RNA Fragments Research Articles

A nanoparticle-based molecular beacon for directly detecting attomolar small RNA from plasma without purification

Molecular beacons (MBs) are DNA-based probes that detect DNA or RNA fragments and hold promise for monitoring diseases and studying protein-nucleic acid interactions. MBs usually use fluorescent molecules as fluorophores for reporting the target detection event. However, the fluorescence of the traditional fluorescent molecules can bleach and even be interfered with the background autofluorescence, reducing the detection performance. Hence, we propose to develop a nanoparticle-based MB (NPMB) that uses upconversion nanoparticles (UCNPs) as a fluorophore, which can be excited by near-infrared light to avoid background autofluorescence and thus enables us to detect small RNA from complicated clinical samples such as plasma. Specifically, we employ a DNA hairpin structure, with one segment complementary to the target RNA, to position a quencher (gold nanoparticles, Au NPs) and the UCNP fluorophore in close proximity, leading to the quenching of the fluorescence of UCNPs in the absence of a target nucleic acid. Only when the hairpin structure is complementary with the detection target, will the hairpin structure be destroyed to separate Au NPs and UCNPs, resulting in the instant recovery of the fluorescence signal of UCNPs and the consequent ultrasensitive detection of the target concentrations. The NPMB has an ultra-low background signal because UCNPs can be excited with NIR light with a wavelength longer than the emitted visible light. We demonstrate that the NPMB can successfully detect a small (22-nt) RNA (using a microRNA cancer biomarker, miR-21, as an example) and a small single-stranded DNA (complementing the cDNA of miR-21) in aqueous solutions from 1 aM to 1 pM, with the linear detection range being 10 aM to 1 pM for the former and 1 aM to 100 fM for the latter. We further show that the NPMB can be used to detect unpurified small RNA (miR-21) in clinical samples such as plasma with the same detection region. Our work suggests that the NPMB is a promising label-free and purification-free method for detecting small nucleic acid biomarkers in clinical samples with a detection limit as low as the aM level.

Led-Seq:ligation-enhanced double-end sequence-based structure analysis of RNA.

Structural analysis of RNA is an important and versatile tool to investigate the function of this type of molecules in the cell as well as in vitro. Several robust and reliable procedures are available, relying on chemical modification inducing RT stops or nucleotide misincorporations during reverse transcription. Others are based on cleavage reactions and RT stop signals. However, these methods address only one side of the RT stop or misincorporation position. Here, we describe Led-Seq, a new approach based on lead-induced cleavage of unpaired RNA positions, where both resulting cleavage products are investigated. The RNA fragments carrying 2', 3'-cyclic phosphate or 5'-OH ends are selectively ligated to oligonucleotide adapters by specific RNA ligases. In a deep sequencing analysis, the cleavage sites are identified as ligation positions, avoiding possible false positive signals based on premature RT stops. With a benchmark set of transcripts in Escherichia coli, we show that Led-Seq is an improved and reliable approach based on metal ion-induced phosphodiester hydrolysis to investigate RNA structures in vivo.

MiR-483-5p offsets functional and behavioural effects of stress in male mice through synapse-targeted repression of Pgap2 in the basolateral amygdala

Severe psychological trauma triggers genetic, biochemical and morphological changes in amygdala neurons, which underpin the development of stress-induced behavioural abnormalities, such as high levels of anxiety. miRNAs are small, non-coding RNA fragments that orchestrate complex neuronal responses by simultaneous transcriptional/translational repression of multiple target genes. Here we show that miR-483-5p in the amygdala of male mice counterbalances the structural, functional and behavioural consequences of stress to promote a reduction in anxiety-like behaviour. Upon stress, miR-483-5p is upregulated in the synaptic compartment of amygdala neurons and directly represses three stress-associated genes: Pgap2, Gpx3 and Macf1. Upregulation of miR-483-5p leads to selective contraction of distal parts of the dendritic arbour and conversion of immature filopodia into mature, mushroom-like dendritic spines. Consistent with its role in reducing the stress response, upregulation of miR-483-5p in the basolateral amygdala produces a reduction in anxiety-like behaviour. Stress-induced neuromorphological and behavioural effects of miR-483-5p can be recapitulated by shRNA mediated suppression of Pgap2 and prevented by simultaneous overexpression of miR-483-5p-resistant Pgap2. Our results demonstrate that miR-483-5p is sufficient to confer a reduction in anxiety-like behaviour and point to miR-483-5p-mediated repression of Pgap2 as a critical cellular event offsetting the functional and behavioural consequences of psychological stress.

MSP1 encodes an essential RNA-binding pentatricopeptide repeat factor required for nad1 maturation and complex I biogenesis in Arabidopsis mitochondria.

Mitochondrial biogenesis relies on nuclearly encoded factors, which regulate the expression of the organellar-encoded genes. Pentatricopeptide repeat (PPR) proteins constitute a major gene family in angiosperms that are pivotal in many aspects of mitochondrial (mt)RNA metabolism (e.g. trimming, splicing, or stability). Here, we report the analysis of MITOCHONDRIA STABILITY/PROCESSING PPR FACTOR1 (MSP1, At4g20090), a canonical PPR protein that is necessary for mitochondrial functions and embryo development. Loss-of-function allele of MSP1 leads to seed abortion. Here, we employed an embryo-rescue method for the molecular characterization of msp1 mutants. Our analyses reveal that msp1 embryogenesis fails to proceed beyond the heart/torpedo stage as a consequence of a nad1 pre-RNA processing defect, resulting in the loss of respiratory complex I activity. Functional complementation confirmed that msp1 phenotypes result from a disruption of the MSP1 gene. In Arabidopsis, the maturation of nad1 involves the processing of three RNA fragments, nad1.1, nad1.2, and nad1.3. Based on biochemical analyses and mtRNA profiles of wild-type and msp1 plants, we concluded that MSP1 facilitates the generation of the 3' terminus of nad1.1 transcript, a prerequisite for nad1 exons a-b splicing. Our data substantiate the importance of mtRNA metabolism for the biogenesis of the respiratory system during early plant life.

Identification of RNA Virus-Derived RdRp Sequences in Publicly Available Transcriptomic Data Sets.

RNA viruses are abundant and highly diverse and infect all or most eukaryotic organisms. However, only a tiny fraction of the number and diversity of RNA virus species have been catalogued. To cost-effectively expand the diversity of known RNA virus sequences, we mined publicly available transcriptomic data sets. We developed 77 family-level Hidden Markov Model profiles for the viral RNA-dependent RNA polymerase (RdRp)-the only universal "hallmark" gene of RNA viruses. By using these to search the National Center for Biotechnology Information Transcriptome Shotgun Assembly database, we identified 5,867 contigs encoding RNA virus RdRps or fragments thereof and analyzed their diversity, taxonomic classification, phylogeny, and host associations. Our study expands the known diversity of RNA viruses, and the 77 curated RdRp Profile Hidden Markov Models provide a useful resource for the virus discovery community.

Rfoot-seq: Transcriptomic RNase Footprinting for Mapping Stable RNA-Protein Complexes and Rapid Ribosome Profiling.

Ribosome profiling isolates ribosome-protected fragments for sequencing and is a valuable method for studying different aspects of RNA translation. However, conventional protocols require millions of input cells and time-consuming steps to isolate translating ribosome complexes using ultracentrifugation or immunoprecipitation. These limitations have prevented their application to rare physiological samples. To address these technical barriers, we developed an RNase footprinting approach named Rfoot-seq to map stable transcriptomic RNA-protein complexes that allows rapid ribosome profiling using low-input samples (Li, Yang, Stroup, Wang, & Ji, 2022). In this assay, we treat a cell lysate with concentrated RNase without complex crosslinking and retained only RNA footprints associated with stable complexes for sequencing. The footprints in coding regions represent ribosome-protected fragments and can be used to study cytosolic and mitochondrial translation simultaneously. Rfoot-seq achieves comparable results to conventional ribosome profiling to quantify ribosome occupancy and works robustly for various cultured cells and primary tissue samples. Moreover, Rfoot-seq maps RNA fragments associated with stable non-ribosomal RNA-protein complexes in noncoding domains of small noncoding RNAs and some long noncoding RNAs. Taken together, Rfoot-seq opens an avenue to quantify transcriptomic translation and characterize functional noncoding RNA domains using low-input samples. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Harvesting and lysing adherent cells Alternate Protocol 1: Harvesting and lysing suspension cells Alternate Protocol 2: Harvesting and lysing primary tissue samples Basic Protocol 2: RNase treatment and footprint purification for low-input samples Alternate Protocol 3: RNase treatment and footprint purification for ultra-low-input samples Basic Protocol 3: Library preparation for high-throughput sequencing Support Protocol: Preparation of dsDNA markers for library size selection Basic Protocol 4: Data analysis and quality control after sequencing.

Targeting Fusobacterium nucleatum through chemical modifications of host-derived transfer RNA fragments

Host mucosal barriers possess an arsenal of defense molecules to maintain host-microbe homeostasis such as antimicrobial peptides and immunoglobulins. In addition to these well-established defense molecules, we recently reported small RNAs (sRNAs)-mediated interactions between human oral keratinocytes and Fusobacterium nucleatum (Fn), an oral pathobiont with increasing implications in extra-oral diseases. Specifically, upon Fn infection, oral keratinocytes released Fn-targeting tRNA-derived sRNAs (tsRNAs), an emerging class of noncoding sRNAs with gene regulatory functions. To explore potential antimicrobial activities of tsRNAs, we chemically modify the nucleotides of the Fn-targeting tsRNAs and demonstrate that the resultant tsRNA derivatives, termed MOD-tsRNAs, exhibit growth inhibitory effect against various Fn type strains and clinical tumor isolates without any delivery vehicle in the nanomolar concentration range. In contrast, the same MOD-tsRNAs do not inhibit other representative oral bacteria. Further mechanistic studies uncover the ribosome-targeting functions of MOD-tsRNAs in inhibiting Fn. Taken together, our work provides an engineering approach to targeting pathobionts through co-opting host-derived extracellular tsRNAs.

On-chip multivariant COVID 19 photonic sensor based on silicon nitride double-microring resonators

Abstract Coronavirus disease 2019 (COVID-19) is a newly emerging human infectious disease that continues to develop new variants. A crucial step in the quest to reduce the infection is the development of rapid and reliable virus detectors. Here, we report a chip scale photonic sensing device consisting of a silicon-nitride double microring resonator (MRR) for detecting SARS-CoV-2 in clinical samples. The sensor is implemented by surface activation of one of the MRRs, acting as a probe, with DNA primers for SARS-CoV-2 RNA, whereas the other MRR is used as a reference. The performance of the sensor is determined by applying different amounts of SARS-CoV-2 complementary RNA. As will be shown in the paper, our device detects the RNA fragments at concentrations of 10 cp/μL and with sensitivity of 750 nm/RIU. As such, it shows a promise toward the implementation of label-free, small form factor, CMOS compatible biosensor for SARS-CoV-2, which is also environment, temperature, and pressure independent. Our approach can also be used for detecting other SARS-CoV-2 genes, as well as other viruses and pathogens.

SARS-CoV-2 viral RNA detection using the novel CoVradar device associated with the CoVreader smartphone app

The COVID-19 pandemic has highlighted the need for innovative approaches to its diagnosis. Here we present CoVradar, a novel and simple colorimetric method that combines nucleic acid analysis with dynamic chemical labeling (DCL) technology and the Spin-Tube device to detect SARS-CoV-2 RNA in saliva samples. The assay includes a fragmentation step to increase the number of RNA templates for analysis, using abasic peptide nucleic acid probes (DGL probes) immobilized to nylon membranes in a specific dot pattern to capture RNA fragments. Duplexes are formed by labeling complementary RNA fragments with biotinylated SMART bases, which act as templates for DCL. Signals are generated by recognizing biotin with streptavidin alkaline phosphatase and incubating with a chromogenic substrate to produce a blue precipitate. CoVradar results are analysed by CoVreader, a smartphone-based image processing system that can display and interpret the blotch pattern. CoVradar and CoVreader provide a unique molecular assay capable of detecting SARS-CoV-2 viral RNA without the need for extraction, preamplification, or pre-labeling steps, offering advantages in terms of time (∼3 h/test), cost (∼€1/test manufacturing cost) and simplicity (does not require large equipment). This solution is also promising for developing assays for other infectious diseases.

On-site treatment of hospital wastewater in a full-scale treatment plant in Germany: SARS-CoV-2 and treatment performance.

The separate, advanced treatment of hospital wastewater might be a promising approach to prevent the dissemination of residual compounds of high environmental concern, like pharmaceuticals, viruses and pathogenic microorganisms. This study investigates the performance of a full-scale, on-site treatment plant, consisting of a membrane bioreactor and a subsequent ozonation, at a German hospital. We analysed the elimination of pharmaceutical residues, microbiological parameters and SARS-CoV-2 RNA fragments. Additionally, we conducted an orienting study on the practicability of implementing targeted wastewater monitoring at a hospital. Our results demonstrate that after 10 years of stable operation, the treatment plant works highly efficiently regarding the elimination of pharmaceuticals and bacterial indicators. Elimination rates for pharmaceutical substances were above 90%, and log reductions of up to 6 log10 units for microbiological parameters were achieved. SARS-CoV-2 RNA could be detected and quantified in the influent but not in the effluent. The RNA load in the raw wastewater showed good correspondence with COVID-19 case numbers in the hospital. We showed that the full-scale on-site treatment of hospital wastewater is technically feasible and contributes to sustainable hospital effluent management and that monitoring biological markers on the building level might be a useful complementary tool for disease surveillance.

Studies on the Proteinaceous Structure Present on the Surface of the Saccharomyces cerevisiae Spore Wall.

The surface of the Saccharomyces cerevisiae spore wall exhibits a ridged appearance. The outermost layer of the spore wall is believed to be a dityrosine layer, which is primarily composed of a crosslinked dipeptide bisformyl dityrosine. The dityrosine layer is impervious to protease digestion; indeed, most of bisformyl dityrosine molecules remain in the spore after protease treatment. However, we find that the ridged structure is removed by protease treatment. Thus, a ridged structure is distinct from the dityrosine layer. By proteomic analysis of the spore wall-bound proteins, we found that hydrophilin proteins, including Sip18, its paralog Gre1, and Hsp12, are present in the spore wall. Mutant spores with defective hydrophilin genes exhibit functional and morphological defects in their spore wall, indicating that hydrophilin proteins are required for the proper organization of the ridged and proteinaceous structure. Previously, we found that RNA fragments were attached to the spore wall in a manner dependent on spore wall-bound proteins. Thus, the ridged structure also accommodates RNA fragments. Spore wall-bound RNA molecules function to protect spores from environmental stresses.

Areas of Crush Nuclear Streaming Should Be Included as Tumor Content in the Era of Molecular Diagnostics

Degenerated tissues are frequently observed in malignant tumors, but are not analyzed. We investigated whether nuclear streaming and necrosis samples could be used for genetic analysis to expand the sample pool. A total of 81 samples were extracted from small cell carcinoma and lymphoma FFPE tissue blocks and classified into three histological cohorts: 33 materials with well-preserved tumor morphology, 31 nuclear streaming samples, and 17 necrosis samples. DNA and RNA integrity numbers, percentage of RNA fragments with >200 nucleotides, and next-generation sequencing quality metrics were compared among the cohorts. DNA quality did not significantly differ between nuclear streaming materials and materials with well-preserved morphology, whereas that of the necrosis samples was inferior. RNA quality decreased in the following order: materials with well-preserved morphology > nuclear streaming > necrosis. The sequencing metrics did not differ significantly between the nuclear streaming samples and materials with well-preserved morphology, and reliable variants were detected. The necrosis samples extracted from resections exhibited sequencing failure and showed significantly fewer on-target aligned reads and variants. However, variant allele frequency did not differ among the cohorts. We revelated that DNA in nuclear streaming samples, especially within biopsies, could be used for genetic analysis. Moreover, degenerated non-tumor cells should be counted when evaluating tumor content to avoid misinterpreting the variant allele frequency.

Molecular insight into the specific interactions of the SARS-Coronavirus-2 nucleocapsid with RNA and host protein.

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) nucleocapsid protein is the most abundantly expressed viral protein during infection where it targets both RNA and host proteins. However, identifying how a single viral protein interacts with so many different targets remains a challenge, providing the impetus here for identifying the interaction sites through multiple methods. Through a combination of nuclear magnetic resonance (NMR), electron microscopy, and biochemical methods, we have characterized nucleocapsid interactions with RNA and with three host proteins, which include human cyclophilin-A, Pin1, and 14-3-3τ. Regarding RNA interactions, the nucleocapsid protein N-terminal folded domain preferentially interacts with smaller RNA fragments relative to the C-terminal region, suggesting an initial RNA engagement is largely dictated by this N-terminal region followed by weaker interactions to the C-terminal region. The nucleocapsid protein forms 10nm ribonuclear complexes with larger RNA fragments that include 200 and 354 nucleic acids, revealing its potential diversity in sequestering different viral genomic regions during viral packaging. Regarding host protein interactions, while the nucleocapsid targets all three host proteins through its serine-arginine-rich region, unstructured termini of the nucleocapsid protein also engage host cyclophilin-A and host 14-3-3τ. Considering these host proteins play roles in innate immunity, the SARS-CoV-2 nucleocapsid protein may block the host response by competing interactions. Finally, phosphorylation of the nucleocapsid protein quenches an inherent dynamic exchange process within its serine-arginine-rich region. Our studies identify many of the diverse interactions that may be important for SARS-CoV-2 pathology during infection.

Genome-wide identification of Kanamycin B binding RNA in Escherichia coli

BackgroundThe aminoglycosides are established antibiotics that inhibit bacterial protein synthesis by binding to ribosomal RNA. Additional non-antibiotic aminoglycoside cellular functions have also been identified through aminoglycoside interactions with cellular RNAs. The full extent, however, of genome-wide aminoglycoside RNA interactions in Escherichia coli has not been determined. Here, we report genome-wide identification and verification of the aminoglycoside Kanamycin B binding to Escherichia coli RNAs. Immobilized Kanamycin B beads in pull-down assays were used for transcriptome-profiling analysis (RNA-seq).ResultsOver two hundred Kanamycin B binding RNAs were identified. Functional classification analysis of the RNA sequence related genes revealed a wide range of cellular functions. Small RNA fragments (ncRNA, tRNA and rRNA) or small mRNA was used to verify the binding with Kanamycin B in vitro. Kanamycin B and ibsC mRNA was analysed by chemical probing.ConclusionsThe results will provide biochemical evidence and understanding of potential extra-antibiotic cellular functions of aminoglycosides in Escherichia coli.

Site-specific analysis reveals candidate cross-kingdom small RNAs, tRNA and rRNA fragments, and signs of fungal RNA phasing in the barley-powdery mildew interaction.

The establishment of host-microbe interactions requires molecular communication between both partners, which may involve the mutual transfer of noncoding small RNAs. Previous evidence suggests that this is also true for powdery mildew disease in barley, which is caused by the fungal pathogen Blumeria hordei. However, previous studies lacked spatial resolution regarding the accumulation of small RNAs upon host infection by B. hordei. Here, we analysed site-specific small RNA repertoires in the context of the barley-B. hordei interaction. To this end, we dissected infected leaves into separate fractions representing different sites that are key to the pathogenic process: epiphytic fungal mycelium, infected plant epidermis, isolated haustoria, a vesicle-enriched fraction from infected epidermis, and extracellular vesicles. Unexpectedly, we discovered enrichment of specific 31-33-base 5'-terminal fragments of barley 5.8S ribosomal RNA in extracellular vesicles and infected epidermis, as well as particular B. hordei transfer RNA fragments in haustoria. We describe canonical small RNAs from both the plant host and the fungal pathogen that may confer cross-kingdom RNA interference activity. Interestingly, we found first evidence of phased small interfering RNAs in B. hordei, a feature usually attributed to plants, which may be associated with the posttranscriptional control of fungal coding genes, pseudogenes, and transposable elements. Our data suggest a key and possibly site-specific role for cross-kingdom RNA interference and noncoding RNA fragments in the host-pathogen communication between B. hordei and its host barley.

Intensity of sample processing methods impacts wastewater SARS-CoV-2 whole genome amplicon sequencing outcomes

Wastewater SARS-CoV-2 surveillance has been deployed since the beginning of the COVID-19 pandemic to monitor the dynamics in virus burden in local communities. Genomic surveillance of SARS-CoV-2 in wastewater, particularly efforts aimed at whole genome sequencing for variant tracking and identification, are still challenging due to low target concentration, complex microbial and chemical background, and lack of robust nucleic acid recovery experimental procedures. The intrinsic sample limitations are inherent to wastewater and are thus unavoidable. Here, we use a statistical approach that couples correlation analyses to a random forest-based machine learning algorithm to evaluate potentially important factors associated with wastewater SARS-CoV-2 whole genome amplicon sequencing outcomes, with a specific focus on the breadth of genome coverage. We collected 182 composite and grab wastewater samples from the Chicago area between November 2020 to October 2021. Samples were processed using a mixture of processing methods reflecting different homogenization intensities (HA + Zymo beads, HA + glass beads, and Nanotrap), and were sequenced using one of the two library preparation kits (the Illumina COVIDseq kit and the QIAseq DIRECT kit). Technical factors evaluated using statistical and machine learning approaches include sample types, certain sample intrinsic features, and processing and sequencing methods. The results suggested that sample processing methods could be a predominant factor affecting sequencing outcomes, and library preparation kits was considered a minor factor. A synthetic SARS-CoV-2 RNA spike-in experiment was performed to validate the impact from processing methods and suggested that the intensity of the processing methods could lead to different RNA fragmentation patterns, which could also explain the observed inconsistency between qPCR quantification and sequencing outcomes. Overall, extra attention should be paid to wastewater sample processing (i.e., concentration and homogenization) for sufficient and good quality SARS-CoV-2 RNA for downstream sequencing.

Preparation of DNA and RNA Fragments Containing Guanine N2 -Thioalkyl Tethers.

This article describes procedures for preparation of deoxyguanosine and guanosine derivatives in which the guanine N2 contains a thiopropyl tether, protected as a tert-butyl disulfide. After incorporation into a DNA or RNA fragment, this tether allows site-specific cross-linking to a thiol of a protein or another nucleic acid. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Preparation of diisopropyl-1-(tert-butylthio)-1,2-hydrazinedicarboxylate (4) Basic Protocol 2: Preparation of the 2'-deoxyguanosine N2 -propyl-tert-butyl disulfide phosphoramidite (12) Basic Protocol 3: Preparation of the guanosine N2 -propyl-tert-butyl disulfide phosphoramidite (20) Basic Protocol 4: Preparation of DNA fragments containing N2 -propyl-tert-butyl disulfide guanine Alternate Protocol: Preparation of RNA fragments containing N2 -propyl-tert-butyl disulfide guanine Basic Protocol 5: Conversion of N2 -propyl-tert-butyl disulfide to the free thiol, disulfide 5-thio-2-nitrobenzoic acid disulfide, or ethylamine disulfide.

Modified Nucleotides for Chemical and Enzymatic Synthesis of Therapeutic RNA.

In recent years, RNA has emerged as a medium with a broad spectrum of therapeutic potential, however, for years, a group of short RNA fragments was studied and considered therapeutic molecules. In nature, RNA plays both functions, with coding and non-coding potential. For RNA, like any other therapeutic, to be used clinically, certain barriers must be crossed. Among them, there are biocompatibility, relatively low toxicity, bioavailability, increased stability, target efficiency and low off-target effects. In the case of RNA, most of these obstacles can be overcome by incorporating modified nucleotides into its structure. This may be achieved by both, in vitro and in vivo biosynthetic methods, as well as chemical synthesis. Some advantages and disadvantages of each approach are summarized here. The wide range of nucleotide analogues has been tested for their utility as monomers for RNA synthesis. Many of them have been successfully implemented, and a lot of pre-clinical and clinical studies involving modified RNA have been carried out. Some of these medications have already been introduced into clinics. After the huge success of RNA-based vaccines that were introduced into widespread use in 2020, and the introduction to the market of some RNA-based drugs, RNA therapeutics containing modified nucleotides appear to be the future of medicine.

Engineered exosome-mediated messenger RNA and single-chain variable fragment delivery for human chimeric antigen receptor T-cell engineering

Background aimsMost current chimeric antigen receptor (CAR) T cells are generated by viral transduction, which induces persistent expression of CARs and may cause serious undesirable effects. Messenger RNA (mRNA)-based approaches in manufacturing CAR T cells are being developed to overcome these challenges. However, the most common method of delivering mRNA to T cells is electroporation, which can be toxic to cells. MethodsThe authors designed and engineered an exosome delivery platform using the bacteriophage MS2 system in combination with the highly expressed protein lysosome-associated membrane protein 2 isoform B on exosomes. ResultsThe authors’ delivery platform achieved specific loading and delivery of mRNA into target cells and achieved expression of specific proteins, and anti-CD3/CD28 single-chain variable fragments (scFvs) expressed outside the exosomal membrane effectively activated primary T cells in a similar way to commercial magnetic beads. ConclusionsThe delivery of CAR mRNA and anti-CD3/CD28 scFvs via designed exosomes can be used for ex vivo production of CAR T cells with cancer cell killing capacity. The authors’ results indicate the potential applications of the engineered exosome delivery platform for direct conversion of primary T cells to CAR T cells while providing a novel strategy for producing CAR T cells in vivo.

ALL-tRNAseq enables robust tRNA profiling in tissue samples.

Transfer RNAs (tRNAs) are small adaptor RNAs essential for mRNA translation. Alterations in the cellular tRNA population can directly affect mRNA decoding rates and translational efficiency during cancer development and progression. To evaluate changes in the composition of the tRNA pool, multiple sequencing approaches have been developed to overcome reverse transcription blocks caused by the stable structures of these molecules and their numerous base modifications. However, it remains unclear whether current sequencing protocols faithfully capture tRNAs existing in cells or tissues. This is specifically challenging for clinical tissue samples that often present variable RNA qualities. For this reason, we developed ALL-tRNAseq, which combines the highly processive MarathonRT and RNA demethylation for the robust assessment of tRNA expression, together with a randomized adapter ligation strategy prior to reverse transcription to assess tRNA fragmentation levels in both cell lines and tissues. Incorporation of tRNA fragments not only informed on sample integrity but also significantly improved tRNA profiling of tissue samples. Our data showed that our profiling strategy effectively improves classification of oncogenic signatures in glioblastoma and diffuse large B-cell lymphoma tissues, particularly for samples presenting higher levels of RNA fragmentation, further highlighting the utility of ALL-tRNAseq for translational research.