Thermococcus kodakarensis to identify its biological targets. A comparative analysis of circular RNA reveals that the Rnl-knockout strain represses circularization of C/D box sRNAs without affecting the circularization of tRNA and rRNA processing intermediates. Recombinant archaeal Rnl could circularize C/D box sRNAs with a mutation in the conserved C/D box sequence element but not when the terminal stem structures were disrupted, suggesting that proximity of the two ends could be critical for intramolecular ligation. Furthermore, T. kodakarensis accumulates aberrant RNA fragments derived from ribosomal RNA in the absence of Rnl. These results suggest that Rnl is responsible for C/D box sRNA circularization and may also play a role in ribosomal RNA processing.RNA ligases play important roles in repairing and circularizing RNAs post-transcriptionally. In this study, we generated an allelic knockout of ATP-dependent RNA ligase (Rnl) in the hyperthermophilic archaeon  Numerous RNA molecules are involved in controlling gene expression to maintain cellular RNA metabolism. In addition to tRNA, mRNA and rRNA, cells also contain a striking diversity of additional RNA types, such as edited RNAs, circularized RNAs, trans-spliced RNAs, and other non-coding RNAs . We hypoATP-dependent RNA ligase (Rnl) catalyzes the formation of phosphodiester bonds between the 5′-phosphate and 3′-hydroxyl termini of RNA . Rnl canPyrococccus abyssi (PabRnl) and Methanobacterium thermoautotrophicum Rnl (MthRnl) have been solved and were shown to catalyze an intramolecular ligation of single-stranded RNA to form a covalently closed circRNA  and analyzed the change in RNA metabolism using high-throughput RNA-Seq technology. We showed that deletion of TkoRnl selectively dissipates circular C/D box sRNAs and other small RNA species. The conserved C/D box sequence elements were not strictly required for ligation activity of the archaeal Rnl. We also found that deletion of TkoRnl produces aberrant rRNA fragments, suggesting that TkoRnl may also participate in rRNA maturation process.Here we generated an allelic knockout of the Rnl gene in T. kodakarensis KUW1 . For RNA isolation and growth analysis, cells were cultured in MA-YT-P medium  which lacks elementary sulfur.The sis KUW1  and genesis KUW1 . ASW-AA sis KUW1 . ElementTK1545 gene along with its 5′- and 3′-flanking regions (∼1.0 kbp) was amplified with forward (5′ACTCTCTCCTTTTCTCCAATTTCGG-3′) and reverse (5′-TCAGGATTTTGCAAA GTACTGACTGG-3′) primers using T. kodakarensis KUW1 genomic DNA as a template for PCR and was cloned into the Hinc II site of pUC118 to obtain pUC-TK1545. The RNL coding sequence in pUC-TK1545 was replaced with a DNA fragment containing pyrF gene (TK2276: orotidine 5′-phosphate decarboxylase) and its promoter element as described (-TK1545 using two outward primers (5′-AGCTGTAAGGGGCCTGTGGACATTTC-3′ and 5′- GATATCACCGAGAAGAGTGGGAGC-3′) complementary to the upstream and downstream sequence of the TkoRNL coding sequence. The amplified plasmid fragment was ligated with a PvuII-PvuII restriction fragment (763 bp) containing the pyrF marker gene derived from pUD2 plasmid  were spread onto ASW-AA-S0 solid medium and incubated at 85°C for 3 days. Only cells that obtained a phenotype exhibiting uracil prototrophy by homologous recombination can grow in the absence of uracil. Single colonies were selected and then cultured in ASW-YT-S0 medium at 85°C for 10 h. Throughout this article, the term wild-type (WT) refers to KUW1 and tk1545 KO refers to TkoRnl deletion in the KUW1 strain. The cells of the tk1545 KO strain were harvested and suspended in distilled water. Genomic DNA was extracted from the cells using phenol-chloroform treatment. The replacement of TK1545 gene with pyrF gene was verified with PCR (A DNA fragment containing the escribed . This wa plasmid . All thewith PCR .T. kodakarensis WT and tk1545 KO (1.6 L) were cultured in MA-YT-P medium at 85°C and harvested when the absorbance at 660 nm reached ∼0.7 (late-log phase). Total RNA was isolated using TRIzol reagent (Invitrogen) following the manufacture’s instruction. The total RNA and small RNA libraries were prepared using standard Illumina protocol by UB Genomics and Bioinformatics Core at The State University of New York (SUNY) at Buffalo, United States. The high-throughput RNA-sequencing was performed using Illumina HighSeq2500 technology . Raw reads were trimmed, and FastQC was used to determine the clean reads. For both WT and tk1545 KO datasets, 80 percent of the reads had Phred quality scores >35. The reads were mapped to T. kodakarensis reference genome (NC_006624.1) using bowtie2-2.2.9 with the option preset set to sensitive and alignment type set to local mode. The reads per kilo-base per million mapped reads (RPKM) were calculated using featureCount  and 3′-adaptor sequence (5′-TGGAATTCTCGGGTGCCAAGG) were trimmed using Cutadapt, with 80 percent of the reads having Phred quality scores of >35. The reads aligned to reference genome as described above. We obtained 22 million pairs of reads from the WT sample, of which 79.94% mapped to the reference genome. From the e genome .tk1545 KO were identified, respectively  in a supplied reaction buffer at 55°C for 10 min  containing total RNA (200 ng) from either WT or E. coli and purified from soluble bacterial extracts using Ni-agarose chromatography as described previously  were produced in eviously . In vitrtk1545 KO were separated using 10% denaturing polyacrylamide gel, and RNA ranging from 70 to 120 nt was isolated by elution from the gel with Tris-EDTA (TE) buffer. RNA was ethanol-precipitated and resuspended to 50 µL with TE buffer. The isolated small RNA was used for the adapter-mediated RNA cloning with SOLiD Small RNA Expression Kit (Ambion). Ligation and reverse-transcription were performed according to the manufacturers’ instructions. cDNA was amplified using Taq DNA polymerase, inserted into TOPO cloning vector (Invitrogen), and transformed into E. coli DH5α. The region of cDNA insertion within the TOPO vector was PCR amplified directly from the bacterial colonies, and product size was analyzed on 1.8% agarose gel. The majority (90%) of the plasmid recovered contained primer-dimer insert. Therefore, PCR product corresponding to >40 bp cDNA insert was regarded as a “positive” clone and was sequenced to reveal the identity of cloned RNA species. PCR product length corresponding to <40 bp insert was omitted, as it likely represented a fragment derived from a primer dimer.Total RNA (15 µg) from WT and TK1545 encoding for T. kodakarensis ATP-dependent RNA ligase (TkoRnl) was removed via homologous recombination using a non-replicating targeting vector carrying orotidine-5′-monophosphate decarboxylase gene (pyrF) flanked by ∼700 bp of upstream and downstream TK1545 DNA sequence. The plasmid was transformed into T. kodakarensis strain KUW1 (∆pryF) and the Ura+ transformants were recovered. Deletion of TK1545 locus was confirmed using PCR analysis  and elevated temperatures (93°C)  , which is (93°C) . Comparetk1545 KO strains were sequenced on an Illumina HighSeq platform sized at 51-bp and mapped uniquely to the annotated genome. The gene expression abundance was normalized using RPKM , phosphate transporter proteins (TK2060 and TK2061), sulfur reductase subunits (TK2071 and TK2072), and Acetyl-CoA acetyltransferase pathway . The genes encoded within each operon were either enhanced or reduced to a similar extent in tk1545 KO. Transcriptome analysis from the small RNA-Seq data is shown in Total and small RNAs isolated from the wild-type and ing RPKM , and sca dataset . Finallyo the WT . Many oftk1545 KO. Our criteria for detecting circular reads from the RNA-Seq data were as follows: 1) the 51-nts RNA-Seq reads containing two segments, and each segment has a minimum 20-nts match to the reference genome sequence; 2) the two matched segments within the read are encoded in the same transcriptional direction, but are positioned in inverse order in the reference genome  e genome . The pree genome . For thee genome .T. kodakarensis, 31 circRNA reads were detected from the small RNA Seq data set, many of which were derived from C/D box sRNAs  and non-coding RNAs designated here as ncRNA01, ncRNA02, and ncRNA03 . Similar results were obtained when RNA-Seq analysis of WT and tk1545 KO were analyzed on a SOLiD sequencing platform (tk1545 KO. All these circRNAs have a similar length (61–71 nts) and homology to the C/D box sRNA. The terminal ends are generally GC-rich, and sequences at the termini can hybridize to form a stem, a structural characteristic found in C/D box sRNA. Secondary structure analysis suggests that hybridization between the two terminal ends could be critical for RNA circularization  (−60). The biochemical activity of MthRnl has been extensively characterized and all amino acid residues found to be essential for the MthRnl ligation activity are conserved in TkoRnl  ; thus, Mn TkoRnl .Incubation of MthRnl with either 5′-monophosphate terminated sR42 or sR31 RNAs generates a circRNA molecule that migrates slower than the linear pRNA on a denaturing PAGE . The cirThe circularization activity was not significantly affected when the conserved C/D box sequence was substituted with different bases ; sR31-b.tk1545 KO, high-levels of circRNA reads were detected from the 16S-23S rRNA operon and tRNATrp intron , 16S circRNA-1  and 23S circRNA-1  are likely cleaved at the Bulge-Helix-Bulge (BHB) motifs by tRNA splicing endonuclease and joined by tRNA ligase RtcB to form a circular rRNA processing intermediate, as reported  located ∼100 nucleotides upstream of the mature 3′-end  near the predicted 5′-and 3′-ends of 23S rRNA  . Consistent with this finding, analysis of small RNA-Seq data reveals that the reads-coverage upstream of H98 are more abundant in the TkoRnl deletion strain than the WT. (Alignment of sequences retrieved from small RNA cloning shows that all the fragments obtained were derived from 16S and 23S rRNAs. There were no overlaps between the fragments recovered from WT and  the WT. . Taken tT. kodakarensis to determine the biological targets of archaeal Rnl. Whereas TkoRnl was not essential for the growth of T. kodakarensis under standard laboratory conditions, we showed that its absence abolishes circularization of C/D box sRNAs. The conserved C/D box sequence element, however, was not sufficient for circularization because not all C/D box sRNAs were circularized in T. kodakarensis. Furthermore, we demonstrated that recombinant Rnl was capable of forming circular C/D box sRNA with a mutation in the conserved sequence element. The archaeal Rnl could not circularize unstructured RNA of a similar length or C/D box sRNAs that have disrupted terminal stem structures. We conclude that archaeal Rnl may preferentially recognize the terminal stem, and the proximity of the two ends could be critical for intramolecular ligation.Here we generated an allelic knock-out of ATP-dependent RNA ligase in T. kodakarensis. As expected, circularization of tRNA intron or rRNA processing intermediates was not affected in the absence of Rnl, which implies that Rnl does not affect the RtcB ligation pathway. Our finding that C/D box sRNAs are prime substrates for archaeal Rnl is consistent with the previous findings that circular C/D box sRNA-Rnl complexes were detected in P. abyssi (We also detected numerous circular RNA-Seq reads derived from 16S and 23S rRNAs in . abyssi .The functional significance of circular C/D Box sRNA is unclear. C/D box sRNAs have been reported to function as a guide RNA for methylating tRNAs and rRNAs. Circular C/D Box sRNA may alter the specificity to guide RNA to regulate rRNA methylation. However, we did not detect significant differences in the expression, or the overall read coverage, of rRNA genes from the whole transcriptome RNA-seq analysis, between the WT and TkoRnl deletion strains. Comparative transcriptomics analysis revealed that TkoRnl may alter that abundance of subset of C/D Box sRNAs . TkoRnl P. furiosus, T. kodakarensis appears to excise H98 from 23S rRNA (T. kodakarensis 70S rRNA (T. kodakarensis accumulates ∼90 nts fragments consisting of a sequence that matches the H98 3′-cleavage site to the predicted 3′-end of 23S rRNA. We speculate that the excision of H98 releases the 3′-end fragment and may have accumulated in T. kodakarensis. Intriguingly, we did not retrieve the same fragments from the TkoRnl deletion strain. Instead, we recovered fragments that mapped upstream of H98 near the SRL. SRL interacts with the translational elongation factors that hydrolyze GTP during translocation (Similar to 23S rRNA , consist70S rRNA . We founlocation , and thelocation .cis-splicing” events near the 3′-end of T. kodakarensis 23S rRNA. Furthermore, TkoRnl is not likely involved in rearranging the 3′- and 5′-ends of the 23S rRNA as observed in P. furious (While it is tempting to speculate that Rnl directly participates in joining the breakage upon excision of H98, we fail to detect any RNA-Seq reads suggesting such “ furious  because  furious . TherefoT. kodakarensis, P. furiosus, P. abyssi, and Methanopyrus kandleri (T. kodakarensis and P. furiosus, H98 is excised evinced by discontinuous RNA-Seq map coverage ((Methanomicrobiales and Archaeoglobales encode Rnl, the helix equivalent of H98 is replaced with a short linker sequence (Haloferax volcanii, Nanoarchaeum equitans, Sulfolobus solfataricus, Sulfolobus acidocaldarius, and Pyrobaculum aerophilum, do not encode homolog of type-3 Rnl. Circular tRNA intron and rRNA intermediates are present in abundance, but only a modest number of circular C/D box RNAs were reported in H. volcanii, S. solfataricu, S. acidocaldarius, and N. equitans (S. acidocaldarius and P. aerophilum rRNAs were shown to retain H98 evinced by a continuous read coverage at the 3′-end (Nonetheless, the phylogenetic analysis suggests a possible link between the archaea Rnl, C/D box sRNA circularization, and H98 processing. A high abundance of circular C/D box sRNA molecules was detected in kandleri . They alkandleri  and possoverage ( and thissequence . In contequitans . The lare 3′-end . Because


Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and Clustered Regularly Interspaced Short Palindromic Repeats-Associated Proteins (CRISPR-Cas) have promising prospects in the field of nucleic acid molecular diagnostics. However, Clustered Regularly Interspaced Short Palindromic Repeats-based fluorescence detection technology is mainly hindered by proteins with conjugated double bonds and autofluorescence, resulting in high fluorescence background, low sensitivity and incompatible reaction systems, which are not conducive to automatic clinical testing. Chemiluminescence (CL) detection technology has been applied mainly owing to its greatly high sensitivity, as well as low background and rapid response. Therefore, we developed a rapid, ultrasensitive and economical detection system based on Clustered Regularly Interspaced Short Palindromic Repeats-Clustered Regularly Interspaced Short Palindromic Repeats-Associated Proteins 13a combined with magnetic beads (MBs) and chemiluminescence (CL) (Cas13a-MB-CL) to detect Influenza A (H7N9), an acute respiratory tract infectious disease. The carboxyl functionalized magnetic beads (MBs-COOH) were covalently coupled with aminated RNA probe while the other end of the RNA probe was modified with biotin. Alkaline phosphatase labeled streptavidin (SA-ALP) binds with biotin to form magnetic beads composites. In presence of target RNA, the collateral cleavage activity of Cas13a was activated to degrade the RNA probes on MBs and released Alkaline phosphatase from the composites. The composites were then magnetically separated followed by addition of ALP substrate Disodium 2-chloro-5-{4-methoxyspiro -4-yl}-1-phenyl phosphate (CDP-star), to generate the chemiluminescence signal. The activity of Associated Proteins 13a and presence of target RNA was quantified by measuring the chemiluminescence intensity. The proposed method accomplished the detection of H7N9 within 30 min at 25°C. When combined with Reverse Transcription- Recombinase Aides Amplification (RT-RAA), the low detection limit limit of detection was as low as 19.7 fM (3S/N). Our proposed MB-Associated Proteins 13a-chemiluminescence was further evaluated to test H7N9 clinical samples, showing superior sensitivity and specificity. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-Associated Proteins (CRISPR-Cas)-based diagnostics have revolutionized in the field of nucleic acid molecular diagnostics . DiffereChemiluminescence (CL) detection technology has completely avoided the shortcomings of high fluorescent signal background and easy to quench, which is becoming the most important area of biological analysis research and development . Also, tAvian influenza A (H7N9) virus is classified into subtypes based on two surface proteins, namely Hemagglutinin (HA) and Neuraminidase (NA). It is a new recombinant virus which has the ability to bind to human receptors, and can invade the epithelial cells of human lower respiratory tract and lung cells, and in mammals . Since hvia external magnetic fields, followed by addition of ALP substrate in the supernatant to generate a chemiluminescence signals. When combined with Reverse Transcription- Recombinase Aided Amplification (RT-RAA) to amplify target RNA, this method showed superior sensitivity and specificity in short times.In this study, we established a method for detecting H7N9 by recording the chemiluminescence signals of ALP utilizing RT-RAA-Cas13a-MB-CL to amplify nucleic acids. The schematic process can be seen in 2), peptone, yeast powder, agarose powder, ampicillin (Amp) and ALP-SA were purchased from Dalian Meilun Biological Technology Co., Ltd. . Tris-base, Tris hydrochloric acid (Tris HCl), Isopropyl β-d-1-thiogalactopyranoside (IPTG) and 4-hydroxyethyl piperazine ethanesulfonic acid (HEPES) were supplied by Sigma-Aldrich . Dynabeads® MyOne™ Carboxylic Acid  and HiScribe T7 Quick High Yield RNA Synthesis kit (New England Biolabs) were also used. RT-RAA nucleic acid amplification kit was purchased from Jiangsu Qitian Gene Biotechnology Co., Ltd. . The RNA probes and H7N9 gene were synthesized by was purchased from GenScript Biotech Co., Ltd. . All the oligonucleotides (Sup. Table 1) and diethylpyrocarbonate (DEPC) treated water were obtained from Sangon . The pC013-Twinstrep-SUMO-huLwCas13a (Addgene plasmid #90097) was provided by Zhang Feng’s laboratory. CDP-Star was served by Sysmex Corporation . All chemical reagents used in the study were of analytical grade and all solutions were prepared with DEPC treated water. All the hemagglutinin (HA) and neuraminidase (NA) sequences of H7N9 used in the present study were downloaded from the NCBI , and the sequences of RNA probe, crRNA and H7N9 virus and other control viruses, including seasonal influenza viruses (A/H1N1 and A/H3N2), 2009 swine-origin influenza virus A/H1N1, and avian influenza viruses (A/H5N1 and A/H9N1) were donated by Jiangsu Provincial Center for Disease Prevention and Control are shown in Sup. Table 1. And nuclease assay buffer (NAB) was prepared using 20 mM HEPES, 60 mM NaCl, 6 mM MgCl2, pH 6.8.2-morpholinoethanesulfonic acid monohydrate (MES), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC·HCl), sodium chloride (NaCl), Tween-20, magnesium chloride (MgClvia Fast protein liquid chromatography (FPLC) . Then the protein bound column was washed three times using a buffer . About 250 Units of SUMO protease (ThermoFisher) was then loaded onto the column and incubated overnight at 4°C with rotation. Finally, the column was washed with lysis buffer again and the collected solution was analyzed by 12% SDS-PAGE. Fractions containing pure LwCas13a were pooled and exchanged the buffer via a Millipore centrifugal ultrafiltration tube (Amicon Ultra) to a storage buffer . The final product was stored at −80°C.Extraction of PCO13-Twinstrep-SUMO-huLwCas13a-plasmid used the Plasmid Mini Kit (OMEGA). 50ng of plasmids were then transferred into E. coli competent cells BL21 (Cwbiotech). After transformation, 100 μL of dilutions of cells were plated on ampicillin (Amp) and Luria-Bertani (LB)-agar plate and incubated overnight at 37°C. The next day, a single colony was picked and plated in a 10 ml of LB medium (Containing 100 μL of Amp), which was used to inoculate 1 L of LB medium and shook overnight at speed of 220 r/min until the OD600 was 0.6 at 37°C. At this time, LwCas13a protein expression was induced by supplementation with IPTG to a final concentration of 500 μM. The culture was maintained at 16°C for 16 h, and the cells were then centrifuged at 4,500 g for 10 min at 4°C. Considering the stability of the Cas13a protein, all steps of protein purification were carried out at 4°C. After centrifugation, the cell pellet was harvested and resuspended in lysis buffer . Then, protease inhibitor (MCE) and lysozyme were added, followed by sonication  with following conditions: 200 W of power, ultrasonic 2.5 s and 10 s of pause for 30 min. Centrifugation was carried at 10,000 g for 60 min at 4°C and filtered the supernatant through a 220 nm filter (Sterile Millex). The filtered supernatant was applied to a 5 ml HisTrapHP nickel ion affinity column  Plasmid containing the HA or NA gene of the H7N9 virus was amplified using PrimeSTAR Max DNA Polymerase (TaKaRa), and then purified using a SanPrep PCR product purification kit (Sangon Biotech). Purified dsDNA was incubated with T7 polymerase overnight at 37°C using HiScribe T7 Quick High Yield RNA Synthesis Kit (New England Biolabs). The RNA was purified by RNA rapid concentration and purification kit (Sangon Biotech) and quantified by One drop  to prepare a gradient RNA standard solution. The crRNA was designed based on the target RNA sequence. Two complementary crRNA-DNA primers (with an appended T7 promoter sequence) were annealed with final concentration 10 μM. Then the dsDNAs were transcribed to crRNA, using the HiScribe T7 Quick High Yield RNA Synthesis kit (New England Biolabs).Taking 10 mg/ml carboxylated MBs was stored in a refrigerator at 4°C, followed by vortex on a vortex shaker for 5 min to mix the MBs. 100 μL of the mixed MBs were then washed twice with 1 ml of 100 mM MES buffer, resuspended in 800 μL of MES buffer. Then an appropriate amount of 1.25 M EDC and RNA probe were added to make a total volume of 1 ml, rotating and incubated overnight at 4°C. The next day, after blocking the MBs twice with 1 ml of TT buffer , the MBs were resuspend with 1 ml of NAB. At last, ALP was added (according to the volume ratio of ALP:MBs = 1:10) to the MBs and rotating for 1 h. The MB-RNA-ALP complexes were washed twice with 1 ml of TT buffer and stored in TT buffer at 4°C.For each test, 2 μL of primary MBs were added to the reaction solution , 100 ng of background total human RNA (purified from human PBMC), supplemented with NAB to 50 μL) and mixed on a roller mixer for 30 min. The activated LwCas13a cleaved the RNA probe and the ALP was released from the MB-RNA-ALP complexes. Non-target RNA or non-Cas13a protein served as a negative control (NC) in the detection system, and MBs complexes supplement 50 μL of NAB buffer served as background value. At the same time, MB-RNA-ALP complexes were incubated with RNase acting as a positive control, to confirm that the ALP could release from complexes and act as a signal molecule. After magnetic separation, 30 μL of the reaction solution and ALP substrate were incubated in a 96-well microplate format and the CL signal value was recorded using a microplate reader (SpectraMax M5) at 42°C for 15 min.About 1 μL of mouse RNase inhibitor, 11.25 nM purified LwCas13a, 22.5 nM crRNA, 100 ng of background total human RNA and various concentrations of target RNA were mixed with MBs complexes and were further supplemented with NAB buffer to a final volume of 50 μL for HA RNA and NA RNA detection.Improved detection assays were performed with RT-RAA nucleic acid amplification kit (Qitian Gene Biotechnology) and HiScribe T7 Quick High Yield RNA Synthesis kit (New England Biolabs) as follows: 0.48 μM forward primer, 0.48 μM reverse primer, 14 mM magnesium acetate anhydrous, 12.5 μL mM rNTP, 2.5 μLT7 Mix, various concentrations of target RNA, 2 μL murine RNase inhibitor and 25 μL Basic E-mix to 50 μL final volume. After completing optimization of the RT-RAA amplification system for the cleaving system, MB-RNA-ALP complexes were added into the reaction solution  and replenished with NAB buffer 50 μL to a final volume of 50 μL for HA RNA and NA RNA detection.H7N9 virus samples and other viruses samples, including seasonal influenza viruses (A/H1N1 and A/H3N2), 2009 swine-origin influenza virus A/H1N1, and avian influenza viruses (A/H5N1 and A/H9N1) donated by Jiangsu Provincial Center for Disease Prevention and Control were tested after amplification by RT-RAA. The testing was conducted using the same procedure as described in In our study, based on the structural features of PCO13-Twinstrep-SUMO-huLwCas13a-plasmid, the LwCas13a protein was purified by the SUMO enzyme. The molecular weight of LwCas13a containing the SUMO site and the marker protein was 155.2 kD, but it was 138.5 kD after digestion with SUMO enzyme. LwCas13a expressed in E. coli was analyzed by SDS-PAGE. Coomassie blue stained gel results showed that the IPTG significantly induced the production of Cas13a protein in E. coli , lane 2.RNase A can degrade RNA probes, so we used RNase A as a positive control to verify the RNase activity of LwCas13a. As shown in Given the non-specific absorption and steric hindrance effect, the density of probes on the surface of MBs may also affect the cleavage of RNA probes by Cas13a protein. About 1 mg MB and five different concentrations of RNA probes  were used to determine their effect on the CL intensity. We used 10 nM target RNA to activate Cas13a. R2 = 0.98) (The schematic of Cas13a-MB-CL detection system was illustrated in  = 0.98)   .The clinical performance of RT-RAA-Cas13a-MB-CL was evaluated using H7N9 virus samples donated by Jiangsu Provincial Center for Disease Prevention and Control. And the CL values of HA and NA in the two clinical samples were greater than that of negative controls , showingIn addition, the detection of H7N9 RNAs by the RT-RAA-Cas13a-MB-CL should be sequence-specific, so we tested seasonal influenza viruses (A/H1N1 and A/H3N2), 2009 swine-origin influenza virus A/H1N1, and avian influenza viruses (A/H5N1 and A/H9N1) donated by Jiangsu Provincial Center for Disease Prevention and Control (Sup. Table 1). The CL detection signals of H7N9 virus samples were significantly higher compared to other virus samples . Above aWe successfully developed a novel method for detection of H7N9 virus based on MBs, CL and CRISPR-Cas13a, overcoming the problem of high fluorescence background in CRISPR combined with fluorescence detection technology. We improved sensitivity of detection methods with RT-RAA, a novel developed amplification technique which can react at constant temperature and is compatible with CRISPR. The LOD was as low as 19.7 fM at 25°C. Compared to the traditional fluorescence detection technology, it is an innovation in the method. Since MB-RNA-ALP complexes can be prepared and stored in advance, the entire detection process can be shortened to 30 min. More importantly, this method combined with magnetic bead method has proven to be applicable for influenza virus RNA detection in complex biological samples although having an incompatible reaction system. This assay has great promise for virus detection and can be adapted to clinical large-scale screening in the future. Thus, it is expected to scale up clinical applications and can be a valuable improvement towards achieving automated testing platforms for application in clinical setups in the near future.


Wine lees are one of the main by-products produced during winemaking. Little is known about the effect of the vinification technique on the phenolic compounds and the biological activity of wine lees extracts. Wine lees collected at varying vinification sources of two grape varieties, Riesling (RL) and Pinot Noir (PN), were analyzed for total phenolic content (TPC), tannin content (TTC), their anthocyanin and phenolic profile, and the antioxidant and antimicrobial activities of their extracts. The results showed a low TPC and TTC in RL lees, which could be attributed to the varietal characteristic of RL grapes and to less skin contact during vinification. Vinification techniques modified the composition of the phenolic compounds in the lees. The results showed a good linear relationship between the antioxidant activities and the TPC and TTC, indicating that PN lees were better sources of phenolics and antioxidant activity than RL lees. The antimicrobial activity of wine lees was related to the phenolic composition rather than the quantity of total phenolics. Knowing the grape and wine processing conditions can provide some insights into the potential composition of wine lees and, hence, determine the potential economic use of the by-product. Significant growth in the New Zealand wine industry started about three decades ago. As of June 2019, the New Zealand wine industry recorded its 24th consecutive year of export growth [According to Lavelli et al. , 100 kg Wine lees are sludge (mud-like) materials generated in various processes, such as after fermentation, during storage or after authorized treatment, and it is also the residue obtained following filtration or centrifugation . Lees coIt is expected that wine lees produced commercially will have different chemical compositions and characteristics due to the fact that lees can be obtained from a range of vinification techniques due to differences in the maceration time, yeast strain, time in oak barrels, malolactic fermentation, and vast grape varieties used in the wine industry. It is reasonable to suggest that different management solutions and the potential usage of wine lees can be devised based on their chemical compositions and characteristics. However, such information is not available, and research is required to benchmark the effects of these factors. To maximize the potential use of this by-product, it is necessary to characterize a wide range of commercial lees. The present study examined the phenolics characteristics of lees from various sources, utilizing a range of the winemaking techniques of two grape varieties to improve the understanding of this material and to devise useful strategies to add value to this waste stream.Wine lees used in this study were kindly provided by Pegasus Bay Winery , Omihi Hills Winery , Felton Road Winery , and Vinpro Winery  of New Zealand between April and August of the wine production season . A totalg for 5 min using a Megafuge 1.0 with a Siehe rotor . The second step of the extraction repeated the procedure above. Supernatants from each step of extraction of the subsampled lees samples were decanted, mixed, and stored at −20 °C. Wine lees and sample preparation were done in triplicate.Frozen lees samples were freeze-dried  at a pressure of 0.5 mbar. Freeze-dried (FD) lees samples were then homogenized with a mortar and pestle and kept in air-tight containers at −20 °C. The lees samples were extracted as described by Mercurio et al. . A two-sMethyl cellulose, gallic acid, and resveratrol were purchased from Sigma Chemical Co. . Folin–Ciocalteu reagent; α, α-diphenyl-β-picrylhydrazyl (DPPH); 2, 2′-azobis (2-amidinopropane) dihydrochloride (AAPH); catechin and epigallocatechin gallate; malvidin; caftaric acid; cinnamic acid; and quercetin were obtained from Sigma–Aldrich Chemical Co. . Hydroxybenzoic acid was purchased from Fisons Scientific Apparatus . Ethanol (100%) was obtained from Fisher Scientific . Methanol (100%) and hydrochloride were obtained from Merck . All reagents and chemicals used in this study were of analytical grade or higher.The total phenol content (TPC) of wine lees extracts was determined using Folin–Ciocalteu reagent as described by Makkar et al. . Gallic The total tannin content (TTC) of extracts of FD wine lees was determined using the methylcellulose precipitable (MCP) tannin assay as described by Sarneckis et al. . Epicateλvis-max) and 700 nm (A 700) against water blank. Monomeric anthocyanin pigments can be calculated using the following equations:λvis-max = 520 nm, and A 700 = 700 nm.The total monomeric anthocyanin content of extracts of FD wine lees was determined using the pH differential method as described by Giusti and Wrolstad  with mod420), 520 nm (A λvis-max), and 700 nm (A 700) against water blank. The percentage of polymeric anthocyanin was calculated as written below:For the determination of polymeric anthocyanin, the method described by Giusti and Wrolstad  was adopThe calculation is based on the absorbance of the control sample, and DF = 30 in the current study.The calculation is based on the absorbance of the treatment sample, and DF = 30 in the current study.Phenolic compounds were determined using the method reported by Kemp et al. . Wine lePhenolic compounds in the samples were confirmed individually by mass spectroscopy and were quantified with a PDA detector at 280 nm using corresponding standards.Antioxidant activity of wine lees extracts was determined using α, α-diphenyl-β-picrylhydrazyl radical (DPPH•) and oxygen radical antioxidant capacity (ORAC) methods.515) was measured at 0, 5, 10, 20, 30, and 60 min of reaction using a spectrophotometer against a water blank. The cuvettes were covered with parafilm and kept in a light-tight container between absorbance measurements.The ability of lees extracts to scavenge the DPPH• was evaluated using method previously described by Sánchez-Moreno et al.  with mod515) against concentration. The equation below was applied to calculate the percentage of remaining DPPH• at the reaction time of 10 min. The percentage of remaining DPPH• was then plotted against the weight (mg) of extract.T is the DPPH• concentration at a plateau; (DPPH•) T=0 is the DPPH• concentration at zero reaction time.The concentration of remaining DPPH• in the reaction medium at each reaction time was calculated from a calibration curve of DPPH• with absorbance at 515 nm . EC50 of different sample extracts were calculated from the regression equation.Results were also presented as ECThe ORAC assay uses 2,2′-azobis (2-amidinopropane) dihydrochloride (AAPH) as a peroxyl radical generator  and repoDiluted antioxidant standard and samples (25 μL) were added to the 96-well microplate followed by the addition of 150 μL of 10 nM fresh fluorescein working solution to each well. The microplate was then covered by parafilm and incubated at 37 °C for 30 min. After incubation, 25 μL of the AAPH (65 mg/mL) solution was automatically injected into each well by the plate reader pump followed by 5 s of shaking.AUC) method was used for the quantification of the ORAC [AUCs of blank and of antioxidant were calculated from the equation below:RFU0 is the relative fluorescence value of time point zero and RFUt is relative fluorescence value of time points. The net AUC can be calculated by subtracting AUC (blank) from AUC (antioxidant). Based on the standard concentration between 0 and 0.05 mg/mL Trolox, the antioxidant activity was calculated and expressed in equivalent Trolox concentration (TE).The area-under-the-curve (the ORAC . AUCs ofS. aureus and Gram-negative. E. coli) and fungi  were incubated in Mueller–Hinton broth (MHB) and Sabouraud dextrose broth (SAB), respectively, as described by Wood and Washington [The antimicrobial and antifungal activities of the wine lees extracts were determined by conducting the minimum inhibitory concentration (MIC) assay in microtiter plates with the nutrient broth. Bacteria  for 20 min and diluted with 0.1% peptone water with reference to the viable counts, and they were photometrically adjusted to achieve an inoculum size of approximately 10w/v) [To perform a minimum inhibitory concentration assay, 100 µL of nutrient broth was firstly pipetted into each well of a microtiter plate. A total of 100 µL of wine lees extracts previously filtered through 0.45 µm filters were pipetted into the 2nd column wells, and serial dilutions were made in the wells along the microtiter plate at 50%, 25%, 12.5%, 6.25%, 3.125%, and 1.5625%. A total of 10 µL of the microorganism was added to each well, and ampicillin and amphotericin B were used as standard antibacterial and antifungal agents, respectively. Stock solutions of antimicrobial agents were prepared at a concentration of 0.8 mg/mL (w/v) .® Synergy 2, Gen 5TM, Winooski, VT, USA) after 24 h of incubation of the mixture at 37 °C for E. coli and S. aureus and after 48 h for C. albicans. Absorbance readings using the microplate reader were set at 600 nm and at 37 °C. All measurements of MIC50 values were performed in triplicate, including three controls with 50% acidified ethanol, MHB, and microorganisms.The MIC values were determined using a microplate reader  of the mean. Pearson correlation coefficients, among the measured parameters, were also calculated. Genstat® software  was used for statistical analysis.Analysis of variance (ANOVA) was carried out to investigate the effects of samples (dependent variable) on the measured parameters. Significant difference at p > 0.05). This might have been caused by the combined statistical analysis of PN and RL samples which generated a high pooled standard error of the means and masked the small differences within the RL samples. Although not statistically significant, the highest TPC value of 11.5 ± 0.8 mg GAE/g DM lees was observed in RL1 lees extracts, and this might be attributed to the application of pre-fermentation maceration which allowed the contact of grape skin with the must.Lower TPC values were observed in RL lees extracts compared to PN lees extracts . The TPCp > 0.05). This may also indicate that the interaction of several factors, such as machine and hand harvest, the fermentation temperature, and pre- and post- fermentation maceration, can remove any significant impact of any one single factor on the TPC of PN lees.Within the Pinot Noir lees samples, PN9 and PN10 had the highest TPC values at 72.6 ± 8.4 and 68.7 ± 4.9 mg GAE/g DM lees, respectively . This hiw/v, 60 °C, 25 min) from wine lees was 58.76 mg GAE/g. In contrast, a higher yield was obtained in the current study with 50% acidified ethanol. Thus, the increasing TPC in wastes generated during winemaking seemed to occur in the following order: skin < lees < seeds.Cheng  reportedp < 0.05) TTC was found in the PN lees compared to the RL lees  of sixteen wine lees extracts were measured, and anthocyanins were only detected in the PN lees extracts. The T-mAcy of the ten PN lees extracts is shown in For The percentage of the polymeric anthocyanin (pAcy%) in the ten PN lees extracts is shown in The identification and quantification of the ten Pinot Noir (PN) lees and the six Riesling (RL) lees are shown in For the PN lees, the gallic acid concentrations ranged from 0.5 to 2.2 mg/g extracts. Gallic acid was the predominant nonflavonoid in PN1 to PN6, which was higher than the total hydroxycinnamic acids measured in the study. In contrast, the gallic acid concentrations were lower than those of the hydroxycinnamic acids in PN7, PN9, and PN10. After fermentation and pressing, PN1 to PN6 were transferred to oak barrels. Oak cooperage has been noted as a source of phenolic acids . For theFlavan-3-ols were the primary flavonoids which were consistent with the composition of grape seeds reported by Bakkalbaşı et al. . The conp < 0.05). This can be attributed to the vinification of white wine in which the contact of skins and seeds with must is avoided. This was consistent with the observation reported in an earlier section of the current study.For the RL lees, the derivatives of hydroxycinnamic acids were the primary nonflavonoid in the lees. White wines are normally stored in stainless steel tanks; thus, there are no contributions of phenolic acids from oak extraction. Additionally, hydroxycinnamic acids are largely found in the flesh of the berries and are readily released upon crushing and pressing, while hydroxybenzoic acids exist in the skins and seeds of the grape berries . In gene50 values of the lees extracts were determined, and extracts of the PN lees exhibited significantly lower EC50 values (from 9.9 ± 1.7 to 17.3 ± 0.1 g FD lees/g DPPH) than those of the RL lees (from 107.3 ± 8.3 to 251.9 ± 13.2 g FD lees/g DPPH) (p < 0.05)  . These rr et al.  reportedr et al. . 50) of Pinot Noir by-products: The seeds (156.8 mg extract/g DPPH) exhibited the highest antioxidant activity followed by the pomace (304.02 mg extract/g DPPH) and skins (1365.7 mg extract/g DPPH) (seeds > pomace > skin). The EC50 values of the RL seeds and skins were 1070 and 22451 mg extract/g DPPH, respectively [50 values of the PN lees observed in the current study were in the range of 3530 to 7200 mg extract/g DPPH, which indicated a lower antioxidant activity than that of the PN seeds, pomace, and skins. Similar results were also found with the RL lees.Cheng  reportedectively . In contResearchers normally use more than one method for the analysis and comparison of the free radical scavenging ability because different methods target different compounds. The results of the ORAC clearly distinguished the differences in the free radical scavenging activity between the white and red wine lees, which was consistent with the observation in the DPPH assay . The res50) and the TPC and TTC of the sixteen wine lees extracts is shown in 50 were negatively correlated with the TPC and TTC of wine lees extracts. The results of the ORAC were positively correlated with the TPC and TTC of wine lees extracts  to 6.25 mg/mL, which was the strongest antimicrobial activity found in the RL lees extracts.The MIC values of the lees extracts ranged from 0.78 mg/mL to 50.00 mg/mL . Both thE. coli. All of the white and three of the red  lees extracts exhibited antibacterial effects. The MIC values for Gram-negative E. coli ranged from 3.13 to 25.00 mg/mL.Growth inhibition was also observed in C. albicans. C. albicans appeared to be more resistant to the lees extracts, and only RL1, RL2, and RL4 inhibited its growth at concentrations that ranged from 12.50 to 50.00 mg/mL. The RL5 lees extracts were found to be the most effective in inhibiting the growth of S. aureus and E. coli, but these extracts had no antifungal activity against C. albicans, indicating that bacteria and fungi exhibit different resistance abilities to bioactive compounds in lees extracts.The PN lees extracts did not have any antifungal activity against S. aureus and C. albicans at concentrations that ranged from 0.39 to 0.78 mg/mL. A similar trend was observed in which PN seed, skin, and pomace extracts were less effective against E. coli. In comparison with a Merlot pomace extract [S. aureus (7.81 mg/mL) and E. coli (62.5 mg/mL).The PN lees extracts might have lower antimicrobial and antifungal activities when compared with the seed, skin, and pomace extracts from the same grape variety. Cheng et al.  investig extract , PN leesS. aureus and E. coli. The RL5 extract showed medium inhibition effects on S. aureus.The antimicrobial activity of plant material extracts in previous studies has been classified as strong (MIC: <500 µg/mL), medium (MIC: 600–1500 µg/mL), and weak (MIC: >1600 µg/mL) ,34,35,36p < 0.05) than that of the RL lees extracts  lead to varying amounts of phenolics being extracted into the wine. It would be expected that the vinification methods that lead to more phenolic extraction into wine or juice would potentially result in higher concentrations of phenolic compounds in lees. This was evident since the use of carbonic maceration (PN4 and PN8), the degree of crushing, and the greater proportion of the whole berry content remaining during red wine fermentation (PN7) limited the extraction of various phenolic compounds compared with the rest of the PN lees samples and since the RL lees have significantly lower phenolic compounds compared to the PN lees.Polymerization will naturally occur given that there is a sufficient amount of phenols present. For example, the highest level of T-mAcys was observed in PN8, which also had the lowest level of pAcy%, TTC, and TPC , indicat50 values and, hence, the highest antioxidant activity measured by the DPPH assay  to the rest of the PN extracts. This was in agreement with a previous study that reported that the correlation between the TPC and antioxidant activity will vary due to the diverse quantifying methods applied to evaluate the antioxidant activity and due to the complexity of the natural mixture of the phenolic compounds . Therefog/g DPPH ,47.50 values) than the PNs with lower TPC values. PN9 and PN10 were processed in stainless steel tanks, which resulted in higher hydroxycinnamic acids  compared to gallic acid  were found to be negatively correlated with the ORAC assay but positively correlated with the FRAP assay (ferric reducing ability of plasma); quercetin-3-O-glucuronide was found to be positively correlated to the ORAC and HOSC  . Bekhit 50 values of the grape seeds, pomace, and skins obtained during the winemaking processes and vinification lees may vary according to vinification techniques.The majority of the free radical scavenging activity of wine by-products comes from phenolic compounds remaining after extraction  during the winemaking process. This is in agreement with Jara-Palacios’  study whB. cereus, S. aureus, Escherichia coli, S. typhimurium, Y. enterocolitica, L. monocytogenes, H. pylori, and C. jejuni [S. aureus) (p < 0.05), which was consistent with the observation that QMG showed the highest concentration in the RL5 lees sample which exhibited the strongest antimicrobial activity against S. aureus  exhibited inhibitory effects against Gram-positive bacteria, Gram-negative bacteria, and fungi, including . jejuni ,53,54,55. jejuni ,54,55. I. jejuni . Quercet. aureus .Various vinification techniques modified the extraction of phenolic compounds. More phenolic extraction to wine means correspondingly less phenolic compounds remaining in the grape marc. This is not true for wine lees. The lees samples in this study were obtained ranging from 1 to 5 months post alcoholic fermentation when the extraction processes were completed. It would be expected that vinification techniques that lead to more extraction into wine or juice would potentially result in  more phenolics and other compounds bounded with dead yeast or yeast residues and would become part of wine lees. This highlights the fact that lees obtained from wines with a high phenolic content can be used as a potential source of phenolic compounds.Knowledge of the characteristics of grape varieties and wine vinification techniques can provide some information on the potential composition and content in wine lees. In this study, the grape variety, harvest method, winemaking stages when the lees sample were obtained, additives, and vessels all seemed to play a part in determining the phenolic compositions of the lees. Further research is required to identify and quantify the influence of each individual vinification factor.The results also showed a good linear relationship between the antioxidant activities and the TPC and TTC, indicating that lees can be a useful source of extracts with appreciable antioxidant activities. The PN lees had significantly higher values in the TPC, TTC, and higher antioxidant activities compared with the RL lees samples , suggest


Glioma is the most common malignant tumor in the central nervous system. The clinical treatment strategy is mainly surgery combined with concurrent temozolomide chemotherapy, but patients can develop drug resistance during treatment, which severely limits its therapeutic efficacy. Epigenetic regulation at the RNA level is plastic and adaptable, and it can induce a variety of tumor responses to drugs. The regulators of RNA modification include methyltransferases, demethylases, and methylation binding proteins; these are also considered to play an important role in the development, prognosis, and therapeutic response of gliomas, which provides a basis for finding new targets of epigenetic drugs and resetting the sensitivity of tumor cells to temozolomide. This review discusses the relationship between the development of adaptive drug resistance and RNA modification in glioma and summarizes the progress of several major RNA modification strategies in this field, especially RNA m6A modification, m5C modification, and adenosine-to-inosine editing. Gliomas are the most common primary central nervous system tumors and similar histological features to normal glial cells , accounting for approximately 80% of malignant tumors of the central nervous system . AccordiIn the clinical setting, the standard treatment for patients with primary GBM is surgical resection with a maximum safety margin, postoperative radiotherapy combined with concurrent temozolomide (TMZ) chemotherapy, and then 6-week of TMZ maintenance therapy . SurgicaTMZ is a synthetic small-molecule alkylating agent , which cThe cytotoxicity and mutagenicity of TMZ mainly cause DNA damage to O6-MeG (methylation of the O6 position of guanine). Regulation of O6-MeG DNA methyltransferase (MGMT)  and an aThere is a growing evidence suggesting that gliomas may evolve adaptive drug resistance through TMZ-induced epigenetic changes, of which Wu et al.  have conThere are more than 170 types of biochemical modifications of RNA in living organisms . These RThe regulatory roles of RNA modifications in the occurrence, development, proliferation, differentiation, invasion, and metastasis of gliomas have been mostly summarized . HoweverTumor chemotherapy resistance is generally divided into primary and acquired drug resistance. Acquired resistance refers to the responses of tumors in the long-term that initially respond to treatment. Tumor heterogeneity and clonal evolution are recognized as important causes of acquired drug resistance. Glioma is highly heterogeneous; during the process of continuous proliferation and variation, tumors form many cell subclones with genomic and epigenetic diversity. The interaction between subclones and the tumor microenvironment (TME) affects the development of tumors . In the Among glioma cell lines, A172, U87, U251, and U373 are more sensitive to TMZ, while LN-18, T98G, and U138 are somewhat TMZ-resistant, with the former having a lower IC50  than the latter. However, sensitive cell lines can also be induced by TMZ to acquire resistance, which to some extent mimics the presence of primary and acquired resistance to TMZ in patients .RNA modification can act indirectly by regulating MGMT gene expression or directly targeting TMZ-related cell death pathways, including drug metabolism and transport, drug targeting, cell death signaling, DNA damage repair, the TME, immunity responses, and maintenance and differentiation of cancer stem cells, among others . Almost . In this section, we focus on RNA modifications of m6A and m5C that play strong roles in TMZ resistance in gliomas and tumor chemoresistance.RNA modifications mainly include N6-methyladenosine (m6A), 5-methylcytidine (m5C), N1-methyladenosine (m1A), N3-methyladenosine (m3A), N7-methylguanosine (m7G), pseudouridine, and adenosine-to-inosine (A-to-I) RNA editing. Each modification is reversible and caused by specific regulators, mainly including methyltransferases, demethylases, and methylation binding proteins. These regulators and their related mechanisms in the regulation of TMZ tolerance are provided in RNA m6A modification occurs in approximately 25% of mRNAs and is a dynamic and reversible process that is coordinated by the activity of methyltransferase writers , demethylase activity erasers (FTO and ALKBH5), and m6A-modified readers (YTHDF2 -3 and YTHDC1) working in concert ; multiplThe RNA methyltransferase m6A, which provides the methyl group from S-Adenosyl methionine (SAM) and transfers it to the substrate adenosine, has more than 200 members, but only a few have been identified by specific structural features and functions . In mammvia an internal tryptophan. RNA reading proteins are mainly divided into three main families, YTHDC1, YTHDC2, and YTHDF. YTHDC1 is the main nucleophile that binds rapidly to mRNA after methylation, regulates mRNA splicing by recruiting pre-mRNA splicing factors, and allows access to the target binding regions of mRNAs  microarray and RNA-sequencing database revealed that both m6A writers and readers were significantly increased, while erasers such as FTO were relatively decreased . Xu et a2 suppressed the growth and self-renewal of GSCs, which are related to the chemo- and radiotherapy-resistance , with S-Adenosyl methionine as the methyl group donor . m5C is Ribosomal RNA in all living organisms shows m5C modifications , a proceThe methylation m5C loci in mRNA has been controversial, tied to methodological limitations and a process of continuous refinement. Squires et al.  first coA decade of technological development has updated the picture of m5C distribution in RNA, with ever-optimized bisulfite transformation protocols and more stringent analytical treatments being more often applied to m5C sequencing in different species and tissue classes. The prevailing view is now that m5C is not as widely distributed in mRNA as initially thought , as showc-Myc, which is most closely linked to tumor development  to avoid death and ultimately reduce patients’ therapeutic sensitivity to TMZ . In addiThe above studies provide clues that RNA m5C modifications affect drug resistance in glioma, but strong direct evidence is lacking. Current research mainly focuses on the perspectives of the m5C methyltransferase NSUN family and m5C-associated lncRNA expression profiles. Maintenance and differentiation of tumor stem cells, adaptive changes in tumor cells under stressful conditions, and alterations in the tumor immune microenvironment are research directions worthy of further exploration.ADAR1  are deaminated to hypoxanthine nucleotides (inosine), also called inosine. During translation, the I on the mRNA is recognized by the ribosome as G (Guanosine), so A-to-I editing alters the amino acid translation codon in the protein-coding sequence, affecting the structure and function of the protein. This pairing also causes a mismatch between RNA sequences and genetic DNA sequences and is used for genomic localization . VertebrADAR1 , ADAR2 /arginine (R) site of the glutamate receptor subunit B to a degree of nearly 100% in the central nervous system, which is essential for normal receptor function . It is wvia multiple pathways. Studies have described relevant pathways in breast cancer , a target of the chemotherapeutic drug methotrexate, enhancing the proliferation and chemoresistance of breast cancer cells . HoweverThe above studies provide clues for exploring the biological significance of A-to-I RNA editing in the drug resistance phase of glioma. Future studies could be conducted in the following directions: 1) combining sequencing and existing findings to uncover differences in A-to-I editing in chemoresistant and chemosensitive GBM cells, exploring their upstream and downstream pathways, and uncovering drug therapy related genes; 2) finding and integrating the interactions between A-to-I editing and other regulatory molecules to reveal the complete post transcriptional regulatory map of gene expression in glioma drug resistance, providing information for personalized medicine; 3) validating whether ADAR is a potential therapeutic target for glioma drug resistance, which can be used for adjuvant therapy after TMZ treatment by developing molecules that regulate ADAR expression or activity; 4) verifying whether A-to-I editing in glioma affects the TMZ effects by influencing genes related to alkylated drug metabolism, which may provide new options for the treatment of patients with GBM by adjusting the route of administration or co-administration of metabolic enzyme inhibitors.The role of other RNA modifications in GBM resistance has not been well described, but there have been some advances in the progression of GBM occurrence and clinical characteristics of patients with GBM. For example, the enzyme pseudouridine synthase 7 is associated with prognosis in patients with GBM  and is iWe have also noticed the essential effects of non-coding RNA in post-transcriptional gene regulation in TMZ resistance. As a typical example, miRNA, small non-coding RNAs consisting of 20–24 nucleotides, can change the expression profiles of glioma cells through binding with target mRNAs and causing instability and translation inhibition . miRNA dvia the MAPK/ERK pathway ( pathway . lncRNA  pathway .Over the past three decades, the relative survival rate for malignancies has increased from 23% to 36%, but GBM has seen only a slight increase of 3% . TMZ, thAdvances in methodology have led to a better understanding of the regulation of gene expression in glioma drug resistance, and the involvement of RNA modification in the formation of a rapid drug resistance phenotype in glioma is an important finding of recent scientific work. Although epigenetic drugs targeting epigenomic regulators and protein modification-regulated enzymes have won approval as a new type of antitumor drugs, with the first clinical applications, there have been no major satisfactory results . The revThe interactions between RNA modification regulators and interactions of RNA modification with non-coding RNA regulation, epigenomics, proteomics, and metabolomics, also deserve attention. These different levels or dimensions of regulatory factors constitute a complex and stable network of gene expression regulation and are often responsible for the suboptimal clinical outcomes of new drugs. Mapping the complete epigenetic map of gene expression in glioma chemoresistance may provide clues for developing rational drug treatment regimens. Moreover, applying the detection of RNA modifying regulatory factors to the molecular pathological diagnosis of clinical patients and developing individualized treatment plans to achieve precision medicine is also a possible future development direction.


Dark-haired dogs are predisposed to the development of digital squamous cell carcinoma (DSCC), suggesting an underlying genetic predisposition which is yet to be explained. Some authors have suggested a correlation between the number of copies of KIT Ligand, a gene associated with cell survival, proliferation, and melanogenesis, among other functions, and the potential predisposition to DSCC in dogs. This was evidenced by the fact that dogs with DSCC had a significantly higher copy number of this gene than those who did not have the neoplasia. For this reason, we evaluated the potential correlation between the number of copies of the KIT Ligand in genomic DNA with the histological grade of malignancy in dogs with DSCC. Our findings reveal a significant correlation between the number of copies of KIT Ligand and DSCC histological grade. This supports previous studies that KIT Ligand may play a role in DSCC development and, additionally, may be involved with the presence of histologically malignant morphological features. This suggests a potential factor in the development of canine DSCC, which may signify a potential advance in personalized veterinary oncological approaches as well as future breeding programs.n = 11); Group 1.a/black non-Schnauzers (n = 15); group 1.b/black Schnauzers (n = 33); group 1.c/black and tan dogs (n = 7); group 2/tan animals (n = 4). The DSCCs were histologically graded. Additionally, KITLG Copy Number Variation (CNV) was determined by ddPCR. A significant correlation was observed between KITLG copy number and the histological grade and score value. This finding may suggest a possible factor for the development of canine DSCC, thus potentially having an impact on personalized veterinary oncological strategies and breeding programs.Dark-haired dogs are predisposed to the development of digital squamous cell carcinoma (DSCC). This may potentially suggest an underlying genetic predisposition not yet completely elucidated. Some authors have suggested a potential correlation between the number of copies KIT Ligand (KITLG) and the predisposition of dogs to DSCC, containing a higher number of copies in those affected by the neoplasm. In this study, the aim was to evaluate a potential correlation between the number of copies of the KITLG and the histological grade of malignancy in dogs with DSCC. For this, 72 paraffin-embedded DSCCs with paired whole blood samples of 70 different dogs were included and grouped according to their haircoat color as follow: Group 0/unknown haircoat color ( The largest reported cohort of 2912 canine toes, conducted by Grassinger et al., classified 52% of their samples as neoplasms, 78% of which were malignant. Of those, approximately 65% were digital squamous cell carcinomas (DSCCs) ,4,5, strThis aforementioned study, coinciding with other publications ,7,8,9,10KIT Ligand (KITLG), also known as stem cell factor , is a geMoreover, Karyadi et al.  suggesteThe objective of this research was to evaluate if there is a correlation between histological features of aggressiveness in canine digital SCC and copy number values in KITLG. From 2014–2019, 72 histological samples of DSCC from 70 dogs were selected retrospectively from the routine diagnostic pool submitted to LABOKLIN GmbH & Co. As a selection requirement, the samples had to contain a clear neoplastic invasive front and known breed. All DSCC from animals only included fragments with neoplasia, and samples with no clear invasive front or without information about the breed were excluded. Furthermore, additional blood samples from these dogs were available from routine diagnostics . As all samples (toes and whole blood) were submitted for routine diagnostic purposes, ethics committee approval was not required (RUF-55.2.2-2532-1-86-5). All the material used was no longer needed for diagnostics.The ages of the dogs ranged from 4 to 16 years, with a median of 10 years. Sex was either female intact (15), female spayed (15), male (22), or male castrated (20). Limb and toe affected, when available, was noted. The breeds included Giant Schnauzer (20), Standard Schnauzer (14), Mixed (8), Briard (5), Labrador (5), Gordon Setter (3), Russian Black Terrier (2), Flat-Coated Retriever (2), Bernese Mountain Dog (2), Rottweiler (2), Belgian Shepherd (1), Havanese (1), Irish Setter (1), Puli (1), German Wirehaired Pointer (1), Hovawart (1), Giant Poodle (1), West Highland White Terrier (1), and Standard Poodle (1).n = 11), composed of animals with unclear haircoat color; Group 1.a/blacks (n = 15), with non-Schnauzer black breeds; Group1.b/Schnauzers (n = 33), with black Schnauzers; Group 1.c/black and tan (n = 7), with black and tan animals; and Group 2/light (n = 4), with dogs with a light blond or reddish haircoat. Additionally, Groups 1.a, 1.b, and 1.c were summarized as “dark coated breeds” (n = 55). Signalment and individual cases with more detailed information can be seen in The majority of these animals were further grouped according to their haircoat color, similar to the previous research , as foll® rapid decalcifier solution for histology; catalogue no. 101736) over a period of 24–72 h, periodically assessing tissue until it was ready to be further processed according to published trimming guidelines [Similar to the previous study conducted by this research group , all digidelines . AfterwaFor more detailed information concerning the adapted gradings performed in the study, please refer to the previous open-source publication carried out by this research group  .As previously performed , NagaminIn a similar manner to previously conducted research by this group , an adapTumor budding was considered when neoplastic aggregates/complexes fewer than 5 cells disconnected from the main neoplasm and infiltrated into the surrounding stroma. These “buds” were assessed in an area of 2.37 mm2 at 40x magnification in high-incidence areas. The point system was granted following the table . The sum® assays specific for the KITLG CNV sequence and proto-Oncogene 1 (ETS1) as reference gene. Measurement was carried out in duplicate, and the mean value was used for further analyses. Intra-assay correlation was 0.85. Copy number was determined using DropletReader  and QuantaSoftware . Genomic DNA extraction and isolation from EDTA blood were performed with the MagNA Pure 96 system using DNA Tissue Lysis Buffer and viral NA Small RNA kit  according to manufacturers´ manual. Similar to that described by Bannasch et al. , the copStatistical analysis between dark-  and light-coated  breeds as well as between the different pre-established subgroups  were performed using the Mann–Whitney U test. Group 0 was excluded because of heterogenic genetic background.All statistical analyses and visualizations were carried out with the statistical framework R version 4.2 (R Core Team 2022). To identify the optimal model, we applied the “step“ R function with direction “both“ and the Bayesian Information Criterion (BIC). The marginal effects of the interaction term were visualized by the function “plot_model” as implemented in the “sjPlot” R package Lüdecke . Robust Out of the 72 samples from 70 different animals, 46 DSCCs were located in the forelimb and 24 in the hindlimb. Two samples were not provided with an exact location.Following a similar model to that provided in previous studies conducted by this research group , the samp > 0.5).When associating the IFGS grade with the haircoat color, there was no significant difference between any of the groups  or betwep < 0.05), with group 2 having a lower grade that any of the other darker groups   may result in significant statistical differences in the degree of tumor differentiation and potential malignant behavior. The value of the copy number (CN) of KITLG was measured for each animal included in the study and ranged between 2.02 and 9.14, with a mean value of 5.6. The value of Group 0/others was 5.7, for Group 1.a/black it was 5.8, for Group 1.b/Schnauzers it was 5.7, for Group 1.c/black and tan it was 5.5, and for Group 2/light it was 4.5. The differences between the groups were not significant.p = 0.001) and grade III (p = 0.007). On the other hand, when comparing KITLG CN with TCBGS, only animals with grade III had a significantly higher KITLG CN value than those animals with grade I (p = 0.042). This suggests that a higher histological grade is correlated with a higher KITLG CN value when comparing low versus high grades in both histological systems applied. A statistical analysis comparing the independent correlation of the TCBGS grade and IFGS grade with the KITLG copy number (CN) value was performed. Comparatively, animals with IFGS grade I had significantly lower KITLG CN than those of grade II (p = 0.006) (p < 0.001) . The identified optimal regression model considers only the KITLG CN value and the IFGS score value, with a strong significant correlation between these two features  A. The TC< 0.001) B. These In veterinary medicine, an effort has been made to try to identify genes associated with cancer or disease in different canine breeds or lineages. Some examples of this include renal cystadenocarcinoma , histiocp = 0.006; TCBGS ~ 0.75 * CNV KITLG; p < 0.001).To this end, this represents the largest collection of canine DSCC along with pairing whole blood samples to date, allowing both histological and genetic assessment of the given animals. The aim of the study was to evaluate a hypothetical correlation between histological features of invasiveness in canine digital squamous cell carcinoma, based on two different adapted histological grading systems (IFGS and TCBGS), and copy number values of KITLG. Animals with lower IFGS and/or TCBGS score values had a significantly lower KITLG copy number . On the other hand, when looking into the IFGS, there were no significant differences between the grade and haircoat color group. Nonetheless, statistical interpretation should be cautious, given the large group size disparities. Similar to previous studies , the degp = 0.006) compared to TCBGS (p < 0.001), but both correlations were robust. The slight correlation discrepancy between grading systems and KITLG CN could be partially explained by the fact that both grading systems pay attention to different histological features, and potentially one of those features may be correlated with a higher (or lower) KITLG CNV. This has been established independent of the haircoat color and/or gender. It must be noted that this estimation may have been a more robust linear regression model if extreme mean CNV values would have been available. Despite this, with the available data, there was still a robust linear regression with comparable results. This may potentially suggest that with a known KITLG CN value, one could potentially predict the histological malignancy of a present DSCC, thus deciding the most appropriate course of treatment.The KITLG copy numbers were compared independently to the histological score values of both the TCBGS and the IFGS systems of the established animal groups. Interestingly enough, both IFGS and TCBGS showed strong correlation between the KITLG CN and their histological score values. In summary, animals with a higher KITLG CN value have more histological features of malignancy of DSCC. IFGS had a slightly inferior correlation with the KITLG CN  and Standard (n = 14). However, the present study did not compare animals of the same breed with and without DSCC, so a relative KITLG CNV could not be established between affected and non-affected animals. It is for this reason that we cannot draw conclusions regarding the comparatively higher KITLG CNV of animals with and without this tumor from the same breed. In order to prove this, further larger studies of dogs of the same breed (to ensure a more homogeneous sample) with and without DSCC in their entire lifetime should be performed to evaluate whether the hypothesis that a higher KITLG CNV is related to animals developing DSCC still stands. This is currently under investigation to allow future comparisons. Additionally, as a correlation between KITLG CNV and malignant histological features are observed, future studies should include KITLG transcription and protein expression level. Currently, there are few studies investigating the molecular expression of KITLG and c-KIT in veterinary medicine, mostly in canine mast cell tumors ,40,41,42This study represents the largest of its kind pairing up canine DSCC with blood samples, allowing a simultaneous histological and genetic assessment. When assessing the results, there was a significant correlation between KILTG CN increase and higher histological score value on both IFGS and TCBGS.It would be interesting to evaluate a large cohort of canine breeds non-predisposed to DSCC to establish a KILG CNV baseline, identifying if there is a significant difference when these same breeds develop a DSCC. With this, KITLG CNV can be seen in a large cohort of animals with and without DSCC. More homogeneous groups, along with the identification of other “protective” or “causative” genes for CDSCC, should be evaluated. Further studies are needed to further characterize a possible genetic test for the predisposition of DSCC in dark-haired breeds, as well as KITLG transcription levels and final protein expression. This could possibly aid in identifying other potential genes that may serve as a protection against this neoplasm or may confer it with more benign or malignant histological features.


From the early days of research on RNA biology and biochemistry, there was an interest to utilize this knowledge and RNA itself for therapeutic applications. Today, we have a series of oligonucleotide therapeutics on the market and many more in clinical trials. These drugs - exploit different chemistries of oligonucleotides, such as modified DNAs and RNAs, peptide nucleic acids (PNAs) or phosphorodiamidate morpholino oligomers (PMOs), and different mechanisms of action, such as RNA interference (RNAi), targeted RNA degradation, splicing modulation, gene expression and modification. Despite major successes e.g. mRNA vaccines developed against SARS-CoV-2 to control COVID-19 pandemic, development of therapies for other diseases is still limited by inefficient delivery of oligonucleotides to specific tissues and organs and often prohibitive costs for the final drug. This is even more critical when targeting multifactorial disorders and patient-specific biological variations. In this review, we will present the evolution of complexity of oligonucleotide delivery methods with focus on increasing complexity of formulations from gymnotic delivery to bioconjugates and to lipid nanoparticles in respect to developments that will enable application of therapeutic oligonucleotides as drugs in personalized therapies. The development of oligonucleotide therapeutics is tightly connected to the history of RNA research, and both have complicated stories. In the late 19th and early 20th centuries, researchers defined the basic differences between DNA and RNA, but a functional characterization of RNA is still an ongoing process, with new functions of RNAs in cellular processes still being discovered , provide cell and tissue specificity, stability in extra- and intracellular environment, fast cell penetration, efficient localization to subcellular compartments, and allow the targeting oligonucleotide to bind specific (on-target/off-target ratio) and stable to the intended target including full compatibility with the cellular machinery and very limited interference with non-targeted cellular processes. The design of oligonucleotide therapeutics will become even more challenging when considering patient-to-patient variation, multiple targets in the same cell and attempts for tuning levels of expression instead of completely silencing them. So far, there is no single delivery system developed yet that can fulfill all the criteria mentioned, but considerable progress has been made in solving some of the challenges. The most convenient patient compliant methods of delivery to the body e.g. pills, ointments, or inhalations, which allow patients to self-administer the drug could expand RNA therapy applications to less severe diseases or more regular interventions. This does not diminish the therapeutic relevance of RNA therapeutics since many RNA therapeutics show very long half-lives and efficacies which require very infrequent dosing which makes different routes of administration clinically very relevant. So far none of the RNA drugs on the market fulfill the criteria for self-administration, but functionalization of RNAs with lipids and other hydrophobic moieties as well as formulation with lipid nanoparticles allows for absorption through the skin or nebulization and thereby lung delivery ,16. The Recent research on extracellular vesicles shows a great promise of developing multifunctional biomimetic nanoparticles of native origin i.e. fully compatible with patients’ immune system. All these strategies will be classified as third generation of RNA therapeutics in the current review, implementing all previous strategies of chemical modifications and lipid encapsulation or lipid membrane anchoring of functional molecules  or enhancement of cell penetration and intracellular distribution (peptides and hydrophobic compounds). Sometimes, conjugation provides additional benefits of stabilizing RNA oligonucleotides against nucleases or adds functionality such as allowing for receptor-mediated endocytosis and subcellular localization. There are multiple strategies to conjugate molecules to ONs that can be divided into in-line conjugation and post-synthesis conjugation. The most common method for synthesis of modified RNA ONs is based on phosphoramidite chemistry, many commercially available phosphoramidites provide convenient access to a broad range of building blocks for internal and terminal incorporation of non-natural functional groups , includiin vivo binding to folate receptors (FRs) which are overexpressed in many cancer cell types [E. coli and S. typhimurium and reduction of expression of target genes, without toxic effects to mammalian cells[Small molecules are monomeric molecules which add new functionality or change biophysical properties, e.g. non-nucleotidic building blocks, receptor targeting ligands, and fluorescent probes. Fluorescent labelling is the most frequently used conjugation with a multitude of applications from fundamental molecular biology studies, diagnosll types . In the ll types . Exploitll types . The dirian cells.tert-butyldiphenylsilyl group at 5’ terminus significantly improved the activity, questioning the effectiveness of glycosylation itself. What is clear, is that glycosylation at both termini effectively prevents degradation by exonucleases, that require unmodified 3’ or 5’ ends and are the most abundant nucleases in cells[in vitro studies. Interestingly, addition of cationic lipids improved silencing to 98%, pointing to inefficient endosomal escape when using glycosylated siRNA alone[Glycosylation of biological compounds is a natural process with a wide range of functions, from molecular recognition and interaction through cell communication to detoxification which turns synthetic carbohydrate-conjugation into a useful tool in developing diagnostic and delivery strategies. Due to the availability of well-established enzymatic and chemical methods, protein and peptide glycosylation is commonly used, but recently glycosylation methods for other molecule classes, including ONs, have been developed. D’Onofrio et al. developed phosphoramidites for conjugation of mono- and disaccharides at the 3’ or 5’ terminus of ASO during automated synthesis. The G-q in cells. Beyond  in cells. TargetiRNA alone. RecentlRNA alone. CompariRNA alone. The terRNA alone,58. The RNA alone,59. CompRNA alone. NeverthRNA alone. The disRNA alone. In anotRNA alone.in vivo stability 16-fold and was significantly accumulated in liver, heart, kidney, and lungs[~99%) when prepared with reconstituted high-density lipoproteins (rHDL), that delivered siRNA to hepatocytes and reduced Pokemon and Bcl-2 protein expression in in vitro and in vivo experiments[ND5 gene[in vitro activity when conjugated with C16 at 5’-terminus, but changing the design to an asymmetric, 3’ blunt ended DsiRNA, showed better silencing of 3’ conjugated C16, although it’s not clear if that is because of an improved DsiRNA conformation or Dicer protein preference[KRAS gene expression regulator in pancreatic cancer cell line (Panc-1) in in vitro studies [th position from 3ʹterminus, that allowed for efficient binding of lipidated G4-decoy ON to the surface of palmitoyl-oleyl-phosphatidylcholine (POPC) liposomes[in vitro experiments saturated and unsaturated fatty acids and found that C16 and C18 carbon length in cis-form are optimal for transformation with lipofectamine, but without lipofectamine only longer carbon chain conjugates, C22, eicosapentaenoic acid (EPA) and Docosahexaenoic acid (DHA), showed significant activity[in vivo they quickly bind to serum albumin which improves RNA stability and bioavailability. Diacyl lipid (C18) conjugated to siRNA showed almost 99% uptake in the mice tumour site and over 40:1, tumour: liver accumulation ratio, which was 1,6 fold better uptake and 13 fold better accumulation ratio than unmodified siRNA compared with an in vivo commercial transfection formulation [in vivo injection it bound to serum albumin and was effectively distributed to cancer cells. Jin et al. have exemplified this by synthesis of lipidated single stranded floxuridine homomers which attach to Albumin and are subsequently transported and accumulated in cancer cells and generate free floxuridine upon lysosomal degradation which interferes with DNA synthesis and inhibits cell proliferation [Huntingtin (Htt) mRNA[Covalent conjugation of lipids and other hydrophobic molecules to RNA significantly changes the biophysical behaviour in aqueous environment by inducing potential self-aggregation depending on sequence design or partitioning to the surface of lipid membranes. RNA lipand lungs. Chol-siperiments. Incorpo[ND5 gene. The hyd[ND5 gene. Similar[ND5 gene. There ireference. Direct reference. Potentireference. That isreference. Spontan studies ,74. Cogoliposomes. This foliposomes. Lipidatliposomes. This opliposomes. Lipidatliposomes. Kubo et activity. Targeti activity. When limulation . ‘Hitchhmulation . In soluferation . That shferation . Godinhoferation . In anotHtt) mRNA. Alpha-tHtt) mRNA. The tisHtt) mRNA.in vitro experiments[4 significantly improved PMOs’ delivery in vitro and measured activity, in the absence of endosomolytic agents [4 vs (RBR)4 showed improved serum stability and higher activity of the β-alanine version peptide [4 showed even better extra- and intracellular stability with superior PMOs antisense activity [4XB to 5’-terminus of PMOs targeting conserved cis-regulatory elements of flavivirus’ RNAs (mosquito-borne and West Nile viruses), showed effective inhibition of viral protein expression and viral RNA replication [4 conjugated to PMO against 5’ terminal or leader-TRS region reduced >95% of viral titres 12–48 h after cell infection [2 conjugated PNAs, which also reduce >95% of SARS-CoV-2 titres, promise to quickly develop and deliver viral-strain-specific drug to the market, in vivo antiviral efficacy was demonstrated by PPMO against several respiratory viruses in previous studies [gyrA gene, significantly inhibit S. pyogenes cell growth, and pioneered a promising strategy of fighting multidrug resistant bacterial strains[trans-membrane transport of bacterial protein toxins allowed for efficient siRNA release from the caveosomal pathway and targeting of the ER-specific perinuclear site in a model endothelial cell line (ECV304) [4 conjugate with PNA shows strong mdx exon skipping efficiency in myotube cells of DMD model mice [8XB and (RXRRBR)2XB showed high accumulation in liver or in quadriceps and heart, respectively, in DMD model mice [2) called PMO internalizing peptide 6a (Pip6a) conjugated to splice-switching PMO, targeted exon 7 of survival motor neuron 2 (SMN2) pre-mRNA and showed significant distribution to the CNS after systemic administration, and resulted in 38-fold extended life expectancy in a spinal muscular atrophy (SMA) mice model [2XB, can also be delivered as PMOs across BBB and substantially increases SMN protein levels, alleviating major SMA symptoms in mice [in vitro transcribed mRNA allowed SCO2 protein complementation in SCO2/COX-deficient primary fibroblast and β-globin production in cells from β-thalassaemic patients[Well-established peptide synthesis strategies and a large number of commercially available natural and non-natural peptide building blocks make peptides attractive for ON-conjugation. Specific peptides can modulate immune response, improve RNA stability, cell targeting and specificity (peptidic ligands), cell penetration (CPPs) and endosomal release (cationic peptides). Peptide synthesis is also compatible with alternative backbone chemistries i.e. PMOs and PNAs, that significantly alter the biophysical properties of oligonucleotides and have already shown to be safe in some clinical applications ,85. CPPsperiments. Conjugaperiments or R6-Peperiments, improvec agents . In simi peptide . Additioactivity . The conactivity . Despiteactivity . The firactivity . Conjugalication . Similarlication , measleslication , infectilication , influenlication . Years olication . (RXR)4 nfection . Impleme studies . Beyond l strains. Targetil strains. Cen et l strains. Conjuga(ECV304) . Similar(ECV304) . Using D(ECV304) . Using a(ECV304) . Currentdel mice . Comparidel mice . Anotherce model . An even in mice . Covalen patients.O-benzylguanine-modified guide RNA (gRNA)[in vivo [G12C-specific siRNA and Cetuximab, allowed efficient KRAS-mutant downregulation in lung cancer cells and their sensitization to the small molecule chemotherapeutic, Gefitinib[Extending peptide conjugation to larger molecules, protein conjugation adds new modalities  to RNA, expands available libraries of antibodies (Abs) and significantly improves cell selectivity. An interesting example is redirecting adenosine deaminase acting on RNA (ADAR) protein to a new target-of-choice, by substitution of N-terminal, dsRNA-binding domains of the ADAR with SNAP-tag domain, that chemo-selectively forms a covalent bond with 5’-NA (gRNA). New gRNNA (gRNA). The speNA (gRNA). In hardNA (gRNA). The posNA (gRNA). Introdu[in vivo . The onlGefitinib.EGFR mRNA in HER2+ breast cancer cells can specifically and efficiently inhibit breast cancer growth in a mice model system. The design used is a noncovalent approach where both HER2 and HER3 targeting aptamers are coupled by hybridization of two complementary 19mer sense strands and anti-sense EGFR siRNA sequences, both linked to the respective aptamers[BCL2 mRNA and inhibition of cell proliferation[Despite functional similarities between antibodies and aptamers , the lat aptamers. They de aptamers. Crossliiferation.sn-3-phosphoethanolamine (DOPE) and dioleoyl phosphatidylcholine (DOPC). Delivery of ONs complexed only by DOTMA, or other cationic lipids, is known as lipofection, and is the most popular in vitro transformation method[in vivo delivery due to high cytotoxicity making liposomal formulations more suitable for therapeutic applications[in vivo applications, this simple formulation can elicit inflammatory response and if unresolved can create entrained allergic reaction or even death[sn-glycero-3-phosphocholine (DSPC):PEGylated lipid (50:38.5:10:1.5), showed a very low polydispersity index (PD) <0.1 with an average diameter of 50 nm, which are very desirable characteristics for in vivo applications. These LNPs with Lipid10 (lipid with a N-methyl-piperazine head group) showed high accumulation in spleen, and after LNP surface modification with anti-integrin β7 mAb using intravenous administration to mice, were able to modulate CD45 expression exclusively in primary lymphocytes [G12D mutant sequence (the specificity or level of off-target effects are not reported), and lipidated Tat peptide as CPP, improving cell penetration to pancreatic ductal adenocarcinoma (PDAC) cells[in vitro studies showed up to 70% of reduction in mutated KRAS protein production. Conjugation of different functionalities to PEG-DOPE allowed Pan et al. to create multifunctional micellar LNPs[Lipid-based nanoparticles are among the most well established nanoparticle systems applied to delivery of RNA. By encaon method. Unfortulications. In morelications. In in vven death. Therefoven death. One of ven death. PEGylatven death. When LNven death. Even ifven death. Similarven death. They fophocytes . ExploitAC) cells. Incubatllar LNPs. They crllar LNPs. Few LNPllar LNPs. Crucialllar LNPs. First, llar LNPs. Later, llar LNPs. LNPs collar LNPs. An ionillar LNPs. Scientillar LNPs. During llar LNPs.HuR), in cancer cells when compared to gymnotic delivery[Huntingtin (Htt) mRNA showed greatly improved in vivo distribution of EVs in mice brain and significant, up to 35% reduction in Htt mRNA[FLOH1, NKX3, DHRS7, and showed more than 3-fold improvement of internalization over gymnotic delivery and desired activity against all targets. Major challenges for practical therapeutic applications of EVs remain, despite their potential of as delivery vehicles. The challenges comprise isolation, upscaling, batch-to-batch variability, shelf live-related stability and the impurity profile depending on the biological source and purification techniques used. Nevertheless, their unique composition, ability to cross biological barriers and current improvements in techniques for loading and generation of EV-hybrid particles may well prove important for future EV-based delivery approaches and enable personalized formulations if patient isolated EVs are used for more specific applications.The natural role of extracellular vesicles (EVs) as ‘transporters’ of biological material between cells, tissues, and organs represents one way of long-distance communication, suggests EVs as very promising vehicles for therapeutic applications. They have very diverse compositions dependent on the cellular origin of the vesicles, which includes DNA, RNA, membrane and soluble proteins, lipids, carbohydrates, and other small molecules and have shown to be able to cross many biological barriers including the blood-brain barrier. Many of these molecules are presented at the surface and can contribute to unspecific or specific cell targeting. This abundance of potential ligand-receptor interactions explains the higher efficacy of EVs internalization compared to synthetic LNPs. The outstanding challenge for therapeutic application is efficient loading with synthetic cargo, such as siRNA or ASO. EVs are characterized by surprising mechanical and chemical stability after isolation but that also limits potential methods for incorporating external molecules. Electro delivery. Similar Htt mRNA. An inte Htt mRNA. Instead Htt mRNA. The res Htt mRNA. This fo Htt mRNA. Diao etin vitro experiments[In comparison to biomimetic LNPs and EVs, using polymeric materials allows to create highly controlled and diverse structures and shapes of nanoparticles. They range from spherical micelles, polyplexes and lipoplexes to cubes, rods, microneedles, and hydrogels. The functionality of formulation is mainly guided by polymer’s characteristics or by combination of various polymers, which allows to precisely finetune the functions. To speeperiments. Limitinperiments. Alternaperiments.in vivo studies they observed significant accumulation of MOFs in the tumour and reduction of fluorescence or slowdown of tumour growth[Metal-organic frameworks (MOFs) show similarities with polymeric particles by encapsulating or condensing ONs. Supramolecular, organic ligands coordinate metal ions forming porous materials that can trap ONs during formation or complex them afterwards. Similarly to polymeric nanoparticles they can form well defined structures with high loading capacity. Their sur growth. Anotherur growth.N-1-methyl-pseudouridine instead of uridine, improved mRNA conformational stability and translation efficiency but did not address susceptibility to reactive oxygen species (ROS) that can act on 2ʹOH group and cause ON chain breaks. Addition of ROS scavengers into LNPs or use of modified lipids at the inner leaflet can alleviate the problem. Alternatively, developing strategies with polymerases that can site-specifically incorporate 2’-modified nucleotides also yield improved stability[The idea to use RNAs as therapeutic agents and target RNAs for therapeutic purposes emerged soon after their discovery but it took decades of research and development to bring the first ON-based drugs to the market. In thisstability. RNA delstability or otherstability, peptidestability,137, pepstability and oligstability,168–170.stability and EVs stability. Still, stability and alsostability. With grstability. In prac