly, EtNa activity drastically decreases at ethanol concentrations beyond 72 % (v/v) ethanol, where the B‐form helix of DNA is converted into the A‐form that (in contrast to ribozymes) seems to be incompatible with the formation of the active DNAzyme conformation. Given that EtNa shows cooperative binding of and activation by Ca2+ (in contrast to Mg2+)2+ levels down to 1.4 μm Ca2+.Zhou et al reported the isolation of the DNAzyme EtNa Figure  from a r6.3The remarkable ability of dehydration to potentiate ribozyme function suggests that such conditions may have been important to the emergence of replicating RNA. Wet‐dry cycles, perhaps driven by day‐night cycles or geothermal activity on early Earth, have been proposed as possible drivers of the emergence of function. Viscous environments formed by water evaporation facilitate non‐enzymatic RNA replication cycles slowing reannealing and thereby circumventing strand inhibition.Wet‐dry cycles can also be produced by the application of thermal gradients at an air–water interface Figure . The res7Exposure to UV radiation presents a challenge to the survival of prebiotic nucleic acids, and is often raised as a major problem in any RNA world scenario due to the elevated levels of surface UV radiation compared to the present day.Despite its deleterious effect of nucleic acids, ultraviolet radiation has been observed to promote prebiotic chemical reactions that yield ribonucleotidesDespite the well‐documented exploration of UV‐induced nucleic acid damage, relatively few insights are available regarding the role of UV exposure on functional RNA (or other nucleic acid) enzymes. This may be in part due to a complex interplay between UV radiation and other factors influencing RNA catalysis, such as the presence of metal ions. When exposed to UV radiation, tobacco mosaic virus (TMV) RNA accumulates lesions in the form of uridine hydrates and pyrimidine dimers. However, in the presence of magnesium the rate of accumulation was approximately one‐third than that in water, implying that folded RNA is more resistant to UV radiation damage than the unfolded random coil.The influence of structure and conformation on nucleic acid UV sensitivity was further demonstrated by Kundu et al., who reported an unexpected discrepancy between the UV sensitivities of dTdT dinucleotides in either RNA or DNA hairpins.Recently, Saha and Chen monitored the function, folding, and kinetics of RNA aptamers that bind conditionally fluorescent ligands in vitro following UV induced photodamage.While UV irradiation has been generally demonstrated to have a detrimental on functional nucleic acids, some examples of UV‐dependent nucleic acid catalysts have been reported. Chinnapen and Sen reported the in vitro selection of a DNAzyme with photolyase activity, UV1C, from a pool of random sequences.8The activities of both ribozymes and deoxyribozymes are compatible with a broad range of potentially prebiotic conditions. Despite being less versatile and powerful than protein‐based catalysis, nucleic acid catalysts are capable of escaping irreversible aggregation, while also tolerating or even benefiting from much harsher conditions such as freezing, drying or dehydration. Moreover, nucleic acid catalysts often require only modest changes in their sequences to adapt to novel challenging conditions such as harsher pH values or higher temperatures, and can often tolerate or adapt to a broad range of different metal ion cofactors. These combined features make them ideal candidates for early biocatalysis, which presumably emerged and remained functional outside the sheltered and constant milieu of the modern cell.Despite the large body of research, further explorations of nucleic acid enzymes under prebiotic conditions may yield yet more unforeseen properties relevant for abiogenesis, and warrant further investigation. For example, selection experiments under prebiotically plausible conditions beyond aqueous solutions in a modern oxygen‐rich atmosphere could reveal further unexpected catalytic properties of ribozymes. In addition to the factors discussed in this review, other environmental factors such as mineral surfaces,Finally, it remains essential to further expand far‐from‐equilibrium scenarios to explore different stages of molecular evolution  experimentally under heterogeneous conditions, such as the continuous provision of chemical fuel and/or pH, temperature, or salinity cycles.The authors declare no conflict of interest.Hannes Mutschler received his M.Sc. in biophysics from the Humboldt University of Berlin. His Ph.D. topic was on bacterial toxin‐antitoxin systems at the Max Planck Institute of Medical Research/Heidelberg University, Germany. For his postdoc on origins of life research, he joined Phil Holliger's lab at the Protein and Nucleic Acid Chemistry Division lab at the MRC Laboratory of Molecular Biology in Cambridge, UK. In 2016, he became a MaxSynBio independent research group leader at Max Planck Institute of Biochemistry in Martinsried, Germany. His current research interests include bottom‐up synthetic biology, cell‐free protein synthesis as well as RNA catalysis under prebiotic conditions.Kristian Le Vay received his M.Sc. in chemistry from the University of Bristol, UK, and completed his Ph.D. at the Bristol Centre for Functional Nanomaterials. During this time, he designed and developed artificial enzyme systems based on photoactive protein‐nanoparticle conjugates. He is currently a postdoctoral researcher at the Max Planck Institute for Biochemistry. Here, his research encompasses both synthetic biology and origin of life studies, focusing on prebiotic RNA catalysis in unusual environments and the development of dynamic model protocell systems.Elia Salibi completed his B.Sc. in Biology at the University of Beirut, Lebanon. He then received his M.Sc. in Molecular Biology and Genetics from the University of Pavia, Italy. His thesis focused on developing and characterizing a synthetic modular CRISPR/dCas9‐based gene silencing toolbox for Escherichia coli. Currently he is a Ph.D. student at the Max Planck Institute of Biochemistry investigating the role of RNA protocells in the context of the emergence of life.Emilie Y. Song received her B.Sc. in Biochemistry and Chemistry from the University of British Columbia in Vancouver, Canada. She then completed her M.Sc. in Molecular Genetics at the University of Toronto screening for novel Streptomyces bacterial natural products with potential bioactivity against parasitic worms. She is currently a PhD candidate in the CRC235: Emergence of Life graduate program investigating RNA stability and catalysis in plausible prebiotic environments.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. 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