In the fight against cancer, early diagnosis is critical for effective treatment. Traditional cancer diagnostic technologies, on the other hand, have limitations that make early detection difficult. Therefore, multi-functionalized nanoparticles (NPs) and nano-biosensors have revolutionized the era of cancer diagnosis and treatment for targeted action via attaching specified and biocompatible ligands to target the tissues, which are highly over-expressed in certain types of cancers. Advancements in multi-functionalized NPs can be achieved via modifying molecular genetics to develop personalized and targeted treatments based on RNA interference. Modification in RNA therapies utilized small RNA subunits in the form of small interfering RNAs (siRNA) for overexpressing the specific genes of, most commonly, breast, colon, gastric, cervical, and hepatocellular cancer. RNA-conjugated nanomaterials appear to be the gold standard for preventing various malignant tumors through focused diagnosis and delivering to a specific tissue, resulting in cancer cells going into programmed death. The latest advances in RNA nanotechnology applications for cancer diagnosis and treatment are summarized in this review. Despite decades of basic and clinical investigation, as well as trials of new therapeutic modalities, cancer remains a substantial cause of mortality worldwide . VariousSimilarly, biodegradable and biocompatible natural and engineered biomolecules  have been broadly examined to synthesize nanostructures ,27. BiomIt is scientifically established that nucleic acids can be manipulated and designed to create many nanostructures . In the Due to its increased thermodynamic stability, the RNA structure can be more flexible while folding into different structures , and it produces diverse building blocks for numerous therapeutic applications , includiNanomedicine has led to the development of a variety of nanoscale therapeutics and diagnostics to treat a variety of diseases, specifically cancer ,44,45,46In the past, numerous RNA-NPs have been prepared by an automated self-assembly process and studied in the context of cancer research ,51. A keThe conjugation of polymeric NPs with RNA molecules, such as RNA aptamers, cause these bio-conjugates to be easily absorbed by specific tumor cells, and therefore, can be considered as a beneficial strategy towards the controlled release of polymeric drug delivery vehicles . In the Using in-vitro and in-vivo experimental models, Yin et al. exploited RNA-based technology to efficiently deliver anti-microRNA to cancer cells derived from breast tissue . GhimireRNA therapies provide new insights for cancer treatment. The escalating growth of RNA nanotechnology in cancer theranostics demands the preparation of an updated review. This article comprehensively discusses recent findings and highlights the promising avenues of RNA nanotechnology implemented to design stable RNA nanostructures for diagnostic and prognostic purposes. Finally, we will discuss strategies to improve and overcome many technical obstacles in this field.Biosensors are diagnostic systems that convert a natural response into a programmable signal . The calRNA NPs are discrete entities, quite different from classical therapeutic RNA, including siRNA, anti-miRNA, miRNA, mRNA, ribosomal-RNA, and viral immune-stimulatory RNA. These traditional RNAs have a broad fundamental history of research in the RNA field. These small RNAs are caught by cells, and they stimulate RNA-sensing pattern recognition receptors (PRRs). Some of these are reported to be immunogenic.d, i.e., 1.2 L/kg of pRNA NPs, indicates the presence of a valuable amount in peripheral tissues, especially in a tumor. The comparatively small amount of clearance (Cl) value indicates the insufficient filtration of the NPs from the kidneys. A specific targeted delivery can be achieved by incorporating traditional RNA into NPs by taking advantage of these properties. These NPs show specific targeted delivery, higher therapeutic efficacy, and an increased in-vivo half-life [There are a lot of benefits of RNA NPs as compared to traditional RNA, for example: (a) enhanced permeability and retention (EPR) effect, (b) the small size offers to promise pharmacokinetic and pharmacodynamic properties, (c) decreased liver accumulation, (d) Small non-coding RNAs or microRNAs serve as scaffolding and active elements in the bottom-up self-assembly of more complex nanomaterials, and (d) untraceable toxicity in-vivo . Multinaalf-life .Cancer nanotechnology faces a serious challenge of non-specific accumulation of delivered NPs in healthy organs, such as lungs, liver, spleen, and kidneys . The nonThe nano-biosensor is an innovative unit that creates nano-conjugated biological systems, which function as signaling mediators to detect the specific contents of medical, biochemical, or physical agents. The related data is transferred in the form of signals using thermometric, piezoelectric, optical, magnetic, electrochemical, and micromechanical methods. The signals produced by these methods depend on the bio-recognition of a cancer cells-related surface or intracellular biomarkers through bio-ligands or antibodies. In a new era of research, nano-biosensors will increasingly be classified based on bio-recognition and signal transduction elements. RNA-based nano-biosensors are displayed in In the basic structure of a sensor, there are two main components: (i) the target analyte that can be a nucleic acid, antibody, drug, protein, or cell-surface molecule; and (ii) the transducer used for altering a signal into a form of energy. It can be detected electrochemically by detecting the energy it produces (voltage and current), (absorption and luminescence), and mechanically (resonance). The exclusive and tunable physicochemical properties of nanostructures, such as enhanced electric conductivity, greater area to volume ratio, high reactivity, unique magnetic properties, and powerful scattering and absorption, make them a fascinating tool for bio-sensing. The nanomaterials’ ability to interact effectively with biologically important analytes and convert those interactions into considerably enhanced signals has enabled a new class of early diagnostic procedures. Gold NPs (AuNPs) and QDs are excellent signal transducers because the existence of a specific analyte regulates the signal generated by the material’s optical properties. Nano and microsensors reduce the size of the receptor to improve their responsiveness. Increased signal-to-noise (S/N) ratio is responsible for this enhanced sensitivity. However, a running device’s success depends on its ability to detect a tremendously low concentration in a reasonable amount of time.The overall sensitivities of a sensor are often affected by mass transport limitations, such as when the substance is transported to the receptor by diffusion mechanism. The limiting factor in saturating the receptor for a specific geometry of the sensor is the time required by the analytes to reach the sensor area. As the receptor area becomes smaller, the time grows higher. The decreased number of analytes obstructs the detection of extremely dilute solutions. Many practical solutions have been tested to concentrate the dilute solution straight onto the sensor surface. For example, Melli et al. formulated a unique solution with a series of micropillars with superhydrophobic surfaces to simplify the management of samples with a low concentration of analytes. A diluted solution can be placed on these micropillars, the target molecule will become concentrated by the evaporation of liquid, and the detection time is significantly decreased .Halo et al. used the same theory to detect colorectal cells by mRNA quantification. They developed a NanoFlare platform comprising a single layer of single-stranded DNA (ssDNA) coated on spherical AuNP. A fluorescent reporter was added to a short DNA counterpart that was hybridized to the ssDNA recognition sequence. The reporter fluorophore was slaked while it was close to the AuNPs, but it broadened when the target mRNA displaced the DNA, allowing the fluorescence reading. The detection limit of the nanosensor was about 100 cancer cells/mL blood .A particular type of RNA aptamer that causes small-molecule fluorophores to emit fluorescence is called a light-up aptamer. Several new light-up aptamers have been developed that can bind to different biocompatible fluorogenic ligands and make their way to the design of modern RNA-based molecular strategies for sensing applications. The Systemic Evolution of Ligands by EXponential enrichment (SELEX) is a combinatorial procedure for identifying such aptamers. RNA aptamer libraries were exposed to recurrent cycles of collection and amplification in this procedure, resulting in RNA aptamers having the strongest selectivity for the target ligand. Various RNA NPs can be coupled with light-up aptamers having programmable sensing capabilities to create dynamic reporting entities .2 group of the RNA fragment, and similarly, thiol to maleimide and alkyne to azide, etc. RNA was directly manufactured, and classical pairing reactive groups, e.g., amino –NH2-COOH, azide, maleimide, alkyne, and thiol, were incorporated by similar methods for the production of polyvalent RNA NPs [RNA/DNA nano-biosensors can measure the responses produced by aptamer hybridization or nucleic acid conversion processes as promising diagnostic tools . As apta RNA NPs . Single- RNA NPs .RNA polymer is conjugated with NPs , and a sequence of research studies used siRNA, pRNA, and phi29 for this purpose. The siRNA was coupled with many nano-based imaging agents to form multifunctional NPs exhibiting diagnostic and therapeutic moieties.apt/3WJ/Bio) theme for the conjugation of pRNA-3WJ to the quantum dot (QD). Firstly, the SEPapt/3WJ/Bio was attached to G-100 resin, and then the streptavidine-labeled quantum dot was linked to SEPapt/3WJ/Bio by streptavidine-biotin coupling on Sephadex G-100 and STV/QD-SEPapt/3WJ/Bio conjugates were subjected to dissociation in the elution buffer. Later, the STV/QD-Bio/3WJb, 3WJa, and SEPapt/3WJc were split by elution buffer. After that, the STV/QD-Bio/3WJb fragment was filtered and reconvened with pRNA 3WJa and thiol-labeled pRNA 3WJc for resistive biomemory application. Hence, the RNA polymer can be easily incorporated with other NPs and used for various diagnostic, therapeutic, and bio-electronic fields [Bhatia et al. established tumor-highlighted peptides (F3) and siRNA coupled to the PEGylated QDs core as a framework. The F3 peptide was conjugated to amine group-modified QD via sulfo-LC-SPDP (sulfosuccinimidyl 6-(3′-(2-pyridyldithio)-propionamido) hexanoate as a heterofunctional cross-linker, and thiol modified siRNA used sulfo-SMCC  cyclohexane-1-carboxylate) for conjugation. The QD-siRNA/F3 conjugate NPs were proficiently transported to HeLa cells and unconfined from their endosomal setup, which provided the demolished EGFP signal. These siRNA-NPs conjugates exhibited both imaging and therapeutic properties. Furthermore, the siRNA was coupled to iron oxide NPs, which showed magnetic characteristics for biomedical applications. The linkage of siRNA to iron NPs exhibited a double response, such as the in-vivo delivery of siRNA and gathering of siRNA in the tumor by MRI and the near-infrared fluorescent (NIRF) in-vivo optical imaging. The amine groups of iron oxide NPs were treated with m-maleimidobenzoyl N-hydroxysuccinimde ester (MBS) for linkage between siRNA and magnetic NPs. After that, the reduced thiol group of RNA was treated, and magnetic NPs were coupled with and near-infrared Cy5.5 dye and membrane translocation peptides. The MRI and NIRF were used simultaneously to see the siRNA-magnetic NP uptake. The coupling of AuNPs with RNA is studied to enhance the accessibility of tethered RNA splicing enhancers. Guo et al., in 2007, produced a pRNA of the phi29 and DNA-packaging motor linkage to AuNPs to study the phage assembly. In this case, the SH-labeled DNA oligonucleotide was merged with 3′ terminal of pRNA to incorporate the thiol (-SH) group into pRNA. Then, the thiol-treated pRNA was conjugated to gold NPs. The pRNA/AuNPs conjugate was attached to procapsid by an in-vitro phage assembly. Guo’s group proved that the RNA polymer was coupled with the AuNPs effortlessly, and this procedure can be used for imaging purposes. Currently, pRNA-3WJ was coupled straight to the quantum dot for resistive biomemory applications. They introduced a sephadex G-100 resin-recognizing RNA aptamer in the biotin-labeled pRNA/3WJ  for the exosomal analysis of GBM. Three functional sections were combined by iMER, i.e., real-time RNA analysis, targeted up-gradation of extracellular vesicles, and on-chip RNA isolation. The up-gradation or enrichment process detached the cancer exosomes immunomagnetically from host-derived exosomes, and the later analysis was executed on enriched populations. Later, a glass-bead filter was used to pass the lysate collected after the lysation of exosomes. RNA was immobilized onto glass beads by forming electrostatic bonds during this method and then extracted and quantified by qPCR. Magnetic microbeads with anti-epidermal growth factor receptor (EGFR) antibodies were used to identify and enrich GBM-derived exosomes. These GBM-derived exosomes were incubated with beads, and the whole surface seemed thickly covered. A large quantity of mRNA was found in these vesicles, having mRNA of nuclear proteins as well .Spherical nucleic acids SNA are a type of NPs that are made up of a NP core treated with oligonucleotide structures and comprised of RNA interference RNAi reporter molecules and therapeutics. The exclusive 3D SNA structures are resilient to nuclease degradation and co-opt into the cells despite the lack of transfection agents. SNAs act as diagnostic agents and have the potential to detect two unique mRNA sites at a time inside of a cell . These SIqbal et al. sequestered an anti-RNA aptamer from purified human protein by creative selection. The authors confirmed the ‘aptamer’s ability to detect and seize murine and human GBM cells after its immobilization on a glass substrate. Aptamer binds with wild-type and mutant EGFR with excellent specificity and affinity (Kd = 2.4 nM). This technique is used for the initial detection and analysis of residual disease. Similarly, Iqbal’s group fabricated a flow-through lab-on-chip tool that used surface-attached aptamer’s affinity for GBM’s overexpressed biomarker, i.e., EGFR, to prove that a microfluidic-based technique can be used to detect and capture GBM cells. Later, the same group took radical steps in diagnostics related to anti-EGFR aptamers and came up with two succeeding articles on tracing the differential dynamics of GBM cell structure on substrates grafted with aptamers. They analyzed the dynamic morphology of GBM in computational single-cell metrics to identify and capture tumor cells ,81.Choulier et al. linked cell-SELEX and protein to separate RNA aptamers. These aptamers have the potential to bind specifically to integrin α5β1, which is an αβ heterodimeric receptor related to cancer angiogenesis and GBM ferociousness. The authors used in histo-fluorescence analysis on patient-based xenografts and a fluorescence-related analysis on cell lines to verify the diagnostic capability of tagged aptamers . RecentlRich et al. designed a pool of DNA aptamers distinguishing TICs with an extremely low dissociation constant (Kd between 0.12 and 3.75), and an aptamer-Cy3-related fluorescence proved the binding. In 2019, Affinito et al. used an RNA library on basic human GSCs to design a 20-F-RNA aptamer A40s, which was explicitly related to the GBM cells. The authors established the detection of A40s-based analysis in GBM cells and GSCSs of human tissue sections . Here, tCurrently, three-dimensional RNA NPs with tetrahedral structures have been designed. RNA tetrahedrons are used in various applications in nanomedicine and nanomaterials because of their structural permanence and mechanical rigidity. The EGFR aptamer was bonded with the RNA tetrahedron structure to target triple-negative breast cancer cells. After IV administration, the drug-loaded NPs face a sequence of biological blockades. NPs face quick opsonization and succeeding sequestration by local macrophages under physiological conditions. As a result, healthy organs, such as the spleen and liver, accumulate very high levels of NPs . To addrIn another study, QD-mi RNA let-7a-gold NPs (QD-RNA-Au NP) were conjugated with Chitosan-based nano-formulation, including negatively charged poly (g-glutamic acid) (PGA) for transfer to breast cancer cells. In the cells, the QDs were separated by dicer-mediated release and showed fluorescence for theranostic applications .Mediley et al. described the use of an AuNP aggregation-related colorimetric sensor for straight cancer cell detection. However, the signal produced by aptamers after binding with cancerous cells was too low to detect CCRF-CM cells due to their weak binding affinity. To solve this problem, Lu et al. used S6 RNA aptamer-linked multifunctional oval-shaped AuNPs and the monoclonal anti-HER2/c-erb-2 antibody for multivalent attachment of AuNPs with target cells for extremely sensitive analysis of SK-BR-3 breast cancer cells. AuNP-linked colorimetric techniques are used for initial and sensitive in-vitro recognition of cancerous cells .Sioss et al. fabricated a nanowire-resonator array sensor for signal detection using oligonucleotide-treated AuNPs to analyze RNA in circulating tumor cells (CTCs). In this method, RNAs were attached to AuNPs previously immobilized on the sensor by hybridization. The resonance frequency of the sensor was changed by AuNPs after adding mass to it. The authors calculated this change in resonance frequency and detected PCA3 RNA, a nucleic acid prostate cancer marker. After measurement of RNA and volume used, they calculated that the level of development was 1 CTC/10 mL blood. The sensors used were extremely sensitive and specific, showing a single-nucleotide inconsistency calculation. AuNPs have a large surface area, and they are used as a support to increase signal formation by enhancing molecular binding events .The prostate-specific antigen (PSA) was spotted in prostate cancer cells and the neovasculature of many types of malicious neoplasms, such as breast cancer , lung 8, and somMohamadi et al. fabricated a microelectrode biosensor based on the PSA mRNA and magnetic NPs-based circulating tumor cells. Another study demonstrated aptamer-based in-vivo targeting by pRNA-3WJ NPs containing the anti-prostate-specific membrane antigen (PSA) RNA aptamer. On the other hand, folate can be coupled with pRNA-3WJ NPs as an innovative strategy for improving nanocarrier distribution.The pRNA-3WJ-folate conjugates have been used in many cancer cells in which FR is overexpressed, such as colorectal, gastric, head and neck cancers, and glioblastoma. The in-vivo distribution of RNA NPs was studied using pRNA-X NPs containing fluorophore or folate. These NPs were injected into athymic mice with KB cells xenografts. Whole-body images were taken at different time intervals and indicated the high accumulation of RNA NPs in cancer cells within 4 h. Specific localization of pRNA-X NPs in the tumor was calculated at the 8th hour of organ imaging, without accumulating in healthy organs .Cationic lipids (CLs) are used for liposome-oriented DNA/miRNA transport. These lipids are made up of a linker to which a hydrophobic part is linked to a cationic head part. The positively charged head group is attached to the negatively charged phosphate group of nucleic acids . Liposom3O4) NPs were used in this study. These magnetic NPs showed highly efficient in-vivo imaging along with increased (human gastric cancer cell line 7901) SGC7901 sensitivity to the drug Adriamycin [Chen et al. developed an MRI-visible system based on superparamagnetic iron oxide NPs (PEG-g-PEI-SPION) and polyethylenimine conjugated to polyethylene glycol (PEG) for the delivery of siRNA to gastric cancer. Despite its use in cancer gene down-regulation, this PEG-g-PEI-SPION has verified itself as an extremely effective contrast agent for in-vivo MRI scanning as well. In the same way, Sun et al. designed Micro-RNA-16-loaded magnetic NPs to solve drug resistance challenges in the mouse gastric cancer model. Polyethylene glycol (PEG)-coated iron oxide  is composed of glycoproteins, elastin, collagen, and hyaluronic for providing solid structural support for cellular processes, i.e., proliferation, cell migration, and growth . MoreoveConventional cancer treatment has failed to mitigate the impact of malignancies . TherefoRNA-based drugs are often entrapped or attached to the surface of different nanovehicles to deliver the cargo to the cells. These nanovehicles can be modified with various moieties, including polyethylene glycol (PEG) and cholesterol, which enhance them by the membrane of target cells, where the nanovehicle can enter the target cells via endocytosis A. MeanwhThe miRNA or siRNA possesses the capability to bind with the enzyme-containing molecule RNA-induced silencing complex (RISC), resulting in the enzymatic cleavage of the target mRNA . TargeteMoreover, all the RNA-based nanoparticles tend to adopt various advanced strategies to provide the targeted and efficacious treatment against various metastatic cancer, as shown in 2O. The precipitated were re-dissolved and purified by the ion-pair reverse phase HPLC in a PTX-labeled RNA for assembling NPs. Finally, an RNA four-way junction NP (4WJ-X nanostructure) with ultra-thermodynamic stability to solubilize and load PTX for targeted cancer therapy was developed by a 3D computational model generated using Swiss PDB Viewer and PyMOL Molecular Graphics System. Assembly of NPs was confirmed using a specified buffer solution. EGFR NPs were also conjugated with one of the aptameric RNA oligomers and 4WJ-X-24 PTXs. It was observed that each RNA NPs was found to be successfully attached via covalent interaction to twenty-four molecules of PTX as a prodrug. The developed RNA-PTX complex was found to be structurally stable and rigid. It was concluded that RNA NPs proved to help increase the water solubility of the BCS class II drug PTX by 32,000-fold, which possesses the issue of low water solubility. This treatment strategy of RNA functionalization in the form of intravenous injections resulted in the specified cancer targeting. RNA ligand proved to dramatically inhibit the growth of breast cancer with non-detectable toxicity and immune responses in mice. Moreover, no mortalities were observed at the LD50 dose of PTX [Cancer of the breast is the deadliest disease in the world . Breast e of PTX .By using nanotechnology for RNA, we are able to overcome many shortcomings of naked RNA molecules, such as poor chemical stability, easy degradation by nucleases, and extremely short half-lives for in-vivo applications. NPs act as a multipurpose and targeted system for the safe transfer of naked RNA molecules. The NP-based protect RNA from enzymatic cleavage and immune system threats. Because of their EPR properties, these nanostructures facilitate excellent RNA accumulation at the tumor site. Nowadays, the nanocarriers used for RNA delivery are lipid-based nanosystems , polymerRNA NPs can easily include targeting ligands, counting RNA aptamers, and chemical ligands to impart specific targeting against proteins, fluorescent chemicals, and cell surface receptors. Chemical ligands, e.g., folate, are attached to the end of the RNA strand while the RNA is synthesized by modified RNA phosphoamidites. These ligands can also be attached to the RNA strand after RNA synthesis by the chemical conjugation method. The RNA aptamer strand, such as EGFR aptamer, can be manufactured by elongation of the scaffold strand.The binding efficiency in-vitro and in-vivo of RNA NPs with ligands is tested by labeling them with radioisotopes and fluorescent dyes . The labFolate receptors are overexpressed in epithelial cancer cells surfaces, permitting the folic acid conjugated NPs to target these cancer cells at a frequency higher than the normal cells.RNA NPs can efficiently target cancer metastasis that is difficult to target because of the spread of cancerous cells to far-off cells and lymph nodes. Folic acid was used as a targeting agent by RNA NPs to simultaneously target colon cancer cells in the chief sites of metastasis, such as lungs, liver, and lymph nodes . The 3WJSmall interfering RNA (siRNA) showed great potential in advanced therapeutics because of its highly sequential ability for silencing genes . One of Furthermore, Derlin 1-siRNA can be adjunct to the formulated conjugate of RGDfC-SeNPs via electrostatic interaction. The RGDfC-Se@siRNA successful conjugation and synthesis was confirmed via size determination by a zeta sizer, transmission electron microscopy (TEM), and Fourier transform infrared spectroscopy (FTIR). Elemental compositions of RGDfC-SeNPs were studied by energy dispersive spectroscopy (EDS). RGDfC-Se@siRNA characterization results proved that it followed Clathrin-mediated endocytosis for specifically reaching HeLa cancer cell lines and exhibited triggered siRNA release in a tumor microenvironment as compared to the biological microenvironment. However, in qPCR and Western blotting assays, both techniques showed eminent chances of gene silencing in HeLa cells. RGDfC-Se@siRNA was found to suppress the tumor invasion and division in HeLa cells via triggering the apoptosis pathway. Moreover, another crucial mechanistic approach of mitochondrial membrane disruption and reactive oxygen species generation (ROS) in HeLa cells was quite convincing in understanding that mitochondrial dysfunction mediated by ROS might play a significant role in RGDfC-Se@siRNA-induced apoptosis. Interestingly, advanced nanotherapeutics also presented substantial antitumor activity in a HeLa tumor-bearing mouse model .Prostate cancer malignancy is one of the significant reasons for mortality worldwide. Prostate cancer is a non-skin malignancy causing the second biggest number of deaths in men when differentiated with different tumors. Conventional therapies are expected to cause erectile dysfunction, libido, obesity, and bone mass loss. Nanotechnology has modernized the field of medication to sidestep conventional therapies against metastatic cancers and different intracellular infections . Xiao di3O4 NPs (SPIONs) [Globally, death from gastric cancer accounts for the majority of deaths. Conventional treatments have not been able to alleviate the consequences of this deadly disease. The development of targeted therapies for gastric cancer requires new technologies . The mos(SPIONs) . SPIONs (SPIONs) .Hepatocellular carcinoma (HCC) is the most common malignancy of the liver and the most common cause of morbidity and mortality ,122. A fTimely diagnosis and therapy are the prime responsibility of the healthcare system . SeveralMesoporous silica NPs (MSNs) have been considered the most promising nanocarriers for attached targeted moieties owing to unique features of tunable pore structure, greater surface area, and pore volume ,127,128.Thanks to their selectivity and enhanced sensitivity, RNA-based theragnostic approaches have tremendously improved current methods for diagnosis and provided versatile delivery systems for the treatment of a variety of cancers. This is important because in-vivo applications of the naked RNA molecule face many challenges that need to be tackled. For instance, the naked RNA molecules have poor chemical solubility, extremely short half-lives, and are easily degraded by nucleases ,130,131.A number of nanovehicles have been studied for their ability to deliver RNA molecules to target locations. For instance, it has been documented that lipid-based nanostructures , polymer-based nanomaterials, inorganic NPs, and bio-inspired nanovehicles can be used for the targeted delivery of nucleic acids, such as RNA. The majority of these nanocarriers have the advantages of easy preparation, good biocompatibility and bioavailability, low cost of production, controlled release, easy modification, and easy uptake. However, they have shown poor solubility, rapid clearance, easy leakage of cargo, dose-dependent toxicity, and stability concerns .The application of RNA technology has a major advantage of exploiting cellular machinery that permits the targeting of complementary transcripts, resulting in a dramatic decrease in the expression of a target gene. The main limitations of this approach are ineffective in-vivo delivery, off-target effects, and the induction of type I interferon responses. .The RNA molecule itself is unable to cross cell membranes. In addition, prompt degradation of the therapeutic agent in the endocytic pathway, called the endosomal escape, could be a great challenge in ligand-mediated endocytosis for the specific delivery of siRNA . A cruciIn this review, we highlighted advancements in the use of RNA nanotechnology in cancer diagnosis and treatment. The development of novel RNA nanotechnology-based tools for cancer diagnosis and treatment has been studied extensively in recent years. Compared to presently available cancer diagnosis and treatment in clinics, a number of RNA-based NPs demonstrated improved sensitivity and selectivity or provided whole new capabilities that could not be attained with conventional techniques. Compared with conventional diagnosis and therapies, an RNA functionalized nanocarrier-mediated anticancer drug delivery leads to high therapeutic efficacy, targeted binding with the ligand, more accurate diagnosis, lower toxicity, and site-specific delivery, resulting in cytotoxicity management and cost-effectiveness. The barriers to the diagnosis and treatment of cancers and the killing of healthy cells have been minimized using biocompatible polymers ligands. Promising advances in cancer treatment and detection, such as RNA-functionalization and biocompatible ligands, are rapidly paved the way in addressing the disadvantages of existing approaches by effectively enhancing the treatment and diagnosis of metastatic tumors. Recently, significant advances have been made in the area of RNA nanotechnology for cancer diagnosis and treatment, and our knowledge of this topic has significantly expanded. However, just a few RNA-based NPs have progressed to clinical trials, and RNA nanotechnology is expected to enter the clinic in the coming years with collaborative efforts among scientists, technologists, and therapists. RNA nanotechnology, with its excellent specificity, accuracy, and multiplexed measuring capabilities, has significant potential for improving cancer diagnosis and treatment, finally leading to a higher cancer patients’ chance of survival.


Escherichia coli, and designed paper-based cell-free colourimetric assays that accurately identified two human immunodeficiency virus (HIV) subtypes (by executing OR logic) in amplified synthetic HIV RNA as well as severe acute respiratory syndrome coronavirus-2 (via two-input AND logic) in amplified RNA from saliva samples. The sequence-independent molecular logic enabled by the integration of multi-arm junction RNAs with motifs for loop-initiated RNA activators may be broadly applicable in biotechnology.Applications of RNA-based molecular logic have been hampered by sequence constraints imposed on the input and output of the circuits. Here we show that the sequence constraints can be substantially reduced by appropriately encoded multi-arm junctions of single-stranded RNA structures. To conditionally activate RNA translation, we integrated multi-arm junctions, self-assembled upstream of a regulated gene and designed to unfold sequentially in response to different RNA inputs, with motifs of loop-initiated RNA activators that function independently of the sequence of the input RNAs and that reduce interference with the output gene. We used the integrated RNA system and sequence-independent input RNAs to execute two-input and three-input OR and AND logic in  Multi-arm junction RNAs integrating motifs for loop-initiated RNA activators enable the execution of molecular logic independent of RNA-input sequence, thus facilitating the design of cell-free diagnostics. RNA molecules designed to fold into diverse secondary structures have been used to tightly regulate gene expression at the transcriptional and translational levels in response to trans-acting RNAs7, small molecules8, proteins9, or specified logic expressions11, and have been used in strand-displacement systems for computing and imaging applications13. Moreover, they have found use in low-cost systems for detection of viruses16, mutations17, and for water testing19. At the same time, the structural diversity of RNA has been harnessed in RNA nanotechnology to generate a variety of RNA-based nanostructures with complex geometries through self-assembly22. These structures are assembled from molecular building blocks featuring hairpins, multi-arm junctions, and other structural elements programmed to fold into prescribed structures through combinations of dangling end, kissing loop, and crossover interactions27. Such assemblies have enabled the production of multivalent nanoparticles carrying siRNA payloads30 and have also been synthesized within living cells enabling enzyme localization32. They provide a wealth of different RNA structures that can be harnessed for programming cellular function.RNA adopts diverse secondary and tertiary structures that enable it to perform a variety of different roles in the cell, from regulating gene expression and catalysing chemical reactions to sensing small molecules and scaffolding proteins34, recent years have seen the development of self-assembly-driven molecular computing systems that operate in living cells and exploit the combined interactions of multiple carefully designed synthetic RNAs37. Such ribocomputing devices act by modulating gene expression in response to specified combinations of input RNAs and take advantage of the predictability of RNA–RNA interactions to enable effective computer-based design. These systems have been used to carry out combinations of AND, OR, NAND and NOR logic with up to a dozen inputs and have operated using complexes formed from as many as five distinct RNAs in living cells36. The ribocomputing devices developed thus far, however, have had several critical limitations that constrain the range of input RNAs that they can monitor and the range of output proteins that they can produce. These systems have relied on hybridization between multiple input RNAs for implementing AND logic, limiting their use against natural transcripts that lack the necessary complementarity between sequences or requiring adapter strands that reduce system output  subtypes and AND logic to accurately identify severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) in clinical saliva samples by targeting two regions of the virus at the same time.Herein we describe a strategy for implementing molecular logic that exploits multi-arm junction RNA structures to regulate gene expression while eliminating input RNA sequence constraints and reducing sequence interference with the output gene for OR logic. These ribocomputing systems make use of loop-initiated RNA activator (LIRA) motifs that bind to input RNAs through extended loop domains and expose downstream functional domains for subsequent reactions. We show that LIRAs can be used as riboregulators to activate gene expression with high dynamic range and orthogonality in les Fig. . The resOur general strategy for regulating gene expression using multi-arm junctions is illustrated in Fig. 7, have relied on single-stranded toehold domains to initiate reactions38, we hypothesized that long loop domains could be utilized to provide similar performance. Such long loops would provide a strong thermodynamic driving force to initiate RNA–RNA interactions and provide an input RNA binding site that is sufficiently labile and unconstrained to offer good reaction kinetics. Figure 7. To enable efficient expression and testing in vivo, four bulges were incorporated into the LIRA hairpin structure to reduce the likelihood of premature rho-independent transcriptional termination and to increase the thermodynamics driving the input–LIRA reaction. A 6 bp clamp domain was also added immediately after the start codon in the LIRA stem to reduce the likelihood of translational leakage  and plasmids were constructed to express the input and LIRA transcripts using T7 RNA polymerase in E. coli BL21 Star DE3 cells  deposited onto 2-mm-diameter paper discs  to amplify low-concentration pathogen RNAs before use in the paper-based assays. In NASBA, a combined reaction featuring reverse transcription, T7 RNA polymerase, RNase H and DNA primers that incorporate the T7 promoter sequence is used to generate multiple RNA copies from a starting RNA template. Synthetic RNA targets from norovirus and yellow fever virus (YFV) were supplied to NASBA reactions at an initial concentration of 200 aM and amplified over 2 h at 41 °C. We found that both pathogen RNAs could be detected in the colourimetric paper-based reactions following NASBA Fig. .In addition, we applied the assay to clinical serum samples positive and negative for the dengue virus. The serum samples were first diluted by 10-fold into water and heated to 95 °C for 2 min to release the viral genome from the capsid. The RNA was then amplified using NASBA and applied to the paper-based LIRA sensors. We found that LIRAs could unambiguously identify the clinical dengue sample through the resulting purple colour. To determine the detection limit of the dengue assay, we carried out a series of NASBA/LIRA reactions with synthetic dengue target RNA concentrations ranging from 200 fM down to 0.2 aM. We found that the dengue transcript could be detected down to concentrations as low as 20 aM in the NASBA reaction, which corresponds to 12 RNA copies per µl of reaction Fig. .41. Within group M, there are nine different subtypes with genetic distances of 25% to 35% and prevalences that vary depending on the geographic region. HIV-1 subtype C causes >50% of infections worldwide and circulates mostly in India and regions of Africa, while HIV-1 subtype B predominates in Europe and the Americas42. We thus aimed to develop a logic system capable of detecting both HIV-1 subtype B and C using a single OR operation, which could be deployed in an area such as Southern Brazil where both subtypes are common43.Diagnostic devices that combine visible readouts with the ability to perform information processing on biomolecular inputs have the potential to improve assay capabilities by expanding the number of pathogens a single test can detect, reducing false positives, and lowering assay complexity and cost. To demonstrate the potential of such logic-enabled paper-based diagnostic devices, we carried out proof-of-concept studies exploiting the logic capabilities of multi-arm junction molecular logic for HIV and SARS-CoV-2 detection. HIV continues to be a major global health threat with HIV-1 group M being the predominant cause of infections worldwideα subunit was produced as evidenced by increased production of the purple cleavage product in the paper-based reactions  gene, 2019-nCoV_N1 and 2019-nCoV_N2. RT-qPCR is the most common method of detection of SARS-CoV-2 given its excellent specificity and sensitivity. However, it requires well-trained personnel and expensive equipment, which makes virus detection more challenging in rural areas with limited medical resources and requires additional time to ship samples to centralized facilities. Previous paper-based cell-free assays have been limited to detecting only a single pathogen target sequence at a time, and parallel assays that detect target RNAs in separate reactions can suffer as a result of differences in riboregulator activation speeds and lead to increased assay cost.Following the US Centers for Disease Control and Prevention (CDC) recommendationsTo overcome these issues, we combined AND logic multi-arm junctions with isothermal amplification reactions to simultaneously detect two different SARS-CoV-2 N gene sequences using a single paper-based readout reaction. The resulting two-input AND gate RNAs contained a hairpin reconfiguration domain to encourage binding between the gate RNA and the input viral RNAs Fig. . Similar48. Using a plate reader, SARS-CoV-2 down to a concentration of 2 aM in the NASBA reaction could be distinguished. We then tested six positive saliva samples from SARS-CoV-2 patients together with six negative ones. Figure We then designed specific NASBA primer pairs for each of the two devices to amplify the input RNAs from the SARS-CoV-2 genome. NASBA reactions were performed using heat-inactivated SARS-CoV-2 virus particles at different concentrations. We found that gate N1*N2* performed better than gate N2*N1 and enabled detection of SARS-CoV-2 down to concentrations of 20 aM in the NASBA reactions when viewed by the naked eye Fig. , a conceE. coli cells. We also applied these systems in paper-based cell-free assays for detection of viruses, including the dengue virus and SARS-CoV-2 from clinical samples. Using multi-arm junctions in paper-based reactions, we produced colourimetric assays that harness OR logic to activate in response to two different subtypes of HIV-1 and AND logic to target two regions of the nucleocapsid gene of SARS-CoV-2 at the same time. Application of the system to a set of positive and negative saliva samples demonstrated accurate identification of SARS-CoV-2 using a two-input multi-arm junction gate RNA.We have implemented a strategy for encoding molecular logic operations in multi-arm RNA junctions for regulation at the translational level. These systems make use of loop-initiated RNA–RNA interactions via LIRA modules to detect input RNAs and direct the programmed unfolding of the multi-arm RNA structures to report on computation results. We have found that LIRAs on their own can operate as riboregulators with wide dynamic range, good orthogonality and low translational leakage using such loop interactions. Moreover, they completely decouple the sequence of their cognate input RNA from the sequence of the output module that they expose, thereby avoiding some of the limitations of toehold-based riboregulators. By incorporating LIRA modules into the multi-arm junctions, we implemented three-input OR and three-input AND operations in living 11. The use of multi-arm junctions for LIRA OR gate RNAs does not require translation through downstream hairpin structures and alleviates the need for long N-terminal peptides to be added to the output protein. These design features for toehold-mediated OR gates result in N-terminal peptides that increase in size by about 24 residues for each additional input detected  reported in this work for visible detection. CRISPR-based visible readout reactions have relied on lateral flow strips and targeted a single viral site in each reaction53. Our strategy provides a simplified procedure by monitoring two SARS-CoV-2 amplicons in the same reaction. Since the paper-based riboregulator assays can be run in array formats14 and monitored directly using cameras without added light sources or filters, readout via colourimetric cell-free reactions could also enable parallel testing of larger numbers of samples than CRISPR-based assays.CRISPR-based molecular diagnostics58 and aptamer-based probes18. Moreover, the strategy for encoding molecular logic using multi-arm junction structures can also be applied to a variety of different forms of RNA output and provide the capacity to respond to multiple input species without sequence constraints. We anticipate that these capabilities will prove valuable for constructing intracellular systems that respond to endogenous RNAs to report and control cell state for biological circuits. In addition, they can be deployed in diagnostic assays to increase specificity and sensitivity, while reducing cost and test complexity to help respond to infectious disease outbreaks.We expect that the in vivo-validated loop-initiated motifs described here, which eliminate any correlation between the input and output sequence, will prove broadly useful for implementing a variety of other forms of RNA-based regulation, particularly those that can require strict sequence constraints, such as conditional guide RNAs39 and selected for experimental testing using procedures reported previously7. Briefly, a set of 337 candidate LIRA devices were generated by NUPACK and the top 60 of these riboregulators were selected on the basis of their ensemble defect levels. All candidate LIRAs shared the same secondary structures but differed in sequence outside of the conserved RBS, start codon and reporter gene regions. Pairwise interactions between all the LIRAs and input RNAs were then computed to determine the expected equilibrium concentration of the LIRA–input complexes formed. Using non-cognate LIRA–input complex formation probability as a crosstalk metric, a Monte Carlo selection algorithm was used to generate a library of 24 LIRAs expected to display the lowest expected overall crosstalk.LIRAs were designed computationally using the NUPACK software packageE. coli strains were used in this study: BL21 Star DE3 (F−ompT hsdSB (rB−mB−) gal dcm rne131 (DE3); Invitrogen), BL21 DE3 (F−ompT hsdSB (rB−mB−) gal dcm (DE3); Invitrogen), MG1655Pro (F− λ−ilvG- rfb-50 rph-1 SpRlacR tetR), and DH5α (endA1 recA1 gyrA96 thi-1 glnV44 relA1 hsdR17(rK−mK+) λ−; Invitrogen). All strains were grown in Luria broth (LB) medium at 37 °C with appropriate antibiotics.The following E. coli DH5α strain and sent out for sequence validation via Sanger sequencing. Backbones used for constructing the plasmids were amplified from the commercial vectors pET15b, pCOLADuet and pCDFDuet (EMD Millipore) via PCR followed by treatment with the restriction enzyme DpnI. The reporter protein for all plasmids is GFPmut3b with an ASV degradation tag unless otherwise noted.Plasmids were constructed via PCR and Gibson assembly. Single-stranded DNAs for expressing LIRAs, gate RNAs and input RNAs were purchased from Integrated DNA Technologies and amplified into double-stranded DNA form via PCR. The amplified DNAs were then connected with plasmid backbones by 30 bp homology domains using Gibson assembly. All Gibson assembly products were transformed in the −1 kanamycin, 50 µg ml−1 ampicillin and 25 µg ml−1 spectinomycin. After 80 min of recovery, isopropyl β-D-1-thiogalactopyranoside (IPTG) was added into each well to a final concentration of 0.1 mM. Flow cytometry measurements were performed after 3, 4 and 5 h of induction.Bacterial colonies transformed with combinations of LIRA or gate RNA and input RNA plasmids were inoculated in 1 ml of LB in triplicate with appropriate antibiotics and grown overnight at 37 °C with shaking. On the second day, 5 µl overnight-cultured medium was diluted 100-fold in 495 µl of fresh LB with 30 µg ml33  equipped with a high-throughput auto sampler . Before running measurements, cells were diluted ~10-fold into phosphate buffered saline (PBS) in 384-well plates. Forward scatter (FSC) was used for the trigger, and ~40,000 individual cells were recorded. Cell populations were gated according to their FSC and side scatter (SSC) distributions as described previouslyd-galactopyranoside , 2.5%; with the remaining volume reserved for LIRA riboregulator or gate RNA plasmids, water and lacZ α peptide added to a final concentration of 2 µM. When testing LIRA riboregulators from a plasmid, the plasmid DNA was added to a final concentration of 30 ng µl−1 in the cell-free reaction mix. For the gate RNA devices test in the paper-based system, the final concentration of the plasmid was 15 ng µl−1.Cell-free transcription–translation systems  were prepared for freeze-drying according to the following recipe: cell-free solution A, 40%; cell-free solution B, 30%; RNase inhibitor , 2%; chlorophenol-red-b-14. The paper discs remained active for at least a month under storage at room temperature.Filter paper  for depositing and freeze-drying of the cell-free system was first blocked with 5% bovine serum albumin overnight. The paper was washed three times in water for 5 to 10 min after overnight blocking, transferred on a hot plate at 50 °C for drying and then cut into 2-mm-diameter paper discs with a biopsy punch. The discs were then transferred into 200 µl PCR strips and 1.8 µl of the above cell-free reaction mix was applied to each of them. Liquid nitrogen was used to freeze the PCR strips containing these paper devices. The frozen paper discs were dried overnight in a lyophiliser. Plate reader tests were carried out on the freeze-dried paper discs 2–4 d later. The systems were stored in a nitrogen environment shielded from light and with the silica gel desiccation packages as described previouslyNASBA experiments were carried out using the following standard protocols: reaction buffer , nucleotide mix , RNase inhibitor , 12.5 µM of each DNA primer (2%), nuclease-free water (2.5%) and RNA amplicon (20%) were assembled at 4 °C. After being incubated at 65 °C for 2 min and a 10 min incubation at 41 °C, 1.25 µl of enzyme mix  was added to the reaction. The reaction took place at 41 °C for 2 h and was then diluted 1:6 into water before applying 2 µl to the freeze-dried paper devices. For the dengue samples, de-identified clinical serum samples positive and negative for the virus were obtained at Salud Digna  and provided as remnant biospecimens. The sample was first diluted 10-fold into water and then heated for 2 min at 95 °C for RNA release. The heat-extracted RNA was then added to the NASBA reaction. Heat-inactivated de-identified saliva samples positive and negative for SARS-CoV-2 were provided by the Arizona State University Biodesign Institute Clinical Testing Lab as remnant biospecimens. Heat inactivation was performed by incubating samples at 65 °C for 30 min. The saliva samples were diluted 1:1 into water and heated at 95 °C for 2 min before spiking in NASBA reactions. A 1 µl aliquot of each sample was transferred into 5 µl NASBA reactions. Each sample was amplified using separate NASBA reactions with the corresponding primer pairs designed for each input RNA. After a 2 h reaction, 1 µl of each pair of NASBA products was combined and diluted with 5 µl water before adding 2 µl to the paper-based cell-free reaction.GFP gene and 16S rRNA, which was used as the internal control (see Supplementary Table Primers were designed for both the Further information on research design is available in the Supplementary InformationSupplementary figures.Reporting SummarySupplementary DataSupplementary Tables 1–11, containing DNA and RNA sequence information.


Local three-stranded DNA/RNA hybrid regions of genomes (R-loops) have been detected either by binding of a monoclonal antibody (DRIP assay) or by enzymatic recognition by RNaseH. Such a structure has been postulated for mouse and human telomeres, clearly suggested by the identification of the complementary RNA Telomeric repeat-containing RNA “TERRA”. However, the tremendous disparity in the information obtained with antibody-based technology drove us to investigate a new strategy. Based on the observation that DNA/RNA hybrids in a triplex complex genome co-purify with the double-stranded chromosomal DNA fraction, we developed a direct preparative approach from total protein-free cellular extract without antibody that allows their physical isolation and determination of their RNA nucleotide sequence. We then define in the normal mouse and human sperm genomes the notion of stable DNA associated RNA terminal R-loop complexes, including TERRA molecules synthesized from local promoters of every chromosome. Furthermore, the first strong evidence of all telomeric structures, applied additionally to the whole murine sperm genome compared to the testes, showed reproducible R-loop complexes of the whole genome and suggesting a defined profile in the sperm genome for the next generation. In the recent period, increased attention has been drawn to R-loop complexes, which appear as frequent features of the eukaryotic genomes. These three-stranded structures are made of the two strands of a DNA molecule, one of them displaced by hybridization with a complementary RNA. Moreover, short-lived intermediates generated during transcription and detrimental effects of stable local R-loops were documented in several instances, while they have also been considered as potentially important players in genome biology ,2. Howevn at the end of all chromosomes and its transcription (UUAGGG)n make telomeres a privileged system to study R-loops [n and sequence elements from the subtelomere [One physiological instance of such a three-stranded hybrid structure has been proposed for the mouse and human telomeres ,7,8,9. R R-loops ,4,5. TERtelomere . TERRA htelomere ).In fact, TERRA are thought to be transcribed from a number of chromosomes’ telomeric repeats . These hHere, we directly investigate the evidence of TERRA in germ cells, as sperm cells are transcriptionally silent with limited RNAs left during spermiogenesis to understand how TERRA containing R-loops formed during the last transcription in spermatids are connected to each chromosome and transferred from within sperm head to an oocyte.Following early reports of a positive DRIP assay ,11, a co−/−Terc animals in generations G1 to G3 were kindly provided by Dr. C. Gunes (Germany) and Dr. A. Londono (Paris) and −/−Tert animals JAX mice  were purchased from The Jackson laboratory, Bar Harbor .The experiments described herein were carried out in compliance with the relevant institutional and French animal welfare laws, guidelines, and policies. Testes, sperm biopsies, and embryos of Mouse spermatozoa were collected from the cauda epididymis. Motile spermatozoa were washed twice in MEM buffer  by centrifugation. Sperm pellets were suspended in phosphate-buffered saline (PBS) and centrifuged again. The pellets were washed twice in 50 mM HEPES buffer pH 7.5, 10 mM NaCl, 5 mM Mg acetate, and 25% glycerol. Samples of human sperm of unknown donors were kindly provided by the fertility clinic (20 September 2011 committee number: 2011/10).32P-labelled oligonucleotide probes.After overnight incubation at 56 °C in Tris buffer 20 mM pH 8, EDTA 50 mM, with 0.5% SDS, 20 µM dithiothreitol and 400 µg/mL Proteinase K, the total nucleic acid fraction was precipitated with ethanol, re-dissolved, further fractionated on Zymo-SpinTM columns  and digested with Msp1 for resection of the telomeric and subtelomeric sequences up to the first CCGG site. After electrophoresis on either 8% acrylamide or 1.5% agarose gels and transfer to a nitrocellulose membrane, DNA and RNA were revealed by hybridization with www.zymoresearch.com accessed on 2 March 2021, ZR-Duet ™ DNA/RNA MiniPrep Plus catalog number D7003). The free RNA was separated, and the fraction that remains bound to the DNA further purified after DNase digestion and purification with Zymo-SpinTM columns .Extraction of total nucleic acids after enzymatic removal of the proteins was followed by fractionation of DNA and RNA with the standard TRIzolTM protocol . Both fraction of nucleic acid aqueous phase (RNA) and chloroform-water interphase separately ethanol precipitated, again, once more fractionated through binding onto Zymo-SpinTM columns  and elution columns or by chloroform extraction (Restriction cleavage: extracts were incubated overnight with the indicated restriction enzymes (Promega) in 50 µL containing 1–5 µg total DNA. For electrophoresis, 25 µL samples were loaded per lane. For extensive hydrolysis, extracts (20 µL) were incubated for 30 to 60 min with RNase 0.5 µg/µL , RNaseH 1 u/µL (which remove only RNA molecules hybridized with the DNA), and DNase 10 u/µL , all provided by Roche Life Science .6 spermatozoa/male and from the total testis (estimated 60% of diploid cells) of a 6 month-old and a 14 month-old male, and two sperm samples from a man at reproductive age, all showing identical electrophoretic profiles of the DNA/RNA complexes. Mouse spermatozoa were collected from the cauda epididymis and ejaculated human sperm kindly provided by the Fertility Clinics of the University. In both cases, the standard protocol was already reported previously by Kianmehr et al. [www.zymoresearch.com accessed on 2 March 2021) see DNA-bound RNAs were prepared from epididymal sperm 10 to 20 × 10r et al. , brieflyAfter DNase digestion, 10–100 ng of RNA were recovered and sent to Eurofins Genomics  for high throughput sequencing on Illumina HiSeq 2500 or Illumina MiSeq. About 10–100 ng of RNA were analyzed by high throughput sequencing on Illumina HiSeq 2500 or Illumina MiSeq . Libraries were generated and designated D1 and D3 from the DRNAs of a 6 month (D1) and a 14 month-old male (D3), with R1 and R3 the corresponding nucleoplasmic sperm RNAs. Totally, 66 million (m) reads were mapped to unique sites in the mouse genome (D1), 69 m (D3), 77 m (R1), and 87 m (R3). From the total testes it was prepared only the DRNA libraries TD1 (70 m reads) and TD3 (82 m), and from the human sperm of a healthy unknown donor, the hD 1 and 4, 50 m (reads), and hR 1 and 4 (88 m). The average length of the reads was about 100 nt. All the primary sequence characteristics for sample libraries of the DNA-bound RNA and free-RNA sequences of germ cells are summarized in http://www.bioinformatics.babraham.ac.uk/projects/fastqc/ accessed on 2 March 2021) quality control of the RAW sequencing reads was performed. Trimming of bad-quality reads was performed using Trimmomatic version 0.36 [7 reads per sample in order to make visualization of different samples comparable. Visualization of alignment was performed using IGV version 2.3.67 [p-value over local regions <0.0001, and FDR adjusted p-value < 0.001). We retrieved the sequences of those peaks located in telomeric regions using UCSC genome browser. Multiple sequence alignment of these peaks was performed using EMBL-EBI Clustal Omega [With FastQC version 0.11.5  and the three tissues analyzed, brain (B), total testis (T), and purified epididymal sperm (S). n used in Here, we generated results in parallel on human and mouse sperm cells. Sperm cells were chosen for the first trial because they have silent transcription, and the majority of cytoplasmic RNAs are already removed at the compacting stage of spermiogenesis, thereby minimizing the risk of contamination by cytoplasmic RNAs. The total nucleic acid fraction recovered from the TRIzol chloroform-water interface was ethanol precipitated and after purification  followed by 45 min in 0.5 N sodium hydroxide , transfer and hybridization b reveale−/−Terc . In the case of the −/−Tert and −/−Terc mutants, they could also be evidenced in embryonic fibroblast cultures from homozygous crosses during the first three generations in which the mutants maintain telomeres [In addition, these complexes did not appear to depend on the telomerase, as they were observed in three generations of telomerase-negative mouse sperm cells Terc−/− c. The saTerc−/− c top, whTerc−/− c bottom,elomeres ,28 and a6 cells/lane) extract was much higher in the testes and brains compared to sperm cells.Complexes with the same electrophoretic profiles were identified in different somatic cell types. To minimize contamination of the cytoplasmic RNA, TRIzol extraction was applied twice on the somatic cells. DNA-associated RNAs are stably maintained with genomic DNA even after twice treatment with TRIzol. Comparable profile and amounts in every mouse cell tested, from embryonic fibroblasts to adult tissues  as well as cultivated cell lines and short-term cultures of differentiated cells, in Properties of the complexes were compatible with the R-loop structure schematically drawn in ® HTB-96™) as a positive control, in which DNA/RNA telomeric hybrids have previously been identified by DRIP [The same results were also generated in parallel on a series of human and mouse cells, ex vivo organ extracts that were mostly normal cells, but included cultured cell lines , and the by DRIP .A key aim of this paper was to establish whether the observed structure was present in only one or a few telomeres or, on the contrary, was a general feature of the genome. It has been generally assumed that R-loops are generated by transcription from adjacent promoters. On the other hand, a major part of TERRA has been reported in cell culture to be transcribed from chromosome 18 in mouse cells and from chromosome 20q in humans ,25 so thWe further ascertained by Illumina high-throughput analysis of the DNA-bound RNA molecules see . StartinDNA associated TERRA found from every telomeric end in mouse and human sperm cells is consistent with the idea that TERRA is expressed from subtelomeric promoters and remained associated at the telomeres ends. TERRAs that remained bound to the sperm genome were generated from the last wave of transcription before the compaction step at the late stage of spermiogenesis. Our results need to be compared with previous reports of major TERRA promoters in C18 (mouse) and 20q (human) chromosomes ,25. TherSince the association of TERRA transcripts with telomeres could be revealed by the proposed method, we also asked if TERRA hybrids located at defined non-telomeric sites could be detected from data generated by Illumina high-throughput analysis of the DNA-bound RNA molecules. In order to try to distinguish stable complexes of R-loops ,33 and tGenome-wide profiling shows representative landscapes of expression signals of chromosomes 2, 10, 12, and 18 see . For eacIn addition, research on known non-telomeric TERRA sites has identified a fraction of TERRA transcripts near the sex chromosome that undergo pairing in both sexes, the inactive X chromosome (Xi) of female cells, and the Y  chromosoIllumina high-throughput RNA-seq analysis of the DNA-bound RNA molecules of sperm and testicles of mice revealed transcripts from the whole reference genome. Here we only analyzed data from mice because DRNA-seq data was available for two types of samples: sperm (haploid) and testis containing both haploid and diploid cells . A general overview of RNA associated with DNA revealed, in addition to TERRA, a wide variety of RNA sequences maintained on the DNA (DRNA fraction), as shown for mouse sperm and testes in Here, we have documented the following results: (i) a fraction of nascent TERRA was stored packed in vivo with the genome in the form of DNA/RNA hybrids in the telomeres of each chromosome in sperm (mouse and human) and other mouse organs (testes and brain); (ii) non-telomeric TERRA repeats transcribed from different regions of the genome were also maintained in their genomic loci as DNA/RNA hybrids; (iii) a fraction of the TERRA repeat-containing transcripts previously described near the sex chromosome were attached as DRNA to Y (Erdr1) or X (Asmt) chromosomes; (iv) other RNAs than the TERRA repeats were found to be attached to DNA fractions with simple repeated sequences; (v) a general profile of transcripts stably attached to DNA throughout the mouse genome and the discovery distinct transcripts profile retained in the sperm DRNA fraction and important differences in the levels amongst the transcripts attached to DNA in sperm and testicular cells. Our results suggest that RNAs associated with DNA could define cell fate and specificities. The approach developed and validated here could be easily applied to any type of cells in normal development and diseases to eventually establish its dynamics.DNA-associated RNA (DRNA) extraction is a straightforward approach without the need for supplementary interacting guide molecules (such as antibody or gene product), inexpensive, highly reproducible, and efficient. Bioinformatic analysis with alignment to the reference genome allowed the generation of genome-wide profiling of stable DNA/RNA hybrid transcripts purified with genomic DNA. Here, our data shows that all of these stable R-loops provide insight into the amount of RNA maintained in the sperm genome. These transcripts are of interest in view of the various reports highlighting a role for sperm RNAs in the epigenetic control of gene expression ,35. We pThe parallel is possible, with S9.6, RNase H, and more recently MapR  establisIn the testes, several studies have already reported genome-wide transcripts, and it is also assumed that from the last stage of elongated spermatids, with the elimination of the majority of cytoplasmic RNAs, a number of transcripts remain in the sperm . We haveGenome-wide profiling detected DNA/RNA hybrids at multiple loci across the genome   compared to free RNAs and the testis DRNA fraction revealed a significantly higher level of intergenic transcripts than found in exon, intron, 3′, 5′, promoter-TSS (transcription start site), TTS (transcription termination site), and non-coding transcripts . Some exIn parallel, both molecular analysis and Illumina high-throughput analysis of the DNA-bound RNA molecules (DRNA-seq) was applied firstly to TERRA complexes of the sperm from human and, in particular, mouse sperm telomeres, provide significant solid information. In fact, in both species, sperm telomeres retained a fraction of the TERRA synthesized directly from promoters adjacent to all chromosomal ends. In addition, this was confirmed by molecular analysis in human somatic cells and in a variety of mouse cells in culture and tissues. With DRIP-seq analysis, such a conclusion could not be completely reached. Here, with both molecular and RNA-sequencing analysis, we reach the same conclusion that DNA/RNA hybrids at the level of telomeres were maintained in all types of cells. In addition, RNA-seq analysis in the testes and sperm of mice confirmed that the TERRA (telomeres) transcribed in the testes of all chromosomes were maintained until the final stage of sperm maturation in the epididymis. Human and mouse sperm TERRA would be thus transferred to the offspring packed with genomic DNA. TERRA as DNA/RNA hybrids in the genome suggests a potential role of these non-coding RNAs on the stability of the telomeres and may be on its expression in the next generation.We addressed that TERRA signals are significantly enriched at telomere sequences; and TERRA repeats are also reported at non-telomeric regions of chromosome 2, 9, 10, 12, 14, 18, and Y see , where wn containing transcripts in the DNA/RNA hybrids fraction of mouse sperm, which were previously established in ES cells [We also identified sex chromosomes PAR-TERRA (UUAGGG)ES cells . The finEvidence of DNA/RNA hybrids for both TERRA and its generalization to genome-associated transcripts in semen underscore ongoing questions about how these hybrids might influence genetic and/epigenetic memory. Among several interpretations, one possibility is a trivial phenomenon unique to sperm: the RNA produced by the spermatid at the time of conversion of the chromatin to its final compact structure can remain associated with the locus, perhaps only the last oligonucleotide segment synthesized, giving the artifactual image of a complete RNA taking into account the sequencing procedure. However, DNA/RNA hybrids in a way in active testicular cells were also observed. In addition, a noticeable difference in the transcripts level from different regions between sperm and testes was visible. Moreover, our less advanced analysis of somatic cells also showed a direct hybrid DNA/RNA structure detectable at the telomeres. These studies demonstrate that some transcripts appear strongly maintained in a stable complex DNA/RNA hybrid structure, and further studies should reveal that its meaning and function may be intentional and selective for a given cell.In conclusion, we believe that the approach designed to identify the telomeric TERRA has wider applications. These results are encouraging and should be extended to different processes and diseases. Future research is needed to reveal specificities, biogenesis, and functions of stably DNA-associated RNAs.


Introduction: Recombinant human erythropoietin (rHuEPO) administration studies involving transcriptomic approaches have demonstrated a gene expression signature that could aid blood doping detection. However, current anti-doping testing does not involve collecting whole blood into tubes with RNA preservative. This study investigated if whole blood in long-term storage and whole blood left over from standard hematological testing in short-term storage could be used for transcriptomic analysis despite lacking RNA preservation.Methods: Whole blood samples were collected from twelve and fourteen healthy nonathletic males, for long-term and short-term storage experiments. Long-term storage involved whole blood collected into Tempus™ tubes and K2EDTA tubes and subjected to long-term  storage and RNA extracted. Short-term storage involved whole blood collected into K2EDTA tubes and stored at 4°C for 6‒48 h and then incubated at room temperature for 1 and 2 h prior to addition of RNA preservative. RNA quantity, purity, and integrity were analyzed in addition to RNA-Seq using the MGI DNBSEQ-G400 on RNA from both the short- and long-term storage studies. Genes presenting a fold change (FC) of >1.1 or < ‒1.1 with p ≤ 0.05 for each comparison were considered differentially expressed. Microarray analysis using the Affymetrix GeneChip® Human Transcriptome 2.0 Array was additionally conducted on RNA from the short-term study with a false discovery ratio (FDR) of ≤0.05 and an FC of >1.1 or < ‒1.1 applied to identify differentially expressed genes.Results: RNA quantity, purity, and integrity from whole blood subjected to short- and long-term storage were sufficient for gene expression analysis. Long-term storage: when comparing blood tubes with and without RNA preservation 4,058 transcripts (6% of coding and non-coding transcripts) were differentially expressed using microarray and 658 genes (3.4% of mapped genes) were differentially expressed using RNA-Seq. Short-term storage: mean RNA integrity and yield were not significantly different at any of the time points. RNA-Seq analysis revealed a very small number of differentially expressed genes (70 or 1.37% of mapped genes) when comparing samples stored between 6 and 48 h without RNA preservative. None of the genes previously identified in rHuEPO administration studies were differently expressed in either long- or short-term storage experiments.Conclusion: RNA quantity, purity, and integrity were not significantly compromised from short- or long-term storage in blood storage tubes lacking RNA stabilization, indicating that transcriptomic analysis could be conducted using anti-doping samples collected or biobanked without RNA preservation. The World Anti-Doping Agency (WADA) was created in 1999 and has been at the forefront of anti-doping ever since . Over th2EDTA tubes, do not contain RNA stabilizing reagents. Once collected, the whole blood samples are sealed in tamper-proof containers, stored upright in a refrigerated state, dispatched with a temperature data logger to the laboratory, and analyzed within 72 h from collection  could impact transcriptomic analysis and 2) to investigate if whole blood collected into K2EDTA tubes and stored for up to 48 h at 4°C, to simulate short-term storage, could impact transcriptomic analysis, to see if leftover blood from standard hematological testing could be preserved and then used for this purpose.The continued success of the storage and re-analysis program is predicated on the inclusion of new technologies as these become available. For example, next-generation sequencing and “omics”-based technologies are increasingly being used in clinical testing, scientific research, and disease diagnoses, including cancer, with immense success . It is allection . As suchllection . Importallection ; when thThe study designs and collection procedures were explained to all participants who provided informed consent. The local ethics committee  approved the study designs, and all collection procedures were carried out according to the Helsinki Declaration. Blood samples were collected by trained phlebotomists.2EDTA tubes).The long-term storage study was designed considering the following: 1) the ideal situation (RNA collection/extraction according to manufacturer instructions) as a reference and 2) the real situation that is faced by anti-doping biobanks, where whole blood samples are stored in tubes without an RNA stabilization reagent . Immediately after collection, the Tempus™ tubes were vigorously shaken for 10 s and incubated at room temperature (RT) for ∼3 h and then stored at −80°C before subsequent analysis. After collection, the K2EDTA tube was gently inverted 10 times, incubated at 4°C for 1–4 h and then stored at −80°C for further analyses. During the 4°C incubation period for the K2EDTA tube, 1.25 mL of blood was pipetted into aliquots of 250 µL into five separate 1.5 mL TubeOne® Microcentrifuge tubes . One of these aliquots was used for RNA extraction from a “fresh” blood sample, and the other four tubes were immediately stored at −80°C.Whole blood was collected from an antecubital vein using a closed vacuette system from 12 male nonathletes : 80.0 ± 13.4; body fat percentage: 12.5 ± 2.9). A 3 mL whole blood sample was collected into a Tempus™ Blood RNA Tube , and a 10-mL whole blood sample was collected into a BD® EDTA tube  the Tempus™ Spin RNA Isolation Kit  and 2) the GeneJET RNA Purification Kit  following manufacturer instructions . The Tem designs . After eRNA quantity and purity were assessed using a Nanodrop® ND-2000 Spectrophotometer . RNA integrity  was assessed using an Agilent® 2100 Bioanalyzer with an Agilent® RNA 6000 Nano Kit .Temp, EDTA + Temp, GJ_aliq, and EDTA + GJ) with the highest quality were selected for the following analysis (GJ_fresh was not included because this did not simulate using frozen/biobanked samples nor was it used in previous studies with anti-doping purposes). One hundred nanograms of purified total RNA was processed using the Affymetrix GeneChip® Human Transcriptome 2.0 Array and a GeneChip® WT Plus Reagent Kit according to manufacturer instructions . Transcriptome Analysis Console (TAC) 4.0 software  performed initial data QC, visualization of array scanning data, and transcript expression analysis. A false discovery ratio (FDR) of ≤0.05 and a fold change of >1.1 or <–1.1 were applied. The ideal situations were considered as reference groups.RNA samples of 8 subjects from four different storage/extraction protocols . dsDNA libraries (with conditions for a 250-bp Insert Size) were created from the rRNA-depleted eluate using an MGIEasy RNA Directional Library Prep Set. dsDNA library quantity was assessed using a Thermo Fisher Scientific Qubit® dsDNA High Sensitivity Assay Kit and a Qubit® Fluorometer . The quality of the fragment size distribution of the dsDNA library was assessed by visual inspection of electropherograms created using an Agilent® DNA 1000 Kit on an Agilent® 2100 Bioanalyzer. Only dsDNA libraries with satisfactory fragment size distributions were carried forward onto the next steps, and dsDNA libraries were re-created for any samples with aberrant electropherograms or low concentrations. dsDNA libraries were circularized and converted into ssDNA libraries using an MGIEasy Circularization Kit. ssDNA library concentration was assessed using a Thermo Fisher Scientific Qubit® ssDNA Assay Kit and a Qubit® Fluorometer. DNA nanoballs (DNBs) were prepared from the ssDNA library pools, with a 40-fmol ssDNA library for each reaction, using an MGI DNBSEQ-G400RS High-throughput Sequencing Set . DNB concentration was assessed using a Thermo Fisher Scientific Qubit® ssDNA Assay Kit and a Qubit® Fluorometer. DNB preparations >8 ng/μL were loaded onto two flow cells  using an MGIDL-200H Portable DNB Loader . The flow cells were placed on an MGI DNBSEQ-G400 sequencer  and subjected to PE100 sequencing.Seven RNA samples with the highest RIN values from each of the groups p ≤ 0.05 for each comparison were considered differentially expressed.Reads were mapped to Human Genome GRCh37 reference (hg19) using STAR 2.5.3. Gene counts were obtained using an annotation model based on RefSeq Transcripts (release 96) and normalized by fragments per kilobase million (FPKM) and by adding 0.0001 to all read counts, to allow statistical testing. Differential gene expression was determined by Partek Gene Set Analysis (GSA) using default settings. Genes presenting a fold change >1.1 or <–1.1 (a FC of 1.1) with 2EDTA tubes. After collection, each of the four tubes was stored at 4°C for 6, 12, 24, or 48 h. At each of these time points, one of the K2EDTA tubes was removed from 4°C and maintained at RT for a total of 60 min to simulate the time required for sample registration and analysis. For the first 15 min of these 60 min at RT, the tube was placed in a sample mixer, and for the remaining 45 min, the tube remained upright at RT. After 60 min, 1.5 mL of whole blood was then removed from the tube and added to a new tube containing 3 mL of Tempus™ Blood RNA preservative, and this tube was shaken vigorously for 10 s. After a final 2 h at RT, the remaining 1.5 mL of whole blood was added to 3 mL of Tempus™ Blood RNA stabilizing reagent in a different tube, and this was shaken vigorously for 10 s. This was to simulate an extended registration and analysis time as can potentially occur in big events with a very high number of samples. All samples were then stored at −80°C until RNA extraction. The experimental design is illustrated in Only one participant from the long-term storage study also took part in the short-term storage study, and sampling was separated by > 12 months. Whole blood was collected from an antecubital vein of 14 male nonathletes, using a closed vacuette system in four separate 3-mL KTotal RNA was extracted from whole blood using a Tempus™ Spin RNA Isolation Kit  following manufacturer instructions . RNA quaFour samples, with the highest RIN value from each of the four time points , which were all incubated at RT for 1 h prior to the addition of RNA preservative, were selected for RNA-Seq and analyzed as described above in the long-term storage study methods section.post hoc. The analyses of RNA quantity and quality were performed using SPSS software (v.23) with alpha at p ≤ 0.05.Descriptive data were presented as mean values and standard deviation (SD). The difference of integrity and purity values between the groups was determined using a generalized linear model, corrected by Bonferroni EDTA + Temp had a statistically significant higher mean RIN than EDTA + GJ (260/280 and A260/230). All groups demonstrated A260/280 > 2, with statistical differences between Temp and GJ_fresh  than inter-kit . The comparison between EDTA + Temp vs. Temp showed the lowest number of differentially expressed transcripts , while EDTA + GJ vs. GJ_aliq demonstrated 6,324 (9.37%) and GJ_aliq vs. Temp revealed 13,678 (20.26%) differentially expressed transcripts.Principal component analysis (PCA)  of microTemp and EDTA + Temp also showed two clusters, according to the sampling tube (The PCA of RNA-Seq data comparing ing tube . The gening tube . None ofing tube  were difp ≤ 0.05).There were no statistical differences for the mean RIN and yield amongst all eight time points . When co260/280 did not present differences among time points  (Ae points . A260/23ctively) .n = 16 samples in total). PCA did not demonstrate clear clusters regarding time of storage but did show clusters regarding samples of the same subjects  > 2  if this ratio presents a value lower than 1.8  extracted sufficient RNA quantities from blood collected in a Ker yield ; however280) > 2 , which i280) > 2 . Althougthan 1.8 . Samples2EDTA tubes were thawed on ice for ∼1.5 h, followed by the RNA extraction protocol. This thawing time could affect RNA integrity as mRNA structures could be degraded by RNase enzymes  genes described in previous studies  have already been shown to be promising blood doping biomarkers even when using blood samples collected by Dried Blood Spot (DBS) technologies , which is expressed in the erythrocyte membrane, involved in the transport of carbon dioxide from tissues to the lungs . The RI enzymes . Neverth enzymes . Of note enzymes  shows th studies   in two 3-mL K2EDTA tubes (2EDTA tubes would be subjected to would impact RNA quality, purity, integrity, and transcriptomic analysis. Unexpectedly, the RIN value of the samples was not significantly different between all the time points, and neither was the RNA yield. However, comparing all samples at RT for 1 h vs. 2 h, the yield was significantly lower in 2 h groups but still sufficiently high for RNA-Seq. RNA-Seq analysis demonstrated that the number of differentially expressed genes increases the longer the sample is stored at 4°C prior to the addition of RNA preservative. However, none of the gene targets described in the gene expression signature of rHuEPO administration (The short-term storage experiments were designed based on the observed logistics of sample collection, storage, transport, registration, and analysis of anti-doping samples during anti-doping testing performed at the IAAF World Championships 2019 in Doha, Qatar. WADA Code guidelines describe that for the analysis of TA tubes . Blood sTA tubes . During TA tubes . Each blTA tubes , and oncTA tubes . After tstration  (list ofstration  presente2EDTA, simulating remaining blood from standard hematological testing, could be used for extracting RNA that has sufficient yield, quality, and integrity for gene expression analyses. The long-term storage experiment also demonstrated that 3 mL whole blood collected in K2EDTA tubes and stored at −80°C can be used for gene expression analysis. In both of the studies, none of the gene targets described in the gene expression signature of rHuEPO administration (The short-term storage experiment showed that 1.5 mL of whole blood collected in Kstration  presente