Contrary to the biosynthetic pathways of many terpenoids, which are well characterized and elucidated, their transport inside subcellular compartments and the secretion of reaction intermediates and final products at the short- (cell-to-cell), medium- (tissue-to-tissue), and long-distance (organ-to-organ) levels are still poorly understood, with some limited exceptions. In this review, we aim to describe the state of the art of the transport of several terpene classes that have important physiological and ecological roles or that represent high-value bioactive molecules. Among the tens of thousands of terpenoids identified in the plant kingdom, only less than 20 have been characterized from the point of view of their transport and localization. Most terpenoids are secreted in the apoplast or stored in the vacuoles by the action of ATP-binding cassette (ABC) transporters. However, little information is available regarding the movement of terpenoid biosynthetic intermediates from plastids and the endoplasmic reticulum to the cytosol. Through a description of the transport mechanisms of cytosol- or plastid-synthesized terpenes, we attempt to provide some hypotheses, suggestions, and general schemes about the trafficking of different substrates, intermediates, and final products, which might help develop novel strategies and approaches to allow for the future identification of terpenoid transporters that are still uncharacterized. Plant specialized metabolites (PSMs), previously referred as secondary metabolites, are small molecules <2000 Da) synthesized and accumulated in specific tissues and developmental stages and/or under particular environmental conditions, which own different functions aiming at generally improving plant fitness, including physiological aspects and responses to biotic and abiotic stresses . These m000 Da syThe transport of molecules between and within cells generally follows different types of mechanisms, based on the nature of the compound. In more detail, gasses and some small lipophilic compounds, including a few volatiles, can pass through membranes by simple diffusion , while cArabidopsis thaliana many members have been functionally characterized ), that is then photochemically converted to artemisinin in the apoplastic space, suggesting the involvement of these transporters in vivo. Finally, a recent study reported the identification of four members of plasma membrane-localized ABCG transporters in Salvia miltiorrhiza Bunge, a plant used in traditional Chinese medicine to treat cardiovascular and cerebrovascular diseases; these transporters could be potentially involved in the export of tanshinone (a lipophilic diterpene) and salvianolic acid (a hydrophilic phenolic compound), which are metabolites highly accumulated in the roots and rhizomes of S. miltiorrhiza ).d27, ccd7, ccd8, and max1 mutants have demonstrated that (a) SLs synthetized in roots can move acropetally; (b) a basal level of biosynthesis is also present in the above-ground tissues; and (c) CL, CLA, and/or its metabolites methyl carlactonoate (MeCLA) and hydroxyl-methyl-carlactonoate (1′-OH-MeCLA) could act as the long-distance signaling molecule/s. Additional studies are necessary to shed light on the nature of the transported molecule/s and if a xylem or a cell-to-cell movement is involved [Plant roots contain relatively high levels of SLs compared to other tissues, and several grafting experiments using a range of combinations of involved .Concerning short-distance signaling, SLs are released into the rhizosphere, providing early plant–AMF interaction signal  and germSolanaceae, relying on the G-type ABC transporter orthologues of P. hybrida PhPDR1 (PLEIOTROPIC DRUG RESISTANCE 1) gene, which was first identified. This transporter was shown to be expressed in the shoot lateral axils, close to the lateral buds, in the root tips, and in the outer lateral membrane of specific non-suberized exodermis cells named hypodermal passage cells (HPCs), that function as entry points for AMF. Moreover, the expression levels were augmented by phosphorus starvation (Pi-) and the application of exogenous GR24, a synthetic SL analogue. Interestingly, the petunia mutant pdr1 showed a reduced amount of orobanchol in root exudates and a high branching phenotype, even if its levels in root extracts was similar to the wild-type ones [pdr1 mutant rootstock can complement the ccd8 mutant, thus suggesting the existence of a PDR1-independent transport route  or substrates [SL exudation appears to be an active process in ype ones . Therefobstrates .PhPDR1 in petunia further confirmed the SL transport activity of this gene. Notably, an increment of total biomass and a reduced leaf senescence phenotype were also observed in the overexpression lines [N. tabacum NtPDR6 has been characterized as a possible orthologue of PhPDR1, with an inducible expression by inorganic phosphate (Pi) and an increase in branching in RNAi lines. However, SLs have not been assessed in this work [Additionally, the overexpression of on lines . Of intehis work .Solanum lycopersicum, the genes ABCG44 and ABCG45 have been knocked-out by the CRISPR/Cas9 technology, and edited lines have been characterized. Overall, both the single mutant abcg44 and the double mutant abcg44/abcg45 showed (i) increased branching and reduced orobanchol content in root exudates, at an even higher extent in the double mutant, and (ii) decreased susceptibility to the parasitic plant Phelipanche aegyptiaca [ABCG44 and ABCG45 on SL transport still needs additional clarifications.Finally, in gyptiaca . HoweverFabaceae exhibit exodermis-free roots and, consequently, they do not have specialized HPCs, the gene MtABCG59 in Medicago truncatula resulted in an orthologue of PhPDR1, with a wider pattern of expression in root compared to petunia. Of note, the root exudates of the mutant line mtabcg59 (retrotransposon insertion) induced a lower germination rate with seeds of the parasitic plant Phelipanche ramosa, thus providing an indirect evidence of SL reduction in root exudates. However, a passive diffusion mechanism of SLs in the rhizosphere was also hypothesized [Interestingly, although thesized ,186.Crocus sativus, in which crocins originate in the stigmas from the cleavage of zeaxanthin by the CsCCD2 enzyme [Gardenia jasminoides, where their synthesis starts from the carotenoids β-carotene, zeaxanthin, and lycopene by the action of GjCCD4a [Buddleja davidii, in which the substrate is zeaxanthin and the enzymes involved are BdCCD4.1/BdCCD4.3 [Crocins are glycosylated apocarotenoid pigments that originate from the oxidative cleavage of carotenoids, such as zeaxanthin, β-carotene, and lycopene. The main source of crocins is saffron, the dried stigmas of 2 enzyme  and unde2 enzyme ,189,190. GjCCD4a , and in BdCCD4.3 ; moreoveBdCCD4.3 ,193.C. sativus, the crocin biosynthetic pathway involves different compartments: the synthesis, indeed, starts in the chromoplast with the cleavage of zeaxanthin by the plastid-localized CsCCD2, leading to the formation of the C20 compound crocetin dialdehyde [C. sativus ABC transporter, CsABCC4a, seems to be capable of transporting crocins with different degrees of glycosylation and stereoisomerism across membranes [trans configuration occurs only at high concentration and in the presence of the cis isomer [N. benthamiana leaves led to crocin accumulation at higher levels compared to that of CsCCD2 alone [N. benthamiana leaves with CsCCD2 and showed that crocins accumulated in the vacuoles, thus demonstrating the existence of endogenous transporters that are able to translocate these compounds in the transformed leaf cells.In aldehyde ,194, conaldehyde , and endaldehyde  and CsUGaldehyde , althougaldehyde . Finallyembranes . This trembranes  were notembranes , thus suembranes , it is ps isomer . This fiD2 alone . This inD2 alone  used a vCrocins are stored in the vacuoles of stigmas until the senescence of the plant leads to the mechanisms of recycling/reusing . InteresCrocus transcriptome were identified and classified into eight subfamilies based on their similarity with A. thaliana transporters. Among them, ABCB and ABCG were the most predominant, although they were mostly expressed in anthers and tepals. Differently, the ABCC, ABCD, ABCE, and ABCF members were expressed mainly in stigma, suggesting a possible role in the transport of saffron apocarotenoids. In fact, one year later, it was demonstrated that CsABCC4a and CsABCC2 transporters were involved in the vacuolar accumulation of crocins [ABCB and ABCC genes showed significant induction in response to hormones, including the diterpene gibberellic acid and the apocarotenoid ABA for ABCB members, and ABA for ABCC, thus providing clues about their possible role in the transport of these hormones. Additionally, they identified a few orthologs of the AtABCG40 transporter that took part in the transport of SLs  and plastidial MEP (a) pathways, different terpenes (b’ and b) are formed in the cytosol or plastid, respectively. These terpenes are then modified into terpenoids (c and d) by different enzymes that localize to the ER and/or cytosol. The translocation of the intermediates from the plastid to these compartments has not yet elucidated, but could involve mechanisms based on hemifused membranes between plastids and the ER [e), secreted into the apoplastic space (f), or imported inside the cells through the action of transmembrane transporters. To date, only members of the ABC, MATE, NPF, AWPM-19, and SWEET families have been reported to be involved in the translocation of terpenoids (indicated in the figure). A sixth class consisting of PUP transporters might also contribute, since they are responsible for the transport of other PSM-chemical types  in plant cells. Picture created with BioRender.com.From the synthesis of the precursors (green dots) through the cytosolic MVA . More is known about the secretion of terpenoids: indeed, many lipophilic compounds with roles in plant defenses are secreted through the plasma membrane and accumulated in the apoplast or in specialized structures, such as glandular trichomes. The preeminent class of transporters involved in the secretion of terpenoids with ecological roles is that of the ABC, subfamily G, transporters ,15,16. IIncreasing the knowledge about ABC transporters, as well as on other classes of PSM transporters, could be useful to generate new idiotypes through the approaches of genome editing and/or metabolic engineering to modify the sequence of transporters and, thus, to alter their substrate specificity. In the perspective of a sustainable agriculture, these approaches might be of great efficacy, especially for ABC transporters involved in the secretion of compounds with symbiotic interaction activities, such as SLs  or the rOne of the more elusive bottlenecks of the identification of transport proteins is related to their functional characterization. In this respect, different approaches have been used to date, but a series of considerations has to be remarked: (i) in vivo approaches, such as silencing, editing, and overexpression, need the availability of genetic information and genetic tools; (ii) the expression in heterologous systems, such as yeast cells, Xenopus oocytes, or insect cells, and in vitro assays often present technical difficulties as gene cloning ; and (iii) a huge diversity of substrates needs to be tested; however, in order to overcome this aspect, transportomic approaches have been developed, consisting of the use of total metabolic extracts, which allows for the simultaneous testing of hundreds of largely diversified compounds . These aOmics technologies  are powerful tools to be exploited for the characterization of PSM transporters . RecentlC. roseus [S. lycopersicum [The present review reveals that, of the thousands of terpenoids identified in nature, only a few of them have been characterized from the point of view of their transport activity . Besides. roseus  and of αpersicum .These findings suggest a possible involvement in the transport of other PSMs, including other terpenoids. Another group of transmembrane transporters that deserve a better extent of investigation as candidate terpenoid transporters are (i) PUPs, which have been reported, to date, to transport alkaloids ,49,50, , or ABAstresses ,210,211,stresses  and posistresses . All of stresses ,214, , muAn additional issue to be addressed is related to the translocation of plastid-localized intermediates of terpenoids that can involve plastidial transmembrane transporters or the interaction between organelles membranes . In thisC. reinhardtii [A last fascinating food for thought is the recent discovery of the so-called ASTER proteins. In mammalian cells, these proteins have been demonstrated to have a role in carotenoid transport , in addinhardtii . This prnhardtii ,220. In nhardtii .In conclusion, although several advances have been achieved on the research area of terpenoid trafficking, many fundamental questions have to be addressed, and we are still far from the final picture. The full elucidation of the transport mechanism of terpenoids are fundamental to finely tune the optimal metabolic engineering strategies for the production of these high-value bioactive compounds in heterologous systems, for their industrial production and exploitation, and for the implementation of the beneficial interactions between plants and useful microorganisms  to finely regulate plant growth in innovative agricultural contexts.


Here, we examined the impact of pCO2 variability on the reef-building coral Pocillopora damicornis originating from reefs with contrasting environmental histories (variable reef flat versus stable reef slope) following reciprocal exposure to stable (218 ± 9) or variable (911 ± 31) diel pCO2 amplitude (μtam) in aquaria over eight weeks. Endosymbiont density, photosynthesis and net calcification rates differed between origins but not treatment, whereas primary calcification (extension) was affected by both origin and acclimatization to novel pCO2 conditions. At the cellular level, corals from the variable reef flat exhibited less intracellular pH (pHi) acidosis and faster pHi recovery rates in response to experimental acidification stress (pH 7.40) than corals originating from the stable reef slope, suggesting environmental memory gained from lifelong exposure to pCO2 variability led to an improved ability to regulate acid–base homeostasis. These results highlight the role of cellular processes in maintaining acidification resilience and suggest that prior exposure to pCO2 variability may promote more acidification-resilient coral populations in a changing climate.Ocean acidification is a growing threat to coral growth and the accretion of coral reef ecosystems. Corals inhabiting environments that already endure extreme diel pCO The increasing frequency and intensity of climate-driven marine heatwaves has provided powerful visual evidence of our changing climate, manifested by mass coral bleaching events and subsequent coral mortality . The imp systems . It is d systems –6. Howev systems ,8. As th2 conditions from which the study specimens were collected, which can fluctuate dramatically on diurnal, seasonal and spatial scales [2 variability promotes increased tolerance to acidification stress.Naturally variable habitats provide a glimmer of hope that climate-resilient coral populations already exist on coral reefs worldwide. Variable habitats or extreme environments, such as tidally dominated back reefs and reef flats, expose organisms to short-term fluctuations in temperature and pH conditions similar to those projected for future reefs ,9–12. Thl scales ,18. As ain situ pCO2 conditions within two habitats, a tidally dominated reef flat and an oceanic reef slope of Heron Island, southern Great Barrier Reef  when exposed to changed pCO2 regimes over a period of two months. Further, we explore the cellular mechanisms involved in coral acid–base homeostasis, how these differ between corals with distinct environmental histories, and if energetic costs are involved in driving these differences.This study examines a suite of physiological parameters to better understand how resistance to natural pH variability influences resilience to ocean acidification in a common hermatypic coral. We characterize the ier Reef , and rec. 2 (a)2 compared to reef slope habitats, and exposes reef flat corals to extreme temperature and CO2 conditions projected for future reefs [2 was recorded over the same season, but in 2016 . Long-term studies show remarkable consistency in pCO2 measurements recorded at the same location between years [2 variability measured within these identical reef habitats over the same time period may be similar across years.The experiment was performed during the austral summer from mid-January to late March 2021 at Heron Island Research Station (HIRS), southern Great Barrier Reef . Heron Reef is composed of five distinct geomorphological habitats characterized by diverse benthic communities and biogeochemical conditions ,20. Thisre reefs ,12,20 (f (b)P. damicornis were collected from the reef flat and slope locations within the same depth range (1–3 m) on 14 and 15 January 2021 (n = 12 per habitat) . One additional chip per colony was preserved in 100% ethanol and kept at −80°C for genetic analyses to confirm the collected coral specimens were all P. damicornis based on the mitochondrial ORF (cf. [n = 12 fragments per tank). To minimize ‘tank effects’, the eight tanks were randomized across one outdoor table (n = 4 per treatment), with each set of coral fragments rotated into an adjacent tank of the same treatment every third day. Tanks and lids were covered with filters  to mimic the light environment at the collection sites  variable pCO2 (2 was continuously recorded in treatment sumps (n = 3 per treatment) were collected for total alkalinity (AT) and pHTotal at midday and midnight. AT was determined via the Gran titration method using 0.1 M HCl and pHTotal was determined via a high-precision glass pH electrode  across replicated 20 g seawater samples [AT and pHTotal measurements using the seacarb package in R [After 7 days of recovery from collection and handling, corals were exposed to two distinct treatments for eight weeks: (i) stable pCOble pCO2 , which wnt sumps  and with samples . Acid coage in R  (table 1 (c)Fv/Fm) of coral fragments were measured six times during the experiment  via buoyant weight and photogrammetry using previously described methods [n = 12) were water-piked on ice to remove coral tissue from the skeleton using 50 ml of 0.1 M phosphate buffered saline solution. The tissue slurry was centrifuged at 4°C once for 5 min at 2500g to sufficiently separate host tissue and the intracellular endosymbiont cells. Host tissue was analysed for host-soluble protein concentration and mycosporine-like amino acids (MAAs) concentrations spectrophotometrically [2), which was determined using the single wax-dipping technique [a were determined via absorbance at 630, 663 and 750 nm using the equations in [3) bulk density, where the skeleton was sealed with a coat of wax, dry weighed and then buoyant weighed [Coral survivorship was assessed visually daily, and only one coral fragment died during the experiment. Net calcification, surface area (a proxy for extension ), volume methods –29 2, PAR and nutrient concentrations were analysed for differences within and between experiment treatments  and reef habitats . The effect of tide  was also explored on nutrient concentrations. The interactive effects of origin and treatment were explored on P. damicornis growth  and physiology  using linear mixed effects (lme) models with colony genotype as a random effect [car package [emmeans package with Tukey HSD adjusted p-values [ggplot2 [Seawater temperature, COm effect . Similar package . Signifip-values . All stap-values , and gra[ggplot2 .. 3 (a)2 (µatm) in situ (measured in 2016) was similar between the reef flat (454 ± 3.0) and reef slope (418 ± 1.9), but the reef flat experienced twice the mean daily pCO2 amplitude than the reef slope  was the same between the reef flat (26.8 ± 0.02) and reef slope (26.8 ± 0.01), the daily amplitude was nearly three times greater on the reef flat than the reef slope . Mean PAR (µmol quanta m−2 s−1) was significantly higher on the reef flat than the reef slope , driven by tidal fluctuations in depth. Seawater nutrient concentrations  were not significantly different between habitats  or between low or high tide  throughout the experimental period .The mean seawater pCO2 dynamics differed significantly between treatments. Specifically, there was a 4.2-fold difference in mean diel pCO2 amplitude (µatm) between the variable (911.3 ± 30.69) and stable (218.3 ± 9.14) treatments across the experimental period in the upstream sumps, which resulted in a mean pCO2 that was slightly higher in the variable treatment (620 ± 3.4) compared to the stable treatment (540 ± 0.9)  conditions in the stable (327.8 ± 10.5) and variable (435.7 ± 25.3) treatments, which were more similar to mean pCO2 conditions measured in situ. Importantly, tank pCO2 was significantly influenced by the interaction between treatment and time , with pairwise comparisons revealing no difference in pCO2 conditions during midday (p = 0.99) but significantly higher pCO2 in the variable treatment during the programmed spike in conditions at midnight . At midday, however, there was no difference in mean pH between treatments (8.15) . This successful maintenance of consistent temperature, PAR and nutrient concentrations between treatments suggests that differences in pCO2 amplitude throughout the experiment are likely drivers of the physiological responses observed.Within the experimental treatments, seawater pCO (b)P. damicornis  or slope (Cladocopium type ‘C33a’) [Cladocopium ‘C33a’. The discrepancy with prior studies may be due to: (i) depth, with reef slope P. damicornis sampled at shallower depths (1–3 m) in this study relative to others (>4 m) [P. damicornis sampled on the open-ocean side of Heron Reef, in contrast to the turbid channel between Wistari and Heron Reef sampled by others . Albeit based on a small sample size, the consistency of Symbiodiniaceae species and genotypic diversity between slope and flat habitats suggests that acclimatization plays a significant role in tolerance to environmental variability. Indeed, holobiont adaptation in response to distinct environmental regimes can occur in the absence of distinct endosymbiont genotypes [All coral colonies used in the experiments were confirmed to be ron Reef . The micron Reef ), contaiatusorum . Our fin ‘C33a’) ,41,43. Ss (>4 m) ,41,43; 2 for two months. For example, symbiont density was significantly influenced by origin , with corals originating from the reef flat containing lower symbiont densities than corals from the slope. This could be a carry-over effect of sublethal stress from the harsh temperature and light regimes of the reef flat habitat . Interestingly, net photosynthesis was slightly higher in corals originating from the reef flat regardless of treatment  , whereas dark respiration , light-enhanced dark respiration  and photosynthesis to respiration (P : R) ratios  displayed no significant patterns between origin or treatment . These observations suggest that autotrophic energy acquisition was not impeded by the daily, extreme, oscillations in pCO2 to which these corals were acclimatized. This is in contrast to corals exposed to simulated seawater acidification and/or in situ thermal stress, which typically exhibit reductions in metabolic rates [a concentrations  or photochemical efficiency (Fv/Fm) between origin  or treatment  , suggesting that pCO2 variability did not have an effect on these parameters. Finally, host protein content, a proxy for biomass, showed no significant differences between origin or treatment  , in contrast to others that have found corals originating from variable habitats tend to have higher tissue biomass than conspecifics from stable habitats due to a prioritization of biomass over calcification in harsher environments [To cope with living under the stress imposed by variable or extreme environments, corals exhibit remarkable phenotypic plasticity ,48. The ic rates ,35. In a−1) was significantly affected by origin, with higher rates in P. damicornis that originated from the reef flat , while the effect of treatment was not significant  (−1), a proxy for extension, showed a significant interaction between treatment and origin . Specifically, corals from the reef flat increased their extension rates in the stable (non- native) treatment, which could indicate a release from stressful pCO2 conditions , but treatment had no effect (2 conditions); however, as net calcification did not change while extension increased in response to novel pCO2 conditions, it is likely that densification correspondingly decreased. Future studies could isolate the new growth area to better resolve morphological changes  resultant from acclimatization to pCO2 variability. The origin-specific differences in CaCO3 density that we observed aligns with habitat-specific patterns observed in earlier studies, where skeletal density was a direct reflection of wave exposure or water motion (e.g. lower CaCO3 density in protected reef flats or lagoons compared to conspecifics from high wave energy reef slopes) [2 [P. damicornis native to the reef flat continuing to exhibit lower density of branches and greater branch thickness than corals native to the reef slope  . Howevernditions . Secondao effect . Lower so effect . Given t slopes) ,48, but opes) [2 ,34. Indeef slope . Similarmicornis , and thu (d)P. damicornis both displaying a positive relationship with increasing seawater pH in both light and dark conditions [2 variability altered coral acid–base homeostasis dynamics by exposing P. damicornis cells to acidification stress (pHe 7.40). Symbiocytes had higher pHi than non-symbiocytes , and overall, coral cells both with and without endosymbionts exhibited an initial acidification of pHi after exposure to acidified seawater, followed by recovery to their initial pHi setpoint, indicative of an active physiological compensatory response in all cell types  (a). Interestingly, the initial change in pHi in response to acidification stress  showed a significant three-way interaction between treatment, origin and cell type  (b). Pairwise comparisons revealed that in non-symbiocytes, there was no difference in acidification magnitude for P. damicornis originating from the reef flat and acclimated to either stable or variable pCO2 treatments (p = 0.6). This suggests corals native to the flat had a robust ability to buffer pHi and maintain acid–base homeostasis in the face of acidification stress regardless of treatment, possibly due to adaptation and/or constitutive expression of regulatory mechanisms to compensate for extreme pCO2 oscillations. While the specific mechanisms were not investigated in this study, it is possible that the upregulation of acid–base homeostasis mechanisms in corals native to the reef flat, such as the proteins involved in pH-sensing [P. damicornis native to the reef flat to cope with acute pH stress. This behaviour of ‘front-loading’ stress response pathways has been seen in coral temperature variability responses [2+-ATPase in the calicodermis, which may uptake protons from the ECM [Maintaining stable intracellular pH (pHi) is critical for cellular physiology  and requnditions . Here, w= 0.049) a. Intere= 0.003) b. Pairwi-sensing , proton -sensing –59, or c-sensing , ultimatesponses , and is esponses ,57. Also the ECM ), wherebP. damicornis native to the reef slope but exposed to the variable conditions showed a three-fold drop in pHi following exposure to acidified seawater relative to slope corals remaining in their native stable pCO2 conditions (p < 0.0001), and this acidification was of significantly larger magnitude than that of corals originating from the flat in either treatment . This large drop in pHi following acute external acidification suggests that corals adapted to stable reef slope conditions have a limited ability to buffer their internal pH after eight weeks of exposure to extreme diel pCO2 variability. Again, the mechanisms behind this response are unknown, but are possibly due to an inability to further upregulate acid–base regulatory machinery during an acute acid challenge following chronic exposure to extreme diel pH variability and/or a lower passive buffering capacity. Furthermore, following the initial acidosis, P. damicornis native to the reef slope exhibited slower pHi recovery rates relative to corals native to the flat , suggesting that corals native to the reef slope have a less robust capacity to compensate for acidification stress than corals native to the reef flat. This investment in acid–base regulation by reef flat corals may be beneficial for coping with ocean acidification, but could also come at a cost. For example, our data appear to demonstrate a trade-off in response to extreme pCO2 variability, where greater investment in acid–base homeostasis leaves insufficient energy to support secondary calcification. If reef flat corals must expend more energy on pHi regulation, such as through the active (i.e. ATP-dependent) removal of protons from their tissues, this could force a trade-off with expensive energy investments such as skeletal infrastructure , suggesting that endosymbionts play a significant role in helping buffer host cells following acidification stress. The mechanisms remain unknown, but could occur through energetic provisioning to the host that supports the energetic demands of acid–base homeostasis , or possibly through their own metabolic activity [2 consumption via photosynthesis, as can occur in the light [2 conditions are able to better cope with acute acidification stress, as climate change intensifies, the interactive effects of ocean warming and acidification could interact to undermine the ability of corals to regulate acid–base homeostasis [3 structures that support entire reef ecosystems and coastal communities [Interestingly, in contrast to corals native to the reef flat, c matrix ; removalc matrix ; transpo the ECM ), possib the ECM , particu the ECM , it becoactivity . Howeverhe light . Despiteeostasis , ultimatmunities ,8.. 42 oscillations, in addition to commonly recognized parameters like temperature, water motion and light, play an important role in influencing phenotypic variability in calcification between extreme environments and suggest that acclimatization to pCO2 variability may promote acidification-resilient populations in the future. However, energy investments into regulating acid–base homeostasis will become more costly in a more acidic ocean [P. damicornis native to stable environments cannot acclimatize to extreme pCO2 oscillations conditions over relatively short time scales. Nevertheless, our results suggest that reef corals may be more resistant to future ocean acidification conditions in extreme environments where diel variation in seawater pCO2 is prominent, which has important implications for reef persistence in a changing climate.Our results highlight that pCOic ocean  and toleic ocean ,11. Whil


Small non-coding RNAs regulate gene expression and are highly implicated in heart failure. Recently, an additional level of post-transcriptional regulation has been identified, referred to as the epitranscriptome, which encompasses the body of post-transcriptional modifications that are placed on RNA molecules. In this review, we summarize the current knowledge on the small non-coding RNA epitranscriptome in heart failure.With the rise of new methods to study RNA modifications, epitranscriptome research has begun to take flight. Over the past 3 years, the number of publications on the epitranscriptome in heart failure has significantly increased, and we expect many more highly relevant publications to come out over the next few years.O-ribose-methylation, adenosine-to-inosine editing, and isomiRs. Their potential role in heart failure is discussed.Currently, at least six modifications on small non-coding RNAs have been investigated in heart failure-relevant studies, namely N6-adenosine, N5-cytosine and N7-guanosine methylation, 2’- Heart failure (HF) affects more than 64 million people worldwide . HF is aAs HF is a major problem in health care worldwide, research into the different physiological, cellular, and molecular aspects of HF is of utmost importance and several animal models have been developed for this purpose. The most commonly used murine model for inducing heart failure is surgical transverse aortic constriction (TAC). In this model, the aorta is partially ligated, inducing increased intracardiac pressure and therefore leading to a pressure overload . SometimA frequently observed feature of HF is fetal gene expression reprogramming and, in recent years, it has been established that several classes of non-coding RNAs (ncRNAs) control this cardiac gene reprogramming in a post-transcriptional manner , 9. LongMicroRNAs are small non-coding RNAs (~ 22 nucleotides long) that regulate protein levels by inhibiting mRNA translation and/or promoting mRNA decay. Recognition of target mRNA occurs via base pairing of the microRNA’s seed sequence, from the 2nd until the 8th nucleotide from the 5’-end of the microRNA, and a complementary sequence in the mRNA transcript, usually located in the 3’ untranslated region (UTR) . FunctioOther classes of small ncRNAs that can play a role in HF include piwi-interacting RNAs (piRNAs), transfer RNA derived fragments (tRFs), and small nucleolar RNAs (snoRNAs), which all have the ability to regulate post-transcriptional gene expression. piRNAs are typically 26–31 nucleotides long and interact with the piwi-subfamily of Argonaute proteins, resulting in the formation of a RISC complex . While mO-ribose-methylation (2’Ome), which occurs on the ribose. Besides methylation, nucleotide editing in the form of adenosine deamination, including adenosine-to-inosine (A-to-I) editing and cytidine-to-uridine (C-to-U) editing, is a widespread phenomenon in microRNAs [Recently, an additional level of post-transcriptional regulation has been identified, referred to as the epitranscriptome. The epitranscriptome describes the body of post-transcriptional modifications that are placed on RNA molecules. Although long understudied, recent studies have demonstrated important regulatory roles for biochemical modifications of RNA , and curicroRNAs . Lastly,In this review, we summarize the current knowledge on small ncRNA epitranscriptomic pathways in heart failure. Epitranscriptome research, especially in the cardiovascular field, is still in its infancy, and therefore, we will focus on the few RNA modifications, on which heart-relevant studies have been performed, namely m6A, m5C, m7G, 2’Ome, A-to-I editing, and isomiRs.Methylation of the N6 position of adenosine (m6A) is one of the most prevalent post-transcriptional modifications of RNA in eukaryotic cells . RNA m6AFrom these studies, it becomes clear that regulation of writer and eraser enzymes has the ability to control the progression of heart failure. In 2020, Gao et al. identified cardiac-hypertrophy-associated piRNA (CHAPIR), a piRNA that is abundantly expressed during cardiac hypertrophy, as a regulator of METTL3 activity. CHAPIR was able to regulate hypertrophy through direct binding of METTL3, thereby inhibiting its activity •. Expres. M6A is detected on snoRNAs, lncRNAs , microRNAs, and other ncRNAs [For instance, changes in methylation status have been shown to affect RNA folding and structure or regulate RNA processing –50. M6A r ncRNAs –53. The r ncRNAs . The seer ncRNAs . Our gror ncRNAs , but impPotential effects of m6A methylation on other small ncRNAs in a cardiac setting are lacking/limited. However, m6A methylation of lncRNAs can alter their local structure, enabling the binding of m6A-reader proteins which consequently influences abundance or function of the lncRNA . Liu et Dnmt2-deficiency in mice led to cardiac hypertrophy, even though cardiac function was preserved, despite the enlargement of cardiomyocytes [The modification of cytosine into N5-methylcytosine (m5C) is well established as a DNA modification in the epigenome , 62. Howmyocytes •.. In the heart, m5C was especially enriched in mRNAs involved in heart and muscle cell development and in mitochondrial function [The distribution of modified cytosines along the RNA is not random; CG-rich regions are enriched in m5C modifications, as are regions immediately downstream of translation initiation sites, untranslated regions, and regions near Argonaute binding sites , 64. Furfunction .Dnmt2-deficiency induced cardiac hypertrophy and the small nuclear RNA (snRNA) Rn7sk [The effect of m5C modifications on small ncRNA in heart failure remains understudied, but in 2016, Ghanbarian et al. found a link between A) Rn7sk •. For trA) Rn7sk , but Rn7A) Rn7sk , 71. GhaThe mechanism behind this increased dissociation may toile in the fact that unmethylated Rn7sk appears less stable and is therefore more readily degraded. Methylation by DNMT2 has proven to protect tRNAs from stress-induced cleavage, interfering with the formation of tRFs . Blanco Shen et al. (2018) looked into the role of these tRFs in cardiac hypertrophy and demonstrated, by small RNA sequencing, that tRFs were extremely enriched in isoproterenol-induced hypertrophic rat hearts. Interestingly, the cleavage patterns indicated that the induced fragments were results of precise cleavage modulations in response to myocardial hypertrophy ••. OvereIn m7G, guanosine is methylated at the 7th position of its base ring. This modification is ubiquitous in mRNA 5’caps, but also widely occurs in tRNAs and rRNAs , 77. MorMembers of the affected let-7 microRNA family are involved in cardiac hypertrophy and heart failure, although they appear to have a dual role –82. On tOn the other hand, Tolonen et al. (2014) investigated the effect of inhibiting let-7c on the progression of postinfarction left ventricular (LV) remodeling in mice . By inhi2’Ome can occur on all four ribonucleosides of RNA and is carried out by methyltransferases like fibrillarin. During 2’Ome, a methyl group is added to the 2’ hydroxyl of the ribose moiety of a nucleoside. Most of these 2’Ome-events are guided by so-called C/D box snoRNAs . Our groOur group confirmed 2’Ome in six integrin pathway mRNAs by a human 14q32 snoRNA, SNORD-113–6. The affected mRNAs showed decreased methylation and increased degradation when SNORD-113–6 was inhibited in human primary fibroblasts ••. As re2’Ome is omnipresent in rRNA, tRNA, and mRNA but has also been observed in small ncRNAs, including microRNAs and even snoRNAs themselves , 88, butWhere methylation mostly affects RNA stability and binding efficacy, RNA editing directly changes the nucleotide sequence. A-to-I editing is catalyzed by adenosine deaminase acting on RNA 1 or 2 (ADAR1 or ADAR2). Although the expression of both ADAR enzymes and the number of A-to-I edited sites in the heart appear to be rather low compared to other tissues , 92, theIn A-to-I editing, adenosine is deaminated, which results in the formation of an inosine. Inosine is interpreted as a guanosine by the cellular machinery, as inosine preferentially binds cytidine . For mRNMicroRNAs are edited in their primary transcripts, as ADARs preferentially target double-stranded RNA structures in the nucleus . The ediMicroRNA editing profiles are tissue specific , 105 andAll RNAs, including ncRNAs, undergo various processing steps after their transcription. For mature microRNAs, this means that they are generated from a primary transcript, which undergoes two consecutive cleavage steps . The looIsomiRs are proven to be functional microRNAs that actively associate with the RISC complex and inhibit mRNA translation of their targets , 118. MoCtgf (connective tissue growth factor) target reporter than the 5’-isomiR, while the 5’-isomiR repressed the Pgam1 (phosphoglycerate mutase 1) target reporter better than canonical miR-133a.Humphreys et al. (2012) identified that the most frequently annotated microRNA sequence that was attributed to miR-133a in murine cardiomyocytes was not the canonical sequence, but a 5’-isomiR ••. That RhoA and Cdc42, proteins implicated in the hypertrophic signaling cascade, and Ctfg, implicated in cardiac fibrosis, as direct targets of miR-133a, proposing a mechanism through which miR-133a exerts its protective effects [Ctfg by the more abundantly expressed 5’-isomiR-133a as mentioned above, the isomiR may prove clinically relevant in heart failure.MiR-133a has been identified as a major cardiac regulator and is highly implicated in heart failure . A decre effects , 127. KeHowever, so far, no one has looked into the effects of this highly abundant 5’isomiR of miR-133a in heart failure. The difference in target selection by isoforms of a microRNA can lead to differences in biological functions. Our group previously demonstrated that the 5’-isomiR of the known vasoactive miR-411-5p is highly expressed in the heart and vasculature. The 5-isoforms of miR-411-5p have highly distinctive targetomes and were shown to be are differentially regulated under ischemia •. Under In conclusion, the available literature shows that post-transcriptional modifications of small non-coding RNAs are likely to play important roles in the regulation of gene expression of many factors involved in the occurrence, progression, and regulation of heart failure. While epitranscriptome research in a cardiac context is still in its infancy, we observed an increase in relevant publications over the past 3 years. The rise of new methods to study RNA modifications is expected to facilitate an exponential increase in more in-depth research concerning the role of the epitranscriptome in heart failure. Given the fact that even with the limited amount of research available now, the implications of the epitranscriptome for heart failure are significant; we can only anticipate exciting and highly important research developments over the next few years.


GCN4, endogenous mRNA MUC4, and lncRNA SatIII were distinctly visualized in single live cells.We reported on an efficient RNA imaging strategy based on a CRISPR-Cas and Tat peptide with a fluorescent RNA aptamer (TRAP-tag). Using modified CRISPR-Cas RNA hairpin binding proteins fused with a Tat peptide array that recruits modified RNA aptamers, this simple and sensitive strategy is capable of visualizing endogenous RNA in cells with high precision and efficiency. In addition, the modular design of the CRISPR-TRAP-tag facilitates the substitution of sgRNAs, RNA hairpin binding proteins, and aptamers in order to optimize imaging quality and live cell affinity. With CRISPR-TRAP-tag, exogenous  RNA-seq technologies have been mushrooming over the years, and they have helped researchers to understand the significant role of RNA even further . While tThere is more room for the improvement of CRISPR–FP, wherein the unbound fluorescent proteins induce an unavoidable fluorescence background signal, which makes this strategy require multiplex fusions of FP to obtain amplified signals. To optimize, the RNA aptamers exhibiting bright and stable fluorescence, when combined with the fluorophore-like synthetic dye , could bMultiple peptides in series interacting with other proteins via an antigen–antibody connection, such as the Suntag system, have been fused into the Cas protein to recruit function proteins for site-specific transcriptome modifications, such as methylation, for DNA as well as for RNA ,24. ThesHence, we suppose that engineering a TAR-aptamer that combines a peptide series will be feasible for introducing a dimension to amplify the signal while possessing the advantage of the aptamer. Here, in this work, we designed a strategy for RNA imaging via the combination of the Tat peptide with a fluorescent RNA aptamer named TRAP-tag . It may To design a stable RNA scaffold that is compatible with the integration of multiple RNA aptamers and TAR, we used the consensus sequence F30 . The detpmRuby3-HSF1, pmRuby3-24 × GCN4, and pHAGE-dPspCas13b-3 × EGFP-2 × NLS-IRES-puro were from Ling-Ling Chen. pHAGE-dPspCas13b-10 × Tat-2 × NLS-IRES-puro were constructed from pHAGE-dPspCas13b-3 × EGFP-2 × NLS-IRES-puro by removing 3 × EGFP and replacing it with 10 × Tat. Pm1374-CIRTS 3 and Pm1374-CIRTS 10 were synthesized by GenScript. pHAGE-CIRTS 3-10 × Tat-2 × NLS-IRES-puro, pHAGE-CIRTS 3-3 × EGFP-2 × NLS-IRES-puro, pHAGE-CIRTS 10-3 × EGFP-2 × NLS-IRES-puro, and pHAGE-CIRTS 10-10 × Tat-2 × NLS-IRES-puro were constructed from pHAGE-dPspCas13b-3 × EGFP-2 × NLS-IRES-puro and pHAGE-dPspCas13b-10 × Tat-2 × NLS-IRES-puro.A series of TAR-Aptamer cassettes were cloned into the pLKO.5 plasmid after the U6 promoter between the BmgBI and EcoRI restriction enzyme cutting sites. Then, we performed Golden Gate Assembly to insert specific spacer sequences into two BbsI sites behind the U6 promoter and before the transcription start sites of single guide RNA cassettes, as shown in detail in v/v) FBS at 37 °C and 5% CO2. For transfection, we used Lipofectamine 3000  to co-transfect 200 ng each of dCas-10 × Tat-2 × NLS and 1 μg each of plasmid DNA for the desired guide RNAs and TAR-aptamer. Additionally, before imaging, the cells were put into another 24–48 h of incubation.Human embryonic kidney cell line 293T  were cultured in 20 mm glass-bottom dishes  with high glucose DMEM , in addition to 10% . The signal-to-noise ratio (SNR) was calculated according to Cells cultured in confocal vessels and transfected with desired plasmids were imaged after the removal of the medium and staining with a fluorophore. Images were acquired with a microscope  that possesses an EMC CD camera  equipped with a 63 × oil objective lens (NA 1.4) and a 2× magnification adapter. Basically, 405, 488, 552, and 638 nm excitation lights were used for the DAPI, Broccoli/Pepper, mRuby3, and smFISH fluorescence excitations, respectively. Correspondingly, 415–478 nm, 498–542 nm, 562–618 nm, and 650–700 nm emission signals were acquired. All of the fluorescence imaging data of the cells were analyzed using Fiji/ImageJ   δ 8.01 , 7.93 , 7.90 , 7.86 , 6.85 , 4.77 , 3.59 , 3.53 , 3.07 . 13C NMR  δ 152.09, 145.93, 140.15, 133.39, 132.43, 125.90, 120.51, 119.37, 119.22, 111.96, 110.08, 100.17, 58.67, 54.27, 39.23.HBC530, H-imidazol-5(4H)-one) DFHBI-1T to produce a green fluorescence, was selected to verify the feasibility of the strategy. In another aspect, we chose the Tat peptide, an ideal object for recruiting TAR RNA with distinct structures [Broccoli, a well-known fluorescent RNA aptamer that can combine and enable the (Z)-4--2-methyl-1--1ructures ,26, to eructures   was fused to the system to bring it into the cell nucleus [GCN4 were confirmed via colocalization with single-molecule FISH (smFISH)  . The enl(smFISH) b, left i(smFISH) b, right,(smFISH) b, demonsSatIII) transcription occurred, and long non-coding RNA Satellite III (SatIII) was gathered to produce nuclear stress bodies (nSBs) [, which indicated the association between the loci of SatIII and HSF to form nuclear stress bodies. By contrast, no gathered signal of TAR-Broccoli was witnessed in the absence of sgRNA  . An RNA s (nSBs) ,35, whicof sgRNA . These rAlthough the Cas13 systems revolutionarily promoted the research on RNA, the huge size and archaea source of these proteins become major obstacles in their applications of both fundamental research and clinical practice. To date, most Cas13 proteins are around 130 kDa. Recently, a strategy named the CRISPR-Cas-inspired RNA targeting system (CIRTS) has been developed to surmount the large size and immunogenicity of present RNA targeting strategies by applying protein parts from the human proteome . SimilarRepresented by the CIRTS replacement of dPspCas13b, as indicated above, the flexibility of the modularized design is one of the advantages of the CRISPR-TRAP-tag. The fluorescence RNA aptamer part of the TAR-aptamer RNA could also be easily substituted using other species of aptamer. We replaced the aptamer component of the CRISPR-TRAP-tag with another species of RNA aptamer named Pepper, attributed to its smaller size and its higher and steadier fluorescence signal, with a red-to-cyan range of emission wavelength  Figure a. HBC530SatIII lncRNA resulted in a similar relative localization with HSF-mRuby as indicated before, which verified the imaging capability of the CRISPR-TRAP-tag with the multiplex RNA aptamer (SatIII targeting (Lastly, benefitting from the advantages of RNA aptamers that have a small size, it may be feasible to introduce a multiplex aptamers array to visualize a target with a brighter fluorescence and a higher SNR. Therefore, we doubled the Broccoli and octupled the Pepper aptamers using connecting frameworks and then connected both of them to the TAR scaffold . TAR con aptamer b. Furtheargeting , all sixGCN4 RNA with high abundance, which might be due to the high backgrounds of 3 × EGFP and probable protein misfolding. However, CRISPR-TRAP-tag-fused TBP/SLBP exhibited a smaller and more accurate fluorescent signal (In summary, CRISPR-TRAP-tag was demonstrated as an ideal RNA localizing and visualizing method in living cells, considering the aspects of its conciseness, dynamic range, sensitivity, and flexibility. The continuous increase of the types of fluorescent light-up aptamers provides more flexibility for the CRISPR-TRAP-tag system for simultaneously imaging multiple RNAs with different colors. This strategy provides a new method to investigate RNA distribution and has several aspects of merit. Firstly, the CRISPR-TRAP-tag system is applicable for RNA imaging, such as cytoplasmic mRNA and intranuclear lncRNA in live cells, providing a multipurpose platform . Secondlt signal . Thus, tt signal . The mult signal . Due to


RNA-based therapy is a promising and innovative strategy for cancer treatment. However, poor stability, immunogenicity, low cellular uptake rate, and difficulty in endosomal escape are considered the major obstacles in the cancer therapy process, severely limiting the development of clinical translation and application. For efficient and safe transport of RNA into cancer cells, it usually needs to be packaged in appropriate carriers so that it can be taken up by the target cells and then be released to the specific location to perform its function. In this review, we will focus on up-to-date insights of the RNA-based delivery carrier and comprehensively describe its application in cancer therapy. We briefly discuss delivery obstacles in RNA-mediated cancer therapy and summarize the advantages and disadvantages of different carriers . In addition, we further summarize and discuss the current RNA therapeutic strategies approved for clinical use. A comprehensive overview of various carriers and emerging delivery strategies for RNA delivery, as well as the current status of clinical applications and practice of RNA medicines are classified and integrated to inspire fresh ideas and breakthroughs. Cancer has always been a major public health problem to be solved, and despite significant advances in existing therapies in recent years, its treatment remains a formidable challenge . Drug thThe recent explosion of RNA research has provided new methods for the synthesis and delivery of RNA . NonetheTo overcome these obstacles and achieve the ideal therapeutic purpose, gene carrier has become the focus of gene therapy research. Different carriers have been developed including cationic polymers ,23, lipoRNA interference (RNAi) technology utilizes double-stranded RNA (dsRNA) to efficiently and specifically degrade intracellular homologous messenger RNA, thereby blocking specific gene expression and resulting in a target gene deletion phenotype. Common effector molecules mainly include small interfering RNAs (siRNAs), micro RNAs (miRNAs), and short hairpin RNAs (shRNAs) ,30. GivesiRNA faces both internal and external barriers. The internal drawback is the short lifespan of synthetic siRNAs, and their ability to regulate gene expression is impaired. Two strategies have emerged to alleviate this dilemma: (i) the introduction of chemical modifications to the backbone of oligonucleotides can extend their half-life and (ii) in vivo processing to short hairpin-like RNA transcripts (shRNA) that express siRNA ,37,38. TMessenger RNAs (mRNAs) play a crucial role in the central tenets of molecular biology developed by Francis Crick. mRNA is defined as the medium through which information flows from DNA into cells ,43. As tTherefore, to overcome a series of obstacles such as nuclease degradation, targeted tumor tissue, endocytosis, and endosomal escape, it is particularly important to realize in vitro and in vivo delivery of gene vector-mediated therapeutic nucleic acid to maximize the therapeutic effect of cancer. The design and development of efficient gene vectors is the focus of research in the field of gene therapy. The reported RNA delivery carrier will be summarized and introduced in the following sections.Polymers have been identified as a potential delivery method for siRNA therapy ,53. ManyDespite the high transfection efficiency of PEI, its cytotoxicity and non-biodegradability are unavoidable. In this case, improving the biocompatibility of PEI is a requisite. Wen and colleagues constructed a pH-responsive carrier polymer for delivery of PD-L1-siRNA using biocompatible hyaluronic acid (HA) combined with polyethyleneimine (PEI) and matrix metalloproteinase-2 (MMP-2). It was confirmed that the synthesized polymer had lower cytotoxicity and faster cell uptake capacity, improved the penetration of PD-L1-siRNA into lung tumor spheres, and effectively down-regulated the expression of PD-L1 in H1975 cells . Luo et To alter biodistribution and further improve the stability, efficacy, and biocompatibility of NPs, PEI-based systems have been modified with liposomes to produce lipo-polymers that combine the properties of both systems . The res6H11O4N [Chitosan is one of the main cationic polymers and the second most abundant polysaccharide in nature .Chitosan6H11O4N . Its sup6H11O4N ,75. In a6H11O4N . In 19956H11O4N . In 19986H11O4N . In 20066H11O4N . So far,6H11O4N ,81.However, the poor solubility of chitosan under neutral and alkaline conditions limits its applicability and application range. It is an effective strategy to improve the solubility of chitosan by chemical modification of its quaternized derivative N, N, N-trimethyl chitosan (TMC) to improve the physicochemical properties . Masjedi+ breast cancer. The final results showed that ML39 SCFV and Tretazicar’s systemic IVT mRNA-loaded EVs at lower EV doses than previously caused almost complete growth arrest of human HER2+ breast cancer xenografts in non-thyroid mice [Extracellular vesicles (EVs) are small membrane vesicles released by cells into the extracellular matrix, which have the characteristics of participating in the exchange of information between cells. Compared with commonly used lipid nanoparticles (LNPs) delivery systems, exosomes have their own unique advantages: (i) It is stable and showing a difficult clearance by macrophages, thus providing better drug protection during drug delivery ; (ii) reoid mice . Kaban doid mice . Exosomeoid mice .Exosomes have more prominent features than LNPs including nucleic acid delivery, protection of therapeutic substances from degradation, elimination by the host immune system, and etc. In addition, the characteristic of exosomes derived from their parental cells give them the potential for targeted delivery and enhance their ability to penetrate the tumor vascular barrier and bioaccumulate at tumor sites, greatly improving their therapeutic bottleneck. More importantly, exosomes have shown amazing feasibility as drug delivery vehicles in many preclinical studies and several clinical trials. From this point of view, the future of exosome-based delivery systems will break new ground in cancer treatment.Tetrahedral framework nucleic acid (tFNA) has excellent mechanical, chemical, and biological properties, which make it a research hotspot in the field of DNA nanomaterials in recent years. Based on these advantages, tFNA has been designed and applied to the oncology therapeutic field ,95. DNA DNA nanostructures have many outstanding features, including excellent biocompatibility and degradability, controllable self-assembly that endow them with structural precision and diversity, and the easy modification and introduction of functional groups, which have laid great potential for RNA delivery applications. But there are still huge problems for clinical application: such as (i) poor stability, (ii) immunogenicity, (iii) assembly efficiency and purification restricting its large-scale production.Compared with organic carriers, inorganic nanoparticles have other excellent physical and chemical properties, including: (i) stable size and shape, which can be precisely controlled in the synthesis process ; (ii) itAmong the many carriers, calcium phosphate (CaP) nanoparticles have received special attention due to their superior physical and chemical properties and have brought broad prospects for siRNA loading. Kara et al. used arginine (Arg) modification to synthesize CaP nanoparticles with different chemical and morphological characteristics as vectors for survivin and Cyclin B1 specific siRNA A,B. The In 2018, the FDA and EMA approved the first lipid nanoparticle (LNP) based RNA interference (RNAi) drug of ONPATTRO TM (Patisiran), confirming that LNP is a valuable means of treating a variety of nucleic acid-related diseases. The use of LNP can overcome the limitations of naked siRNA in clinical applications, including their rapid degradation in biological fluids, limited biological distribution, and cellular absorption. However, factors of LNP lipid component diffusion and protein adsorption on the LNP surface caused the low potency. To alleviate these deficiencies, the strategy of using sphingomyelin (ESM) to replace the auxiliary lipid in cholesterol was proposed by Sato et al. The prepared LNP has a diameter of about 22 nm, and the gene silencing activity is greatly improved . An iBL0Proteins or peptides are produced from various combinations of 20 naturally occurring amino acids with different 3-D conformations, charge, polarity, hydrophobicity, and hydrophilicity. These unique sequences can demonstrate a variety of properties, including siRNA binding, membrane penetration, endosomal destruction, targeting, etc. All these features are very helpful for RNA delivery. Moreover, the natural biological properties enable the direct screening of large proteomic libraries for a variety of interesting functional peptides .Cell-penetrating peptides (CPPS) are amphiphilic oligomers with positively charged residues (arginine or lysine) composed of fewer than 30 amino acids that enable them to penetrate cells and drive a variety of organisms’ active substances . In most cases, the natural helical folding properties of some CPPS when interacting with biological membranes assist improve the intracellular delivery of peptides. Simon’s team have carefully designed and synthesized alpha-helical peptide sequences consisting of short helical peptides with only eight amino acids and two cationic charges, and can efficiently encapsulate siRNA and induce gene silencing in the nanomolar range  Figure A,B. Xu eAs a more precise and effective emerging treatment strategy, gene therapy has gradually emerged in the field of clinical application in the past two decades. Several RNA-based products have been successfully used in the clinic, and many more are in various stages of development . The appRNAi-based nucleic acid drugs have been used in many clinical trials to inhibit tumorigenic genes and inhibit cancer development. Most clinical drugs use liposome nanoparticles as siRNA delivery vectors. For example, nucleic acid drugs such as Atu027, TKM-080301, and EPHARNA are all delivered by liposome nanoparticles. Local Drug EluteR is a miniature biodegradable polymer matrix that is well tolerated and safe in combination with chemotherapy encapsulated in the LODER extended delivery system (targeting KRASG12D), showing significant efficacy in the treatment of pancreatic cancer . There iIn addition to RNAi-based therapies, mRNA anticancer vaccines have made significant clinical advances. mRNA drugs currently under clinical investigation for cancer treatment have vaccine-like properties, promoting the production of tumor-specific and tumor-associated antigens on the surface of cancer cells, further activating the host immune system. A particular advantage of mRNA cancer vaccines is that they can be customized to the patient, thereby improving the efficacy of the vaccine. The mRNA-4157 vaccine, an mRNA vaccine developed by Moderna, uses a novel gene-based technique to identify tumor-specific changes to DNA by comparing the normal cell DNA sequence of a patient with the normal DNA sequence of the tumor. By combining with pembrolizumab, the researchers hypothesized that the vaccine could trigger the immune system to respond more sensitively to PD-1 inhibitors and reduce the risk of cancer recurrence.Although the first generation of RNA drugs have been approved for clinical use and more are in the development, the long-term effects of RNA therapy are still unclear, and some clinical trials have even been stopped because of unclear mechanisms. The safety risks of RNA therapy are the important issues . AlthougIn this review, we summarized RNA therapeutic-based nucleic acid delivery strategies for cancer therapy. After decades of development of RNA therapeutics, the targeting and delivery efficiency have improved significantly. RNA therapies alter the expression of target genes by regulating internal cellular mechanisms, which offers a wide range of potential applications and provides a high degree of flexibility to manipulate previously “unusable” targets. Therefore, siRNA can modulate the expression of various genes which has raised prospect for the new therapeutic strategies. Nowadays, RNA-based cancer treatment strategies are receiving increasing attention, mainly due to the flexibility of targeting previously unmanageable disease genes and modulating a wide range of targets. As summarized in the manuscript, we highlight effective strategies for RNA delivery by different types of carriers , and describe the advantages and disadvantages of each. Meanwhile, the clinical translation progress, challenges, and application prospects of RNA in cancer therapy were reviewed. Although various RNA drugs have been developed, only a few have completed phase 1 clinical trials, clinical application will take more time due to the challenges of nucleic acid stability and unclear delivery strategies and mechanisms.An ideal nanocarrier for RNA delivery should meet the conditions of high safety, biocompatibility, biodegradability, and a high gene transfection rate. Advances in nanotechnology will help address these issues, and nanomaterials hold promise as a potential delivery vector to address the challenges and drawbacks of RNA therapeutics. With the application and development of lipid-based nanoparticles in the clinical scenario of COVID-19 mRNA vaccines, nanotechnology has shown great effectiveness and availability in RNA delivery strategies. According to the previous summary, an effective RNA nanodelivery vector formulation should have these characteristics: (i) low toxicity and serum-stable RNA loading methods, (ii) accurate targeting of target genes, and (iii) a high degree of uptake by target cells and endosomal escape. Through simple structural modification and packaging of the basic nanomaterial carrier, such as the use of specific cell membrane vesicles to make it targeted and biosafe, the carrier can achieve multifunctional requirements containing the above characteristics. The optimization of nanomaterials will provide a safe and effective method of RNA delivery, which will make RNA have a broader application prospect in cancer treatment.Although there are still some unsolved problems, it is believed that with the deepening of research, the continuous improvement of material understanding, and the increasing exploitation of new materials, safe and efficient RNA delivery solutions will be applied to the clinic in the near future. It is also hoped that RNA therapy will be more widely adopted as a new class of therapies in combination with other therapies or drugs. At present, the mechanism of RNA therapy is also constantly revealed and explored, which is expected to shine in cancer treatment.


Known ribozymes in contemporary biology perform a limited range of chemical catalysis, but in vitro selection has generated species that catalyze a broader range of chemistry; yet, there have been few structural and mechanistic studies of selected ribozymes. A ribozyme has recently been selected that can catalyze a site-specific methyl transfer reaction. We have solved the crystal structure of this ribozyme at a resolution of 2.3 Å, showing how the RNA folds to generate a very specific binding site for the methyl donor substrate. The structure immediately suggests a catalytic mechanism involving a combination of proximity and orientation and nucleobase-mediated general acid catalysis. The mechanism is supported by the pH dependence of the rate of catalysis. A selected methyltransferase ribozyme can thus use a relatively sophisticated catalytic mechanism, broadening the range of known RNA-catalyzed chemistry. O6-methylguanine to an adenine N1 in the target RNA and propose a catalytic mechanism based upon proximity, orientation and general acid catalysis.The authors present the crystal structure of the MTR1 ribozyme that transfers the methyl group from  RNA catalysts (ribozymes)2 exist in contemporary biology but catalyze a relatively narrow range of reactions that are limited to phosphoryl transfer reactions and peptide bond formation. Clearly an RNA world would have required ribozymes that facilitate more challenging reactions, such as C–C and C–N bond formation. Yet, the extent of RNA catalysis will be limited by the relatively narrow range of chemical space of RNA compared to proteins restricted to four chemically similar heterocyclic nucleobases, ribose together with its 2-hydroxyl group and a charged phosphodiester linkage with attendant hydrated cations. However, in principle, the chemical functionality of RNA could be greatly expanded by binding small-molecule co-reactants and coenzymes5. RNA exhibits highly selective binding of ligands; this is exemplified by riboswitches7, a number of which bind powerful coenzymes, and we have discussed a possible evolutionary relationship between riboswitches and ribozymes5.The metabolism of a putative RNA world9 and aldol condensations10, alkylation reactions11 and Michael addition12. These species offer proof of principle that RNA can expand its range of chemistry, but there has been very little mechanistic investigation of the way in which this is achieved, and the structures of relatively few of these ribozymes have been determined14. The natural ribozymes that catalyze phosphoryl transfer reactions in general do so by using either metal ion catalysis  or general acid–base catalysis , variously using nucleobases, 2′-hydroxyl groups and metal ion-bound water molecules21. At the present time, there is very little evidence that selected ribozymes performing a wider range of catalysis use any of these strategies.The potential for RNA catalysis of a much broader range of chemistry has been demonstrated by in vitro evolution of catalytic RNA. By these means, ribozymes have been selected that catalyze a range of bond-forming reactions, including Diels–Alder24 and is thought to be an ancient process. Nucleobases and 2-hydroxyl groups are methylated, generally accepting a methyl group from S-adenosyl methionine (SAM), catalyzed by a broad range of methyltransferase enzymes. There is a wide range of SAM-binding riboswitches, possibly indicating an ancient connection between RNA and methylation25. It has been noted that the manner of ligand binding in the SAM-I riboswitch suggests that it could be converted into a methyltransferase ribozyme relatively easily25, and thus the riboswitch may have evolved from an ancient ribozyme. Recently, Flemmich et al.26 have shown that the prequeuosine1 (preQ1) riboswitch can exhibit some methyltransferase activity, whereby the methyl group from O6-methyl preQ1 becomes transferred to cytosine N3 with an observed rate on the order of 0.001 min−1. Murchie and colleagues27 have also very recently selected an RNA that transfers the methyl group of SAM to N7 of a specific guanine of the RNA and determined the crystal structure. Höbartner and colleagues28 used in vitro RNA selection to generate a new RNA species called MTR1 that can catalyze the transfer of the methyl group from O6-methylguanine to N1 of a specific adenine in the RNA , with hairpin loops at the ends of two helices  under ID number 7V9E, and crystallographic statistics are tabulated in Supplementary Table We screened a large number of in vitro-transcribed RNA constructs for crystallization in the presence of ces Fig. . In this29, this is a HS1HS5HS8 junction. Reactant binding and the active site of the ribozyme are contained within the core of the junction. The secondary structure of MTR1 is shown in graphical form in Extended Data Fig. MTR1 has the global structure of a three-way helical junction Fig. . The thrcis-Watson–Crick base pair, but progressing toward the junction, it is followed by C15•A34, a cis-Watson–Crick base pair connected by a single hydrogen bond from C15 N4 to A34 N1   is directed out of the helix and forms a Helical junctions have two chemically distinct sides with major and minor groove characteristics. Views of each side are presented in Fig. cis-Watson–Crick base pair and the C11•G41 cis-Watson–Crick base pair, respectively. The third plane forms where exogenous guanine makes hydrogen bonds to nucleobases C10, U45 and A63  is coplanar with the nucleobases of C10, U45 and A63 , 2′-deoxyguanosine32, ppGpp33 and cyclic di-GMP34 riboswitches. There is more variety in the nature of interactions with the sugar edge, and the Hoogsteen edge is rather less frequently contacted.The structures of 12 distinct guanine-binding sites have been determined in numerous riboswitches, examples of which are shown in Extended Data Fig. N1-methyladenine gives an excellent fit to the electron density  had little effect on activity, neither of which make interactions observed in the crystal. They also showed that the exocyclic 2-NH2 group on the exogenous O6-methylguanine was required for activity. This makes hydrogen bonds to both C10 and U45 in the crystal structure.Scheitl et al.28 using a two-piece ribozyme–substrate complex with a separate 13-nt substrate strand corresponding to the J31 strand . This permits the formation of three hydrogen bonds between the cytosine nucleobase and O6-methylguanine, including that from C10 N3 to O6-methylguanine N1. Methyl transfer (step 3) involves a coordinated movement of the methyl group to A63 N1 and the proton flipping from C10 N3 to guanine N1 within the hydrogen bond, restoring neutral unprotonated C10 and leaving N1-methyladenine with a positive charge at position 63. In principle, the ribozyme could then be restored by release of guanine, although this would require an exchange of the J31 strand, as the N1-methyladenine cannot react further. In addition, there is no evidence that product release occurs from the reacted ribozyme, and this was not required by the selection strategy. To summarize, we propose that the reaction is accelerated by the ribozyme using two catalytic strategies, propinquity/orientation and general acid catalysis. Although we have drawn the reaction in three separate steps for clarity, it is likely that these steps could occur in a coordinated manner, and in particular the protonation of C10 and the binding of O6-methylguanine might be essentially simultaneous. The formation of the base pair would be expected to raise the pKa of the cytosine from its normal value of 4.2, as discussed for structured RNA by Moody et al.35. The proposed role of the nucleobase of C10 in both substrate binding and general acid catalysis is consistent with our activity data. MTR1 U45C exhibits a low level of activity, but activity in the C10U variant is completely undetectable. This suggests that C10 is doing more than just binding O6-methylguanine, consistent with an additional role in general acid catalysis.Methyl transfer from O6 requires restoration of a single bond between N1 and C6 and thus protonation of N1 in the resulting guanine. This indicates a requirement for a general acid to protonate N1, and the only candidate for this is C10. We therefore propose a possible mechanism for the reaction based on the structure observed in the crystal Fig. . We suggO6-methylguanine to the Watson–Crick face of C10 would be stronger if the cytosine nucleobase were protonated at N3 and that the transfer of that proton to N1 of O6-methylguanine could be an integral part of the catalytic mechanism. This leads to the prediction that the rate of methyl transfer should increase at lower pH, reflecting the pKa of C10. We therefore sought to test this prediction by measuring the observed rate of reaction as a function of pH. We devised a new fluorescent assay using the fluorophore Atto488 attached to guanine at O6 via an O-(4-(aminomethyl)-benzyl) linkage  the proposed catalytic mechanism involving proton transfer from N3 of C10, as suggested by the observed structure.The dependence of reaction rate on pH is plotted in Fig. 38, and the MTR1 junction has followed this general behavior. The structure has relatively few long-range contacts, and the extended P2 helix contains consecutive unpaired nucleotides, forming a bubble. These features are not typical of natural RNA species that are the product of extended evolution in the wild, indicative of a somewhat naive structure and perhaps showing the limits of in vitro selection. The critical region of the RNA lies in the core of the junction, where four planes of nucleobase interaction lie between the J12 and J23 strands, creating the environment that binds the exogenous O6-methylguanine in such a way that it acts site specifically as a methyl donor. The coaxial stacking of the P2 and P3 helices and the divergence of the P1 helix makes a pronounced kink in the J31 strand at the position of A63, ‘presenting’ this nucleobase for attack. The local structure immediately suggests a catalytic mechanism for the ribozyme that we discuss below.It is interesting to examine the structure of an RNA that has been selected on the basis of catalytic activity. The overall architecture is that of a three-way helical junction, which was to be expected because the P1 and P3 helices were essentially predetermined by the nature of the seed sequence used in selection. The P2 helix formed during the selection process, with Watson–Crick base pairing at the junction distal end. The junction proximal end remains quasihelical, continuing into the core of the junction where the active center is located. Natural three-way helical junctions tend to adopt a structure in which two arms are coaxial and the third is directed away from the axisGuanine can donate up to four protons and has three potential hydrogen bond acceptors. In MTR1, guanine forms seven hydrogen bonds to the RNA and is bound predominantly on its Watson–Crick edge by C10 and its sugar edge by U45. In the MTR1 ribozyme, the nucleobases of C10 and U45 hold the exogenous guanine tightly, directing the O6 on the Hoogsteen edge toward the target A63. This is an important part of the ribozyme action, as we discuss further in the following section.O6-methylguanine. This is strongly suggested by the structure of the complex in the crystal and supported by the pH dependence of the reaction rate. To our knowledge, there is no other example to date of a selected ribozyme using general acid catalysis, for example, in the three other structures known for selected ribozymes27.Extrapolating the structure of the complex of the ribozyme with exogenous guanine to the expected prereaction complex shows that a methyl group attached to O6 of guanine would be almost perfectly aligned for nucleophilic attack by N1 of A63. Part of the catalytic rate enhancement therefore arises from effective local concentration and orientation. A second element in the catalysis likely comes from general acid catalysis by the cytosine nucleobase of C10 that transfers a proton attached to its N3 to N1 of the exogenous sp2 orbital) to overlap the lowest unoccupied molecular orbital of the carbon that is the σ* orbital of the C–O bond. In the structure of the product observed in the crystal, the distance between the A63 N1 and G O6 atoms is 4.1 Å, so that the distance from O6-methylguanine methyl carbon to A63 N1 will be ~ 3 Å. Initiation of the reaction requires a closer approach for effective sp2–σ* orbital overlap. The exogenous guanine (and therefore the O6-methylguanine) appears be held in place so rigidly that it seems more probable that the nucleobase of A63 would be required to move closer while remaining in plane. A63 lies at the junction between P1 and P3, and A63 is consequently unstacked on its lower face. Its O2′ donates a hydrogen bond to A37 N3, but deletion of this hydroxyl group led to no loss of activity28. On the upper side, A63 is not directly stacked onto G62 but rather on A40, which is part of the triple base interaction. It therefore seems that the environment of A63 may be more flexible than that of the O6-methylguanine. As a working hypothesis, we propose that the methyl transfer reaction might be coupled to a local conformational change that allows the N1 of A63 to move closer, perhaps by a combination of rotation and translation of the nucleobase and potentially involving a reorientation of the P1 helix at the junction.The intramolecular nature of the reaction raises a question about how the nucleophilic attack is initiated. This requires the highest occupied molecular orbital of the nitrogen nucleophile  was obtained from Macklin. Atto488 NHS-ester was obtained from Atto-Tec, and O-(4-(aminomethyl)-benzyl)guanine was obtained from Biosynth Carbosynth.A 69-nt MTR1 RNA for crystallization  GCGGGCUGACCGACCCCCCGAGUUCGCUCGGGGACAACUAGACAUACAGUAUGAAAAUACUGAGCCCGC28 was transcribed in vitro using T7 RNA polymerase at 37 °C. The RNA was purified by electrophoresis in a polyacrylamide gel in the presence of 7 M urea. The full-length RNA was visualized by UV shadowing, excised and electroeluted  in 0.5× TBE buffer for 12 h at 200 V at 4 °C. The RNA was precipitated with isopropanol, washed with 70% ethanol and dissolved in water.that was based on the CA13 selected ribozymetert-butyldimethyl-silyl (t-BDMS) phosphoramidite chemistry39, as described in Wilson et al.40. This was implemented on an Applied Biosystems 394DNA/RNA synthesizer using UltraMILD ribonucleotide phosphoramidites with 2′O-t-BDMS O2′-protection42 (Link Technologies).Oligonucleotides were synthesized using N1-methyladenine (Glen Research), were deprotected using anhydrous 2 M ammonia in methanol (Sigma-Aldrich) for 24 h. Unmodified RNA was deprotected in 1:1 ammonia:methylamine at 60 °C for 20 min. Dried oligoribonucleotides were redissolved in 115 μl of anhydrous DMSO, 60 µl of triethylamine and 75 μl of triethylamine trihydrofluoride to take off t-BDMS groups and shaken in the dark at 65 °C for 2.5 h before butanol precipitation.Ribo-oligoribonucleotides, including 1A)GCCCGC (m1A denotes the position of N1-methyladenine).The following RNA sequences were used in the kinetic assay of methyltransferase activity: MTR1, GCGGGCUGACCGACCCCCCGAGUUCGCUCGGGGACAACUAGACAUACAGUAU; MTR1 C10U, GCGGGCUGAUCGACCCCCCGAGUUCGCUCGGGGACAACUAGACAUACAGUAU; MTR1 U45C, GCGGGCUGACCGACCCCCCGAGUUCGCUCGGGGACAACUAGACACACAGUAU; MTR substrate, AUACUGAGCCCGC; MTR product, AUACUG, 0.08 M KCl, 0.02 M BaCl2, 30% (vol/vol) (+/–)-2-methyl-2,4-pentanediol and 0.012 M spermine tetrahydrochloride, and crystals were grown at 18 °C by hanging drop vapor diffusion. Crystals appeared after 3 d and were frozen by mounting in nylon loops and rapid immersion into liquid nitrogen.A solution of 0.5 mM MTR1 RNA (69 nt) in 5 mM HEPES (pH 7.5) and 100 mM KCl was slowly cooled from 95 °C to 20 °C followed by addition of MgCl43. The resolution cutoff for the data was determined from the the Pearson correlation coefficient CC1/2 and the density map44. Crystals grew in space group P2221 with unit cell dimensions of a = 50.38 Å, b = 52.5 Å and c = 99.9 Å. Eleven barium(II) sites were identified and exploited in single-wavelength anomalous dispersion phasing using the AutoSol function of PHENIX45. Density modification using RESOLVE led to an interpretable electron density map . Models were adjusted manually in Coot 0.9.6 (ref. 46) and underwent several rounds of adjustment and optimization using Coot46, phenix.refine and PDB_REDO47. The geometry of the model and its fit to electron density maps were monitored with MOLPROBITY48 and the validation tools in Coot. Atomic coordinates and structure factor amplitudes have been deposited at the PDB under accession code 7V9E. Crystallographic statistics are presented in Supplementary Table The structure was determined in PHENIX 1.19 ) by rapid cooling from 80 °C, to which was added 2 µl of 400 mM MgCl2. This was equilibrated at 37 °C, and the reaction was initiated by addition of an equal volume of 100 µM O6-methylguanine in 120 mM KCl, 5 mM NaCl and 50 mM HEPES, pH 7.5. Reactions were incubated under mineral oil to prevent evaporation, and 2-µl aliquots were taken at time intervals and added to 13 µl of 95% formamide and 50 mM EDTA stop mix. Samples (5 µl) were electrophoresed ~35 cm in a 20% denaturing polyacrylamide gel, band intensities were quantified by phosphorimaging, and reaction progress was fitted to the single exponential functionA is the amplitude of the reaction, k is the rate of reaction, and t is the elapsed time. The rates and uncertainties given in the text are the mean and s.d. of three or more independent experiments.Single-turnover assays were performed following the method of Sheitl et al.λmax = 500 nm; εmax = 9.0 × 104 M−1 cm−1) attached to guanine at O6 via an O-(4-(aminomethyl)-benzyl) linkage (Atto488-BG). The reaction transfers the Atto488 to A63 in the substrate strand, generating a fluorescent product band that can be quantified by fluorimaging using excitation by a 473-nm laser and a fluorescein isothiocyanate (FITC) filter to collect emission -benzyl)guanine and 0.25 µmole of Atto488 NHS-ester were dissolved in 20 µl of DMSO, and 0.5 µl of triethylamine was added. Following overnight incubation at room temperature, 480 µl of 0.1 M TEAA in water was added, and the reactants were separated by HPLC on a C18 reversed-phase column using an acetonitrile gradient. The reaction was quantitative as judged by the absorbance of the fluorophore. Fractions containing Atto488-BG were dried, dissolved to 200 µM in water and stored at −20 °C.To synthesize Atto488-BG, 1.5 µmole of 2 was added; the reaction was initiated by addition of an equal volume of 100 µM Atto488-BG in 100 mM KCl and 50 mM buffer. The buffers used were MES (pH 5.0 to 6.5), MOPS (pH 7.0 and pH 7.5) and TAPS (pH 8.0 and pH 8.5). The rates and uncertainties for each pH are the mean and s.d. of at least four independent experiments.Single-turnover assays were performed using longer RNA strands such that helices P1 and P3 each had 12 base pairs. Reactions were performed as described above, except that 20 pmol of ribozyme strand was annealed to 20 pmol of target RNA in 8 μl of 125 mM KCl and 10 µM Flu-DNA by rapid cooling from 80 °C, to which 2 µl of 400 mM MgClkint is the intrinsic rate of the reaction, A is the pKa of a moiety where protonation is required for activity , and B is the pKa of a moiety where the deprotonated form is active. While kinetic ambiguity prevents the assignment of a particular function to each ionization constant, in this case, we have assumed the higher ionization constant results from C10 acting as a general acid because its pKa shift is readily explicable.The observed pH dependence of the reaction was fitted to a two-ionization modelFurther information on research design is available in the Any methods, additional references, Nature Research reporting summaries, source data, extended data, supplementary information, acknowledgements, peer review information; details of author contributions and competing interests; and statements of data and code availability are available at 10.1038/s41589-022-00982-z.Supplementary InformationSupplementary Fig. 1 and Table 1.Reporting Summary