CAS Content Collection, we provide a landscape view of the current state and outline trendsin RNA research in medicine across time, geography, therapeutic pipelines,chemical modifications, and delivery mechanisms.In the past decade,there has been a shift in research, clinicaldevelopment, and commercial activity to exploit the many physiologicalroles of RNA for use in medicine. With the rapid success in the developmentof lipid–RNA nanoparticles for mRNA vaccines against COVID-19and with several approved RNA-based drugs, RNA has catapulted to theforefront of drug research. With diverse functions beyond the roleof mRNA in producing antigens or therapeutic proteins, many classesof RNA serve regulatory roles in cells and tissues. These RNAs havepotential as new therapeutics, with RNA itself serving as either adrug or a target. Here, based on the  While human RNA medicinehas faced many challenges in terms of efficacy and immunogenicity,the recent success of mRNA vaccines against COVID-19 and the approvalof new RNA-based drugs provide new momentum to the field. Many classesof RNA play important regulatory roles in cells and tissues, beyondthe obvious role of mRNA in protein synthesis. Scientific research,clinical development, and commercial production now focus on exploitingthe many roles of RNA for use in biotechnology and medicine. Advancesin understanding RNA structure and function are combined with a robustproduction pipeline to develop clinically effective RNA-related applications.13 In the next decade,the 5′-cap on mRNA was discovered,15 the first liposome-entrapped RNA was delivered into cells,16 and antisense oligomers (ASOs) were used toinhibit Rous sarcoma virus (RSV).17 Inthe 1980s, in vitro transcription from engineeredDNA templates using a bacteriophage SP6 promoter and RNA polymerase18 allowed the manufacture of mRNA and expressionof other types of RNA in cell-free systems. Later in the 1980s, thefirst cationic-lipid-mediated mRNA delivery was achieved.20 The discovery of RNA interference (RNAi)21 and the approval of the first antisense RNA drug in the late 1990s22 were key to the development of RNA therapeutics.For their pioneering work on RNAi and the RNA-induced silencing complex(RISC),23 Fire and Mello21 were awarded the Nobel Prize in Physiology and Medicinein 2006. During the 2000s, the discovery of the importance of pseudouridinemodification24 and further research onmRNA led to the first human trial of an mRNA vaccine against melanomain 2008.25 In 2010, a pivotal human clinicaltrial showed that siRNA could target specific human genes,10 and subsequent pre-clinical research and developmentled to the approval of the first siRNA drug in 2018.26 Most recently, Doudna and Charpentier were awarded theNobel Prize in 2020 for CRISPR-Cas9 gene editing. In a CRISPR-Cas9system, a small piece of RNA with a short “guide” sequenceattaches to a specific target sequence of DNA in a genome; the RNAalso binds to the Cas9 enzyme—thus, the modified RNA is usedto recognize the DNA sequence, and the Cas9 enzyme cuts the DNA atthe targeted location.27 Two human mRNAvaccines against COVID-19 received Emergency Use Authorization in2020, and one of them was finally approved in 2021.30 These key milestonesand achievements are captured in Many key discoveries have contributed to the advancing ofRNA medicineswe have today. Early research in the 1960s on nucleic acids led tothe discovery of mRNA.31 Drugs to treat many other diseases, includingcancer, hepatic and renal diseases, cardiac diseases, metabolic diseases,blood disorders, respiratory diseases, and autoimmune diseases arecurrently in various stages of clinical studies, some showing promisingresults.RNA technology provides an innovative approach for developing newdrugs for rare or difficult-to-treat diseases. Since 2014, severaldrugs have been approved to treat macular degeneration, Duchenne musculardystrophy (DMD), polyneuropathy, and amyotrophic lateral sclerosis.32 this paperfocuses on chemical modifications to the base, backbone, sugar, and5′ or 3′ ligations to other molecules. While chemicalmodifications increase RNA stability, complexes of RNA within nanoparticlesprovide further protection. Except for aptamers that bind to a cellsurface target, RNA must be delivered into the cell by a carrier.After the RNA is internalized, it must be released from the membrane-boundvesicle or endosome to the cytosol. Without a doubt, the deliverysystem has been an important research topic for RNA medicine.Compared to other biomolecules, RNA molecules are unstableandtransient. Foreign RNA molecules, when introduced to the human body,have limited protein expression levels in cells and often triggerimmunogenicity in the body. However, these practical problems canoften be mitigated by various optimizations on RNA molecules, includingchemical modifications, mRNA cap/codon/tail optimization, etc. Leveragingthe unique aspects of both the chemical and biological informationof the CAS Content Collection,32 Wethen appraised different types of RNA in medical applications: theiradvantages, challenges, and research trends. Subsequently, we assessedthe development pipelines of RNA therapeutics and vaccines with companyresearch focuses, disease categories, development stages and publicationtrends. Finally, we discussed RNA chemical modifications and deliverysystems in detail, as they are critical to the success of RNA medicine.We thus revealed the sustained global effort that propelled this fieldto the cusp of realization for novel medical applications of RNA inmany diseases. We hope this review can serve as an easy-to-understandoverview so that scientists from many different disciplines can appreciatethe current state of the field of RNA medicine and join in solvingthe remaining challenges for fulfilling its potential.In this paper, we reviewed naturally occurring RNAs with theircellular functions and the associated research trends based on theanalysis of CAS Content Collection.34 transfer RNA (tRNA),34 small nuclear RNA (snRNA),37 small nucleolar RNA (snoRNA),45 long non-coding RNA (lncRNA),52 short hairpin RNA (shRNA), micro-RNA (miRNA),59 transfer messenger RNA (tmRNA), small interfering RNA (siRNA),71 small activating RNA (saRNA), piwi-interacting RNA (piRNA),78 circular RNA (circRNA), ribozymes, and exosomal RNA.83 The cellular localizations of different types of RNA are illustratedin RNA, a versatile macromolecule that is specializedfor many functions,can be broadly defined as coding or messenger RNA (mRNA) and non-codingRNA (ncRNA). There are several different types of ncRNA includingribosomal RNA (rRNA),mRNA, which was thefirst RNA to be characterized,is the initial transcription product of a protein-coding gene andincludes both protein-coding exons and non-coding introns. The mature,translatable mRNA must be spliced to remove the introns and, in transcriptsthat can go through alternative splicing, one or more potential exons.The mature RNA has a 5′-7-methylguanosine cap, a 5′-untranslatedregion, a start codon (unique sequence of 3 bases) for the translatedregion of the gene, a stop codon that ends the translated region ofthe gene and starts the 3′-untranslated region, and a 3′-polyadenosinetail. Gene expression, which is often regulated by the amount of mRNAfor the gene, is controlled by the balance between synthesis and degradationof mRNA. Although mRNA is a critically important RNA, it makes uponly 1–5% of the RNA in a cell.34ncRNAs, in contrast to mRNA, are the final functionalproducts of the DNA. Although it was thought initially that ncRNAswere non-functional junk RNA, in the 1950s, in the same paper thatintroduced the phrase “Central Dogma”, Francis Crickcorrectly hypothesized that the ncRNA might function in the translationof mRNA into protein.84 In 1955, GeorgePalade identified ribosomes as a small particulate component of thecytoplasm that contains RNA, and in 1965, Robert Holley purified atRNA from yeast and determined the structure.86 In the past half-century, many types of ncRNA with various functionshave been identified; many are involved in regulating transcriptionand protein expression in the cell.94rRNA, which constitutes up to 80% of the RNAin anactive cell, comprises three rRNAs  complexedwith many proteins to form the large subunit of the ribosome and onerRNA (the 18S) complexed with proteins to form the small subunit ofthe ribosome. There are also two mitochondrial rRNA genes (the 12Sand 16S) which, along with many proteins, form the mitochondrial ribosome.rRNA in the ribosome, acting as a ribozyme, catalyzes peptide bondformation between two amino acids. Synthesis of the large amount ofrRNA occurs in the nucleolus, a heterochromatic region found in mostnuclei.34tRNA, which makes up10–15% of the RNA in thecell, translates the mRNA codon sequence for each amino acid. Themany different tRNAs, which are usually 75–95 nucleotides,all fold into very similar three-dimensional structures. The 3D structureexposes three unpaired nucleotides that serve as the anti-codon tobase pair with the mRNA. Specific amino acids are covalently boundto a tRNA by aminoacyl tRNA synthetases. The specificity between theanti-codon and the bound amino acid is the basis of translation. EachtRNA anticodon can bind to several different mRNA codons. This pairingis based on the wobble rules for the third nucleotide position ofthe anti-codon, which is often post-transcriptionally modified toallow for wobble-pairing. Common modifications at the third positioninclude 5-methyl-2-thiouridine, 5-methyl-2′-O-methyluridine,2′-O-methyluridine, 5-methyluridine, 5-hydroxyuridine, hypoxanthine,and lysidine.34snRNAs (∼150nucleotides) are components ofthe small nuclear ribonucleoproteins (snRNPs) of the spliceosome.They act as catalysts that splice mRNA into its mature form and theyare important in the selection of alternative splicing sequences.37snoRNAs (60–300 nucleotides) are boundto fourcore proteins and act as guides to correctly target modificationsfor the maturation of rRNA. They comprise two classes of RNA. C/DsnoRNAs participate in the 2-methylation of targeted nucleotides,while H/ACA snoRNAs participate in the modification of uridine topseudouridine. They help guide the protein to the specific target,rather than catalyzing the reaction directly. As participants in rRNAmaturation, snoRNAs are found in the nucleolus.45siRNAs are products of double-stranded lncRNAs and are central to RNA interference, which negativelyregulates gene expression. Double-stranded RNA (dsRNA), either fromgenomic lncRNA or dsRNA viruses, is recognized and cleaved by theendonuclease Dicer into 20–24 base-pair sections with shortoverhangs on both ends. These siRNAs bind the Argonaute protein toform the pre-RISC (RNA-induced silencing complex). Argonaute selectsthe less thermodynamically stable strand of the siRNA and releasesthe other strand to form the mature RISC. RISC recognizes mRNA complementaryto the single-stranded siRNA, and the Argonaute endonuclease cutsthis targeted mRNA, thereby downregulating the gene product. The bindingof a RISC to a target mRNA also prevents efficient ribosome bindingand translation, further downregulating the gene product. Active RISCsmay also affect the transcription of target genes by inducing chromatinreorganization through epigenetic modifications. This can be a defensemechanism against dsRNA viruses or an endogenous gene-regulatory mechanism.71miRNAs are closely related to siRNAs but areformedfrom pri-miRNAs (primary microRNAs), which are long, imperfectly pairedhairpin RNA transcripts. The pri-miRNA is processed first by Droshanuclease into a ∼70-nucleotide imperfectly paired hairpin pre-miRNA(precursor microRNA) that is then, like siRNA, processed by Dicerto produce the 21–23-bp, mature, double-stranded miRNA thatbinds to Argonaute to form the RISC. Alternatively, some miRNAs aremade from introns in mRNAs. After splicing, that intron is a pre-miRNAthat is processed by Dicer to form a RISC. miRNAs form negative generegulatory networks and intronic miRNAs may regulate and balance potentiallycompeting pathways.59piRNAs, like siRNA and miRNA, negatively regulategene expression, but they interact with the Piwi class of Argonauteproteins. Unlike siRNA and miRNA, piRNAs (24–31 nucleotides)are produced from long, single-stranded RNA transcripts through anuncharacterized Dicer-independent mechanism. Mature piRNAs bind toPiwi proteins to form RISCs that act primarily as epigenetic regulatorsof transposons (genetic elements that move around the genome) butmay also regulate transposons post-transcriptionally through the ping-pongpathway.78saRNA, like siRNA, is a ∼21-bp dsRNA longthatinteracts with Argonaute proteins to form a RISC. Unlike siRNA, saRNAupregulates target gene expression by an unknown mechanism, perhapsactivating transcription by targeting the promoter region of the gene.saRNA may be produced endogenously or artificially to strongly activatethe target gene.101lncRNAs comprise a mixed group of RNAs >200bp, whichdifferentiates them from short ncRNAs such as snoRNA, siRNA, miRNA,piRNA, etc. lncRNAs have a wide variety of functions including regulationof chromosome architecture and interactions, chromatin remodeling,and positive or negative regulation of transcription, nuclear bodyarchitecture, and mRNA stability and turnover.52circRNAs are lncRNAs with 5′ and 3′ends linked covalently to form a continuous circle. circRNAs are broadlyexpressed in mammalian cells and have shown cell-type and tissue-specificexpression patterns.102 Neither the mechanismsleading to circularization of the RNA nor the function of circRNAis known, but the leading hypothesis is that they may serve as miRNAsponges. Many circRNAs contain large numbers of miRNA target sitesthat may competitively antagonize the ability of miRNA to silenceits target genes.Exosomal RNAs are mRNAs, miRNAs,siRNAs, and lncRNAsthat are packaged and exported from the cell through the exosomalpathway. Although they are poorly understood, exosomal RNAs may serveas signaling molecules to regulate gene expression in target cells.These circulating RNAs, especially miRNAs, may serve as diagnosticand/or prognostic targets for various diseases such as cancers.83Antisense RNA (asRNA), also referred to as antisenseoligonucleotide (ASO), is a single-stranded RNA that is complementaryto a protein-coding messenger RNA (mRNA) with which it hybridizes,and thereby blocks its translation into protein.10332 is the largest human-curated collection of published scientificknowledge, used for quantitative analysis of global scientific publicationsagainst variables such as time, research area, formulation, application,disease association, and chemical composition. We searched the title,abstract, or CAS-indexed terms using RNA-related keywords and theirsynonyms to identify relevant published documents. The CAS Content CollectionTo better reveal the rising trendsof those recently emerged typesof RNA, the percentage of document publications of a specific yearwas calculated within the given type of RNA over the time 4. AlthoumRNAtranscripts can act as therapeutic RNAs, diagnostic biomarkers, ortherapeutic targets. Translation of an mRNA in the cell can producea therapeutic protein to replace a defective or missing protein. Inthe case of vaccines, mRNA translation can generate antigenic targetsfor the immune system, such as the spike glycoprotein of SARS-CoV-2in the COVID-19 mRNA vaccines. mRNA may also serve as a therapeutictarget for ASOs, siRNA, miRNA, aptamers, and suppressor tRNAs.104 Because a single miRNA binds to multiple mRNAs, miRNAsserve as regulatory check points. The cellular processes regulatedby miRNAs include those involved in many diseases, such as cardiovasculardisease, cancer, and disease-related metabolic pathways.106 Thus, they can serve as biomarkers for disease diagnosis, as potentialdrugs, or as attractive targets for other regulatory RNAs.In the cell, miRNAs bind to the 3′-untranslated region ofmRNAs and target them for degradation by the RISC.104AlthoughsiRNAs, like miRNAs, use the RISC to degrade their targetmRNAs, siRNAs bind to specific areas in the mRNA coding region. Thistarget specificity makes them attractive as potential drugs, but off-targeteffects can negate this advantage. In order to minimize off-targeteffects, siRNAs are modified to decrease their thermal stability,increase their target specificity, and decrease the stability of theirbinding of the siRNA to mRNAs that are not an exact match to the intendedtarget mRNA. These usually include 2′-O-methyl and 2′-MOEribose modifications that introduce steric hindrance to decrease bindingaffinity ,the antibody targets the site of interest while carrying an ASO ora siRNA that acts on the targeted region.6 The ability of the CRISPR-Cassystem to create directed double-stranded breaks in DNA allows therepair of genetic mutations. Changing the endonuclease activity ofCas can convert CRISPR-Cas to a system that nicks a single strandof the DNA or that deaminates a specific nucleotide.109 If the Cas endonuclease is inactivated, the system cansimply bind to DNA to regulate transcription.104The CRISPR-Cas system uses a guide RNAthat is either a combinationof a trans-activating CRISPR RNA (tracrRNA) and a CRISPR RNA (crRNA)or a joined single guide RNA (sgRNA). The guide RNA directs the CRISPRcomplex containing a Cas endonuclease to a specific site in the genomefor cleavage.In vitro systematic evolution of ligands by exponentialenrichment (SELEX) is used to identify single-stranded RNA or DNAoligonucleotides with a high affinity for a target. Because theirbinding depends on their 3D structure, aptamers can bind a wide rangeof targets, including proteins, cells, microorganisms, chemical compounds,and other nucleic acids.110 Aptamers mayalso serve as delivery agents for siRNA in nanoparticles for cancertherapy.111Aptamers are structure-based rather than sequence-basedligandsthat neither hybridize with other nucleic acids nor produce proteins.They can be RNA, DNA, RNA/DNA combinations, or even proteins. 32 miRNA and mRNA, the two most populartherapeutic RNAs in the journal and patent literature, can serve asdrugs, disease biomarkers, and drug targets. Together with siRNA,they represent most of the therapeutic RNA patent activity. ApprovedRNA drugs include mRNAs, siRNAs, ASOs, and aptamers; these RNAs alongwith CRISPR RNAs and AOCs comprise most of the clinical candidates.To measure the distributionof research effort using differenttypes of RNA as therapeutics, vaccines, or diagnostics, related documentswere extracted accordingly from the CAS Content Collection, and journaland patent publications percentages for each type of RNA were determinedas shown in 32 shows a steadyincrease in the number of journal articles and patents related toRNA applications in medicine  shRNA; (b) lncRNA,an RNA whose role in gene regulation is generating increasing interest;(c) circRNA, once thought to be a byproduct of RNA splicing, thatmay have a role in regulation as a miRNA sponge; (d) saRNA, whichregulates gene transcription; and (e) exosomal RNA, which is containedin naturally occurring lipid vesicles called exosomes, which can crossthe blood-brain barrier and appear to be vital for cell-to-cell communication,and which transport mainly miRNA.32 only mRNA has substantial applications in vaccines and lateral flow immunoassays.ts power 7; howeveThereare several important advantages to RNA therapeutics. They are: (a)target specific, (b) modular with easy-to-switch sequences, (c) predictablein terms of pharmacokinetics and pharmacodynamics, (d) economicalin comparison to antibodies or protein drugs since they are synthesizedfrom widely available synthons on an automated synthesizer, and (e)relatively safe, as most of them do not alter the genome.120In vitro transcription using commercial kits121 produces longer RNAs, i.e., mRNAs and lncRNAs.The sequence of the RNA can be changed easily, providing custom moleculestargeting different proteins or genes. Thus, the development of RNAtherapeutics and vaccines that target disease-specific genes or proteinsis relatively fast and straightforward. This was demonstrated by thedevelopment, testing, and administration of the COVID-19 mRNA vaccineswithin a year of the isolation and sequencing of the SARS-CoV-2 viralgenome.108Proteinsmust be designed for synthesis from genes in plasmids, then optimizedfor expression and purification, and these processes may be differentfor each protein and challenging to optimize. In contrast, RNA canbe easily synthesized and purified by established methods using commerciallyavailable reagents and equipment. Small RNAs, such as aptamers, siRNAs,miRNAs, and ASOs can be synthesized using solid-support chemistryin commercial oligonucleotide synthesizers.104Most RNAs regulate transcription,post-transcriptional processing,and translation, but they do not alter the genome. The exception isthe CRISPR-Cas system in which the guide RNA and Cas endonucleasecan edit the genome. RNA aptamers, which mimic ligands, regulate post-translationalprotein activity. The rapid degradation and predictable pharmacokineticsof RNAs give them a safety advantage over gene therapies.104 Although designed for specifictargets, therapeutic RNAs can have off-target effects, limiting theirusefulness as drugs. Several of these limitations can be mitigatedby chemically modifying the RNA to increase target specificity, lowernuclease susceptibility, and improve cellular uptake.108Despite the attractiveness of the plug-and-playconcept of RNAtherapeutic drug design, they require testing to determine their efficacyand safety, and cell delivery is difficult because RNA is easily degraded.The therapeutic RNA must penetrate the cell membrane and escape endosomalentrapment.32 for journal publications and patents on RNAsas therapeutics, vaccines, or diagnostic agents for diseases and foundthat 50% of the publications are associated with cancer diagnosisor treatment, although lung, liver, and metabolic diseases are alsohighly represented . Infectious diseases and cancer haveshown the greatest growth and are the most frequent diseases treatedby RNA, followed by eye and cardiovascular diseases, which grew inthe first decade of this century and remained relatively stable inthe second decade .Among the top 15 RNA therapeutics companies , each mostly focuses on one type ofRNA to develop novel RNA therapeutics for treating diseases that rangefrom very rare to common. mRNA and siRNA are the most common RNAsused by the top 15 companies, followed by ASO, CRISPR, and aptamers.Many of these companies are among the top patent assignees for RNAtherapeutics, vaccines, and diagnostics in the commercial sector  Alnylam Pharmaceuticalsand Dicerna are headquartered in Massachusetts, USA. patents 11. Dicer patents 11. Sirnaapeutics 11. Silenapeutics 11.132 and Sarepta Therapeutics in Massachusetts,USA.133 Ionis has developed over 40 therapeutics were then categorized basedon their targeting diseases and development status .Disease types along with their RNA therapeuticsare examined andsummarized below 1–11. TheCardiovascular diseases, which account for 32% ofall deaths, are the leading cause of death worldwide, taking an estimated17.9 million lives each year.138 Cardiovasculardiseases include disorders of the heart and blood vessels. Over 80%of cardiovascular disease deaths are due to heart attacks and strokes,and one-third of these deaths occur prematurely in people under 70years of age.138139 It is a trivalent N-acetylgalactosamine -conjugated siRNA with threeGalNAc molecules clustered and conjugated to one siRNA molecule.140Leqvio is an approvedtherapeutic for the treatment of the cardiovasculardisease, familial hypercholesterolemia 1. Leqvio141 By targetingthe LPA gene, SLN360 lowers the levels of LpA, decreasing the riskof heart disease, heart attacks, and strokes.131 Alnylam is also evaluating the siRNA product Zilebesiranthat targets angiotensinogen for sustained reduction of hypertension.142 Interim phase I results show >90% reductionin serum angiotensinogen (AGT) for 12 weeks at a dosage of 100 mgor greater given quarterly or biannually.143 This dosing regimen and the continued efficacy and safety of thedrug are being evaluated in a phase II study initiated in June 2021(NCT04936035).143 Zilebesiran also usesAlnylam’s trivalent GalNAc-conjugated siRNA delivery platform.140 AstraZeneca and Moderna are collaborating onthe mRNA drug AZD8601 to treat ischemic heart disease.144 AZD8601 targets vascular endothelial growthfactor-A (VEGF-A).145 When AZD8601 is injectedinto the epicardium, VEGF-A is produced close to the damaged heartmuscle, allowing cardiac regeneration.146In additionto this approved one, there are several more promisingRNA therapeutics for cardiovascular diseases currently in the clinicaltrial stages 1. SilencMetabolic diseases affect over a billion peopleworldwideby causing too much or too little of essential substances in the body.149 Diabetes alone affects 422 million people worldwide,causing 1.5 million deaths annually.150 IONIS-GCGRRx is an ASO product developed by Ionis Pharmaceuticalsthat treats diabetes by reducing the production of the glucagon receptor(GCGR) , received European Union(EU) approval in 2019 as a treatment for familial chylomicronemiasyndrome (FCS)  reduces its mean plasmalevels.154r(GCGR) 2.151 Glume (FCS) 2.153 FCS155 Mutations in the SERPINA1 gene cause a deficiency of AAT in the blood, leadingto toxic effects in the lungs and the accumulation of high levelsof AAT in the liver that cause liver damage.155 ARO-ATT, a siRNA product developed by Arrowhead Pharmaceutical,targets the SERPINA1 mutation, and in pre-clinicalstudies has shown promise in reducing AAT liver disease  is a hereditarymetabolicdisease. Alpha-1 antitrypsin (AAT) is a glycoprotein produced in theliver that travels through the bloodstream to protect the lungs frominflammation. disease 2.156 Bea159 MMA is caused by mutationsin the MMUT, MMAA, MMAB, MMADHC, and MCEE genes. The mutationin the MMUT gene accounts for about 60% of MMA cases.Moderna has developed an mRNA therapeutic mRNA-3705 that targets the MMUT mutation  is a hereditarymetabolic diseasein which the body is unable to metabolize certain proteins and lipidscorrectly.mutation 2.144 mRNLiver diseases affect morethan 1.5 billion peopleworldwide164 and account for over 2 milliondeaths per year.165 The siRNA therapeuticGIVLAARI, developed by Alnylam, received FDA approval in 2019 forthe treatment of acute hepatic porphyria , reducing its expression.151 Mutation of PNPLA3, whichproduces a protein that accumulates on the surface of intracellularlipid droplets, is strongly associated with an increased risk forNASH.169Non-alcoholic steatohepatitis(NASH) is an accumulation of fatin the liver that causes liver damage. The antisense therapeutic ION839/AZD2693from Ionis/AstraZeneca entered phase I trials 3 in 2020Cancer includesa large group of diseases that arecharacterized by abnormal cell growth in various parts of the body.It is the second leading cause of death globally, accounting for anestimated 10 million deaths per year with over 19 million new casesdiagnosed in 2020.171 Currently there areno approved RNA therapeutics for cancer treatment. NOXXON Pharma’slead aptamer candidate NOX-A12 is in development as a combinationtherapy for multiple types of cancers ,172 which, with its receptors,acts as a link between tumor cells and their environment, promotestumor proliferation, new blood vessel formation, and metastases, andinhibits cell death.174 cancers 4.172 It 175 targets TGF-β1and COX-2 mRNAs  phase II studies with newly diagnosed patients.178 Descartes-11 contains autologous CD8+ T cells engineered with RNA chimeric antigen receptors (CARs) thatbind to B-cell maturation antigen (BCMA).177 BCMA is highly expressed in all myeloma cells, and Descartes-11binds and destroys BCMA-positive myeloma cells.177The anti-cancer siRNA product,STP707 by Sirnaomics,-2 mRNAs 4. A pre-ma cells 4.177 It Infectious diseases are caused by bacteria,viruses,fungi, or parasites.183 The COVID-19pandemic brought the first approved mRNA vaccine to market. BioNTech/Pfizer’sCOVID-19 BNT162/Comirnaty vaccine was given U.S. FDA emergency useauthorization in 2020 and approved by the FDA in 2021  therapeutic specifically designed to reduce all hepatitis Bvirus (HBV) antigens, including hepatitis B surface antigen (HBsAg)5.187 HBs144 mRNA-1010targets influenza lineages recommended by the World Health Organization(WHO) for the prevention of influenza, including seasonal influenzaA H1N1 and H3N2 and influenza B Yamagata and Victoria.189 CureVac has a mRNA prophylactic vaccine forrespiratory syncytial virus (RSV), a common respiratory virus thatcan cause serious illness in infants and older adults, in their pipelineunder pre-clinical development for the treatment of spinal muscular atrophy  andis used for the treatment of hATTR amyloidosis-polyneuropathy, a diseasedue to mutations in the gene encoding TTR that leads to abnormal amyloiddeposits on nerves.201 Onpattro by Alnylamis another approved RNA drug for the treatment of hATTR amyloidosis-polyneuropathyby targeting hepatic production of transthyretin , that are in pre-clinicalstudies ,the second most common and best understood genetic cause of ALS.Amyotrophic lateral sclerosis (ALS) is a progressiveneurodegenerativedisease that affects nerve cells in the brain and spinal cord.l trials6.151 Tof205 AOC 1001 has an ongoing phase I/IItrial in adults with myotonic dystrophy type 1 (DM1). DM1 is a progressiveneuromuscular disease that impacts skeletal and cardiac muscle. DM1is caused by an abnormal number of CUG triplet repeats in the myotonicdystrophy protein kinase gene (DMPK), reducing muscleblind-like protein(MBNL) activity and disrupting muscle development.206 AOC 1001 is designed to reduce DMPK levels and CUG tripletrepeats so that MBNL can perform normally.205 Avidity Biosciences has also developed an AOC to treat DMD.205 The oligonucleotides are designed to promoteskipping of specific exons to produce dystrophin in patients withDMD. Their leading DMD drug candidate, AOC 1044, can induce exon skippingspecifically for exon 44 (205Avidity Biosciences has a pipeline of antibody oligo-nucleotideconjugate (AOC) therapeutics focused on neuromuscular diseases. Theirleading candidate, AOC 1001, is a siRNA conjugated with a monoclonalantibody (mAb) 6.205 AOC exon 44 6, and clEye diseases including visualimpairment affect 2.2billion people globally.210 Macular degeneration,which causes loss in the center of the field of vision and is irreversible,affects 196 million people.211 Macugen,developed by Gilead Sciences  protein in the eye, reducing the growth of blood vesselsto control the leakage and swelling that cause vision loss.213 As age-related macular degeneration (AMD) progresses,geographic atrophy (GA), a chronic progressive degeneration of themacula, can develop.214 Zimura by IverBio is another RNA aptamer therapeutic and is currently in a phaseIII clinical trial  whichresults from trinucleotide repeat expansion mutations in the Transcriptionfactor 4 (TCF4) gene.217Sciences 7, was thal trial 7. ZimuraKidney disease affects over 850 millionpeople worldwide.218 Primary hyperoxaluriatype 1 (PH1) is a genetickidney disease characterized by the overproduction of oxalate, whichleads to kidney stones, kidney failure, and systemic oxalosis.218 The siRNA therapeutic Oxlumo was developedby Alynlam and approved by the FDA in 2020 (219 Oxlumo targets the hydroxyacid oxidase 1 (HAO1) gene, which encodes glycolateoxidase.219 Oxlumo reduced urinary oxalateexcretion in patients with progressive kidney failure in PH1.220 The majority of patients had normal or near-normal levels of oxalateafter 6 months of treatment. Autosomal dominant polycystic kidneydisease (ADPKD) is caused by mutations in the PKD1 or PKD2 gene.221 Itis characterized by cysts within the kidneys, often leading to kidneyfailure. To treat ADPKD, Regulus designed RGLS4326, a second-generationoligonucleotide that inhibits miR-17 (222 Immunoglobulin A (IgA)nephropathy is a chronic kidney disease that is caused by depositsof protein IgA inside the glomeruli of the kidneys.223 Overproduction of complement factor B (FB) is associatedwith increased IgA nephropathy. Ionis has partnered with Roche todevelop a ligand-conjugated ASO therapeutic, IONIS-FB-LRx, which targetsFB to reduce the production of IgA and alleviate the symptoms of IgAnephropathy  to the forefront of medical news during thepandemic. ARDS is a severe inflammatory lung disease with a mortalityrate of over 40%.228 Inflammation leadsto lung tissue injury and leakage of blood and plasma into air spaces,resulting in low oxygen levels and often requiring mechanical ventilation.228 Descartes-30 is an engineered mRNA cell therapyproduct by Cartesian Therapeutics  in the lungs and airways  protein, resulting from mutations inthe rapeutic9 that by airways 9. IncreaBlood diseases affect one or more componentsof blood.Sickle cell disease is a type of blood disease that causes red bloodcells to become misshapen and break down.238 Beta-thalassemia syndromes are a group of blood diseases that resultin reduced levels of hemoglobin in red blood cells.239 BEAM-101 , which could alleviatethe effects of mutations causing sickle cell disease or beta-thalassemiasince the fetal hemoglobin does not become misshapen.240 Beam plans to initiate a phase I/II clinicaltrial to assess the safety and efficacy of BEAM-101 for the treatmentof sickle cell disease.240 BEAM-101 usesan electroporation delivery system.157 SilenceTherapeutics has RNA therapeutics for blood diseases such as thalassemia,myelodysplastic syndrome, and rare iron-loading anemias.241 Their siRNA therapeutic SLN124 targets transmembraneserine protease 6 (TMPRSS6)  10, a genKeloid scarring is atype of pathological conditionthat forms abnormally thick scarring after a skin injury. STP705,from Sirnaomics, is a siRNA therapeutic in phase II clinical trialfor the treatment of keloid scars  is the inability to controlalcohol use despite adverse social, occupational, or health consequences.According to the World Health Organization, AUD affects over 283 millionpeople globally.248 Alcohol is metabolizedin the liver to acetaldehyde via alcohol dehydrogenase and then toacetic acid by aldehyde dehydrogenase 2 (ALDH2).249 Inhibiting ALDH2 results in unpleasant symptoms due tothe increased acetaldehyde when alcohol is not fully metabolized.250 The siRNA therapeutic DCR-AUD by Dicerna knocksdown ALDH2 protein expression (250pression 11 in thed-ribose through a β-N-glycosyl bond between the ribose and a pyrimidine base (uracil andcytosine) or a purine base (adenosine and guanosine). The nucleosidesare connected by a phosphodiester bond using the phosphate betweenthe 3′ and 5′ carbons on adjacent ribose molecules .254 In addition, modifications improve the performanceof therapeutic RNA. Replacing uridine with the modified base 1-methylpseudouridine. Another modification at the C-2′position is the LNA in which a 2′-O,4′-C-methylene bridge connects the 2′ position to the4′ position on the ribose.257The hydroxylgroup on the C-2′position of the ribose destabilizes RNA compared to DNA. Modificationof this hydroxyl protects against nuclease digestion and can lowerthe thermal stability of duplexes formed with a target RNA. 254In addition, the 5′ and3′ ends of RNA can be modifiedusing cleavable linkers attached to GalNAc groups or lipophilic moietiesto target the therapeutic RNA to the desired tissue. The linkers arecleaved by acid pH, redox potential, or degradative enzymes in cellsbut not in serum or blood. Cleavable linkers include acid-cleavablegroups, ester-based cleavable groups, and peptide-based cleavablegroups.258Modifications to the phosphategroup in the sugar–phosphate backbone can improve the resistanceof therapeutic RNAs to extracellular and intracellular nucleases.In addition, the negative charge on the phosphate group interfereswith the delivery of the RNA into the cell through the lipid bilayermembrane, which is impermeable to polar molecules. Thus, replacingthe oxygens on the phosphates with neutral groups or complexing thephosphate groups to cations like sodium can improve RNA delivery.259 RNA molecules with phosphorothioate linkagesat the ends resist exonucleases, whereas RNA molecules with phosphorothioatelinkages within the RNA resist endonucleases. However, the sulfuron the phosphate group creates stereogenic α-phosphorus atomsresulting in diastereomers with different functional properties thatcan affect duplex formation. Careful spacing of the phosphorothioatelinkages within the RNA can ameliorate this problem.258A widely used backbone modification,phosphorothioate, as shown in d-ribose witheither an l-ribose or a non-ribose moiety (260 In peptide nucleic acids (PNAs)the sugar–phosphate backbone is replaced with a flexible N-(2-aminoethyl)glycine polymer with the nucleobases attachedvia a methylene carbonyl linkage.261 PMOs,GNAs, and PNAs all resist nuclease degradation. PNA forms duplexeswith complementary DNA or RNA with higher affinity and specificitythan unmodified DNA–DNA or DNA–RNA duplexes. However,duplexes containing PNAs, PMOs, and LNAs resist RNase H degradation,inhibiting gene knockdown through targeted mRNA degradation.257Other backbone modifications replacethe e moiety 20. Phosp262 from publisheddocuments and stored in the CAS Content Collection.32 To better understand the chemical modification trends onRNA molecules, we extracted ∼170,000 modified RNA sequencesfrom the CAS Content Collection. Sequence datafor RNAs and RNA modifications are annotated and collected with CASRegistry Sequence Guidelinesin vitro transcription. The mostcommon modifications contained in the longer sequences are triphosphatesand 7-methylguanosines, suggesting that they are mRNAs with 5′end caps consisting of 7-methylguanosine linked to the 5′ endof the mRNA with a triphosphate group. In addition, modified adenosineis observed in longer sequences, particularly in sequences from 901to 1000 nucleotides; this would include the mRNA modification, 6-methyladenosine.264 Since therapeutic mRNAs are translated by the ribosomes to producean active protein, excessive modification might provide steric hindrancethat inhibits translation.Chemical modifications were furtheranalyzed using this data setwith specific types of modifications and sequence lengths. Out of145 curated sequence modifications, 117 sequence modifications wereidentified in the modified RNA data set. A heatmap 22 coveri5U) and 5-methylcytosine(m5C). The rare base pseudouridine appeared frequentlyin the database, followed by the rare bases dihydrouracil and inosine.Bases in which the N-7 position is converted to a carbon (7-deazaguanineand 7-deazaadenine) appeared with some frequency, as did the replacementby sulfur of the oxygen double-bonded to the N-4 or N-2 position ofuracil or the N-2 position of cytosine. Less common modificationsincluded benzoyl groups attached to N-3 in cytosine and the nitrogensin adenosine.Common nucleobase modifications for small RNAs (those <200 nucleotidesin length) are 5-methyluridine  at sequence lengthsof 21 and 42 is because thymidine at the 3′ end of artificialsiRNAs protects them from exonucleases. Artificial siRNAs are predominantly21-mers, and since they are double-stranded, they are highly representedat both 21 and 42 nucleotides.l-ribosein place of d-ribose, is a relatively rare modification,as are replacement backbones, such as PMO, GNA, and PNA.Phosphorothioate is the mostcommon phosphate modification forthe sugar–phosphate backbone, although phosphorodithioate alsooccurs. Altered ribose stereochemistry, such as Figures S3 and S4). Overall, thereis little correlation between the type of RNA and the targeted diseases, suggesting any RNA can beexplored to target any disease.Heatmaps for modifications versus disease targets show littlecorrelation between the disease and the modifications on the therapeuticRNA  residues on both RNA strands to protect the siRNA from exonucleases.siRNAs have a moderate numberof phosphorothioate linkages, with2′-fluoro and 2′-O-methyl modification of the ribose,and are often modified on their 3′ ends. Three of the approvedtherapeutic siRNAs, Leqvio, Givlaari/Girosiran, and Oxlumo, are 3′-glycosylatedwith the trivalent GalNAc conjugate104 The single FDA-approvedaptamer, Macugen, has extensive 2′-O-methyland 2′-fluoro modifications, a 3′-to-3′ linkto thymidine at its 3′ end, as well as double PEGylation atits 5′ end  on their 5′ends. Because aptamers bind to their targets as a result of theirtertiary structure and do not rely on nucleic acid hybridization forfunction, they have fewer constraints on modifications compared toother therapeutic RNAs.s 5′ end 23.The mRNA vaccines BNT162/Comirnaty and mRNA-1273/Spikevaxhave7-methylguanosine caps connected to the 5′ end of the mRNAby a triphosphate group and have all their uridines replaced with1-methylpseudouridine, which improves mRNA stability and translation.In addition, both contain a 2′-O-methyl modification to thenucleotide immediately following the 7-methylguanosine cap to improvestability to base and nuclease digestion.271In summary, chemicalmodifications protect therapeutic RNAs fromexonucleases, endonucleases, and the cellular environment, and theyenhance pharmaceutical activity. The choice of the backbone determineswhether an ASO blocks cellular processes such as translation, transcription,or splicing, or targets an RNA for nuclease digestion. The 2′-ribosemodifications on siRNAs mitigate off-target effects by lowering thethermal stability, thereby enhancing target-specific binding. 1-methylpseudouridineimproves stability and translation of therapeutic mRNAs. Since therapeuticRNAs are extensively modified, they frequently are not designatedas RNA vs DNA but have names such as ASO (antisense oligonucleotide)and siNA (short interfering nucleic acid) for siRNAs.11 While biological barriers such as immunogenicityand nucleases are usually addressed by modifying the RNA chemically,encapsulation of RNA into nanocarriers can both protect and deliverRNA to cells.272 Nanomaterials with biodegradability,biocompatibility, and low toxicity are used as RNA carriers. Theseinclude lipids, chitosan, cyclodextrin, polyethylenimine (PEI), poly(lactic-co-glycolic acid), dendrimers, magnetic nanoparticles, carbonnanotubes, gold nanoparticles, silica nanoparticles, and others , are in the CAS Content Collection.carriers 25.276 The advantages of lipid systems include ease of production, biodegradability,protection of entrapped nucleic acids from nuclease degradation andrenal clearance, promotion of cellular uptake, and endosomal escape.277 Liposomes have been recognized for a long timefor their role as immunological adjuvants in vaccines. As early as1974 it was reported that they enhance the immune response to liposome-entrappedtetanus toxoid.278 Subsequently, this wasshown to apply to many other antigens including mRNA vaccine products,such as the spike protein of the coronavirus.281Lipid nanoparticles, comprisingstable complexes between synthetic cationic lipids and anionic nucleicacids, are currently the most widely used non-viral delivery systemfor nucleic acid drugs and vaccines.19 Cationic lipidsdiffer from natural lipids by having an ionizable (cationic) headgroupin place of the zwitterionic or anionic headgroup of the natural lipids.They include two hydrophobic alkyl chains or a cholesterol moiety,a positively charged polar headgroup, and a linker joining them. Ionizablelipids are positively charged inside the cell but are neutral in thebloodstream due to pH differences, and they are less toxic than non-ionizablecationic lipids.282 Examples of cationiclipid structures used as vectors for gene delivery are provided in 32 is available.276Since the first cationic lipids successfully delivered plasmidsinto cells in 1987, many more have been synthesized and tested asnucleic acid carriers.283 as well as pyridinium,piperazine, and amino acid headgroups such as lysine, arginine, ornithine,and tryptophan285 have also been used. Headgroupswith multiple cationic charges such as DOSPA, DOGS286 may be more efficient than single-charged lipids287 since the multiple charges of the headgroupscondense nucleic acids. However, multivalent cationic lipids bindstrongly to the nucleic acid, preventing intracellular release, andthey tend to form toxic micelles.288 Combinationsof quaternary amines and polyamines enhance transfection efficiency.The first cationic lipid to combine these two functionalities, Lipofectamine,is the long-standing gold standard in nucleic acid delivery efficiency.It comprises DOSPA formulated with the helper lipid dioleoylphosphatidylethanolamine(DOPE).289The head groups of cationic lipidsare typically amine derivativessuch as primary, secondary, and tertiary amines ,quaternary ammonium , combinationsof amines , and amidinium salts 26. Guani290 These vaccines deliver mRNAencoding the SARS-CoV-2 spike protein into the cytoplasm of host cells,which is then translated and acts as an antigen for the developmentof an immune response. Both vaccines contain ionizable cationic lipidsenabling RNA complexation. The hydrocarbon chains of the proprietarycationic lipids of both these vaccines are optimized to enable safeclearance and mRNA delivery efficiency. They also contain a PEGylatedlipid to confer “invisibility” from antibody opsonizationand phagocytosis. Packing of RNA into lipid nanoparticles is aidedby distearoylphosphatidylcholine and cholesterol.Recently, LNPs have beenin the global spotlight as a vital componentof COVID-mRNA vaccines, playing a key role as an immunological adjuvant,in effectively protecting and transporting mRNA to cells.291 which are associated with the efficacy of cationiclipid carriers in delivering nucleic acids into cells.292 Despite their benefits, lipid nanoparticlesshow cell toxicity and stimulate the release of systemic inflammatorycytokines, and lipid aggregates may accumulate in the liver and spleencausing hepatotoxicity.293Complexationwith cationic lipids stabilizes nucleic acids, protectsthem from nuclease degradation, and facilitates delivery to the targetcells. Lipid nanoparticles carrying nucleic acids adsorb to the cellmembrane, are endocytosed, and release the nucleic acids into thecell. Cell membranes have negative charges that attract the cationiclipid nanoparticles and drive the adsorption and fusion of the lipidnanoparticle with the cell membrane. The anionic lipids of the cellsare thought to neutralize the charge of the cationic lipid carriers,thereby eliminating the electrostatic interactions between the lipidcarriers and the nucleic acids, and facilitating the release of thenucleic acids. Neutralization of the cationic lipids also destroysthe nanoparticles by promoting the formation of non-lamellar structures,295 Although their functions are largely unknown, they are believedto be associated with important physiological processes such as theregulation of intercellular communications and signaling, and of transmissionof macromolecules between cells.296 Exosomes,which are secreted by most cells, may be important in intercellularcommunication and signaling, and in the transport of proteins, lipids,and nucleic acids between cells. Since they are rich in mRNAs andsmall RNAs and can transport their contents to recipient cells, theyare considered good candidates for RNA delivery. Exosome-mediateddelivery of β-secretase-1 siRNA  to the brain resultedin highly efficient β-secretase-1 gene knockdown in the mousebrain cortex.297 Electroporation has beenapplied to load exosomes with exogenous siRNA.299Nearly 20 yearsafter scientists first prepared liposomes (thefirst-generation lipid nanoparticles), they found similar ∼100nm extracellular lipid vesicles in most eukaryotic cells. They areexosomes that form naturally in the endosomes and are released fromcells normally or as a result of some pathologies. They are formedby invaginations of the endosomal membrane and are subsequently releasedin the extracellular space via fusion with the plasma membrane.300 and hence less likely to be toxic or to cause immune responses.Moreover, exosomes can cross biological barriers such as the blood-brainbarrier. The unique membrane composition of exosomes is key to theirability to enter target cells. The membranes of exosomes are enrichedin cholesterol and phosphatidylserine.301 However, vesicles comprising only the lipids from the exosomal membraneare unable to fuse with cells, indicating that exosomal membrane proteinsare also important for their activity.301 A notable advantage of the exosomes as compared to other nanoparticledelivery vehicles is that they do not lead to a harmful accumulationof therapeutic RNAs in the liver.303 In additionto exosomes, other biological carriers obtained from living organismsmay include red blood cells, extracellular vesicles, platelet membrane-coatingvehicles, red blood cell membrane coating nanoparticles, cancer cellmembrane-coated nanoparticles, and macrophage-based vehicles.305 Red blood cells are appropriate as carriers because they lack bothmitochondrial and nuclear DNA, so the risk of horizontal gene transferis avoided.306 Macrophages are attractivecarriers for solid tumor targeting RNA delivery due to their naturalcapacities to reside into tumors throughout tumor progression.307Exosomes exhibit certain advantages over conventional deliverysystems. They are natural transporters308Polymerscomprise the secondlargest group of nucleic acid delivery vehicles after lipids. Cationicpolymers form stable complexes (polyplexes) with anionic nucleic acidsand offer a versatile, scalable, and easily adjustable platform forefficient nucleic acid delivery, while minimizing immune responsesand cellular toxicity.309 PEI, poly(l-lysine)(PLL), poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA), poly(2-aminoethylmethacrylamide) (PAEMA), poly(amidoamines) (PAAs), and poly(β-aminoesters) (PBAEs) have been studied for drug delivery  (PPI) dendrimers 27. Branc315PAMAM dendrimers, the most widely used dendrimer for nucleicaciddelivery, comprise a hydrogen-bonding amide and tertiary amine groupsin their cores and primary amine end groups in their coronas. Structuralmodifications have been attempted to lessen potential toxicity, enhancecirculation time, and/or enhance targeting.316 Chitosan, alinear cationic polysaccharide, has been formulated into polyplexesto deliver miRNA to osteopathic tumors, for multiple myeloma, andfor bone regeneration.319 An advantage of chitosan isthat nucleic acids easily dissociate from the polymer upon cellularinternalization.320 Cationic collagen derivativeshave been used for nucleic acid delivery to articular cartilage, forbone regeneration, and for treating tumor metastases.323Natural polymers including proteins and polysaccharides, particularlycationic collagen derivatives and chitosan, have been studied as nucleicacid delivery vehicles.324 that haveshown clinical success.325 CDs are biocompatiblecyclic oligosaccharides with a hydrophilic exterior surface and ahydrophobic interior cavity. Cationic CD derivatives assemble withsiRNA via electrostatic interactions into stable gene delivery nanostructuresof 50–200 nm.326Cyclodextrins (CDs) are natural carbohydrate polymers forthe deliveryof therapeutic nucleic acids327 and areinternalized by endocytosis.328 Followingendocytosis into early endosomes, polyplexes move through the progressivelymore acidic endosomes (pH 6.0–4.8). Escaping endosomal confinementis critical for nucleic acid delivery; therefore, scientists are developingand modifying polymer-based systems to promote endosomal escape.331Similar tocationic lipid nanoparticles, polymeric nucleic acidcarriers bind non-specifically to the negatively charged cell membranevia electrostatic interactions333 Amphipathic CPPs have been developed using hydrophilic and hydrophobicdomains to provide both nucleic acid complexation and membrane interactions.334 CPPs are promising, non-viral alternativesto lipid- or polymer-based carriers of therapeutic nucleic acids.12Peptides are structurallyand functionallyversatile, biocompatible, and can target cells; thus, they are attractiveRNA carriers. Peptides that penetrate the cellular membrane and transferto the cytoplasm, CPPs, are used most frequently for RNA delivery13. CPPs 338 The positively charged oligoarginine peptidespromote internalization by forming hydrogen bonds with the membranesulfates and the nucleic acid phosphate groups.340 The histidine-rich peptide is efficient in siRNA delivery.341 The skin permeating and cell entering (SPACE)peptides facilitate the penetration of conjugated cargoes into theepidermis and dermis.342 Recently, fusion peptides were developed comprising SPACE and cationicoligoarginine linked by a GCG sequence.343 Nanocomplexes of fusion peptides and siRNA exhibited enhanced cellularuptake, gene silencing, and retention, possibly due to the synergisticeffect of the oligoarginine and SPACE peptides. Oligoarginine electrostaticallyattracts siRNAs to form nanocomplexes, and the SPACE peptide interactswith the cellular membrane via hydrogen bonding.The Tat peptide originated from the Tat protein ofthe human immunodeficiencyvirus (HIV).345 The hydrophilic domain of positively charged amino acids, such asarginine, lysine, and histidine, provides a net positive charge ofat least +8 that condenses the RNA and promotes hydrogen bonding withthe anionic cellular membrane to stimulate cellular uptake.346 The hydrophobic peptide domain of tryptophanand phenylalanine enhances interactions with the lipid bilayer. Terminalmodification of the cationic peptides with hydrophobic molecules suchas cholesterol, stearic acid, or cholic acid increases the hydrophobicity.348 The other terminus of the peptide can be modified with hydrophilicmolecules such as PEG. These peptides form a micelle-like structurewith siRNA and deliver them efficiently to target cells.348Non-covalentcomplexation of cationic peptides that comprise hydrophilicand hydrophobic domains with RNA provide efficient gene silencing.349 Protamine–RNAcomplexes show limited efficacy, possibly due to the tight interactionbetween the protamine and RNA.350Protamines are naturally occurring cationic peptides involvedinthe condensation of chromatin during spermatogenesis. Comprising >65%positively charged arginines, protamines form non-covalent electrostaticcomplexes with nucleic acids and protect them from enzymatic degradation.Although protamines are intrinsically disordered peptides, in thepresence of nucleic acids they switch from a random coil to a structurewith one or more α-helices.352 Two approaches forthe design of AuNP nucleic acid carriers are covalent attachment andsupramolecular assembly. To covalently conjugate RNA with the AuNPcore, a thiol group is introduced onto the RNA353 to create RNA monolayers by linking RNAs directly to theAuNP surface via Au–thiol bonds. The dense shell of oligonucleotideson the surface of the AuNPs inhibits nuclease degradation. Since AuNP–RNAconjugates can be delivered transdermally, they may be useful forgene therapy to treat cutaneous tumors, skin inflammation, and otherskin disorders. To electrostatically attract the negatively chargednucleic acids, AuNPs have been functionalized by structurally modifyingthem, adding positively charged molecules such as polymers and aminoacids.354Gold nanoparticles (AuNP) carriers arebiocompatible, protect RNA from degradation, and have tunable shape,size, and optical properties.355 Since they are resistant to nuclease degradationand are taken up efficiently by cells they show promise as therapeuticagents.356 Despite their high negativecharge , they rapidly andefficiently enter cells by caveolin-mediated endocytosis and regulategene expression. Immunomodulatory spherical nucleic acids that stimulateor regulate immunity by engaging toll-like receptors are a promisingimmunotherapy for cancers including triple-negative breast cancer,prostate cancer, and melanoma.357Spherical nucleic acidsare 3D nucleic acid nanostructures that are functionalized, packeddensely, and oriented spherically around a nanoparticle core.359 The nanoparticles need functionalization to enhance their performance as a magnetic gene delivery vector. Theearliest magnetic core–shell nanoparticles for gene deliverywere iron oxide nanoparticles stabilized with high molecular weightPEI, with a hydrodynamic diameter of ∼200 nm and positive zetapotential.360 Nucleic acids with magneticnanoparticles increased transfection efficiency by about 1000-foldcompared to non-magnetic vectors. A variety of formulations of PEI-coatedsuperparamagnetic iron oxide nanoparticles complexed with nucleicacids have been reported.361 Other polymersand lipids have also been used to functionalize iron oxide nanoparticlesfor nucleic acid delivery.366 These include biodegradable polylactide magnetic nanoparticles containingoleate-coated magnetite and surface-modified with PEI-oleate for nucleicacid binding,367 and mesoporous silicananoparticles decorated with magnetite nanocrystals to yield highlyefficient transfection agents.368Magnetic nanoparticles deliver nucleic acid by magnetofection,which is the application of a magnetic field.369 Mesoporous and solidsilica nanoparticles are synthesized using the sol–gel methodin which a silica precursor is hydrolyzed and then condensed to generatespherical particles. Tetraethyl orthosilicate, the usual silica precursor,provides good control by modification of the synthesis conditions.Silica nanoparticles with a spiky nanotopography can enlarge the surfacearea for binding RNA molecules. To bind the negatively charged nucleicacids and enhance cellular uptake, silica particles are modified withpositively charged compounds such as PLL or PEI, or with branchedPEI to enhance the binding of the nucleic acids.370 After cell entry by endocytosis, the acidic pH in the endosomeschanges the charge of PEI resulting in the dissociation and releaseof the RNA.Mesoporous silica nanoparticles (MSNs) are hollow particles thatcan encapsulate a wide range of drugs after surface modification.Silica nanoparticles allow slow release of the cargo as they degradeslowly in the body to non-toxic byproducts. The advantages of MSNsas drug delivery vehicles include a large surface area, tunable poresizes, simple surface modifications, and efficient encapsulation ofcargo molecules.371 There is particular interestin the use of calcium phosphate nanoparticles with RNAi to regulategene expression in bone tissue engineering because of their osteoinductivity,osteoconductivity, and affinity for nucleic acids.372 Calcium phosphate nanoparticles are endocytosed by cellsand dissolve in the acidic endosomes and lysosomes, resulting in therelease of the nucleic acid cargo.373 Calciumphosphate nanocarriers are biocompatible, biodegradable, non-toxicand non-immunogenic, inexpensive, and easily synthesized. However,they become unstable over time because of the growth of crystals,371 and they provide only limited nuclease protection.Surface functionalization of calcium phosphate particles can facilitatetheir endocytosis and subsequent endosomal escape.374Calcium phosphate nanoparticles are promising non-viralvectorsfor gene therapy, an emerging strategy for effective bone repair andregeneration. The calcium phosphate naturally present in bones makescalcium phosphate nanoparticles a good choice for RNA delivery forbone repair.375 However, RNA itself can self-assemble intocomplex programmable structures.377 Ligand-conjugatedRNA nanoparticles based on bacteriophage phi29 packaging RNA providea targeted ribozyme delivery system.378 RNA nanoparticles can also carry CpG DNA to macrophages.377 Origami-like RNA nanostructures are stableand efficient as drug carriers for controlled drug release.380Although many vehicles or ligands have been used for the deliveryof nucleic acids, they may exhibit low immunocompatibility, toxicity,poor stability, inefficient drug release, or restricted tissue accessibility.381 VLPs produced in vivo are used for various medical applications such asdrug and vaccine delivery,382 includingthe COVID-19 vaccine Novavax (NVX-CoV2373) currently in phase IIItrials.383In vivo and in vitro cargo loading has been demonstrated for VLPs.384Virus-like particles (VLPs) are organized protein complexesresemblinga native virus capsid. VLPs are either naturally occurring empty virusshells or are synthesized by the expression of viral structural proteinsthat self-assemble into a virus-like structure. VLPs are attractivedrug delivery platforms due to their biocompatibility, biodegradability,and targeting ability.385 QDs emit narrow wavelength bands under a wideexcitation range, can be appropriately functionalized, and are desirablevectors for imaging-guided therapy. Since they can efficiently deliverRNAs into target cells and can be used to track the RNA distributionin cells,386 they may serve as an effectivetheranostic RNA delivery agent.388 The applicationof theranostic agents with appropriate targeted, controlled deliveryand imaging capabilities has the potential to significantly advancegene therapy.Quantum dots (QDs) are semiconductorcrystalline nanoparticleswith unique tunable optical properties including nearly 100-fold greaterbrightness and 1000-fold better stability against photobleaching versusorganic dyes and luminescent proteins. Their fluorescence emissioncan be adjusted by the particle size, known as the quantum size effect.389 Amino-functionalized multiwalled CNTs deliverproprietary toxic siRNA to human lung carcinoma cells.390 Appropriately designed amino-functional segmentson the CNTs promote internalization in the cell and gene silencing.391 Cationic single-walled CNTs deliver siRNA toLewis lung carcinoma cells.392Carbon nanotubes (CNTs)—tubes made of carbonwith nanosizeddiameters—have attracted increasing attention in biomedicinebecause of their unique structures and properties, being the strongestand stiffest materials yet found in terms of tensile strength andelastic modulus. With functionalization, CNTs have been used as nanocarriersfor nucleic acids including siRNA, oligonucleotides, and RNA/DNA aptamers.in vivo,393 including delivery of mRNA by intramuscular,subcutaneous, or intradermal injections; the latter can promote woundhealing by in situ expression of specific proteinsin the skin.398 Local injections circumvent complications associated with systemicadministration such as clearance from the bloodstream.398 Naked mRNA administered subcutaneously producesa more efficient translation of the protein than mRNA-loaded nanoparticles.400Despitethe widespread use of carriers for RNA delivery, nakedRNA has also been delivered 401 and it also improves the surface propertiesof biomolecules and drug delivery systems. It blocks surface accessby steric hindrance, increasing the circulation time in blood.402 Thus, it may improve pharmacokinetic propertiesand enhance the efficacy of drugs, including RNA therapeutics.403 PEGylation of the RNA nanocarriers reducesnon-specific interactions with serum proteins and prevents recognitionby the immune system, increasing the blood circulation time. PEGylationstabilizes RNAs, with longer PEGs stabilizing oligonucleotides betterthan shorter ones.404 The aptamer drugMacugen comprises an oligonucleotide modified with branched 40 kDaPEG at the 5′ terminus, thus enhancing the nuclease resistanceof the PEG-aptamer conjugate. PEGylation markedly improves the pharmacokineticsand boosts the neutralizing activity of anti-IL-17A aptamers, an importantadvance in the development of therapeutic aptamers.405PEGylation allows protein drugs to avoid the immune response,The advantages of the various RNA carriers are summarizedin Ourunderstanding of the types and functions of RNA has increaseddramatically over the past 50 years. Originally identified simplyas coding or non-coding, RNA was recognized early as central to theprocess of transcribing and translating a gene into a protein. Overthe last few decades, many new types of RNA have been discovered withspecific functions, including snRNA, snoRNA, shRNA, miRNA, tmRNA,siRNA, saRNA, piRNA, and circRNA. These RNA molecules participatein a complex network that regulates how RNA is used in the cell andmodifies gene expression. The multiple functions of RNA provide multiplepathways for exploiting RNA as a therapeutic molecule.invitro transcription or solid-support synthesis. TherapeuticmRNAs, which are translated in vivo to deliver atherapeutic or vaccine protein, require extensive replacement of uridinewith 1-methylpseudouridine to decrease the immune response to themRNA and improve the translation. ASOs and therapeutic siRNAs havephosphorothioate and 2′-ribose modifications to protect theRNA from degradation and to improve target specificity. Aptamers,whose 3D structure rather than primary sequence determines their function,are heavily modified. For all RNAs, the degree of modification canbe adjusted to enhance the effectiveness of the therapeutic RNA.RNA canbe engineered for its intended therapeutic functions by There are now approved RNA medicines for cardiovascular, metabolic,liver, infectious, neurological, neuromuscular, kidney, and eye diseases,with many more in the research phase. Although there are many typesof therapeutic RNAs, ASOs dominate the approved drugs, followed bysiRNAs. CRISPR and AOC therapeutics currently have high research interests,still in the pre-clinical stage. The top RNA medicine companies suchas Moderna, Ionis, BioNTech, Alnylam, and Sirnaomics all specializein one type of RNA, but research and treat as many as nine differentdiseases. RNA therapeutics have the potential to treat a wide rangeof diseases, from the most common to the extremely rare.RNAcarriers are important in overcoming the physiological barriersof systemic administration of RNA-based drugs. However, they providea challenge in the translation of RNA medicines to the clinical setting.After the recent success of the lipid nanoparticle-based mRNA vaccinesagainst COVID-19, lipid carriers have become the primary RNA deliveryvehicle. However, since lipid nanoparticles can be toxic to cellsand stimulate the release of systemic inflammatory cytokines, interestin natural transporters such as exosomes is gaining momentum. Othernon-toxic and non-immunogenic carriers, such as structurally and functionallyversatile peptides, are also attracting attention. Novel technologies,including carbon nanotubes and other inorganic biocompatible polymers,carbon nanodots, and functionalized or hybrid systems, are also excitingavenues of exploration and evaluation.Our growing understandingof the many types and functions of RNAhas been combined with the ability to synthesize modified RNAs withimproved stability and pharmaceutical activity. Nanotechnology-basedsystems to deliver those RNAs to the cell have resulted in an explosionof new therapeutic options for diseases ranging from viral infectionsto cancer. This arsenal of multiple specifically targeted RNA therapieshas the potential to revolutionize the treatment of human disease.


Clear cell renal cell carcinoma (ccRCC) is a common malignant tumour of the urinary tract. The major causes of poor prognosis are the lack of early diagnosis and metastasis. Accumulating research reveals that circular RNAs (circRNAs) can play key roles in the development and the progression of cancer. However, the role of circRNAs in ccRCC is still uncertain.n = 4) was performed to investigate the circRNAs with differential expression in ccRCC tissues. The candidate circRNA was selected based on the cut‐off criteria, such as circRNA expression abundance, circRNA size and the design of divergent primers. The circ‐transportin‐3 (TNPO3) levels in ccRCC tissues were tested by quantitative real‐time (qRT)‐PCR (n = 110). The characteristics and subcellular localization of circ‐TNPO3 were identified via RNase R assay, qRT‐PCR and fluorescence in situ hybridization (FISH). Then, we explored the biological roles of circ‐TNPO3 in ccRCC via the function experiments in vitro and in vivo. RNA pull‐down, RNA immunoprecipitation, bioinformatic analysis, RNA‐FISH assays and rescue assays were applied to validate the interactions between circ‐TNPO3, insulin‐like growth factor 2 mRNA‐binding protein 2 (IGF2BP2) and serpin family H member 1 (SERPINH1) to uncover the underlying molecular mechanisms of circ‐TNPO3.The circRNAs microarray (n = 110). The lower circ‐TNPO3 expression was found in ccRCC patients with distant metastasis, higher World Health Organization/International Society of Urologic Pathologists (WHO/ISUP) grade and more advanced tumour T stage. In vitro and in vivo, circ‐TNPO3 significantly suppressed the proliferation and migration of ccRCC cells. Mechanistically, we elucidated that circ‐TNPO3 directly bound to IGF2BP2 protein and then destabilized SERPINH1 mRNA. Moreover, IGF2BP2/SERPINH1 axis was responsible for circ‐TNPO3's function of inhibiting ccRCC metastasis. Epithelial splicing regulatory protein 1 (ESRP1) was probably involved in the biogenesis of circ‐TNPO3.We detected the obvious downregulation of circ‐TNPO3 in ccRCC compared to matched adjacent normal tissues (Circ‐TNPO3 can suppress ccRCC progression and metastasis via directly binding to IGF2BP2 protein and destabilizing SERPINH1 mRNA. Circ‐TNPO3 may act as a potential target for ccRCC treatment. Circ‐TNPO3 is significantly downregulated in ccRCC.Circ‐TNPO3 can suppress ccRCC metastasis by directly binding to IGF2BP2 protein and destabilizing SERPINH1 mRNA, in turn, regulating the SERPINH1‐SNAIL/SLUG axis.ESRP1 is involved in the biogenesis of circ‐TNPO3 by targeting the flanking intron. This was downregulated in ccRCC tissues and associated with ccRCC distant metastases, World Health Organization/International Society of Urologic Pathologists (WHO/ISUP) grade and tumour T stage. Functional assays indicated that circ‐TNPO3 suppressed the migration and proliferation of ccRCC cells both in vivo and in vitro. Mechanistically, circ‐TNPO3 could bind to IGF2BP2, thereby synergically downregulating SERPINH1 expression and inhibiting ccRCC migration. In conclusion, circ‐TNPO3 is a potential target for the treatment of patients with ccRCC.22.1A total of 114 pair ccRCC tissues and matched paracancerous tissues from ccRCC patients treated by radical nephrectomy or nephron‐sparing surgery were collected at the Southwest Hospital, Chongqing, China (2019–2021). Clinical characteristics are listed in Tables 2.2Four pairs of fresh ccRCC tissues and their matched paracancerous tissues were prepared. RNA extraction, quality identification and analysis of differentially expressed circRNAs were performed by Shanghai Genomics Corporation  as described previously.2.3−ΔΔCT method were performed to evaluate the transcript levels. qPCR procedure was shown as follows: pre‐denaturation at 95°C for 30 s, denaturation at 95°C for 10 s, annealing at 58°C for 10 s and final extension at 72°C for 10 s. A total of 40 cycles were performed from denaturation to extension. The primer sequences information is given in Table TRIzol  was applied to the extraction of RNA from ccRCC cell lines and tissues. Isolation and extraction of RNA from nucleus and cytoplasm were performed using a PARIS Kit . RNA was reverse transcribed to cDNA using the PrimeScript RT Reagent Kit . The expressions of circ‐TNPO3, IGF2BP2, SERPINH1 and other RNAs were analysed by quantitative real‐time (qRT)‐PCR with an SYBR Premix Ex Taq kit . The expression of microRNA was measured using TaqMan MicroRNA Assays . U6 or GAPDH was used as the normalizing control. The Bio‐Rad CFX96 real‐time PCR system and 22.4The Cy3‐labelled circ‐TNPO3 probes targeting the circ‐TNPO3 back splice site were synthesized at GenePharma . ccRCC cells were seeded on a culture plate . Localization of circ‐TNPO3 in ccRCC cells was detected by RNA‐fluorescence in situ hybridization (FISH) assay using the FISH kit . Images were acquired with a Confocal Microscope . The information on probes is presented in Table 2.5siRNA constructs targeting circ‐TNPO3, IGF2BP2, SERPINH1, epithelial splicing regulatory protein 1 (ESRP1) and their non‐targeting controls were synthesized by GenePharma . The sequence information on these siRNAs is listed in Table 2.6The interaction of circ‐TNPO3 with candidate proteins and microRNAs was verified by an RNA pull‐down assay. We constructed a plasmid containing full‐length circ‐TNPO3 and MS2bs sequences in the pLC5‐ciR vector background. The MS2 binding sites sequence is as follows: TACTCCTAGTGGGTACATGGAAGGGACTAGT GGGTACAGACGTCCAGCTGAGATCTTTTGTACTCCTAGTGGGTACATG. The pLC5‐circ‐TNPO3‐MS2 and pMS2‐GFP  plasmids were then co‐transfected into HEK293T cells. The RNA immunoprecipitation (RIP) assay was conducted with GFP antibody, which could specifically pull‐down endogenous RNAs and proteins associated with circ‐TNPO3; The negative control is IgG.2.77 cells/reaction density and lysed by adding lysis buffer containing protease and RNase inhibitors for 5 min. The magnetic beads were combined with 5‐μg antibody against IGF2BP2 , N6‐Methyladenosine  or Flag , or control IgG at indoor temperature, and the corresponding cell lysates were added; the samples were incubated overnight at 4°C. The samples were subjected subsequently to RNA purification and protein extraction, and the RIP efficiency was verified by Western blot assay; the enrichment of circRNAs or mRNAs was verified by qRT‐PCR assay.RIP experiments were performed with the Magna RIP Kit . The cells were processed at 1 × 102.8To perform an immunofluorescence assay, ccRCC cells were grown on a μ‐Slide 8 Well  culture plate and incubated with antibodies specific to IGF2BP2  overnight at 4°C. Further, the cells were stained using DAPI (300 nmol/L) and IgG antibody . For immunohistochemistry assay, paraffin sections were dewaxed and hydrated, following which they were incubated with the corresponding antibody overnight at 4°C. Subsequently, the sections were stained using haematoxylin and DAB. The antibodies used for immunohistochemical experiments were as follows: Ki67 , SERPINH1 , IGF2BP2 , SLUG  and SNAIL .2.9To detect differentially expressed genes in Caki‐1 cells after IGF2BP2‐RIP and circ‐TNPO3 silencing, RNA sequencing was performed by Shanghai Genomics Corporation , including library construction and computational analysis. To screen the target genes of IGF2BP2, total RNAs of Caki‐1 cells with IGF2BP2 knockdown or controls were isolated. mRNA sequencing was carried out by Tsingke Biotechnology Co., Ltd .2.10t‐test, Fisher's exact test, chi‐square test or Mann–Whitney U test were conducted to verify the differences among the two groups. Pearson correlation analysis was applied to determine the correlation between the two groups. Differences were considered statistically significant when p < .05.Statistical analysis was performed via GraphPad Prism 8 software. Student's 33.12FC│≥1, average fluorescence signal value ≥7, and p < .05 as the cut‐off criteria, 3749 differentially expressed circRNAs were identified, including 2210 upregulated and 1539 downregulated circRNAs between ccRCC and matched noncancerous tissues , 12 upregulated and 17 downregulated circRNAs were identified  was the most dysregulated according to its fold change value . The decrease in circ‐TNPO3 level was observed in 89 (80%) of the 110 ccRCC tissues relative to the correspondingly matched noncancerous tissues  according to the WHO/ISUP stage, tumour T stage and distant metastasis to compare the circ‐TNPO3 levels among these groups. The results suggested that lower levels of circ‐TNPO3 were associated with ccRCC of higher WHO/ISUP grade , more advanced tumour T stage  and distant metastasis (Yes)   and high expression groups . Circ‐TNPO3 levels were significantly lower in the Ki67 ≥ 10% group relative to the Ki67 < 10% group  Figure . Moreovep Figure . The abo3.2circ‐TNPO3 (chr7: 128655032–128658211), located on chromosome 7, is derived from exon 2 to exon 4 (432 bp) of the host gene TNPO3 via back‐splicing. By designing divergent primers and Sanger sequencing, we confirmed the predicted junction of circ‐TNPO3 Figure . To veri3.3In order to identify the potential function of circ‐TNPO3, we first constructed siRNAs which can target the back‐splice junction and overexpression vector of circ‐TNPO3. Then we confirmed successful interference and overexpression of circ‐TNPO3 in RCC‐JF and Caki‐1 cells; the mRNA and protein levels of linear TNPO3 remained unaffected Figure . The resTo investigate the impact of circ‐TNPO3 on cellular proliferation, we established lentivirus‐based stable sh‐circ‐TNPO3‐Caki‐1 and sh‐circ‐TNPO3‐RCC‐JF cell lines Figure . Colony 3.4To further elucidate the function of circ‐TNPO3 in an animal model of a tumour, we generated a 786‐O cell line with stably knocked‐down circ‐TNPO3 as previously described. We confirmed the knockdown of circ‐TNPO3 in sh‐circ‐TNPO3‐786‐O cells, and these cells showed increased migration ability Figure . In the 3.5https://circinteractome.nia.nih.gov/) databases. Next, six candidate miRNAs were identified through the overlap of predicted results and expression analysis of miRNAs in ccRCC using TCGA database. However, we could not confirm the binding between candidate miRNAs and circ‐TNPO3 by luciferase reporter assays and RNA pull‐down assays  and found that it was significantly downregulated in human ccRCC tissues  and demonstrated that SERPINH1 mRNA expression was significantly increased in ccRCC tissues (Figure We investigated the role of SERPINH1 on the migration of ccRCC cells and determined the functional interplay between circ‐TNPO3 and the IGF2BP2/SERPINH1 axis. We compared the expression of SERPINH1 in ccRCC tissues relative to matched adjacent noncancerous tissues (s Figure . We confs Figure . In vitrs Figure . CCK8, cs Figure . Additios Figure . Moreoves Figure . In summ3.9Given that some splicing factors were able to regulate the biogenesis of circRNAs, we tried to identify which splicing factor regulates the transcription of circ‐TNPO3. Among the circRNAs‐related splicing factors, we analysed their expression in ccRCC tissues compared with adjacent noncancerous based on the TCGA database. We found three splicing factors with differential expression in ccRCC, including ESRP1, ESRP2 and RBM47 as the candidate regulators for circ‐TNPO3 expression. Then we verified the expression of circ‐TNPO3 was evidently reduced when silencing ESRP1 in ccRCC cells, whereas other splicing factors did not show the effect on the expression of circ‐TNPO3 Figure . Next, w4Recently, several circRNAs have been implicated in tumourigenesis and the development of cancer.To date, the precise mechanism through which circRNAs modulate the expression of target genes is uncertain. Herein, we tried to uncover the interaction between tumour‐associated RBPs and circ‐TNPO3. circRNAs can bind to various proteins and act as protein decoys, sponges, scaffolds or recruiters.In mammals, the three proteins of the IGF2BPs family share similar structures. IGF2BP2 is composed of two RRMs domains and four KH domains and play regulatory roles in post‐transcriptional processes through the IGF2BP2‐RNA complex.IGF2BP2 modulates tumour progression by serving as a post‐transcriptional regulatory factor for the target mRNAs of MYC,Notably, IGF2BP2 can promote target expression by enhancing mRNA stability. In our study, we found that IGF2BP2 could inhibit the stability of SERPINH1 mRNA. This is the first report on IGF2BP2 destabilizing its target mRNA, thus indicating the complexity of the regulatory role of IGF2BP2. As another member of the IGF2BP family, IGF2BP3 reportedly promotes eIF4E‐mediated translational activation by decreasing the stability of EIF4E‐BP2 mRNA, which in turn enhances the proliferation of tumour cells.cis‐ and trans‐regulatory factors further regulate the efficiency of circRNA back‐splicing, and these factors include regulatory RBPs, ICSs flanking circle formation exons and core spliceosome components. In addition, circRNA turnover is also closely related to its expression.The regulation of circRNAs biogenesis is complex. First, Pol II binds and transcribes pre‐mRNAs that produce circRNAs, turning on the regulation of circRNA biogenesis. Second, 5In summary, we verified the downregulation of circ‐TNPO3 in ccRCC and its negative correlation with distant metastasis, WHO/ISUP grade and tumour T stage. Functionally, circ‐TNPO3 suppressed the proliferation and migration of ccRCC cells in vivo and in vitro. Mechanistically, circ‐TNPO3 directly interacted with IGF2BP2 and downregulated IGF2BP2‐mediated SERPINH1 expression by decreasing the stability of SERPINH1 mRNA. ESRP1 is probably involved in the biogenesis of circ‐TNPO3 by targeting the flanking intron. In the future, circ‐TNPO3 may become a potential target for the diagnosis and treatment of ccRCC Figure .The authors declare that there is no conflict of interest.Supporting InformationClick here for additional data file.Supporting InformationClick here for additional data file.


For further understanding of this observation, we performed systematic studies on the efficiency of RNase H against a series of B-ASOs models. The results of kinetic analysis showed that pyrimidine-enriched B-ASO oligomers activated RNase H more efficiently than non-modified ASO. The presence of a single FESAN unit at a specific position of the B-ASO increased the kinetics of enzymatic hydrolysis of complementary RNA more than 30-fold compared with unmodified duplex ASO/RNA. Moreover, the rate of RNA hydrolysis enhanced with the increase in the negative charge of the boron cluster in the B-ASO chain. In conclusion, a “smart” strategy using ASOs conjugated with boron clusters is a milestone for the development of more efficient antisense therapeutic nucleic acids as inhibitors of gene expression.Boron cluster-conjugated antisense oligonucleotides (B-ASOs) have already been developed as therapeutic agents with “two faces”, namely as potential antisense inhibitors of gene expression and as boron carriers for boron neutron capture therapy (BNCT). The previously observed high antisense activity of some B-ASOs targeting the epidermal growth factor receptor (EGFR) could not be rationally assigned to the positioning of the boron cluster unit: 1,2-dicarba- Antisense oligonucleotides (ASOs) have been widely developed for the treatment of various human diseases, including those that were previously incurable. Following the approval of the first ASO drug, Fomivirsen, in 1998, the U.S. Food and Drug Administration has approved ten ASO-based drugs to date. The first antisense drug was designed to inhibit the translation of mRNA encoding key proteins of the early region of cytomegalovirus and was used for the treatment of retinitis caused by this patogen . In 2013More recently, ASO have been used to combat severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is the cause of the current 2019 coronavirus pandemic (COVID-19) causing a global health emergency. The most advanced ASO approach is to knock down angiotensin-converting enzyme 2 (ACE2) which is the entry receptor of SARS-CoV-2. Using splice-switching oligonucleotides (SSOs), it should be possible to modulate alternative splicing of the ACE2 gene to remove critical regions required for SARS-CoV-2 entry into host cells ,13,14.FDA-approved ASOs based on the RNase H mechanism include such drugs as Mipomersen, Inotersen and Volanesorsen, the latest approved by the European Medicines Agency (EMA) in 2019 for the treatment of familial chylomicronemia syndrome (FCS) .ASOs are short synthetic single-stranded oligo-2′-deoxyribonucleotides (13–25 nucleotides), that have the potential to modulate the expression of target genes commonly associated with the development of a variety of diseases . SeveralThe most commonly used antisense mechanism involves recruitment of the ribonuclease H (RNase H) to the heteroduplex consisting of the single-stranded ASO (DNA) and the mRNA of the target protein, followed by cleavage of the RNA within such a duplex. This endoribonuclease is able to recognize any DNA/RNA heteroduplex longer than 5 base pairs . Once th2+ and Mn2+ or in the presence of sulfhydryl reagents, whereas RNase H type 2 is activated only by Mg2+ and inhibited by Mn2+ or sulfhydryl reagents  , [Fe(C2B9H11)2]- (B1), 1,2-dicarba-closo-dodecaborane  (B2), or dodecaborate , [B12H12]2- (B3), which were endowed with an alkyl azide  functional group suitable for copper-catalyzed azide-alkyne cycloaddition (“click chemistry” reaction mode), , .11a, 21a, 31a, 11b, 11c, 11d, 11e, and 12a, 12b, 12c containing a uridine moiety labeled with FESAN, 1,2-DCDDB, or DDB , 3 (for 11b), 7 (for 11c), 11 (for 11d), or 3 and 11 (for 11e). Since 2 has only one thymidine residue (at position 2), in addition to the corresponding BU-containing oligomer 12a, oligomers 12b and 12c were prepared in which the 2′-deoxycytidine at position 11 was replaced by a B1U unit.The resulting oligonucleotides 1 and 2 were designed to be iso-sequential with two fragments of a previously studied 22-nt antisense oligonucleotide (ASO-22), targeting EGFR mRNA -RNA in the reaction mixtures of ASO/RNA duplexes treated with RNase H was determined after 0, 5, 15, 30, 60, and 90 min by PAGE analysis (20% polyacrylamide/7 M urea). Densitometric analysis of intact 5′-[32P]-RNA substrate from three independent PAGE analysis experiments showed that purine-enriched ASO oligomer 2 in an ASO/RNA duplex 2/4 was readily able to activate RNase H, resulting in hydrolysis of 4 with a t1/2 = 11 min  increased RNase H activity, but to a lesser extent than in 11a, 11c, and 11d. Here, the RNA substrate 3 was completely consumed within the first 60 min. In contrast, two B1U units at positions 3 and 7, as in 11e, completely blocked RNase H activity toward complementary RNA. This result is not surprising and is likely due to steric hindrance caused by the presence of two boron cluster units in the B-ASO/RNA duplex. Interestingly, the specificity of the enzyme differed depending on the position of the boron cluster unit in the ASO strand. The B1U unit introduced into 1 at position 1, 7, or 11 produced a single cleavage product of 3 after 90 min of incubation, whereas the B1U unit at position 3 resulted in the formation of two 32P-RNA products , , , and as activity b.1/3 decorated with different boron clusters, we incorporated the B1U, B2U or B3U units into the first position at the 5′-end of the pyrimidine-rich oligonucleotide 1. The B-ASO/3 duplexes containing 11a, 21a, and 31a B-ASO, respectively (B1U) at the 5′-end of B-ASO significantly increased the rate of RNase H-assisted hydrolysis of 3 compared with the native 1/3. In the present experiments, duplexes with the B1U (FESAN) and B3U (DDB) units were better recognized and hydrolyzed than those with the B2U unit  existed less than half the time (t1/2 = 22 min) compared to the duplex with 11a (t1/2 = 50 min) , whereas in the duplex with 21a decorated with a neutrally charged 1,2-DCDDB boron cluster (charge 0), the half-life exceeded 240 min and 3 was only slightly degraded under the same conditions. Moreover, for reaction times longer than 120 min, we observed that hydrolysis of RNA 3 in duplex with 31a  reached an equilibrium state, in contrast to B-ASO 11a , in which RNA 3 substrate was completely hydrolyzed. In this experiment, we have shown that the charge and nature of the boron cluster are critical for RNase H activity. It is an extremely valuable observation for future B-ASO drug development.To evaluate the activity of RNase H against duplex ectively , were teectively , while tectively a. It wasUB2 unit a,b. We d10B), which are necessary for boron neutron capture therapy (BNCT)  in HeLa cervical cancer cells − > carboranes (C2B10H12) >> dodecaborate anion [B12H12]2−. Accordingly, metallacarboranes first interact specifically with the binding cavity of albumin and then, with increasing concentration, interact non-specifically with the protein surface ate (-1) cesium salt (ferra(III) bis(dicarbollide), ([C2B9H11)2]−), FESAN), was purchased from Katchem . Radiolabeled adenosine 5′-triphosphate ([γ-32P]-ATP) was purchased from Hartmann-Analytik , and T4 polynucleotide kinase and kinase buffer were purchased from BioLabs . Autoradiography double-coated films Medical X-ray blue (MXBE film) were purchased from Carestream . The developer reagent and fixer reagent were purchased from Kodak Processing Chemicals . RNase H , an RNA-specific endonuclease that cleaves the RNA within RNA/DNA hybrids was purchased from EURx, .Standard nucleoside phosphoramidites and 5′-dimethoxytrityl-2′-propargyluridine 3′-O-propargyl-5′-DMT uridine with DMT protecting groups on their 5′-hydroxyl moieties, and their exocyclic amine functions were protected with acetyl, benzoyl, or iso-butyryl groups for dC, dA, and dG, respectively. The commercially available nucleoside-linked solid support LCA-CPG and 0.07 M solutions of the monomers in CH3CN were used. Oligonucleotides were synthesized at 0.1 µmol scale using an automated DNA/RNA synthesizer H6 GeneWorld  under standard conditions for DNA oligonucleotide synthesis , N3-alkyl-FESAN, [Fe(C2B9H11)2]−) (1B), 1,2-dicarba-closo-dodecaborane  (2B) and dodecaborate  (3B)  function suitable for the alkyne–azide click reaction. Reactions were performed according to the standard copper sulfate procedure used in the chemistry of oligonucleotides [v/v), (2) 0.1 M copper sulfate in water, (3) 0.2 M tris(3-hydroxypropyltriazolylmethyl) amine (THPTA) in DMSO/tBuOH and (4) 520 mM solutions of azides 6–8 (v/v). Then, the THPTA and copper sulfate solutions were mixed in a 1:1 ratio and degassed in an argon stream. Fifty molar equivalents of the alkyl azide-coupled boron cluster (3.34 µL) and 25 molar equivalents of the copper sulfate/THPTA solution (9.19 µL) were added to a vial containing the oligonucleotide (4 OD) in water (40 µL). The mixture was vortexed, and when a precipitate formed, approximately 80 µL of DMSO /tBuOH   was added. Then, 40 equivalents of freshly prepared 0.1 M sodium ascorbate in water (14.4 µL) was added to the reaction mixture. The final solution was briefly degassed with a stream of argon and stirred for 4 h at room temperature [Oligonucleotides 2−) (B3)  equippedleotides . First, ides 6–8  in DMSO perature ,42,43.18, 250 mm × 4.6 mm column with buffer A (0.1 M CH3COONH4/H2O) and buffer B (100% CH3CN) at a flow rate of 1 mL/min. The buffer gradient was as follows: (1) 0–2 min 0% buffer B; (2) 2–25 min 0–48% buffer B; (3) 25–30 min 48–60% buffer B; (4) 30–35 min 60–0% buffer B; and (5) 35–38 min 0% buffer B . The molecular mass of each synthesized oligonucleotide was confirmed by MALDI-TOF MS or ESI-Q-TOF mass spectrometry  or 4 (0.72 nmol) , T4 polynucleotide kinase   and 2 µL of manufacturer’s kinase 10x reaction buffer (700mM Tris-HCl (pH 7.6 at 25 °C). The reaction mixture, with a total volume of 20 µL, was incubated for 1 h at 37 °C. The mixture was then inactivated by incubation at 80 °C for 3 min and used for further studies without additional purification.A 0.10 OD RNA 32P-radiolabeled substrate RNA 3 or 4 (2 µL of the stock solution after phosphorylation) was mixed with 1a–1e  or 2a–2c B-ASO  in 20 mM Tris-HCl buffer (pH 8) containing 50 mM NaCl and 10 mM MgCl2 in a total volume of 7 µL. The mixture was heated at 75 °C for 5 min and slowly cooled (1 h) to room temperature. Reaction buffer containing 200 mM Tris-HCl, 500 mM KCl, 200 mM DTT, 50 mM MgCl2 (3 µL) and Milli-Q water (19 µL) was added 10x to each sample at 4 °C, and then ribonuclease H   under the conditions recommended by the manufacturer. All compounds were cleaved from the solid support, deprotected using standard procedures, and then purified by RP-HPLC . In the eriments .Therapeutic nucleic acids hold great promise as gene silencing agents due to their functionality and diverse applicability. Recently, during the global pandemic caused by the SARS-CoV-2 virus, new strategies based on ASOs have been developed, providing hope for effective disease control.In this work, we have deepened the scientific understanding of the properties of boron clusters and demonstrated that they can enhance the efficiency of recognition and hydrolysis of B-ASO/RNA heteroduplexes recruited by the endonuclease RNase H. This phenomenon was demonstrated on single-stranded DNA oligonucleotides enriched in pyrimidine and coupled to FESAN. The rate of hydrolysis of the RNA substrate depended on the nucleotide sequence and the position of the modification with the boron cluster. The affinity of FESAN for proteins, as already shown in the literature , confirm