Nothocrax urumutum) visiting mineral licks for the first time. We found seasonal trends in visitation for the black agouti (Dasyprocta fuliginosa), red howler monkey (Alouatta seniculus), blue‐throated piping guan (Pipile cumanensis), red brocket deer (Mazama americana), collared peccary (Pecari tajacu), and tapir (Tapirus terrestris). Lunar trends in visitation occurred for the paca (Cuniculus paca), Brazilian porcupine (Coendou prehensilis), and red brocket deer. The probability of recording groups  at mineral licks was seasonal and related to lunar brightness for tapir. Overall, our results provide important context for how elusive species of birds and mammals interact with these key ecological resources on a landscape scale. The ecological importance of mineral licks for these species can provide context to seasonal changes in species occupancy and movement.Mineral licks are key ecological resources for many species of birds and mammals in Amazonia, providing essential dietary nutrients and clays, yet little is known about which species visit and their behaviors at the mineral licks. Studying visitation and behavior at mineral licks can provide insight into the lives of otherwise secretive and elusive species. We assessed which species visited mineral licks, when they visited, and whether visits and the probability of recording groups at mineral licks were seasonal or related to the lunar cycle. We camera trapped at 52 mineral licks in the northeastern Peruvian Amazon and detected 20 mammal and 13 bird species over 6,255 camera nights. Generalized linear models assessed visitation patterns and records of groups in association with seasonality and the lunar cycle. We report nocturnal curassows ( Many species of animals in the Amazon visit mineral licks, consuming soil to supplement their diet. Using camera trap data, we show that visits to mineral licks are seasonal for many species. These trends in visitation are likely due to species‐specific factors such as reproduction, predator avoidance, seasonal diet shifts, and resource use. They are visited by a diverse array of species, including large‐bodied mammals such as the Brazilian tapir (Tapirus terrestris) and red brocket deer (Mazama americana), rodents such as the paca (Cuniculus paca) and black agouti (Dasyprocta fuliginosa), and arboreal mammals such as the red howler monkey (Alouatta seniculus) and Brazilian porcupine (Coendou prehensilis)  and Spix's guan (Penelope jacquacu)   , which can cause species to be especially vigilant when they are at mineral licks , a 391,039‐hectare protected area in Loreto, Peru  in the Sucusari River Basin at a sample of 52 mineral licks that were identified with the assistance of Maijuna hunters. Starting in August 2018, we visited all mineral licks, obtained GPS coordinates, and placed camera traps in a series of four deployments, each lasting at least 60 days to achieve even coverage of the whole basin . We set cameras to record three rapid‐fire images at each motion trigger with a delay of 2 min between each set of images to avoid expending the camera's batteries. Cameras were set at a minimum of 50 cm from the ground, facing the active face and entrance to the mineral lick, following Tobler et al. . We deteWe identified all medium‐ and large‐sized mammal and bird species (weight > 1 kg) in camera trap images Blake, , removed2.3To assess visitation at mineral licks, we assessed the visit frequency and group size of medium‐ and large‐bodied birds and mammals at mineral licks. We calculated the mean visit frequency for terrestrial bird and mammal species which were recorded at least ten times during the study period. Mean visit frequency was calculated as the number of independent visitation events per night of camera trapping for those mineral licks where the species visited at least one time. Not all mineral licks were considered in visit frequency calculations under the assumption that not all mineral licks are active for each species at all times, due to changes in occupancy, diet shifts, or reproductive periods.densityPlot function in the overlap package . For example, if cameras were active at 10 mineral licks on 10 August 2018, then that date was recorded in 10 different samples, each at a different lick. For each day, a 1 was recorded if the species visited that mineral lick, and a 0 recorded if it did not visit. Visitation was used as the binary response variable, and the covariates included were the month of the visit, the size of the lick in m2, the lick type (face present or not present), elevation in m, slope in degrees, distance the closest river or stream in m, distance from the closest hunting camp in m  and red howler monkey (Alouatta seniculus), was seasonal or related to the lunar cycle. The collared peccary and red howler monkey were analyzed separately because they commonly travel in groups larger than two individuals. Each visit to a mineral lick was considered a sample  and each visit to a mineral lick was a sample. Full models were constructed and tested for overdispersion. We proceeded with the model selection process as described above.glmer function in the lme4 package  species as the mean number of species visiting a mineral lick over the duration of the study period.Over all rotations, the average number of camera traps placed in each mineral lick was 1.2 cameras, with a range of 1–3 cameras per lick. Camera traps captured a total of 319,926 photographs over 6,255 camera nights during the study period. The number of camera nights at each mineral lick was highly variable, with a range of 10 days to 265 days, since many cameras malfunctioned, and several mineral licks flooded or experienced some disturbance . Mineral licks that had fewer than 55 camera nights of data recorded at the end of the study period were excluded from the analyses. After all empty photographs were removed, 143,497 photographs remained from 52 mineral licks. These photographs collectively described 5,210 independent visitation events by mammals and 1,264 visitation events from birds (Table 3.2Pipile cumanensis) was the most common large‐bodied bird visitor to mineral licks, with a mean visit frequency of 20.35 (95% CI 0–41.79) visits per 100 camera nights but only visited four of the sampled mineral licks (Table Psophia crepitans), nocturnal curassow (Nothocrax urumutum), and Spix's guan (Penelope jacquacu) (Table The blue‐throated piping guan (Mazama gouazoubira), and Linnaeus's two‐toed sloth (Choloepus didactylus) were also frequent visitors, with mean visit frequencies greater than 12 visits per 100 camera nights  visits per 100 camera nights, followed by the paca and agouti with mean visit frequencies of 60.17 (95% CI 8.67–111.68) and 56.00 (95% CI 0–130.47)  and 60 times (9.54% of visits), respectively. Only one individual was recorded in most visits by red brocket deer, tapir, and paca, but pairs of red brocket deer were recorded 59 times (3.31% of visits), pairs of tapirs 17 times (4.40% of visits), and pairs of paca 56 times (6.01% of visits).3.5Six of the eight species analyzed exhibited seasonal mineral lick visitation and visitation was related to the brightness of the moon for three species. Optimal generalized linear mixed‐effects models of mineral lick visitation showed that visitation was related to both the month and lunar phase for the red brocket deer Table . Red broOptimal models for the tapir, black agouti, red howler monkey, collared peccary, and blue‐throated piping guan showed that visitation at mineral licks was related to the month of the year Table . The tapSeveral environmental covariates appeared in optimal models of visitation, but the combination of relevant covariates varied among species Table  includin3.6Only one species, the tapir, exhibited temporal variability in the probability of recording groups at mineral licks. The optimal model for the tapir included both month of the year and brightness of the moon as covariates Table . Groups As above, several environmental covariates appeared in optimal models of grouping, but the combination of relevant covariates varied among species Table  includin4Our results describe new patterns associated with visitations of species at mineral licks and associations between visitations, seasons, or lunar phase for a majority of the species that frequently visit mineral licks. In addition, we describe mineral lick visitation for the nocturnal curassow, which has not previously been reported to frequent mineral licks but was recorded 24 times during our study. Our study builds upon results reported by Blake et al. , who inv4.1None of the species recorded visited all mineral licks in the study. For example, the red brocket deer, which was recorded during over 1,700 independent visitation events, visited 88.46% of mineral licks in the study. The red howler monkey visited only 30.77% of mineral licks. Thus, not all mineral licks may be active for all species at all times, potentially because of the mineral composition of the lick and/or the geographic location. If a species is only active at a few mineral licks, and access to those licks is limited by seasonal changes, seasonal trends would appear in model results.Overall, 50% of the species for which activity patterns were analyzed exhibited diurnal activity, 17% exhibited crepuscular activity, and 33% exhibited nocturnal activity patterns. For the diurnal species, most activity peaked at 12.00 hr, but some variation existed between species. For the nocturnal species, there was lots of variation in activity, but most activity occurred between 20.00 hr and 03.00 hr. Our activity data for mammals including the tapir, paca, collared peccary, red howler monkeys, and red brocket deer line up with other studies from the Amazon , and jaguars are all present in the MKRCA and were recorded visiting mineral licks periodically during this study  was in season. Similarly, Sekulic (Dasyprocta punctata) varied seasonally as well, in response to availability of fruit resources. In addition, the red brocket deer avoids flooded forest during the wet season, and those located in floodplain forest shift their diet to include woodier foods during that time due to resource scarcity . The diet of the tapir is also made up of fruit and foliage  , with visitation dropping in the rainy season. Here, we found a similar result for the blue‐throated piping guan, where model results showed that visitation to mineral licks rapidly increased in the months leading up to May. The breeding season for the blue‐throated piping guan in the wild is thought to be from May to November ; formal analysis (lead); funding acquisition (lead); investigation (lead); methodology ; visualization (lead); writing – original draft (lead). Mark Bowler: Conceptualization ; methodology ; resources ; supervision ; writing – review & editing . Michael P. Gilmore: Conceptualization ; funding acquisition ; investigation ; methodology ; resources ; writing – review & editing . David Luther: Conceptualization ; formal analysis ; supervision ; writing – original draft ; writing – review & editing .Tables S1‐S2Click here for additional data file.


The use of ultra-thermostable RNA nanoparticles to deliver chemical prodrugs addresses issues with RNA unfolding and nanoparticle dissociation after high-density drug loading. This finding provides a stable nano-platform for chemo-drug delivery as well as an efficient method to solubilize hydrophobic drugs.Paclitaxel is widely used in cancer treatments, but poor water-solubility and toxicity raise serious concerns. Here we report an RNA four-way junction nanoparticle with ultra-thermodynamic stability to solubilize and load paclitaxel for targeted cancer therapy. Each RNA nanoparticle covalently loads twenty-four paclitaxel molecules as a prodrug. The RNA-paclitaxel complex is structurally rigid and stable, demonstrated by the sub-nanometer resolution imaging of cryo-EM. Using RNA nanoparticles as carriers increases the water-solubility of paclitaxel by 32,000-fold. Intravenous injections of RNA-paclitaxel nanoparticles with specific cancer-targeting ligand dramatically inhibit breast cancer growth, with nearly undetectable toxicity and immune responses in mice. No fatalities are observed at a paclitaxel dose equal to the reported LD Although paclitaxel is widely used as a chemotherapy, it suffers from poor solubility and toxicity issues. Here, the authors develop thermostable RNA nanoparticles and report the RNA-paclitaxel complex to display improved stability, drug loading capacity and solubility for improved targeted cancer therapy and reduced immune responses. Effective therapeutic dosages of PTX cause potential organ damage, myelosuppression, immunotoxicity, hypersensitivity, and neurotoxicity3. These undesired effects have challenged cancer chemotherapy. Due to low water solubility and permeability, PTX was categorized as the lowest bioavailability Class IV drug according to the Biopharmaceutical Classification System (BCS). The first formulation of PTX for intravenous (i.v.) administration was a blend of Cremophor EL (castor oil derivative) and dehydrated ethanol at 1:1 (v/v) as a solvent4. However, subsequent studies revealed that castor oil and its derivatives are toxic and caused severe side effects such as non-linear plasma pharmacokinetics due to entrapment and delayed clearance in the liver6.Historically, chemotherapy plays a significant role in cancer treatment. Many chemotherapeutic drugs, including paclitaxel (PTX), have been used clinically in cancer patient care, but their toxicity and water-insolubility have raised serious concerns10. Nanoparticle-based delivery of small chemotherapeutics via biochemical conjugation, complexation, or prodrug formulation has demonstrated to be a promising strategy for cancer therapy, due to their capacity of refining the poor physicochemical properties of chemo-drugs11. Over the years, the field of RNA nanotechnology has emerged rapidly13. RNA was widely used as a unique and biocompatible material for constructing nanoparticles via bottom-up self-assembly19. The versatility of RNA nanostructures, including composition, structure, and functionality, can be precisely controlled. Typically, RNA nanoparticles can be conveniently functionalized with tumor-targeting ligands to achieve selective targeting and specific delivery to tumor21. This advantage allows the maximum utilization of therapeutics after systemic administration, as well as minimizing side effects caused by non-specific accumulation in other organs. As a result, therapeutic modules such as siRNA and anti-miRNA were conjugated to RNA nanoparticles and exhibited efficient tumor inhibition in various cancer models21. In addition, recent studies show that the immune responses of RNA nanoparticles, based on the phi29 packaging RNA, are size, shape, and sequence-dependent22. Thus, RNA nanoparticles can be tuned not to trigger immunostimulation, or to induce strong immune responses that can be potentially used for cancer immunotherapy or as vaccine adjuvant24. Taking these advantages into consideration, small chemotherapeutic drugs utilizing RNA nanoparticles as a drug delivery platform could exhibit tumor-specific therapeutic effects and reduce the side effects and toxicity that is currently observed.Functional nanomaterials constructed via molecular self-assembly show enormous potential in biotechnology and biomedicine applicationsG°, required for complex formation, is important in RNA nanotechnology12. We previously discovered a thermodynamically stable phi29 pRNA three-way junction (3WJ) motif which has been used as a scaffold to construct multifunctional RNA nanoparticles25. This multivalent RNA can be modified with various chemical moieties26, which makes the covalent conjugation of chemo-drugs to RNA nanoparticles achievable27. However, increasing the drug loading capacity remains challenging for the current pRNA-3WJ nanoparticles. The limited drug loading capacity can be caused by steric hindrance that interferes with assembly, unfolding of RNA 2D or 3D structures after conjugation, or disassociation of RNA nanoparticles due to reduction of thermostability.The concept of thermodynamic stability, defined as the free energy, Δ25 with much higher thermodynamic stability, displaying an extraordinary annealing temperature (Ta) higher than 80 °C, far superior to pRNA-3WJ. Therefore, it completely addresses the concerns of reduced assembly efficiency as well as unfolding and dissociation over time after drug conjugation. The 4WJ-X nanostructures covalently conjugated with and without PTX are successfully formed with high structural stability and rigidity, as revealed by single-particle cryo-EM and 3D reconstruction. The resulting RNA-PTX complex retains its tertiary folding and independent functionalities both in vitro and in vivo, as demonstrated in the inhibition of triple negative breast cancer (TNBC) growth in animal trial. Currently, the major challenge in cancer therapy using PTX is not the anticancer drug efficacy, but its high toxicity. In this study, the RNA-PTX nanoparticles display undetectable toxicity or immune stimulation in mice. Our finding highlights the strong potential for using thermostable RNA-PTX nanoparticles as an efficient anticancer drug carrier.To address these concerns, we deliberately redesign our RNA vehicle to further elevate its thermostability and maximize the number of drugs that can be incorporated in the nanoparticle. An RNA four-way junction (4WJ-X) is constructed to achieve high-yield loading of drugs, as well as significantly solubilize the hydrophobic PTX for tumor inhibition. The engineered RNA 4WJ-X nanostructure is evolved from the pRNA-3WJ motifTa) alteration. Thus, using the previous RNA nanoparticles to carry sufficient chemo-drugs for cancer regression is challenging, due to reduced assembly efficacy and thermostability of the anticipated nanostructure after high-density loading with the drugs. As a means to improve RNA nanotechnologies’ capability in delivering high payloads of chemotherapeutic drugs, we intentionally evolved our nanostructure from pRNA-3WJ25 to 4WJ28 to maximize its thermodynamic stability as well as its drug loading capacity. The RNA nanoparticle used in the present work is a synthetic RNA 4WJ motif, with a hypothetical “X” shaped structure, composed of four RNA oligomers  was found to be 22 °C higher than that of pRNA-3WJ  -modified prodrugs without significant influence on assembly efficiency and thermostability. We then evaluated the capability of this ultra-thermodynamically stable RNA 4WJ-X nanostructure to carry high payloads of drug.Attachment of chemicals or bioconjugation of foreign payloads to an RNA oligomer tends to induce steric constraints, and physical hindrance leading to annealing temperature  modified at pyrimidines to prevent enzymatic degradation in blood circulation and offer high chemical stability for in vivo applications. The successful PTXs multi-conjugation to RNA oligomer was evidenced by slower migration of RNA-multi-PTXs products in gel, due to the increased molecular size and hydrophobicity . Thus, each RNA oligomer was site-specifically conjugated with six PTXs along the ribose-phosphate backbone Fig. . This deely Fig. . When thely Fig. . Quantit6. RNA is a highly water-soluble biomaterial by nature. Dissolving RNA-6 PTXs conjugate in double-distilled water yielded clear solutions at increasing concentrations from 0.4 to 12.8 mg ml−1 30. This significant enhancement demonstrates that RNA can greatly solve the water-insolubility problem of PTX and other chemo-drugs for administration in clinical trials.The unsatisfactory water-solubility of PTX has led to unfavorable pharmacokinetic/pharmacodynamic (PK/PD) profiles, and undesired side effects or toxicityl−1 Fig. . In contn = 3 independent samples, mean ± SD)  Fig. . Step-wiSD) Fig. , slightlSD) Fig. . The zetSD) Fig. . In addiSD) Fig. , which c34. This technique was used to evaluate the structures of 4WJ-X and 4WJ-X-24 PTXs nanoparticles in vitrified solution. The cryo-EM images for both nanoparticles clearly revealed individual particles without any obvious aggregation . Typically, PTX displays a large molecular structure and contains more aromatic rings, compared with ribonucleotides , the 4WJ-X-24 PTXs nanoparticles displayed a very high Tm of 79 °C  has been reported to specifically bind EGFR on the surface of MDA-MB-231 breast cancer cells37. To equip our nanoparticles with tumor targeting capability, this anti-EGFR aptamer was incorporated on one of the 4WJ-X strands to construct multifunctional 4WJ-X-24 PTXs-EGFRapt nanoparticles , 4WJ-X-EGFRapt (8.3%), and free PTX (24.6%)  is associated with high cancer cell proliferation and high risk of recurrence in patients receiving treatmentsles Fig. . The Aleles Fig. . Confocaups Fig. . To eval nM Fig. , while 46%) Fig. . Since aapt-incorporated RNA nanoparticles were evaluated after systemic injection through the tail vein. Ex vivo images of tumor and healthy organs harvested from mice 8 h post-injection showed that the 4WJ-X-EGFRapt nanoparticles strongly accumulated in tumor, with low and no accumulation in liver and other organs, respectively , or PBS served as control groups. Upon the treatment with 4WJ-X-24 PTXs at a dose of 8 mg kg−1 (PTX per mouse weight) every 2 days for a total of five dosages, the results showed a promising inhibitory effect on tumor growth in mice as monitored by tumor volumes, whereas control groups formed faster-growing tumors , and chemokines were evaluated upon systemic administration of 4WJ-X-24 PTXs nanoparticles in mice. The results showed that i.v. injection of 4WJ-X-24 PTXs nanoparticles at the dose of 5 mg kg−1 induced undetectable or negligible tumor necrosis factor α (TNF-α) and interleukin 6 (IL-6) as well as type I IFN-α and type II IFN-γ production  of free PTX and no fatalities were found, confirming that RNA nanoparticles conjugated with PTX are safe and reduce side effects. The results were ascribed to the fact that RNA nanoparticles not only eradicate the need of toxic solvents as excipients and remarkably improve the water solubility, but also take full advantage of the favorable pharmacokinetic nature of the RNA nanoparticles.Toxicological and immunological evaluations are one of the most important drug evaluations prior to the use in the clinicion Fig. . In contion Fig. . Severalion Fig. . These rAn RNA 4WJ-X nanostructure with ultra-thermodynamic stability was constructed for high-yield loading of PTX for targeted cancer therapy. Each RNA nanoparticle loaded with 24 copies of covalently linked PTX was used as a prodrug, while remaining ultra-thermostability for in vivo applications. The water-solubility of PTX with RNA as the carrier was greatly enhanced in comparison to free PTX. Single-particle imaging by cryo-EM clearly showed the formation of “X” shaped RNA nanoparticles at the sub-nanometer scale. Animal trials in triple negative breast cancer model revealed that the RNA nanoparticles incorporated with anti-EGFR aptamer can specifically target tumor and significantly inhibit tumor growth after systemic injections. Currently, the hurdle in using PTX for cancer patient care is not the issue of PTX’s efficacy but its toxicity. The RNA-PTX nanoparticles here displayed extremely low or undetectable toxicity or immune responses in mice. This work demonstrates an effective method to address the insolubility of hydrophobic drugs in patient care. More importantly, it demonstrates a promising nano-platform for chemotherapeutic drug delivery by using thermostable RNA nanoparticles with the capabilities of high-yield drug payload, tumor-specific targeting, and reduced adverse effects for cancer therapy.3 prodrug was synthesized by a reaction mixture of paclitaxel (Alfa Aesar), 6-azido-hexanoic acid (Chem-IMPEX), N,N′-dicyclohexyl-carbodiimide (Acros Organics), and 4-(dimethylamino) pyridine (Sigma-Aldrich) at 1:2:2:1 equivalent ratio in 20 mL dichloromethane as solvent. The reaction was run at room temperature with stirring for 36 h, followed by filtration and rotary evaporation to yield crude product. The crude product was purified by silica gel chromatography using n-Hexane:ethyl acetate as eluent. RNA-6 alkynes oligomers  were synthesized via standard solid-phase RNA synthesis26 using 5′-hexynyl phosphoramidites (Glen Res. Corp.) and 2′-propargyl phosphoramidites (ChemGenes Corp.). RNA oligomers were purified by desalting using Glen Pak purification cartridges (Glen Res. Corp). The RNA sequences  are:3WJ a: 5′-uu*G ccA uGu* GuA uGu* GGG-3′3WJ b: 5′-c*cc AcA u*Ac uuu Gu*u GAu cc*-3′3WJ c: 5′-GGA u*cA Auc* AuG Gc*A A-3′4WJ-X a: 5′*-uuA GGu* AAA Gc*c Acc uGc AGG uGc uAc c*GA uGu* AAu uc*A A-3′4WJ-X b: 5′*-uuG AAu* uAc Au*c GGu AGc AcG GGc uGu Gc*G AGG c*uG AAc* AG-3′2: 5′NH2-C6-u*uG AAu* uAc Au*c GGu AGc AcG GGc uGu Gc*G AGG c*uG AAc* AG-3′4WJ-X b-5′NH4WJ-X c: 5′*-cuG uuc* AGc cu*c GcA cAG ccA GcA c*Gc Acc* uGA Au*A GG-3′apt: 5′*-cuG uuc* AGc cu*c GcA cAG ccA GcA c*Gc Acc* uGA Au*A GGu Gcc uuA GuA AcG uGc uuu GAu Guc GAu ucG AcA GGA GGc-3′4WJ-X c-EGFR4WJ-X d: 5′*-ccu Auu* cAG Gu*G cGu Gcu GGG cuG cAG Gu*G Gcu u*uA ccu* AA-3′PTX-N2O) were thoroughly mixed with PTX-N3 that dissolved in 3:1 (v/v) dimethyl sulfoxide/tert-butanol, followed by adding freshly prepared “click solution” (a blend of copper(I) bromide/Tris[methyl]amine (Sigma-Aldrich) at a 1:2 molar ratio) at a final 1:15:20 molar ratio of RNA:PTX:Cu+. After reaction at room temperature overnight, the product was characterized by 16% (w/v) 8 M urea PAGE in TBE buffer  at 200 V for 1 h, and stained by ethidium bromide  and visualized on a Typhoon FLA 7000 . Gel images were analyzed by ImageQuant TL. The reaction was subsequently diluted with 1/10 volumes of 0.3 M sodium acetate and 2.5 volumes of 100% ethanol and incubated at −20 °C overnight for RNA precipitation. The precipitates were re-dissolved in DEPC-H2O and purified by Ion-Pair Reverse Phase HPLC in an Agilent PLRP-S 4.6 × 250 mm 300 Å column. PTX-labeled RNA was separated from unreacted RNA-6 alkynes in an H2O/Acetonitrile (ACN) ramp. HPLC data was collected by OpenLAB CDS. Fractions of RNA-6 PTXs were combined, dried and resuspended in DEPC-H2O for nanoparticle assembly.Conjugation of PTX to RNA was performed using copper(I)-catalyzed alkyne-azide cycloaddition (“Click chemistry”). RNA-6 alkynes resuspended in diethyl pyrocarbonate-treated water  was incorporated with an anti-EGFR aptamer37 at the 3′-end during synthesis.The 3D computational model of 4WJ-X nanostructure was generated using Swiss PDB Viewer and PyMOL Molecular Graphics System. The 4WJ-X composed of four RNA strands is designed to lock into a four-strand assembly stabilized by base-pairing complementarity which generates four helix domains arranged around a nanoparticle core. 4WJ-X-24 PTXs nanoparticles were assembled by mixing four RNA-6 PTXs oligomers at equimolar concentrations in TES buffer , followed by denaturing at 95 °C for 5 min and gradual cooling to 4 °C over the course of 1 h. Nanoparticle assembly was confirmed using a 12% (w/v) native PAGE in TBE buffer  at 150 V for 1 h, and stained and visualized as described above. To construct 4WJ-X-24 PTXs-EGFRThe apparent hydrodynamic diameter and zeta potential of the RNA nanoparticles in 1 μM in TES buffer were determined using a Zetasizer Nano-ZS  at 25 °C. The laser wavelength was 633 nm. Results were plotted with data points using Origin. For size comparison of 4WJ-X, 4WJ-X-24 PTXs, and PTX, three independent samples were measured and average particle size were obtained.−1 as an annealing process. Annealing curves of RNA nanoparticles were measured by Roche LightCycler® 480 RT-PCR machine, and were plotted with data points using Origin. Three independent samples were measured over three independent experiments with similar results, and representative annealing curves are shown. Ta was determined by LightCycler 480 Software using the first derivative of the annealing profile.Preassembled RNA nanoparticles were added to a 96-well plate with a final concentration of 2.5 μM and 1 μM. SYBR Green II dye (Invitrogen) was added to each well as a reporter dye for RNA nanoparticles formation at a final concentration of 20×. Samples were heated to 95 °C for 5 min as a denaturing process followed by a slow cooling ramp to 20 °C at a rate of 0.11 °C s2 for apo and PTX-bound state, respectively. A total of 2780 movie stacks for apo state and 3800 movie stacks for the PTX-bound state were collected with a defocus range of 1.5–3.5 μm.Two samples of 4WJ-X nanoparticles, apo state (4WJ-X) and PTX-bound state (4WJ-X-24 PTXs), were diluted to a final concentration of ~20 µM. Three microliters of sample were applied onto glow-discharged 200-mesh R2/1 Quantifoil grids. The grids were blotted for 3 s and rapidly cooled in liquid ethane using Vitrobot Mark IV (ThermoFisher) at 22 °C and 100% humidity. The samples were screened using Talos Arctica cryo-electron microscope (ThermoFisher) operated at 200 kV and then imaged in a Titan Krios cryo-electron microscope (ThermoFisher) with GIF energy filter (Gatan) at a magnification of ×215,000  for apo state and at a magnification of ×130,000  for PTX-bound state using EPU (ThermoFisher). Micrographs were recorded with a Gatan K2 Summit direct electron detector, where each image is composed of 30 individual frames with an exposure time of 6 s and a specimen dose rate of 10 and 7.6 electrons per second per Å39 and CTF was determined using CTFFIND440. All particles were autopicked using NeuralNet option in EMAN241 and further checked manually, yielding 336,340 particles from selected 2618 micrographs for the apo state, and 406,408 particles from selected 3496 micrographs for the PTX-bound state. The particle coordinates were then imported to RELION42, where four rounds of 2D classification were performed to remove poor 2D class averages. The initial model was built using the “Ab initio 3D reconstruction” option in CryoSPARC43, and the 3D classification was performed in RELION. The final 3D refinement was performed in RELION using 106,775 particles for apo state and 125,909 particles for PTX-bound state, resulting in the corresponding maps with resolutions of 9 and 7 Å, respectively, based on the FSC of two independent particle data sets at a threshold of 0.143. Figures and movie were prepared using Chimera44.All micrographs were motion-corrected using MotionCor2Tm values were defined as the temperature at which 50% of the loaded nanoparticles dissociated.Preassembled RNA nanoparticles were run in a 12% (w/v) native PAGE in TBE buffer at 100 V for 10 min at room temperature. TGGE (Biometra Gmbh) was subsequently performed by applying a gradient temperature perpendicular to the electrical current and run for 60 min at 20 W. The gel was stained and visualized as described above. Quantified values of bands for each nanoparticle were divided by the sum of the total values in corresponding lanes, calculated by ImageJ. Melting curves were plotted with quantified data points using Origin. Preassembled RNA nanoparticles were incubated at the final concentration of 1 μM in cell culture medium containing 50% (v/v) FBS at 37 °C for different time points through 48 h. The resulting samples were examined using a 3% agarose gel run at 100 V for 40 min and stained and visualized as described above. Quantification analysis was performed using ImageJ to calculate the percentage of intact nanoparticles (intensity of the band at a time point/intensity of the band at 0 h) for each time point. Enzymatic degradation curve was plotted with quantified data points using Origin.−1 penicillin at 37 °C in humidified air environment containing 5% CO2.Human MDA-MB-231 cells were obtained from American Type Culture Collection (ATCC). Cells were grown and cultured in DMEM/F-12 medium (ThermoFisher Scientific) containing both 10% (v/v) FBS and 100 U ml@ Gold Antifade Reagent (Life Technologies Corp.) containing DAPI for cell nucleus staining and assayed on Olympus FV3000 confocal microscope (Olympus Corp.). Data were collected using Fluoview FV31S-SW.MDA-MB-231 cells were seeded on glass coverslips and cultured at 37 °C overnight. Alexa Fluor 647 labeled RNA nanoparticles were incubated with cells at a final concentration of 100 nM for 4 h at 37 °C. After washing twice with PBS buffer, cells were fixed with 4% formaldehyde and washed again, followed by treatment with 0.1% Triton X-100 (Sigma-Aldrich) in PBS buffer for 5 min and subsequent cytoskeleton staining with Alexa Fluor 488 phalloidin (ThermoFisher Scientific) for 30 min at room temperature. After rinsing with PBS buffer, the cells were mounted with ProLong4 MDA-MB-231 cells were seeded on a 24-well plate overnight. RNA nanoparticles and free PTX were added into cells at a final concentration of 1 μM. After 24 h incubation at 37 °C in a humidified 5% CO2 environment, cells were trypsinized to single cell suspension, washed with PBS buffer twice, and resuspended in 100 μL 1 × Annexin V-FITC binding buffer. Five microliters of Annexin V-FITC and 5 μL of PI were added into each sample and incubated at room temperature for 20 min. The samples were then added into flow tubes that contained 200 μL of 1 × binding buffer for FACS analysis by FACSCalibur™ flow cytometer within 1 h. Data were analyzed by FlowJo.Cell apoptosis was studied using FITC Annexin V Apoptosis Detection Kit (BD Pharmingen) following manufacturer instructions. Briefly, 5 × 103 MDA-MB-231 cells were seeded on a 96-well plate overnight. RNA nanoparticles and free PTX were added into each well in triplicates at indicated concentrations. After incubation at 37 °C for 48 h in a humidified 5% CO2 environment. Fifteen microliters of MTT Dye Solution was added to each well and the plate was incubated at 37 °C in the dark for 4 h. Next, 50 μL Solubilization Solution/Stop Mix was added to each well for dissolving the crystal in the dark. The plate was incubated for 2 h at room temperature. Finally, the crystal in each well were fully dissolved to a uniformly colored solution and their absorbance at 570 nm was measured by Synergy 4 microplate reader (Bio-Tek). Data were collected by Gen5, and normalized data was plotted using Origin.Cell cytotoxicity was studied using CellTiter 96 Non-Radioactive Cell Proliferation Assay (Promega) following manufacturer instructions. Briefly, 5 × 106 MDA-MB-231 cells/site resuspended in sterile PBS into the mammary fat pads of nude mice. When the tumor nodules had reached a volume of 75 mm3, the mice were used for tumor inhibition studies.All animal procedures were performed in accordance with the Subcommittee on Research Animal Care of The Ohio State University guidelines approved by the Institutional Review Board. Mice are housed in sterile environment, with all supplies  autoclaved or irradiated. To generate the xenograft model, female athymic nu/nu mice, 4–8 weeks old, were purchased from Taconic BiosciencesFarm. TNBC tumor xenografts were established by injecting 2 × 10apt nanoparticles  were systemically administered via the tail vein into MDA-MB-231 tumor bearing mice. PBS-injected mice were used as fluorescence negative controls. The whole-body imaging of mice was conducted at 8 h using an IVIS system (XMRS) with excitation at 640 nm and emission at 680 nm. The mice were sacrificed at 8 h post-injection by the inhalation of CO2 followed by cervical dislocation, and major organs were collected and subjected to fluorescence imaging for the assessment of biodistribution profiles. The fluorescence imaging data of average radiant efficiency ([p s−1 cm−2 sr−1] [μW cm−2]−1) were quantitative by IVIS system (XMRS) program.Alexa Fluor 647 labeled 4WJ-X-EGFRn = 5 biologically independent animals). Samples were administrated by i.v. injection in a total of 5 doses  every other day. Tumor volume, calculated as (length × width2)/2, and mouse weight were monitored every other day. Curves were plotted with data points using GraphPad Prism. On day 14, the mice were sacrificed followed by tumor extraction. Tumors were weighted, and result was plotted with data points using Origin. Data were statistically analyzed by two-tailed unpaired t-test and presented as mean ± SD; *p < 0.05; **p < 0.01; ****p < 0.0001.Mice with established tumor nodules were randomly divided into four groups  via i.v. injection at 5 mg kg−1 (PTX per body weight). Blood samples were harvested from mice 3 h post-injection by cardiac puncture and centrifugated at 12,800 × g for 10 min. Concentrations of cytokines in serum supernatant were examined in triplicates using Mouse ELISA MAX Deluxe sets (BioLegend) for TNF-α, IL-6, and IFN-γ, and using Mouse IFN-α ELISA Kit (R&D Systems) for IFN-α, following manufacturer provided protocols . Results were plotted using Origin. Data were statistically analyzed by two-tailed unpaired t-test and presented as mean ± SD; *p < 0.05; **p < 0.01; ***p < 0.001.CD-1 mice (4–5 weeks old) were purchased from Charles River Laboratories. 4WJ-X-24 PTXs nanoparticles and control groups were administered into mice . Statistical differences were evaluated using two-tailed unpaired Further information on research design is available in the Supplementary InformationDescription of Additional Supplementary FileSupplementary Movie 1Reporting Summary


Pseudomonas putida KT2440 harbors three known members of the CsrA/RsmA family of post-transcriptional regulators: RsmA, RsmE and RsmI. We have carried out a global analysis to identify RNA sequences bound in vivo by each of these proteins. Affinity purification and sequencing of RNA molecules associated with Rsm proteins were used to discover direct binding targets, corresponding to 437 unique RNA molecules, 75 of them being common to the three proteins. Relevant targets include genes encoding proteins involved in signal transduction and regulation, metabolism, transport and secretion, stress responses, and the turnover of the intracellular second messenger c-di-GMP. To our knowledge, this is the first combined global analysis in a bacterium harboring three Rsm homologs. It offers a broad overview of the network of processes subjected to this type of regulation and opens the way to define what are the sequence and structure determinants that define common or differential recognition of specific RNA molecules by these proteins.Post-transcriptional regulation is an important step in the control of bacterial gene expression in response to environmental and cellular signals.  Pseudomonas, expression of these regulatory RNAs is controlled by the two-component system GacS/GacA , have opposing effects on surface motility and biofilm formation; deletion of the three genes abolishes swarming motility and stimulates bacterial attachment and biofilm formation, although the biofilms formed by a triple rsm mutant are more labile and easily dispersed than wild type biofilms : ampicillin (Ap) 100; kanamycin (Km) 25; streptomycin (Sm) 50 (E. coli) or 100 (P. putida); (Gm) gentamycin 50; tetracycline (Tc) 10 or 20. Cell growth was followed by measuring optical density at 660 nm (OD660).The bacterial strains, plasmids and oligonucleotides used in this study are listed in ida mt-2 . PseudomB medium , M9 or MB medium  supplemeeviously . Unless Pseudomonas cells was performed as previously described  or SM10 λpir (pUX-BF13), the latter when miniTn7 transposase was required for intergenic site-specific insertion of miniTn7 derivatives near glmS  of wild type KT2440 harboring each construct were inoculated in 500 ml of LB medium. Three biological replicates were run in parallel. Cultures were incubated at 30°C under shaking until an OD660 of 0.8 was reached. At that point, expression of each His-tagged-Rsm protein was induced by the addition of IPTG to a final concentration of 0.5 mM. Cultures were allowed to grow for six additional hours. Aliquots of 1.5 ml were then harvested by centrifugation, instantly frozen with liquid nitrogen and stored at −80°C for total RNA purification. Cells from the remaining culture volume were also harvested and pellets were stored at −80°C until use. His-tagged Rsm-RNA complexes were isolated using Ni-NTA Fast Start purification kit (Qiagen). Three replicate extractions were done for each culture replica. Elution aliquots were analyzed by SDS-PAGE.Previously constructed derivatives of expression vector pME6032 harboring each rsm gene  were useTotal RNA and RNA from Rsm-RNA complex was extracted using RNA isolation kit (Macherey-Nagel) following the manufacturer’s instructions. RNA samples were subsequently treated with RNase-free DNase I  to remove DNA traces, as specified by the supplier. Total RNA quality was assessed using Agilent RNA 6000 Nano Kit (Agilent Technologies) in the Agilent 2100 Bioanalyzer. RNA concentration was measured using Qubit RNA BR assay kit (Life Technologies). 1 µg of RNA was used for rRNA depletion using Ribo-Zero rRNA Removal Kit (Illumina). One of the biological replicates of RsmA and one of RsmI did not meet the required quality and quantity standards and were not used in further analysis.The generation of cDNA libraries was carried out using NEBNext Ultra Directional RNA Library Prep kit for Illumina (NEB). Dual Index Primers Set one was used to generate bar-coded multiplex libraries (NEB). Library QC was performed using bioanalyser HS kit (Agilent biotechnologies). cDNA libraries were quantified using qPCR (Kapa Biosystems). Libraries were pooled at the desired concentrations, denatured and loaded for sequencing according to manufacturer’s instructions. Sequencing was performed on the Illumina MiSeq Benchtop Sequencer to generate 2 × 75 bp reads. The number of reads obtained ensured a minimum of 76× coverage of the whole genome for control RNA and a minimum of 60× coverage for Rsm-bound RNA.P. putida KT2440  with Bowtie v2  of each peak  was added to induce the expression of the proteins. After 6 h of further growth, cells were harvested by centrifugation and pellets subsequently stored at −80°C. Protein purification was carried out using QIAexpress Ni-NTA Fast Start Kit (Qiagen), following the manufacturers’ instructions.For purification of Rsm proteins, overnight cultures (10 ml) of 032-rsmI  were usein vitro using the MAXIscript T7 kit (Life Technologies). The RNA obtained was visualized by hybridization of an ATTO700-labeled DNA primer to the 3′ extension of the RNA. Rsm proteins were incubated with target gene RNA (5 or 10 nM) in 1 × binding buffer [25]. Binding in the absence or presence of unlabeled competitor RNA (100-fold excess) was carried out for 30 min at 30°C. Then Bromophenol Blue was added  before electrophoresis on 6% (w/v) non-denaturing polyacrylamide TBE gel at 4°C. Imaging was performed using a 9201 Odyssey Imaging System (LI-COR Biosciences).Electrophoretic mobility shift assays (EMSA) were carried out following a method described previously . DNA temrsmIEA mutant with different nitrogen and carbon sources. Cultures grown overnight at 30°C in M9 minimal medium with glucose as carbon source were used for inoculation in the plates at an initial OD660 of 0.05. Turbidity was measured at different times over 24 h in a Tecan Sunrise microplate reader. Further experiments were done in 96-well plates using M9 or M8 with the indicated carbon or nitrogen sources, at a final concentration of 5 mM. Growth was followed for 24 h at 3°C with continuous shaking in a Bioscreen apparatus C MBR equipped with a wide band filter (420–580 nm).BIOLOG plates  were used for initial assessment of growth of KT2440 and the triple ∆5 CFU/strain) of KT2440Tn7-Km, as the wild type, and the wild type or mutant derivatives tagged with miniTn7Sm by triparental conjugation as described above. Inocula sizes were monitored by plating on LB-agar supplied with kanamycin or streptomycin. After 7 days, bacteria were recovered from the rhizosphere or the root tip as specified (t-test for independent samples (p < 0.05).Surface sterilization, germination of corn seeds, and bacterial inoculation of the seedlings were performed as described previously . Brieflypecified  and the pecified . SigmaStwww.ncbi.nlm.nih.gov/geo/) and are available under accession number GSE154204.Raw and processed data files have been deposited in the Gene Expression Omnibus Database , was carried out to identify genes that could potentially be regulated at the post-transcriptional level by RsmA, RsmE or RsmI in ruginosa  and is sp-value (PV) data–shown as -10×log10 for ease of representation–in each case  of this transposase, based on their sequence identity with ISPpu8 flanking regions. Whether Rsm proteins influence the activity of this mobile genetic element in KT2440 is an interesting issue that deserves further study.Additionally, several intergenic sequences were found among the targets for the three proteins. These correspond to regions adjacent to the different copies of the gyrB), which is among the above-cut-off targets for RsmA but slightly below the FE and/or p-value cut-off for the other two proteins. Other targets are only enriched in association with one of the Rsm proteins and are not present in the RNA population associated to either of the other two, indicating they are truly specific for that Rsm homolog, e.g. PP_1656 (relA) for RsmA, PP_0168 (lapA) for RsmE, or PP_1803 (wpbV) for RsmI. Identifying the molecular basis for such specificity will require detailed bioinformatics analysis combined with in vitro and in vivo assays.As mentioned above, a significant number of targets seem to be exclusive for one of the three proteins. A few of these correspond to enriched peaks that were below the established cut-off parameters in some samples, and therefore could represent common targets showing different affinities for each protein, with a strong preference for one of them. Such is the case, for example, of PP_0013  are below the established cut-off. This could indicate that binding of RsmA to the mRNA of cfcR in vivo is hampered by competition with the other two Rsm proteins, a possibility that has been previously suggested, based on in vivo expression data of cfcR compared to the high affinity observed for the RsmA/cfcR mRNA interaction in vitro  as target of the three proteins, and the rest , as targets of RsmE. Of these, the proteins encoded by PP_0563 and PP_2505 present GGDEF domains, characteristic of diguanylate cyclases. The first corresponds to GcbA, a diguanylate cyclase conserved in Pseudomonas, which has been reported to influence initial attachment and swimming motility  is among the targets for RsmA in KT2440  and hisP  are targets for RsmE; artJ  and amaD  are targets for RsmA, and amaB  is a target for both RsmE and RsmI (www.genome.jp/kegg/), AmaB is involved in catabolic pathways for L-lysine and L-arginine in P. putida, participating in the conversion of aminobutanal, N4-acetyl-aminobutanal, and 4-trimethyl-ammoniobutanal into their corresponding butanoate derivatives.These results evidenced that the Rsm system may play a regulatory role in metabolism and/or uptake of nutrients, particularly of basic amino acids, in KT2440. Hence, potential Rsm-enriched targets explaining these divergences were explored. A survey of the identified RNA molecules related to these processes indicated that those corresponding to and RsmI . Based oE. coli between CsrA and the stringent response (relA (PP_1656) mRNA is also a target for RsmA  plants with the wild type and single , double  and the triple (ΔIEA) rsm mutants (rsmA caused a slight reduction in competitive colonization of the whole root and a much larger effect when root tip colonization was evaluated. This phenotype was not observed in the other single mutants, nor in the ΔIE double mutant. However, while the results with the ΔIA double mutant were similar to those obtained with the ΔA single mutant, a cumulative effect could be observed in the ΔEA strain, which showed a significant decrease in colonization of both the whole root and the root tip. This phenotype was very similar to that observed in the triple mutant. In this set of experiments the Sm resistance marker seemed to confer a slight advantage over the Km resistance marker, according to the results obtained with the wild type derivatives tagged with each mini-Tn7 (rsm mutations might be larger than observed here.Previous reports have established that n plants . In addin plants . These f mutants . For thamini-Tn7 . This coThe loss of fitness in the rhizosphere of the ΔEA and ΔIEA mutants may result from the overall influence of Rsm proteins on different metabolic processes, including amino acid transport, while the reduction in root tip colonzation could correlate with the previously observed decrease in swimming motility in the ΔEA and ΔIEA strains and the lack of swarming motility of all the rsm mutants, with the exception of ΔI, which still retained some surface motility . It willP. putida. Besides exposing its complexity and the vast influence that post-transcriptional regulation is bound to have in this bacterium, ranging from amino acid metabolism to potential transposon-mediated DNA rearrangements, the information obtained leads to a relevant question to be analyzed in detail, i.e. the characteristics by which an RNA molecule constitutes a shared target for the three Rsm proteins or is selectively bound by only one or two of them.This work represents the first effort to define the global regulatory network commanded by Rsm proteins in


Comprehensive mRNA sequencing is a powerful tool for conducting unbiased, quantitative differential gene expression analysis. However, the reliability of these data is contingent on the extraction of high-quality RNA from samples. Preserving RNA integrity during extraction can be problematic, especially in tissues such as skin with dense, connective matrices and elevated ribonuclease expression. This is a major barrier to understanding the influences of altered gene expression in many preclinical pain models and clinical pain disorders where skin is the site of tissue injury.This study developed and evaluated extraction protocols for skin and other tissues to maximize recovery of high-integrity RNA needed for quantitative mRNA sequencing.Rodent and human tissue samples underwent one of the several different protocols that combined either RNA-stabilizing solution or snap-freezing with bead milling or cryosectioning. Indices of RNA integrity and purity were assessed for all samples.Extraction of high-integrity RNA is highly dependent on the methods used. Bead-milling skin collected in RNA-stabilizing solution resulted in extensive RNA degradation. Snap-freezing in liquid nitrogen was required for skin and highly preferable for other tissues. Skin also required cryosectioning to achieve effective penetration of RNA-stabilizing solution to preserve RNA integrity, whereas bead milling could be used instead with other tissues. Each method was reproducible across multiple experimenters. Electrophoretic anomalies that skewed RNA integrity value assignment required manual correction and often resulted in score reduction.To achieve the potential of quantitative differential gene expression analysis requires verification of tissue-dependent extraction methods that yield high-integrity RNA. Thus, as the reliance on RNA-seq increases, it is critical that pain researchers meticulously validate their methods to maximize data set reliability.Comprehensive, unbiased RNA sequencing (RNA-seq) has become a widely used approach with more than 100 different methodological applications, the most common being differential gene expression (DGE) analysis, which determines quantitative expression changes that occur to the active transcriptome.9 RNA degradation can have striking effects on the identification of true differentially expressed genes, with one study citing 4% and 26% of genes changing as RNA integrity values shifted from 9.3 to 7.9 and 6.2, respectively.15 Transcripts also are susceptible to differential degradation in a tissue-specific manner, which can further confound the reliability of DGE analyses.20 Therefore, it is imperative for experimenters to rigorously validate their RNA extraction procedures to minimize the confounding influence of degradation bias.Importantly, the accuracy, and thus reliability, of the DGE analyses is largely dependent on sample RNA integrity. Sample mRNA must be highly intact to accurately reflect ongoing gene expression; otherwise, degradation bias will increase intragroup and intergroup variances and ultimately false discovery rates.27 Still, certain tissues are considerably more challenging to extract than others. One of the particular importance to the pain field is skin, because of its high endogenous expression of ribonucleases and its densely fibrous, yet tensile nature.40 Although several methods have been reported for skin,43 RNA integrity remains a primary issue. Given that paw skin is used in many inflammatory and neuropathic pain models, validation of a robust extraction protocol is important. A reliable extraction method for skin would certainly benefit future transcriptomic studies, especially those involving human biopsies. Therefore, the purpose of this study was to develop a reliable, reproducible extraction protocol that preserves high-integrity RNA from skin. Using indices for RNA yield, purity, and integrity, we evaluated results obtained from human and rodent skin biopsies as well as several other tissue types commonly used in pain research, including dorsal root ganglia (DRG), trigeminal ganglia (TG), spinal cord, and muscle. Collectively, this work emphasizes the critical importance of verifying extraction methods before proceeding forward with quantitative mRNA-seq.The extraction of high-integrity RNA from samples for sequencing experiments has become more routine, notably since the advent of RNA-stabilizing solutions and better homogenization strategies.Regulatory approval was obtained from the Human Subjects Institutional Review Board at the University of Texas Health Science Center at San Antonio (#20160027HU). Informed consent was obtained from patients according to protocol guidelines set by the Institutional Review Board. Samples were from both male and female participants of older than 55 years. The study was performed in accordance with the Declaration of Helsinki.later  for 4 to 6 hours, or (2) immediate snap freezing on liquid nitrogen. Cryotubes were stored at −80°C until processing.Skin punch biopsies were taken 10-cm proximal to the lateral malleolus. Marked biopsy sites were cleaned with alcohol swabs and then injected with 2% lidocaine/epinephrine  solution to anesthetize the skin. Single-use circular punches  were used with rotational motion and slight pressure to biopsy to a depth of 4 mm. The edge of each biopsy was lifted with forceps to remove the underlying fatty tissue. Biopsy sites were covered with pressure bandage for 24 hours. Biopsies were collected in sterile 2-mL cryotubes and preserved by (1) immersion in ice-cold RNAm+/+Leprdb/J aged 16 weeks ,17 male athymic nude mice aged 18 weeks , and male BALB/cJ mice aged 12 weeks (Jackson) were used in this study. Rats were housed in groups of 3 and mice in groups of 4 to 5. Animals were maintained on a 12-hour light–dark cycle with ambient temperatures between 20 and 22°C, with food and water freely available. Animal protocols were approved by the Institutional Animal Care and Use Committee of the University of Texas Health Science Center at San Antonio and conformed to International Association for the Study of Pain and federal guidelines.Male Sprague–Dawley rats  aged 20 weeks, male and female C57BL/6J mice aged 8 to 20 weeks (Jackson), male BKS.Cg-Dock7later or in empty cryotubes that were immediately snap frozen. Other tissues of interest were then dissected as quickly as possible and either immersed in RNAlater or snap frozen. All tissues were stored at −80°C until processing.At sacrifice, the following tissues were collected: glabrous hindpaw skin, lumbar DRG, TG, spinal cord, tongue, incisor apical papilla, and/or masseter muscle. Skin biopsies (4 mm) were always performed first, within 60 seconds of sacrifice and either collected into RNASpecimens underwent extraction according to one of the following protocols:later were first thawed on ice. Meanwhile, the processing chamber of a Bead Ruptor 24  was precooled to 0°C with the BR-Cryo Cooling Unit (Omni). Reinforced screw-cap microtubes (2-mL) with 2.8-mm ceramic beads (Omni) were filled with 900 μL QIAzol reagent (Qiagen). Thawed samples were transferred to QIAzol-filled bead tubes then placed on the tube carriage. Samples were homogenized for 40 seconds at 7.10 m/s and then placed on ice for 5 minutes. For complete homogenization, DRG and TG required one cycle, spinal cord and teeth required 2 cycles, and skin required 3 cycles. Afterwards, samples were transferred to sterile 1.5-mL tubes and centrifuged for 3 minutes at 8000g to remove debris. Supernatants were collected into sterile 2-mL tubes on ice before RNA isolation/purification.Samples in RNAPreviously snap-frozen tissues were placed on dry ice from cryostorage. Bead-filled tubes and the Bead Ruptor were then prepared as above. Samples were homogenized using either the 1-, 2-, or 3-cycle bead-mill protocol described above. Homogenates were centrifuged to remove debris and then transferred to 2-mL tubes on ice before RNA purification.Snap-frozen tongue samples were homogenized with a different bead-milling protocol. First, 2-mL tubes were filled with 1-mL QIAzol and a 7-mm stainless steel ball bearing (Qiagen). Tongue samples were transferred to QIAzol-filled tubes and loaded onto a 12-tube adapter (Qiagen) for the TissueLyser LT bead-mill (Qiagen). Samples were milled at 50 Hz for 45 seconds, placed on ice for 5 minutes, and then repeated to ensure complete homogenization. Homogenates were centrifuged to remove debris and transferred to sterile 2-mL tubes on ice before RNA purification.First, a 34°/80 mm MX35 Premier + cryostat blade , forceps, and aluminum cylinders  were sprayed with RNaseZap (ThermoFisher), wiped and then acclimated in a HM550 cryostat (Microm) held at −21°C. Samples were transferred from −80°C to the cryostat along with sterile 2-mL tubes. Aluminum cylinders were placed atop optimal cutting temperature (OCT)–coated chucks and filled with 60 μL RNase-free water. Once frozen, tissue specimens were positioned within the cylinder followed by another 60 μL RNase-free water and then quick-frozen with a 3-cm stainless steel heat extractor. Ice-encased samples were separated from the cylinder and mounted onto the chucks with OCT. Approximately 20 cryosections (16 μm) were cut and then carefully brushed into cold 2-mL tubes with precooled forceps. This was repeated until the entire sample was processed. Tubes were handled only by the open cap to minimize heat transfer. After cryosectioning, 1 mL QIAzol was quickly added followed by vortexing for 60 seconds. Samples were then placed on ice until RNA purification.later were thawed on ice and then processed according to the cryosectioning protocol described above. After vortexing, samples were placed on dry ice until RNA purification.Samples in RNAg. The upper aqueous phase (∼600 μL) from each sample was transferred to new 2-mL microtubes. After, 600 μL of 70% ethanol was added to extracts with vigorous mixing, transferred to spin columns, and then centrifuged at 12,000g for 30 seconds. Transfer and centrifugation were repeated, and then, the spin columns were washed once with 700 μL RWT buffer and twice with 500 μL RPE buffer. Spin columns were placed into 1.5-mL microtubes, and then 30 μL of RNase-free water was carefully applied to the membrane of each column followed by centrifugation at 14,000g for 60 seconds. Eluates were reapplied to their corresponding membrane and recentrifuged. RNA concentrations and purity were determined with a Nanodrop 2000 spectrophotometer (ThermoFisher).Extracts underwent isolation and purification using the RNeasy Plus Universal Mini Kit (Qiagen). Briefly, 100 μL gDNA eliminator solution and 180 μL chloroform were dispensed into extracts, with shaking after each. After 5 minutes, extracts were centrifuged at 4°C for 15 minutes at 14,00042 ProSize 3.0 (Agilent) was used to manually correct misidentified rRNA peaks in electropherograms where degradation affected the fragment baseline. Identification of 18S (∼1.9 kb) and 28S (∼4.7 kb) peaks were checked first, and if incorrect, the user redesignated the appropriate region. Peak integration boundaries were then validated. Start- and end-points were manually repositioned to only bound the points meeting the fragment baseline. RNA integrity number values were then updated after correction.Samples were delivered to the Genome Sequencing Facility at the Greehey Children's Cancer Research Institute for RNA integrity assessment. RNA integrity number (RIN) or RNA quality number were determined with a Bioanalyzer  or Fragment Analyzer (Agilent), respectively. Concentrated samples (>50 ng/μL) were processed using regular/NanoChip kits (Agilent), whereas dilute samples (<50 ng/μL) required high-sensitivity/PicoChip kits (Agilent). High-integrity RNA was defined with RIN/RNA quality number values ≥ 7.0.Modifications to images and electropherograms (for size) were made using OmniGraffle 7.11.3 .t test or one-way analysis of variance with Tukey's or Sidak's post hoc tests for pairwise comparisons, where appropriate. Pearson correlation coefficients were determined to establish the linear dependence between RIN values and 28S/18S rRNA ratios for all samples included in this study. Linear regression confirmed the correlation value and 95% confidence bands from the best-fit line. All statistical tests were two-sided with the significance threshold established with α = 0.05. Analyses were performed in GraphPad Prism 8.0. Multidimensional scaling plots were generated from selected samples with R v3.5.2.41Group mean values were compared using Student's 40 Successful preservation of RNA from human tissue samples, including skin, has been reported previously with the use of RNAlater.27 Thus, our initial collection protocol included the use of RNAlater to inhibit ribonucleases and preserve transcript integrity. Accordingly, patient skin biopsies were immersed in ice-cold RNAlater for several hours upon collection followed by cryostorage. RNA was extracted using a 3-cycle bead-milling homogenization strategy in combination with silica membrane-based purification. This extraction protocol yielded high RNA concentrations , this particular sample received a higher RIN value of 8.3 compared to a skin sample with minimal degradation (28S/18S = 1.2) that received a RIN of 8.0 Fig. D. These 13 necessitating great care during extraction to minimize the risk of degradation bias with quantitative DGE analysis. Although there is no consensus on the amount of RNA degradation permissible for DGE analysis, more studies are demonstrating the impact of degradation on sequencing data sets.42 Samples with poor RNA integrity have been shown to exhibit stochastic, disproportionate degradation of transcripts that inaccurately represented the active transcriptome.45 Degraded samples inevitably contain fragments that unevenly cover gene length, increasing intersample variance. Degradation also impacts signal/noise ratios, which are critical for subtracting background from sequencing reads.28 Altogether, these confounds increase false discovery rates as well as duplications.38 Alternative to poly(A)-enrichment used for mRNA-seq is ribodepletion for total RNA-seq, which can address some of these issues, especially with degraded samples, by specifically targeting rRNAs for removal. However, one major trade-off due to inclusion of nonpolyadenylated, intronic RNA is lower resolution of exonic reads, which consequently requires greater read depth and often more samples to achieve the same statistical power as mRNA-seq for DGE analysis.47 Still, degradation bias affects sequencing results regardless of enrichment approach; therefore, rigorous optimization of RNA extraction procedures can only benefit the accuracy of quantitative end applications such as DGE analysis.RNA is a labile biomolecule with pronounced susceptibility to chemical- and ribonuclease-mediated hydrolysis,Here, we developed a reliable protocol for high-integrity RNA extraction from skin samples intended for quantitative DGE analysis through unbiased mRNA-seq. We determined over the course of several iterations that extraction of high-integrity RNA from skin requires 2 steps: (1) immediate snap freezing upon collection and (2) homogenization through cryosectioning before extraction. Neither RNA-stabilizing solution nor bead milling preserved RNA integrity in skin. However, bead milling effectively extracted high-integrity RNA from other tissue types, including DRG, TG, spinal cord, tongue, and periapical lesions from teeth. During RNA integrity assessment, we observed subtle electrophoretic anomalies in ∼20% of samples that ultimately compromised RIN value assignment. Manually correcting for these anomalies typically reduced integrity scores, with several samples dropping below the designated cutoff. Collectively, these findings emphasize the need for pain researchers to rigorously optimize sample RNA extraction to accurately reflect gene expression changes used in DGE analysis.37 Utilization of quantitative mRNA-seq with skin holds much promise to better define the molecular bases of clinical pain conditions, including the contributions of keratinocytes and other non-neuronal cell types to peripheral sensitization.21 However, extracting intact RNA from skin has proven challenging.44 For our first iteration, we established that the primary obstacle in preserving RNA integrity in skin was the high ribonuclease expression, especially in epidermal keratinocytes undergoing terminal differentiation and cornification.11 After its failure, we deduced that RNAlater was incompletely inhibiting the ribonucleases and thus switched to snap freezing. If snap freezing alone had been sufficient, sample RNA would have remained intact after bead milling. Our results clearly show this not to be the case, instead indicating that ribonuclease inhibition is lost somewhere before completing RNA extraction. Chemical inactivators could not maintain ribonuclease inhibition, as neither immersion in RNAlater at collection or in QIAzol after snap freezing–preserved RNA integrity. Similar ineffectiveness was also reported with articular cartilage.23 Collectively, these indicated that a second major obstacle, the dense, interwoven connective tissue, impedes rapid chemical inactivation of skin ribonucleases. Thus, we needed an approach that maintained the cold-induced inactivation of ribonucleases while simultaneously permitting mechanical breakdown of skin. Remarkably, cryostat sectioning of skin biopsies was sufficient to preserve RNA integrity through extraction with QIAzol. Although slightly more time consuming, this method is easy to learn and inexpensive. Plus, cryostats are virtually ubiquitous in biomedical research institutions.Skin biopsies are an important resource, which includes their usefulness in clinical diagnostics and preclinical pain research.19 we observed a significant positive correlation between RIN values and rRNA ratios from all samples tested in this study. Interestingly, we identified 42 samples that oddly exhibited high RIN values yet low rRNA ratios. None of the 60 snap-frozen skin samples that underwent cryosectioning were among these abnormal samples. Rather, ∼90% were samples homogenized with the Bead Ruptor, suggesting that bead milling risks additional RNA degradation. Despite the potential of mechanically shearing transcripts and heat-induced oligomerization,31 the majority of bead-milled samples (∼70%) still exhibited normal RIN values and rRNA ratios. Alternatively, these anomalies may have arisen as a consequence of extended dissection. Most of the abnormal samples were in fact DRG, which are collected through a notoriously slow dissection process. We determined through re-examination of the electropherograms that the automated software frequently, but not always, misidentified the 28S peak and overestimated the 18S peak width. We communicated these findings to Agilent technical staff who also guided our correction approach. Of the 42 manually corrected electropherograms, 30 were reassigned lower RIN values, with 6 samples falling below the cutoff. Careful evaluation of the electropherograms at this stage is critical to identify problematic samples before sequencing.As noted previously,35 Despite its status as the current standard metric, we do recognize the growing claim that RIN values may not adequately reflect sample mRNA integrity. This is largely based on the premise that the RIN algorithm heavily weighs rRNA integrity, which recently was shown only to weakly associate with sample transcript quality.20 Considering tissue-specific differential RNA degradation and its impact on DGE analysis, alternative transcript integrity metrics, such as transcript integrity number45 or quality surrogate variable analysis,20 should also be considered for future sequencing efforts.In this study, we used RIN value assignment as the primary metric for RNA integrity and deemed RIN values ≥7.0 as acceptable for mRNA-seq.36 Through adherence to meticulous RNA-processing standards, especially with patient samples, quantitative DGE analysis will be an extremely powerful approach that advances new discoveries in pain research.We developed and validated a robust extraction protocol that combines snap freezing with cryosectioning to routinely yield high-integrity RNA from rodent and human skin biopsies. We also validated RNA extraction protocols for other tissues commonly used in the pain field. Nevertheless, pain researchers must rigorously validate their RNA extraction protocols and abide by strict quality control standards. We also advocate for better reporting standards whereby authors detail methodological information and all data pertaining to their RNA-seq workflows, the extraction approach and sample RINs notwithstanding.The authors have no conflicts of interest to declare.This study was supported by NIH grants: T32DE14318 , F32DK118841 (P.M.L.), UTHSCSA Endodontics Departmental Funds, and the USAA President's Council Endowment. RNA integrity data were generated in the Genome Sequencing Facility, which is supported by UT Health San Antonio, NIH-NCI P30CA054174 , NIH 1S10OD021805-01 (Shared Instrument grant), and CPRIT Core Facility Award (RP160732).


Viruses have developed a number of strategies to suppress host innate immunity and ensure efficient transcription and translation for the replication of new virions. One such viral strategy is to use host m5C RNA methyltransferases to modify viral RNA and thus to affect antiviral host responses. Here, we summarize the latest findings concerning the roles of m5C RNA methyltransferases, namely, NOL1/NOP2/SUN domain (NSUN) proteins and DNA methyltransferase 2/tRNA methyltransferase 1 (DNMT2/TRDMT1) during viral infections. Moreover, the use of m5C RNA methyltransferase inhibitors as an antiviral therapy is discussed.Eukaryotic 5-methylcytosine RNA methyltransferases catalyze the transfer of a methyl group to the fifth carbon of a cytosine base in RNA sequences to produce 5-methylcytosine (m Nuc. Nuc5C Rnd 50nt) ,21 as wend 50nt) . NSUN2 m(vtRNAs) ,21,23,24RNA(Met) ,26. MitoRNA(Met) . NucleolRNA(Met) ,29,30. CRNA(Met) ,32,33. Necursors  and 48ntRNA(Met) ,36.−/− mice, testis size is decreased and sperm differentiation is inhibited [−/−NSun2−/− embryos were smaller and lighter compared to their wild-type littermates. Newborn double-knockout mice also appeared smaller at birth, failed to develop visible milk spots, showed a reduced thickness and organization of the cerebral cortex and an immature skeleton with incomplete ossification [−/−NSun2−/− mice were also characterized by changes in cellular lipid storage [−/−) was significantly retarded [Diverse phenotypic features have been reported in mice lacking selected NSun proteins. NSun1 deletion induced apoptotic cell death, affected lineage specification and decreased the pools of RNA during mouse embryonic development . In NSunnhibited . Moreovefication . Dnmt2−/ storage . It has  storage . The groretarded . Moreoveretarded . In contretarded . Nsun5 kretarded . It has retarded . Mice wiretarded .5C) in the Sindbis virus 26S RNA obtained from infected host cells was revealed and the putative role of m5C during viral infection was postulated [6-methyladenosine (m6A) and a minor fraction of m5C were detected. However, the authors were unable to identify the presence of m5C in the viral RNA particles.In 1975, the presence of 5-methylcytosine  contains higher levels of m6A, m5C, and 2′O-methylated (Nm) ribonucleotides than cellular mRNA [5C and 2′O-methylations have been observed in human immunodeficiency virus 1 (HIV-1) RNA compared to these noticed in typical cellular mRNAs [5C accounted for 0.6–1.4% of cytosines in HIV-1 RNA (approximately 11 m5Cs per HIV-1 genomic RNA) [Recent studies using ultrahigh-performance liquid chromatography (UHPLC), tandem mass spectrometry (UPLC-MS/MS), and photo-cross-linking-assisted mlar mRNA . Similarar mRNAs . Courtnemic RNA) .It is worthwhile to mention that methylation occurs the most frequently in CpG regions of DNA and RNA viruses ,49,50. M5C signals in viral RNA of Coronaviridae family [5C modification occur at consistent positions between subgenome-length mRNAs [Coronaviridae family, is not well established and requires further comprehensive studies.The analysis of RNA modification using direct RNA sequencing also revealed me family ,54. Moreth mRNAs . Thus, oth mRNAs , namely,th mRNAs ,58. Howe5C RNA methyltransferases may play an important role during the recognition of viral CpG and the inhibition of replication process of selected types of viruses. For example, fruit fly m5C RNA methyltransferases Dnmt2 has been reported to directly interact with Drosophila C virus (DCV) RNA being a part of antiviral defense strategy as an evolutionarily conserved innate immune response [It is known that experimental increase in the pools of CpG dinucleotides in CpG-deficient viral genomes may lead to significant decrease in viral replication and virulence ,60,61. Tresponse ,63  are deaminase enzymes (cytidine-to-uridine editing enzymes) that allow for editing of RNA/ssDNA sequences and may promote diversity is mRNA editing [APOBEC3A, APOBEC3B, APOBEC3C, APOBECDE, APOBEC3F, APOBEC3G, and APOBEC3H [5C, while APOBEC3G is much weaker in its ability to deaminate m5C [5C RNA methyltransferases may protect some viruses against mutagenic activity of cytidine deaminase that may limit cytidine deaminase-mediated lethality.To the best of our knowledge, little is known about the effects of m editing . ApolipoAPOBEC3H . APOBEC3APOBEC3H . In partAPOBEC3H ,67. APOBAPOBEC3H . APOBEC3nate m5C . More renate m5C . This munate m5C . This maThe transcriptome of host cells infected with several RNA viruses, e.g., Zika virus (ZIKV), dengue virus (DENV), HCV, poliovirus, and HIV-1, has been reported to be post-transcriptionally modified . This ma5C RNA methyltransferases can be exploited to sustain viral transcriptional and translational activity by the incorporation of 5-methylcytosine into viral mRNA and/or host tRNA and rRNA [5C RNA methyltransferases in RNA modification-based regulation of transcription and translation upon viral infections.Viral infections are also accompanied by cellular stress responses in the host cells that may affect proper transcription and translation of viral mRNA and proteins, respectively . Thus, mand rRNA . Indeed,5C methylation of TAR RNA [It has been postulated that NSUN1 (NOP2), a member of ribosomal large subunit assembly complex, may play the role in the regulation of the cell cycle and in the nucleolar activity associated with cell proliferation . Kong et TAR RNA   that modulates cellular metabolic responses. eRNA methylation stabilizes eRNA-bound protein complex and enhances RNA polymerase II activity [Depletion of NSUN7 decreased the levels of mactivity . This maactivity   database showed that expression levels of both 5-methylcytosine rRNA methyltransferase genes in cells infected with Ebola virus (EBOV) (GSE17509), influenza A virus (IAV) subtype H5N1 (A/H5N1) (GSE43302), SARS-BatS RBD (GSE47960), and HCV JFH-1 strain (GSE20948) were altered suggesting that NSUN3 and NSUN4 proteins are active members of the cellular viral response pathway.Here, we hypothesize that mitochondrial NSUNs are essential for the maintenance of mitochondrial RNA stability and loss of methylation may lead to the transfer of nonfunctional mtRNAs into the cytoplasm. mtRNAs can be recognized by MDA5 and bind to MDA5, a well-known pattern-recognition receptor for RNA that induces a type I interferon response . This hyMoreover, NSUN3 may have distinct roles beyond the methylation of mt-tRNA(Met) I ,26,80. CNSUN2, a multifunctional methyltransferase, is also required for biogenesis of tRNA-derived non-coding fragments (tRFs)  Figure . tRFs inIn addition, C(5)-methylation of mRNAs regulates mRNA export . MethylaNSUN2 controls processing of vault ncRNAs into small regulatory RNAs with microRNA functions such as the three vtRNAs . vtRNA cDuring SARS-CoV infection, small noncoding RNAs were also detected. The analysis of deep sequenced RNA isolated from the lungs of infected mice showed three 18–22 nt small viral RNAs (svRNAs). The three svRNAs were derived from the nsp3 (svRNA-nsp3.1 and -nsp3.2) and N (svRNA-N) genomic regions of SARS-CoV . These o5C RNA methyltransferases in the antiviral response, one can ask whether the inhibitors of these enzymes can be considered as antiviral drugs. Nucleotide and nucleoside analogs are examples of broad-spectrum antiviral drugs that inhibit transcription and/or replication of different RNA and DNA viruses [Taking into account the involvement of m viruses . Pharmac viruses .Interestingly, DNA demethylation responses of azacytidine and decitabine drugs are not restricted to the inhibition of DNA methylation . SchaefeZebularine (pyrimidin-2-one ribonucleoside), a cytidine analog that acts as a DNA demethylase inhibitor as well as a cytidine deaminase inhibitor, can be also considered as an antiviral drug . HoweverSinefungin (A9145) and its related metabolite, A9145C, are analogs of the methyl donor SAM and inhibitors of RNA and DNA methyltransferases . SinefunTea polyphenol epigallocatechin-3-gallate (EGCG) is also another promising inhibitor of DNA/RNA methyltransferases. It was shown that EGCG can inhibit DNA methyltransferase and reactivate methylation-silenced genes in cancer cell lines .5C scaffold.Halby et al. developed a convergent synthetic pathway, starting from a protected bromomethylcytosine derivative, to synthesize transition state analogs of DNA methyltransferases . However5C RNA methyltransferases can be used in the future to modulate virus-mediated effects in host cells. However, several issues need to be addressed, namely, efficiency, selectivity, bioavailability, and biocompatibility of putative inhibitors in biological systems. The side effects of their use can be also a serious problem since these compounds can inhibit the enzyme activity of both virus-infected and virus-free cells. For instance, the use of 5-aza-2′-deoxycytidine can result in thrombocytopenia and anemia and less frequently can lead to congestive cardiac failure and tachycardia (http://sideeffects.embl.de). Moreover, it has been documented that high dose of azacytidine or 5-aza-2′-deoxycytidine may cause renal dysfunction [5C in the binding to the catalytic sites of enzymes and the analogs are better matched structurally that may result in permanent inhibition and degradation of these enzymes [5C RNA methyltransferases can be improved by the addition of functional groups to the analogs [5C analogs based on the mechanism of cationic carriers that would allow for more efficient transport. Techniques for obtaining the oligonucleotide analogs with cationic backbone linkages were previously proposed [5C analogs was also proposed, namely mesoporous silica nanoparticles [5C RNA methyltransferase inhibitors can be improved by the use of nanocapsules that may serve as a promising technology for future antiviral therapies. The unique structure of nanocapsules may allow for proper loading of nucleoside analogs, oligonucleotides with nucleoside analogs, tag-like ligands, receptors, additional dyes, or other compounds that may support the monitoring and control of drug release. More studies are needed to better characterize the molecular bases of the biological action and therapeutic effects of m5C RNA methyltransferase inhibitors and their putative side effects in vitro and in vivo.In summary, we hypothesize that inhibitors of mfunction  and sperfunction  as well function . Efficie enzymes . Another analogs . A good proposed . Anotherproposed ,121. Theproposed . Improviproposed . An altearticles . In this


The electrochemical measurements are performed between the potentials at −0.3 V and +0.3 V with 1 mM [Fe(CN)6] −3/−4. After dropping the cuco2o4 on the SCPE (screen carbon printed electrode), the sensor is turned on due to the high electrochemical properties. Then, p19 proteins move into the hallow structure and inhibit the exchange of electrochemical reactions between the shells and the sensor is turned off. Then, adding the duplexes of RNA/miRs cause to increase the electrochemical property of p19 due to the change of p19 conformation and the system is turned on, again. So, for the first time, a virus-like hallow structure has been used to detect the 21miR in the human serum, MCF-7, Hella cells, with high sensitivity, specificity, and reproducibility in few minutes.Electrochemical sensors are the tools to detect the accurate and sensitive miRs. There is the challenge to increase the power and sensitivity of the surface for the electrochemical sensor. We design a virus-like hallow structure of cuco So, these molecules have been important as diagnostic and prognostic tools in many cancers3. A wide range of nanoparticles, nanorods, nanowires, nanoclusters, nanocomposites, etc, have been used in the electrochemical biosensor fabrication to detect of miRNA5. Hollow nanostructures offer promising potential for advanced energy storage and electrochemical applications. The higher weight of the multi-shelled hollow nanostructures can cause to increase the energy density7. They are designed with a hybrid of internal architectures such as core(s)-in-hollow- shell(s), multishell and multi-chambers9. P19 protein is a suppressing factor in the silencing pathway as a common form of antiviral defense. This protein is used as a nanotechnology tool for micro RNA quantitation11. This protein naturally develops from the CIRV . Cryo-EM of the CIRV particle showed that they have internal shells with the separates section. The outer surfaces of the shells have a negative charge, and the inner surfaces have a positive charge due to pH-induced morphological changes of proteinaceous viral shells13. It was demonstrated that p19 and other proteins staying in the virus based on the changes in the charges14. There is some research about core-shell structures via virus mimicking for medical applications15. In this research; we have constructed the nanomaterials based on the inspiration of the inherent structure of CIRV to use as biosensor applications. This structure consists of a mixture of metals such as cobalt and copper and oxygen (cuco2o4) which generate shells with positive and negative charges that they can create a strong bed surface. For the first time, these structures have been used in the design of electrochemical sensors to the identification of miR with high sensitivity. After dropping the cuco2o4 hallow structures on the SCPE-GNP (screen carbon printed electron-gold nanoparticle) electrode, the chemical reaction has increased significantly due to the cobalt/copper-emitting conductive structure with inner shells and outer. Adding the p19 protein to this sensor cause to move the shells via pore by mimicking of CIR virus. These proteins prevent the electrochemical exchange of ions into the shell and outer space, and the sensor resistance is reduced and switched off. Then, we add a duplex of 21miR/RNA which they moved into the shells and connect with p19 tightly. The change in p19 conformation induced more electrochemical reactions and the sensor is on, again. Because of the high p19 content, the sensitivity of this sensor is very high (1 aM). We use this sensor to detect 21miR in a real sample . So this virus-like sensor can identify the miRs with high sensitivity, reproducibility, and specificity.MicroRNAs are endogenous, single-stranded, non-coding small RNA with a length of ~22 nucleotide (nt). The alterations in the expression of these molecules have been linked to many diseases and cancers16. Screen-printed carbon electrode modified with gold nanoparticles on the ceramic substrate was purchased from DropSens Inc. This electrode consisted of a GNP-carbon working electrode; a carbon counter electrode and a silver reference electrode.All of the chemical materials such as analytical grade potassium ferrocyanide, potassium ferricyanide, sulfuric acid, hydrogen peroxide, sodium chloride, and potassium chloride and cobalt and copper were purchased from chimera co. Glutaraldehyde and Trizol were provided from nano alvand Co. Synthetic oligonucleotides were prepared by MW GE biotech, Ebersberg, Germany. We used as extraction RNA kit and commercially human serum from cinnagen Co (IRAN). The following sequences were used for RNA sensing experiments Probe: 5p′-ACAACAUCAGUCUGAUAAGCUA-3′ (21) miR-21: 5′- UAG CUUAUCAGACUGAUGUUGA -3′ (RNA) mismatch RNA: 5′- GUA GACAAGCAUGACGUACCUG -3′(RNA). P19 was prepared from New England Bio Labs Inc and used without further purification. P19 siRNA Binding Protein (10 units/ml) was stored at −20 °C6]−3/−4 in phosphate Buffer (PS) buffer in the potential window −0.3 V to+0.3 V at a scan rate 50mVs 1. The frequency range used for impedance measurement was 100 kHz to 1 Hz of [Fe (CN) 6]−3/−4 in PBS pH = 7.417. Analysis of EIS spectra was done using equivalent circuits using ZSimpWin 3.22 (Princeton Applied Research) and results reported in Nyquist plots. The AFM and TEM images were gathered using TecnaiG20 instruments from FEI Company and Hillsboro, USA. Particle sizes and zeta potentials were achieved from Horiba nanoparticle size analyzer, Malvern nano SZ-100 which uses green laser light at wavelength 532 nm.In this study, we used as SP-300 Instruments (SP-300) Texas to detect all of electrochemical analyzes such as electrochemical impedance spectroscopy (EIS) and differential pulse voltammetry (DPV). DPV measurements were done in the presence of 1 mM −3/−4 redox couple. To evaluation the sensitivity of miR, increasing concentrations (1 a.M to 500 p.M) of miR21, were incubated with 1 μM of RNA probe in a 100 μL total volume of PBS at 37 °C for 1 h after incubation of 10 μL of the reaction mixture with cuco2o4/p19 sensor for 1 h at room temperature, and then washing 3 times with 500 μL of phosphate buffer (PB) and electrochemical reactions were done16. To examine the effect of the amount of p19 in limit of detection of 21miR, we dropped 0.4 µl of p19 on the sensor with the increasing concentrations (1 a.M to 500 p.M) as the same of conditions.Identification of the specificity of the sensor was done for three electrodes. So, three separate electrodes were incubated with 1 μM of the RNA probe (for detection miR21) with 1 μM of the miR-21 and 1 μM of RNA (mismatch) and one electrode as bare which they incubated in the buffer for 24 h at 4 °C under similar experimental conditions. DPV and EIS techniques were used for monitoring the electrochemical reactions in PBS buffer (pH 7.4) in the presence of the −3/−4 redox probe in PBS buffer on the GNP-SCPE , which was an effective method for the characteristic of surface-modified electrodes20. Nyquist showed the electron transfer by impedance spectra include a semicircle portion. The model was used for this sensor is R (Q(RW))(QR). Figure 2o4/electrode, (c) cuco2o4/p19, (d) negative control sample in 10 mM [Fe(CN)6]−3/−4 and 0.1 M KCl at open circuit potential with frequencies varying from 0.1 Hz to 100 kHz. As shown in Fig. et value of approximately 210 Ω. When cuco2o4 was added on the electrode, the Ret value increased by about 330 Ω  92 mV in the [Fe (CN)6]−3/−4 redox probe 6]−3/−4 to the electrode surface when it connected with the miR/RNA duplex. Since the resistance of the sensor in this stage is very high, it may become saturated p19 in every cuco2o4 structure. Therefore, it can connect with the duplexes of 21miR/RNA. Results of the unit value of the electrochemical tests were reported in the different stages in Table (s.1). On the hands, there are many cuco2o4 structures on the SCPE-GNP which they can behave as electrochemical reservists  was examined by field-emission scanning electron microscopy (FESEM) and transmission electron microscopy (TEM). Figure 2O4). It is visible that the samples have homogeneous microspheres morphology with a diameter of about 900 nm. The rough and wrinkle on the surface of the microspheres demonstrate a large-scale porous and the formation of a thin shell composed of small nanoparticles. As shown in Fig. 2o4 /p19 nanocomposite also prove that there is no interaction of external factors on the surfaces was observed and no change about CuCo2O4 sizes .The morphology of bimetallic  decreased and RCT increased. In Fig. −2) and attachment with non-complementary (target II) of 21 miR caused an 18.1%, increase in the j value (9.32 μAcm−2), respectively. DPV images of hybridization with mismatch RNA of miR21-probe indicated 22.9%, 22.32% increase in the j value (10.78 μAcm−2). The most important power of this study is the high diagnostic detection. Each nanocomposite structure of cuco2o4 can have a reservoir of p19 in the shell. In this study, we optimized the concentration of p19  to reaction with duplexes of 21mir at the same ranges . The current concentration curve showed a linear relationship in the range of 0.1 μl to 0.5 μl of P19 concentration, with a sensitivity of 0.2 μl of 21miR (9033.4 μA mM−1 cm−2). Our result showed that in the least concentration of p19 (0.1 μl), there is a high detection of 21miR . Results showed in Fig. 2 = 0.976) was obtained, where y is the j value in μA cm−2 and x is the logarithmic concentration of 21miR in aM. As shown in Fig. 2 = 0.976) was obtained, where y is the j value in μLA cm−2 and x is the logarithmic concentration of miR21 in aM. As shown in Fig. 23. These results may be due to the qualification of p19 connection with RNA/miR. Because of the p19 is stayed into the cuco2o4 structure in this model, the probability of the connection between the duplex and p19 is more than other p19-biosensors when they fix on the sensor  isolated from these cells. The hallow structure biosensor response was significantly different for MCF-7 and MCF-10A cells, serum human RNA, thus indicating that miR-21 is highly expressed only in the metastatic cell lines (MCF-7), and not in the MCF-10A and HeLa cell lines . We also evaluated the discrimination efficiency towards the tested sequence 1 m by spiking RNAt (1.0 mg) extracted from MCF-10A cells and with the synthetic target  and 1 m oligonucleotides  and MCF-7 cells RNA with the (17.4) fmol of miR-21 per microgram of RNA and 16.1 fmol of miR-21 per microgram of RNAt extracted from Hella cells. Results show the best signal detection of cuco2o4 biosensor is for human serum RNA > MCF-7 cells RNA > Synthetic 21 miR > MCF-10 cells RNA = Hela cells  related to the sensitivity of other p19-biosensor34. On the hand, we evaluated this sensor in the real samples and we get the same acceptable results. These structures can open a new window for a wide range of medical purposes. As a result, the sensor has been able to detect miR with very high sensitivity and stability and high repeatability. In the next research, the other hallow structure should be studied as biosensor applications in the diagnosis and therapy.The large studies were researched about the design of the electrochemical sensors for identification micro RNAsSupplemental information.