They were the key for our current mechanistic understanding of ribosomal peptide bond formation and of phosphodiester cleavage in recently discovered small ribozymes, such as twister and pistol RNAs. Here, we present a comprehensive study on the impact of c3A and the thus far underinvestigated 3-deazaguanosine (c3G) on RNA properties. We found that these nucleosides can decrease thermodynamic stability of base pairing to a significant extent. The effects are much more pronounced for 3-deazapurine nucleosides compared to their constitutional isomers of 7-deazapurine nucleosides . We furthermore investigated base pair opening dynamics by solution NMR spectroscopy and revealed significantly enhanced imino proton exchange rates. Additionally, we solved the X-ray structure of a c3A-modified RNA and visualized the hydration pattern of the minor groove. Importantly, the characteristic water molecule that is hydrogen-bonded to the purine N3 atom and always observed in a natural double helix is lacking in the 3-deazapurine-modified counterpart. Both, the findings by NMR and X-ray crystallographic methods hence provide a rationale for the reduced pairing strength. Taken together, our comparative study is a first major step towards a comprehensive understanding of this important class of nucleoside modifications.Deazapurine nucleosides such as 3-deazaadenosine (c C3 RNA) . Similar3 RNA) . These f7A) (3A) (1A) (7G) (3C) (3G) and 1-deazaguanosine (c1G) would be highly useful for RNA atomic mutagenesis, however, such studies are very rare (Suitable deazanucleosides for informative RNA atomic mutagenesis experiments are 7-deazaadenosine (c7A) ,15,16, 37A) (3A) ,20, 1-de3A) (1A) ,12,14,201A) (7G) ,15 and 37G) (3C) ,21. Moreery rare  because ery rare ,23. In gery rare . Besidesery rare .3G containing RNA beforehand. We here describe the impact of c3G and c3A on RNA properties. Based on UV-spectroscopic melting experiments, a detailed thermodynamic analysis of duplex and hairpin stabilities is provided and the effects on base pairing are discussed in the light of the sequence context. Furthermore, solution NMR spectroscopy sheds light on base pair opening dynamics. In addition, we have solved the X-ray structure of a c3A containing RNA at atomic resolution to disclose crucial structural features, such as ribose puckers, hydrogen-bonding networks, and hydration patterns of the deazanucleoside, and to correlate them to base pairing properties.Therefore, we set out to accomplish a thorough chemical and biophysical analysis of 3-deazapurine containing RNA, along with the improvement of the synthesis of c® SIL G/UV254 plates. Silica gel 60 (mesh size 0.04 – 0.063 mm) for column chromatography was purchased from Macherey-Nagel. The procedures for chemical synthesis of phosphoramidites 8 and 9, and the 6-chloro-3-deazapurine 14 and their characterization data are available in the Supporting Information . 1H, 13C, and 31P NMR spectra were recorded on a Bruker Ultrashield™ 400 Plus spectrometer. Chemical shifts (δ) are reported relative to tetramethylsilane (TMS), referenced to the residual solvent signal . The following abbreviations were used to denote multiplicities: s = singulet, d = doublet, t = triplet, q = quadruplet, m = multiplet, b = broad. Signal assignments are based on 1H–1H-COSY, 1H–13C-HSQC and 1H–13C-HMBC experiments. High resolution mass spectra were recorded in positive ion mode on a Thermo Scientific Q Exactive Orbitrap, ionized via electrospray at 3.7 kV spray voltage.Reagents were purchased in the highest available quality from commercial suppliers  and used without further purification. All reactions were carried out under argon atmosphere, unless otherwise noted. Analytical thin-layer chromatography (TLC) was performed on Macherey-Nagel Polygram2N-Tfa, 2′-O-Tbs and 2N-Tfa, 2′-O-CEM; >98% coupling yield), 3-deazadenosine , 7-deazaguanosine  and 7-deazaadenosine . 2′-O-TOM and acetyl protected nucleoside phosphoramidite building blocks and 2′-O-Tbs 1000 Å CPG solid support (>15nt) were purchased from ChemGenes, Primer supportTM 5G (<15nt) was purchased from GE Healthcare. All oligonucleotides were synthesized on an ABI 391 or ABI 392 Nucleic Acid Synthesizer following standard methods: detritylation (90 sec) with dichloroacetic acid/1,2-dichloroethane (4/96); coupling (5.0 min) with phosphoramidites/acetonitrile  and benzylthiotetrazole / acetonitrile ; capping (2 × 25 s) with Cap A/Cap B (1/1) for c3A, c7G & c7A modified RNA, Cap A: 4-(dimethylamino)pyridine/acetonitrile (500 mM), Cap B: acetic anhydride/sym-collidine/acetonitrile (2/3/5) and Cap A mild/Cap B mild (1/1) for c3G modified RNA, Cap A mild: phenoxyacetic anhydride/acetonitrile (100 mM), Cap B mild: N-methylimidazole/sym-collidine/tetrahydrofuran (0.160/0.265/10); oxidation (60 sec) with iodine (20 mM) in tetrahydrofuran/pyridine/H2O (35/10/5). Solutions of phosphoramidites, tetrazole and Cap were dried over activated molecular sieves (3 Å) overnight.Standard phosphoramidite chemistry was applied for RNA strand elongation and incorporation of 3-deazaguanosine  and aqueous ammonia  for 15 min at 65°C or 3 h at 37°C. The supernatant was removed and the solid support was washed twice with 0.5 ml tetrahydrofuran/H2O (1/1). Combined supernatant and washings were evaporated to dryness and the residue was dissolved in a solution of tetrabutylammonium fluoride in tetrahydrofuran  and incubated for 14 h at 37°C for removal of 2′-O-silyl protecting groups. The reaction was quenched by addition of triethylammonium acetate/H2O . Tetrahydrofuran was removed under reduced pressure and the sample was desalted with size-exclusion column chromatography  eluting with H2O; collected fractions were evaporated and the RNA dissolved in H2O (1 ml). The crude RNA was purified by anion exchange chromatography on a semipreparative Dionex DNAPac® PA-100 column (9 mm × 250 mm) at 80°C with a flow rate of 1 ml/min . Fractions containing RNA were diluted with 0.1 M triethylammonium bicarbonate solution, loaded on a C18 SepPak Plus® cartridge (Waters/Millipore), washed with H2O and eluted with acetonitrile/H2O (1/1). Crude and purified RNA were analyzed by anion exchange chromatography on a GE Healthcare Äkta Explorer HPLC System containing a Dionex DNAPac® PA-100 column (4 mm × 250 mm) at 80°C with a flow rate of 1 ml/min. For RNA shorter or equal to 15 nucleotides, a gradient of 0–40% B in 30 minutes and for RNA longer than 15 nucleotides a gradient of 0–60% B was used; Eluent A: 6 M urea, 25 mM Tris·HCl, pH 8.0; Eluent B: 500 mM NaClO4, 6 M urea, 25 mM Tris·HCl, pH 8.0. HPLC traces were recorded at UV absorption by 260 nm. RNA quantification was performed on an Implen P300 Nanophotometer.For basic deprotection of natural, c2H2(EDTA)/H2O (5/4) for a total volume of 30 μl, injected onto a C18 XBridge column  at a flow rate of 0.1 ml/min and eluted with 0–100% B gradient at 30°C . RNA HPLC runs were analyzed on a Finnigan LCQ Advantage Max electrospray ionization mass spectrometer with 4.0 kV spray voltage in negative mode. Characterization of the c3G modified 47mer was performed on a 7 T FT-ICR-MS . Sample preparation: 2 μM RNA in 1:1 H2O/CH3OH with 20 mM imidazole and 20 mM piperidine (pH ∼ 10). The sample was measured in negative ion mode at a flow rate of 90 μl/h and a skimmer potential of –50 V for ESI and –60 V for CAD  were diluted with 40 mM Na2HPO4 (pH 7.0) containing 150 mM NaCl) and transferred into UV permeable high precision cells made of quartz SUPRASIL® with a light path of 10 mm or 1 mm. UV melting profiles were recorded at 250 and 260 nm on a Varian Cary-100 spectrophotometer equipped with a multiple cell holder and a peltier temperature control device. Each RNA was measured at five different concentrations ranging from 1 to 100 μM. Tm values were determined by calculating the first derivative. Thermodynamic parameters were determined according to . The error limits for ΔG, ΔH and ΔS reflect the standard deviation of three (duplex) or five (hairpin) independent measurements for a confidence interval of 95%.RNA samples were lyophilized as triethylammonium salts, dissolved in 800 or 330 μl buffer (10 mM Na2(CH3)2]·3H2O, 25 mM NaCl, 3 mM NaN3, in H2O/D2O 9:1, pH 6.5) and transferred into 5 mm NMR tubes. Sample concentrations varied between 0.05 and 0.3 mM and experiments were run at 298 K unless otherwise stated. All NMR experiments were conducted on a Bruker 600 MHz Avance II+ NMR or a 700 MHz Avance Neo NM both equipped with a Prodigy TCI probe.RNA samples were lyophilized as triethylammonium salts, dissolved in 500 μl NMR buffer (15 mM Na[AsO2(CH3)2]·3H2O, 25 mM NaCl, 3 mM NaN3, in H2O/D2O 9:1, pH 6.5. The same buffer stock solution was used for all samples to rule out changes in imino proton—bulk water exchange rates due to differences in the buffer composition. The CLEANEX-PM pulse sequence is available from the Bruker standard experiment collection (zgcxesgp) with an excitation sculpting water suppression element. A standard excitation sculpting water suppression experiment (zgesgp) was used as the reference experiment. For the determination of the water T1 relaxation times under the experimental conditions a saturation recovery experiment was used and the longitudinal water relaxation time ranged between 3.03 s (c3G RNA) and 3.13 s (unmodified RNA). The following experimental parameters were used: spectral width 24 ppm, o1p: 4.7 ppm, number of scans 2048, dummy scans 32, interscan delay 1.5 s. Shaped pulse parameters were set via the getprosol command and the hard 90° 1H pulse. The mixing times were set to 5, 25, 50, 50, 100, 100, 150, 200, 300, 400, 400 and 500 ms for both duplexes. The NMR spectra were processed in TOPSPIN 4.0.9 using a line broadening factor of 10 Hz . The absolute peak intensities of the CLEANEX-PM experiments and the reference experiments were determined and used to obtain the relative intensities. The data was then exported to MATLAB and the build-up curves were fitted to the following equation:I/I0 relative peak intensity, k imino proton–bulk water exchange rate (s−1), R1,water longitudinal water exchange rate, mix CLEANEX-PM mixing times (s) and R1,A is a combination of the longitudinal and transverse relaxation rate of the imino proton resonance (floating parameter during fitting). Errors in exchange rates were estimated from replicate experiments and obtained from 1000 Monte-Carlo runs.The CLEANEX-PM NMR experiments were carried out in the following buffer: 15 mM Namethyl (TOM) phosphoramidites and the oligomers were assembled on controlled pore glass (CPG) supports (O-silyl groups (and the 2′-O-Cem group in case of 9) was carried out with tetra-n-butylammonium fluoride trihydrate in tetrahydrofuran for 14 hours at 37°C. The reaction was quenched by the addition of triethylammonium acetate buffer at pH 7.4. Salts were removed by size-exclusion chromatography. Analysis by anion exchange chromatography under strong denaturing conditions usually gave a major peak for the desired RNA which was further purified by anion exchange chromatography on a semipreparative column. The molecular weights of the purified RNAs were confirmed by LC–ESI-MS (The incorporation of building blocks supports . CleavagC–ESI-MS  and the C–ESI-MS  (SupplemC–ESI-MS .3A nucleoside building block has been merged from two routes that we published earlier (Scheme 15 and benzoyl-protected 1-O-acetylribose (N6-benzoyl protection (N6-(di-n-butyl)amidine protection amidine counterparts) because of the significantly larger gap in retention times. Furthermore, in this work we also optimized the access to 6-amino-3-deazapurine 15 and its precursor 6-chloro-3-deazapurine 14 on large scale and starting from cheap materials  and thermodynamic parameters .To the best of our knowledge, hardly anything is known about the impact of 3-deazapurine nucleosides on base pairing in regular RNA. We therefore set out to analyze the influence of c3G (in Ia) and c3A (in Ib) respectively. Interestingly, in this sequence context, the corresponding 7-deazapurines (c7G in Ic and c7A Id) have negligible effect on the Tm and thermodynamic parameters. For the hairpin RNA (type II), the destabilization of both 3-deazapurines is significant, reflected in a decrease of the Tm-value by 9.1 and 7.9°C, for c3G (in IIa) and c3A (in IIb) respectively, when compared to the native hairpin II. In contrast to type I RNA, the monomolecular system (type II) reveals a slight destabilizing effect of 7-deazapurines as well (c7G in IIc and c7A IId).Table 3G the reduction in both palindromic RNAs was dramatic, reflected in –13.5/–13.0°C reduced Tm values (IIIa and III’a), accounting for –6.8/–6.5°C destabilization per single modification which is more than twice of the destabilization that we observed for a single c3G-C base pair in the bimolecular 9 bp duplex Ia. The destabilizing nature was also very pronounced for c3A in both palindromic RNAs (–9.8°C in IIIb and –13.0°C in III’b). In contrast, only little destabilization was found for both 7-deazapurines in both palindromic RNAs .To further elucidate the impact of 3- and 7-deazapurines on base pairing, we investigated the thermodynamic base pairing properties of 3- and 7-deazapurines in short palindromic RNAs. As mentioned above, the effects become intensified because the self-pairing results in two modified base pairs with regularly Watson-Crick–paired regions next to them that are below the required number of base pairs needed for successful double helix nucleation ,46. Inde3G was investigated only within the context of 2′-OCH3–RNA; c3G caused destabilization when paired to C  (N2-acetyl-3-deazaguanine) the native pairing strength was restored  . Interesrestored .Furthermore, a single paper points at the strong destabilizing effect of 3-deazaguanine on DNA base pairing . In cont1H NMR spectra of the palindromic duplex 5′-GGUCGACC III that were collected as a function of temperature, over the range 5–25°C. The signal assignment was based on 2D 1H/1H-NOESY spectroscopy (The NMR resonances for the hydrogen-bonded protons of Watson–Crick base-pairs (‘imino protons’) directly reflect the double helical segments within folded RNA. The chemical shifts of these signals are characteristic for A-U (>14 ppm) and C-G base pairs (∼12–13 ppm), and the linewidths reflect proton exchange with the solvent. Clearly, they are very sensitive to modifications, in particular, if they concern the nucleobases. Figure troscopy . With in3G imino proton resonance of the modified duplex IIIa was shifted highfield  of almost 3 pKa units  by the application of 1D Phase-Modulated CLEAN Chemical EXchange Spectroscopy (CLEANEX-PM) NMR experiments  of 1.68 s−1 was observed for the A-U base pair next to the c3G–C modification. Also for the c3G–C base pair itself the rate k(H2O ex.) was increased compared to the unmodified duplex (1.11 s−1 versus 0.76 s−1), while the G–C base pair two positions away almost the same exchange rates k(H2O ex.) for modified and unmodified duplexes were observed (0.72 s−1 versus 0.62 s−1). This is consistent with a local structural perturbation induced by the 3-deazapurine hotspot.To investigate these effects in more detail, we quantified the water imino proton exchange kinetics of a cs Figure  53–55)..3G–C basE. coli 23S rRNA sarcin−ricin loop (SRL) which is a robust and well-behaved crystallization scaffold  of 0.12 Å (within the errors on coordinates of 0.13 Å). Overall, the structure revealed that the 3-deazanucleobase does not significantly affect the base pair geometry and the SRL RNA fold. However, what is different is the local hydration network in the minor groove of the c3A–U base pair  and the H-acceptor  of ΔpKa 5.5 and 5.4, respectively, which appears optimal for efficient base pairing (7A–U (ΔpKa 3.9), c3A–U (ΔpKa 2.4), c7G–C (ΔpKa 6.2) and c3G–C (ΔpKa 8.2) deazapurine base pairs.Another aspect that was documented earlier is that natural Watson–Crick base pairs A–U and C–G are characterized by a p pairing ,58. ThisKa of the heterocycles (as listed above) contribute to macroscopically observed double helix stabilities. This might be the reason why concepts for predicting hydrogen-bond strengths from acid−base molecular properties  cannot be directly applied to nucleic acid base pairing strengths, although being promising for an integrated theoretical and experimental approach to solve this long-standing problem in the future  affecting the minor groove, and 7-deazapurines (c7G and c7A) affecting the major groove. In particular, the first solved crystal structure of a c3A modified RNA and the comparison to the unmodified counterpart reveal that the characteristic water molecule that always docks on the purine N3 atom and often spans further to the ribose 2′-OH in native RNA double helices  exhibits N7-innersphere coordination to a Mg2+ cation that additionally coordinates to the oxygen atom of a sequence-distant phosphate backbone unit and thus molds a critical part of the binding pocket. Replacement of this adenine by c7A abolishes binding to NAD+  demonstrated that an initial suspect (N3 of A2451) did not actively participate in proton transfer to catalyze peptide bond formation, but instead, the 2′-OH of the very adenosine assists in positioning of the substrate ,18,20,70For atomic mutagenesis experiments using deazanucleobases, a profound knowledge of the intrinsic properties of these heterocycles in an RNA context is imperative to avoid misinterpretations. Functional assays to evaluate ribozyme or riboswitch activity in response to a specific purine-deazapurine replacement are usually designed to impair a potential H-bond or innersphere coordination to a divalent metal ion, as suggested by the observed distances in the three-dimensional structure. Provided such an interaction is crucial for activity, it can thus be easily identified and the mechanistic interpretation becomes straightforward. Importantly, in all of the examples mentioned above, the purine/deazapurine finds itself in an exposed and unpaired position. Clearly, in the light of our present study, functional assays that would involve atomic mutagenesis with 3-deazanucleobases in double helical regions would encounter severe limitations because of thermodynamic destabilization of the stem containing the modification. This aspect is, however, less critical for atomic mutagenesis using 7-deazanucleobases which do not or only minimally destabilize double helices.3A modified RNA to visualize the hydration pattern of the minor groove. The structure points out that the hydrophobic minor grove face of c3A impairs hydration because the water molecule that is usually hydrogen bonded to the N3 atom becomes extruded from the 3-deaza-modified base pair. Taken together, our comparative study sheds light on Watson–Crick base pairing of 3- and 7-deazapurines and thus represents a first important step towards a comprehensive understanding of the intrinsic properties of these RNA modifications that are critical for their proper application in atomic mutagenesis experiments of biologically relevant RNA.In this study, we synthesized and comprehensively analyzed the properties of 3-deazaguanine and 3-deazaadenine modified RNA. When these nucleobases are embedded in a double helical environment we found that they can decrease the stability of base pairing. Interestingly, the effects were much more pronounced for 3-deazapurines compared to the isomeric 7-deazapurines. Moreover, destabilization is more severe in short stems interfering with double helix nucleation, while a more modest decrease is observed if located in the center of an extended double helix. We furthermore found that the imino proton exchange for deazapurine-modified Watson Crick base pairs is significantly higher compared to their native counterparts and simultaneously induces enhanced opening of the very neighbor base pair. These perturbations in local structural dynamics provide a rationale for the observed reduced base pairing strength. Additionally, we solved the X-ray structure of a cAtomic coordinates and structure factors for the reported crystal structures have been deposited with the Protein Data bank under accession numbers 7L3R.gkab256_Supplemental_FileClick here for additional data file.


Persons with disabilities can have physical, mental, intellectual, or sensory impairments which can hinder their social participation. Despite Sustainable Development Goals call for “universal access to sexual and reproductive health (SRH)”, women with disabilities (WwDs) continue to experience barriers to access SRH services in Nepal. This study evaluated factors affecting the utilization of SRH services among WwDs in Ilam district, Nepal.A mixed-method study with 384 WwDs of reproductive age was conducted in Ilam district, eastern Nepal. Quantitative data were collected using a structured questionnaire. Relationships between utilization of SRH services and associated factors were explored using multivariate logistic regression analysis. Qualitative data were collected from focus groups with female community health volunteers and interviews with WwDs, health workers and local political leaders. They were audio-recorded, translated and transcribed into English and were thematically analyzed.Among 384 respondents , only 15% of them had ever utilized any SRH services. No requirement (57%) and unaware of SRH services (24%) were the major reasons for not utilizing SRH services. A majority (81%) of them reported that the nearest health facility was not disability-inclusive (73%), specifically referring to the inaccessible road (48%). Multivariate analysis showed that being married , having perceived need for SRH services  and perceived susceptibility to SRH related disease/condition  were positively associated with the utilization of SRH services. Qualitative findings revealed that illiteracy, poor socioeconomic status, and lack of information hindered the utilization of SRH services. WwDs faced socioeconomic , structural , and attitudinal  barriers to access SRH services.Utilization of SRH services among WwDs was very low in Ilam district, Nepal. The findings of this study warrant a need to promote awareness-raising programs to WwDs and their family members, sensitization programs to health service providers, and ensure the provision of disability-inclusive SRH services in all health facilities.The online version contains supplementary material available at 10.1186/s12913-021-07382-4. Persons with disabilities are “those who have long-term physical, mental, intellectual, or sensory impairments which, in interaction with various barriers, may hinder their full and effective participation in society on an equal basis with others” . AccordiSexual and Reproductive Health (SRH) is an essential component of health and one of the pillars for sustainable development. SRH services are necessary for all people including persons with disabilities, both married and unmarried. All too often, the SRH of persons with disabilities has been overlooked by the disability community as well as those working on SRH. Persons with disabilities have the same needs for SRH services as the rest of the general population. Persons with disabilities may have greater needs for SRH education and care than their counterparts due to existing barriers and their increased vulnerability to abuse. Research shows that persons with disabilities are as sexually active as persons without disabilities. Despite this, too often their sexuality has been ignored and their reproductive rights denied .Access to SRH services is essential for people with disabilities. Growing evidence in North America, Europe and Australia indicate that women with disabilities have poor pre-conception health status  ; and havDespite Sustainable Development Goals (SDGs) explicit call for ensuring “universal access to sexual and reproductive health and reproductive rights” and specific articles in the United Nations Convention on the Rights of Persons with Disabilities (CRPD) , the SRHNepal has a National Guideline for Disability Inclusive Health Services 2019  which guThere is very little data available on the SRH of women with disabilities in developing countries like Nepal. Though there are many surveys and studies on SRH of women in Nepal, the majority of them have not disaggregated their data by disability. A cross-sectional study conducted in six (out of 76) districts of Nepal showed that 76% of young persons with disabilities had knowledge of major components of SRH such as family planning, safe abortion and sexually transmitted infections (STIs). About 22% of the young persons with disabilities reported difficulty in communicating with the SRH service provider .The utilization of SRH services among women with disabilities and its associated factors were inadequately explored in Nepal. There is an urgent need to identify these barriers in Nepal’s health service context. Thus, this study was carried out with the main objective to assess the utilization of SRH services among women with disabilities and explore factors affecting it in Ilam district, Nepal.2 and 290,254 population. It consists of six rural municipalities and four urban municipalities. Ilam district was selected purposively for the study as the prevalence of disability is 2.63% in Ilam, which is higher than the national disability prevalence rate of 1.94 [The study was conducted in Ilam, a hilly district situated in province number one of eastern Nepal with an area of 1703 km of 1.94 , a non-gThe study employed a mixed-method study design, with a cross-sectional survey in the quantitative arm and semi-structured interviews and focus group discussions in the qualitative arm. The study was conducted between September 2018 and February 2020.The sample size was calculated using Epi Info version seven. Since the prevalence of SRH services utilization among women with disabilities was not available, it was considered to be 0.5. Furthermore, with a margin of error of 5 % at a 95% confidence level, the estimated sample for the quantitative study was 384.Women of reproductive age (15–49 years),Had disability ID card or in the process of issuing ID card, andFell under the disability category of the “Washington Group Short Set of the questionnaire” Do you have difficulty seeing, even if wearing glasses?Do you have difficulty hearing, even if using a hearing aid?Do you have difficulty walking or climbing steps?Do you have difficulty remembering or concentrating?Do you have difficulty (with self-care such as) washing all over or dressing?Using your usual (customary) language, do you have difficulty communicating, for example, understanding or being understood?The “Washington Group Short Set of the questionnaire”  was usedA complete list of women with disabilities  aged 15–No - no difficultyYes – some difficultyYes – a lot of difficultiesCannot do at allEach respondent was requested to respond on the following response scales:Only those respondents who replied “Yes, a lot of difficulties” or “Cannot do it at all” in at least one domain of the Washington Group Short Set questionnaire , were inDisability was grouped based on the disability ID card. For those without a disability ID cards, disability was grouped with the support of community-based rehabilitation facilitators. As per the Nepal disability rights act 2017 , disabilAmong 384 respondents, one-third 31%) of them had a physical disability, 7 % had disability-related to vision, 16% had disability-related to hearing, 7 % had disability-related to voice and speech, 12% had mental/psychosocial disability, 9 % had intellectual disability, and 18% had multiple disabilities. Among 318 disability ID cardholders, 14, 40, 25 and 21% of them had profound, severe, moderate and mild disabilities respectively  and local political leaders  were conducted. The samples for the qualitative study were selected purposively.For the quantitative study, a structured questionnaire was developed based on the study objectives and variables in the guidance of subject experts. The questionnaire included information related to sociodemographic and socioeconomic characteristics, type and severity of disability , women eFor the qualitative study, a codebook guide was prepared by adopting the Health Belief Model . InterviFace-to-face interviews of women with disabilities were conducted to collect quantitative data using a pretested structured questionnaire. Support from the caretaker was sought for those who were unable to respond (27%). A total of 10 public health undergraduates were trained as enumerators. Data collection was completed over 2 months starting from 23rd May to 21st July 2019.Qualitative data were collected from semi-structured interviews and focus groups to supplement the findings, facilitate triangulation as well as explore in detail the underlying elements that might have affected the service utilization from both the women with disabilities and the service providers’ perspectives. The interviews were taken by two teams each comprising of a lead female interviewer and a female note taker. They were public health undergraduates, trained in qualitative study and familiar with the local language and cultural context. All interviews and focus groups were conducted with the help of a codebook guide prepared by adopting the Health Belief Model .Two women with disabilities,Two health workers  providing basic health services including SRH services in the local government health facilityTwo elected chairpersons  of local governmentTwo focus groups with female community health volunteers (11 respondents in group one and eight in group two)The list of the participants for the semi-structured interview and focus groups is mentioned below:The respondents for the qualitative study were selected purposively. None of the respondents refused to participate and there were no dropouts. Focus groups with female community health volunteers were conducted in a separate hall of the health post. While the in-depth interviews of women with disabilities were conducted in a separate room of their house, interviews of health workers were conducted in a quiet room of the health post where they work. Likewise, the interviews of the local political leaders were conducted at their offices in a preferred/quiet room. Permissions were taken from the participants for the recordings. An audio recording was done with an audio recorder and field notes were taken during the interviews and focus groups. It took 30–45 min for the focus groups and 15–20 min for other interviews. Sampling for qualitative study was stopped once saturation level  was met Ethical approval was obtained from Nepal Health Research Council . ApprovaFor the quantitative study, data was analyzed using Statistical Package for Social Science (SPSS) software  version Utilization of SRH was the dependent variable and was defined as the use of any of the following SRH services: maternal and newborn care; contraceptive information and services; prevention and appropriate treatment of infertility; safe abortion and post-abortion care; combatting HIV/AIDS and other sexually transmitted diseases; prevention of gender-based violence, care for victims and information, education and counselling on sexual violence; actions to eliminate harmful traditional practices such as early and forced marriage; and comprehensive sexuality education and youth-friendly services.Independent variables of the study consisted of sociodemographic characteristics , socioeconomic characteristics , disability (type and severity of disability ), women Wealth index was measured as a composite indicator of economic status constructed by principal component analysis of more than fifty variables including household related variables such as main fuel for cooking, type of toilet facility, the main source of drinking water, possession of land, the main material of wall, roof and floor; some household assets such as radio, television, computer, mobile, landline phone, refrigerator, sofa, cupboard, fan, motorcycle, cow/bull/buffalo, horse/donkey/mule, goat, pig/wild boar and hen/duck. The wealth quintiles (from lowest to highest) are ranked into five equal categories, each comprising 20% of the study population.Involvement in household decision-makingThis indicator included three decisions: access to health care, household purchasing and freedom to visit relatives. The responses were coded into three categories. Those respondents who participated in all three decisions received a “two” score, those who participated in one or two decisions received a “one” score, and those who did not participate in any decisions received a “zero” score.Membership in community groupsThose respondents who were a member of any community group, such as a mothers’ group, saving group, women’s group and others, were scored as “one” and those who were not involved in any group, were scored as “zero”.Cash earningsThose respondents who earned cash only or both cash and in-kind were given a score of “one” and those who did not earn cash at all were given a score of “zero”.Ownership of house/landThose respondents, who owned a house, land, or both alone or jointly with their husband, received a score of “one” and those who did not own any house, land or both, received a score of “zero”.EducationIt was measured on an ordinal scale under the following categories: No education , primary level (up to grade five), some secondary level (from grade six to ten), and higher secondary and above.Those respondents who have attained secondary or higher education were scored “two”. Those respondents who have attained primary level education were scored “one” and those who did not attend school at all were scored “zero”.Women’s empowerment was assessed using the Women’s Empowerment Index (WEI) , which wThe total scores were ranged from zero to seven. Those respondents who received one to two scores in aggregate were grouped in the low empowerment level. Those respondents who scored three or four were categorized as moderately empowered and those who scored five to seven were categorized as highly empowered.Knowledge of SRH was assessed using composite indicators adapted from Measure Evaluation for Sexual and Reproductive Health Knowledge  comprisiThe total score attained by each respondent was converted into a percentage. The level of knowledge was determined based on the following scoring mentioned below Table .Table 3Sp < 0.05) at the 95% confidence interval (CI) in the bivariate analysis after checking collinearity.For the quantitative study, a cross-tabulation using the Chi-square test (or Fisher’s exact test) was carried out to check for associations between dependent and independent variables. Multivariate analysis was carried out for those variables, which were significant  of them could not read/write. More than half of them were Hindu (56%), never married (64%), belonged to a joint family (59%) and were unemployed (59%). The average household size was 5.1. About one in seven (15%) had ever utilized any SRH services.Age, type of family, family size, marital status, educational status of the study population and their caretaker were significantly associated with the utilization of SRH services. Women aged 35 and above , belonged to the joint family , had less than five family sizes , could read/write , had caretakers who could read/write , and ever married  were more likely to utilize SRH services compared to their counterparts ”Though socioeconomic status was not found to be significantly associated with the utilization of SRH services, the qualitative finding showed that poor socioeconomic status was one of the barriers to the utilization of SRH services among women with disabilities. Socioeconomic status was linked with education and access to information. Those women with disabilities who belonged to families with poor socioeconomic status were deprived of education and knowledge on SRH, which ultimately must have led to less utilization of SRH services.The utilization of SRH services among women with disabilities was low (15%). Only 12% had ever received maternal and newborn care, 11% had ever utilized contraceptive information and services, 0.3% had ever received prevention and appropriate treatment of infertility services, and 0.5% had ever utilized safe abortion and post-abortion care. None of them had ever utilized other SRH services related to HIV/AIDS and other sexually transmitted diseases; gender-based violence, elimination of harmful traditional practices such as early and forced marriage; and comprehensive sexuality education and youth-friendly services. No requirement (57%) and unaware of SRH services (24%) were the major reasons for not utilizing SRH services.Among 384 respondents, 121 (32%) had ever experienced pregnancy and childbirth. Among them, only half 51%) had received antenatal checkups, more than two-thirds (67%) had delivered their child at home mainly with support from family members (52%). Moreover, only 34% of them had received postnatal care  and mild disability  were three times more likely to utilize SRH services compared to their counterparts ”Family support is essential for all including persons with disabilities. The qualitative findings pointed out the need for assistance to women with disabilities but their family members were found to be often occupied in their household chores and livelihood activities. Lack of support from family members was reported as a barrier to the utilization of SRH services among women with disabilities.Among 384 respondents, about two-thirds (61%) did not participate at all in three key household decisions . Only half of them (52%) were members of the community groups. Less than a quarter (22%) earned cash or in-kind and only 12% owned any house or land either alone or jointly with someone else.About two-thirds (63%) of them were less empowered, a quarter (26%) were moderately empowered and only 11% were highly empowered.Those women with disabilities who participated in the household decisions , were a member of the community group , earned cash/in-kind , and were moderately/highly empowered  were more likely to utilize SRH services compared to their counterparts ”The qualitative findings showed consistency with the quantitative findings revealing the lack of empowerment as a barrier to access SRH services by women with disabilities. The study showed that women with disabilities were not empowered enough to express their SRH needs or problems. Moreover, they were also prone to violence.“Many women with disabilities are not getting any support from home and they are not aware of SRH. And (sigh) those women with disabilities, who are bedridden, are prone to sexual violence. There are some instances of rape and forced marriage among women with disabilities in our locality. ”The qualitative findings also highlighted the issues of sexual violence and forced control on reproduction among women with disabilities.“We found that some family members are providing Depo-Provera injection to their daughters with disabilities. The family members have to go outside for work. They feel that girls with disabilities are prone to sexual violence in their absence. So, they provide Depo-Provera injection to prevent them from being pregnant. ”The media exposure of women with disabilities seemed quite low. Those respondents who often listened to radio/FM were two times more likely to utilize SRH services compared to their counterparts  (Table A majority (72%) of respondents had heard about SRH. Radio/FM (40%) followed by the teacher (37%) were found to be the major source of SRH related information. More than two-thirds 69%) of them had very poor knowledge of SRH , had perceived the need for SRH services , perceived themselves susceptible to SRH related disease/condition , perceived SRH related disease/ condition as severe , and reported competent to utilize SRH services  were more likely to utilize SRH services compared to their counterparts ”Knowledge of SRH was found to be significantly associated with the utilization of SRH services. The qualitative findings also show that those women with disabilities who had knowledge of SRH and had access to information were aware of SRH. Those who knew about the availability of SRH services and their benefits utilized the SRH services. The study also highlighted the need to increase access to SRH information by intervening at the household level.“Some women with disabilities have access to information on SRH through media, female community health volunteers and their neighbours/friends. They are aware of SHR services and utilize them. While other women with disabilities stay at their homes. They lack access to information, education and communication. We need to make SRH information materials available in every home of women with disabilities and also intervention at the family level. ”The findings from the qualitative study supported the quantitative findings. Perceived severity and susceptibility contributed to the utilization of SRH services. Those women with disabilities who did not perceive themselves susceptible to SRH related disease/condition and who did not consider SRH related disease/condition as severe had less threat to SRH related disease/condition leading to lower utilization of SRH services.A majority (73%) of respondents reported that the nearest health facility was not disability-inclusive  especialAmong 384 respondents, less than half (47%) could access SRH services within 30 min. The average time taken to visit the nearest health facility for SRH services was 45 min. On average, the distance between home and the nearest health facility with SRH services was nine kilometres. The quantitative study does not show any significant association between the accessibility of health facilities and the utilization of SRH services among women with disabilities Table .Table 12Access refers to ensuring persons with disabilities can access health services and facilities on an equal basis with others in a way that promotes their dignity and independence. Structural barriers are obstacles in natural or man-made environments that prevent access or hinder persons with disabilities from moving around independently .“Persons with severe and profound disabilities are deprived of utilizing SRH services in remote areas as health facilities are not accessible for them and the health providers are unable to reach their home. ”Though the quantitative findings did not show the significant association between the accessibility of health facilities and utilization of SRH services among women with disabilities, the qualitative findings showed that structural barriers such as distant health facilities and lack of accessible infrastructure  hindered the utilization of SRH services among women with disabilities.One of the most significant barriers to effective participation and inclusion of persons with disabilities are negative attitudes and stereotypes. People often see persons with disabilities as incapable, dependent or weak. This perpetuates their segregation and exclusion from society .“The situation has been changed. Now, people treat us (women with disabilities) well. But (pause) some health workers still use inappropriate words and are rude. I feel uncomfortable expressing my concerns and query while visiting the health facility. ”Health service providers’ attitudes that women with disabilities should not be sexually active denies them access to sexual rights and services. In this study, local leaders, health workers, female community health volunteers and women with disabilities themselves perceived that many women with disabilities might be sexually active and need SRH services. However, the study showed the practice of providing information on SRH based on the marital status of women with disabilities that highlighted the need to sensitize health service providers on the SRH needs of women with disabilities.“During the home visits, I provide information about family planning to married women. How can I talk about family planning and abortion service to unmarried women? (chuckle). Unmarried women with disabilities are not interested and they do not need it. ”The qualitative finding showed that the behaviour towards women with disabilities had changed over time. But still, stigmatization and bad behaviour of health service providers persisted.“Women with disabilities may have the desire to get married. But if they get married and have children, then there will be a big problem. They are unable to provide adequate care to their children. As they have an impairment, how can they raise their children properly? ”Fifteen characteristics that exhibited significant association with utilization of SRH services at 95% CI in bivariate analysis were further subjected to multivariate analysis. There was no problem of collinearity among independent variables as the highest variance inflation factor was 2.009.Those women with disabilities who were ever married , perceived the need for SRH services  and perceived themselves susceptible to SRH related disease/ condition  were more likely to utilize SRH services compared to their counterparts Table .Table 13Health Belief Model is a social psychological health behaviour change model developed to explain and predict health-related behaviours, particularly regarding the uptake of health services . As HealThere were various factors responsible for the utilization of SRH services. The first factor was an individual perception, which was related to perceived susceptibility and perceived severity. Perceived susceptibility referred to subjective assessment of the risk of developing a health problem. The combination of perceived severity and perceived susceptibility was referred to as perceived threat . The stuThe modifying variables affect health-related behaviours indirectly by affecting perceived seriousness, susceptibility, benefits, and barriers . PerceivA cue, or trigger, is necessary for promoting engagement in health-promoting behaviours . The stuAccording to the Health Belief Model, the likelihood of action is the result of perceived benefits minus perceived barriers . The stuOur findings provide good evidence that utilization of SRH services among women with disabilities was very low in Ilam district, Nepal. Among 384 women with disabilities of reproductive age, only 15% had ever utilized any SRH services. The finding is similar to the finding from Cameroon , which sThe study showed that the educational status of women with disabilities  and their caretaker  were positively associated with utilization of SRH services. Education and health awareness programs were influencing factors while lack of family support and stigmatization were barriers to utilization of SRH services. The finding contrasts with the study from Cameroon, which showed that education level was not associated with the experience of difficulties in using SRH services . Anotherp = 0.3).The study showed a positive relationship between empowerment and the utilization of SRH services. Employed  and empowered  women with disabilities were more likely to utilize SRH services. However, the cross-sectional study from Cameroon  did not The study showed that women having physical disabilities  and mild disability  were more likely to utilize SRH services than their counterparts. The qualitative finding showed that women with physical disabilities could move independently using assistive devices such as crutches/wheelchairs. Those women having intellectual disabilities or severe disabilities needed someone to escort them to health facilities, which was not possible as family members themselves were busy with their household chores and livelihood related activities. The finding is similar to the study conducted in three countries , which also highlighted that people with disabilities often need to travel with an assistant to help them manoeuvre around obstacles they encounter on the way. This brought additional complications due to the difficulty of finding someone prepared to give up their time and be publicly seen .The study showed that women with disabilities who have knowledge of SRH  were more likely to utilize SRH services. Illiteracy and lack of information hindered the utilization of SRH services. The finding is similar to a qualitative study from Uganda  and a liThough Nepal has a national guideline for disability-inclusive health service  which guStudies from Uganda  and IndiThe findings of the study are also similar to a literature review of barriers to healthcare services for people with disabilities in developing countries , where iThis study highlighted the importance of individual perception for the utilization of SRH services among women with disabilities. Individual perception such as the perceived need for SRH services  and perceived susceptibility to SRH related disease/ condition  were found to be positively associated with utilization of SRH services among women with disabilities.Though SRH services should be provided irrespective of one’s marital status, the study showed that those ever-married women with disabilities were more likely to utilize SRH services compared to their counterparts . The qualitative findings also showed that health service providers hesitated to talk about SRH services to unmarried women with disabilities.These findings call for a need to promote awareness-raising programs to women with disabilities and their family members, sensitize health service providers on SRH needs of women with disabilities and ensure the provision of disability-inclusive SRH services in all health facilities.The study was conducted in one of the hilly districts of province number one of Nepal, so it does not represent the national scenario. Furthermore, because of its cross-sectional design, causality cannot be established. Although the sample size in this study was based on the standard calculation, a few sub-samples representing variables in the regression models had low numbers and may have generated a high standard error. Future studies with a large sample size for all sub-variables can make a robust statistical assessment. Moreover, current trends of SRH was not under the scope of the study. Likewise, the themes were pre-determined from the Health Belief Model for the qualitative arm of the study. However, after categorizing the data using the deductive approach, additional categories and themes were added, inductively, to incorporate the emerging themes from the transcripts.The utilization of SRH services among women with disabilities was very low (15%) in Ilam district, Nepal. Among 121 women with disabilities who had ever experienced pregnancy and childbirth, only half (51%) had received antenatal checkups, more than two-thirds (67%) had delivered their child at home mainly with support from family members (52%) and only 34% had received postnatal care. No requirement (57%) and being unaware of SRH services (24%) were the main reasons for not utilizing SRH services. A majority (81%) of them reported that the nearest health facility was not disability-inclusive (73%) specifically referring to the inaccessible road (48%). SRH services were mostly utilized by those women with disabilities who were married, who felt the need for SRH services and felt susceptible to SRH related disease/conditions. Qualitative findings revealed that illiteracy, poor socioeconomic status, and lack of information inhibited the utilization of SRH services. Furthermore, the study showed that women with disabilities faced socioeconomic barriers , structural barriers , and attitudinal barriers  to access SRH services. These findings call for a need to promote awareness-raising programs to women with disabilities and their family members, sensitize health service providers on SRH needs of women with disabilities, and ensure the provision of disability-inclusive SRH services in all health facilities.Additional file 1.


Long noncoding RNA transcripts (lncRNAs) are being revealed by complementary DNA cloning and genome tiling array studies in animals. Differently from structured RNAs , lncRNAs have been named regulatory RNAs , 2. WithLncRNAs are relatively long RNA sequences (more than 200 nucleotides) long that impact the cellular gene expressions and functions in ways that are just beginning to be explored. The metastasis of human cancers as well as acquired resistance against the administered therapeutics are two major factors responsible for the cancer-associated mortality. In a small number of cases lncRNAs are translated into short peptides, and in this case both RNAs and peptides may have a functional role. The big question is whether these transcripts are relevant. To this aim, there are today technologies available such as transient RNA silencing, gene overexpression, and high-throughput CRISPR/Cas genome editing methods that in principle could generate cell lines with all the possible combinations of silenced genes.In search of novel biomarkers as well as potential therapeutic targets of cancer metastasis and cancer drug resistance, the focus in recent years has turned to lncRNAs. What started with studies on relative expression of lncRNAs with the aim to define their utility as diagnostic biomarkers, has evolved into explorations of the possible functional role that these ncRNAs possibly play. One of the relatively more explored functions of lncRNAs is their ability to sponge and regulate the expression of microRNAs (miRNAs), the other ncRNAs that are much shorter in length. This indirectly opens the flood gates to endless possibilities of regulation of gene expression, as miRNAs represent a much better explored subtype of ncRNA. However, regulation through miRNAs is perhaps not the only mode of lncRNAs functionality, as being revealed through continued interest and evaluations.in vitro or in vivo models and RNA silencing techniques.The list of lncRNAs known to cancer researchers is ever expanding, as is the evidence linking their possible role as diagnostic as well as prognostic markers. Concerning lncRNAs, there are published reports on lncRNAs in almost all cancers that affect humans. There is evidence supporting the role of specific lncRNAs in metastasis in general as well as organ-specific metastasis of different cases. Also, lncRNAs seem to be involved in acquired resistance against a majority of chemical therapeutics as well as radiotherapy. This Research Topic collection includes research and review articles focusing on lncRNAs in cancer types, in metastasis and therapy resistance models. The 38 papers in this Research Topic describe lncRNAs deepening on mechanistic insights using lncRNAs in Cancer Metastasis and Therapy Resistance”, and wish that new research work will get insight from this collection to continue to produce data and information on the large set of human lncRNAs in cell physiology and in the pathology of cancers.We thank the editorial team at Frontiers for their support in the process of reviewing and publishing this Research Topic, “PP and AA wrote the manuscript. SU reviewed it. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The discovery of nucleic acids in blood has rapidly led into the development of methods for their isolation, characterisation and validation in the context of liquid biopsy. In this emerging field, miRNAs are promising biomarkers for the detection of solid tumours in the body fluids of cancer patients. This review gives an overview of the current methods of isolating circulating free and exosomal miRNAs, the advantages and disadvantages of the methods and the different techniques for miRNA quantification. At the end, the clinical relevance and market value of RNA-based liquid biopsy are explored and discussed with respect to the field of precision medicine.There is unequivocal acceptance of the variety of enormous potential liquid nucleic acid-based diagnostics seems to offer. However, the existing controversies and the increased awareness of RNA-based techniques in society during the current global COVID-19 pandemic have made the readiness of liquid nucleic acid-based diagnostics for routine use a matter of concern. In this regard—and in the context of oncology—our review presented and discussed the status quo of RNA-based liquid diagnostics. We summarized the technical background of the available assays and benchmarked their applicability against each other. Herein, we compared the technology readiness level in the clinical context, economic aspects, implementation as part of routine point-of-care testing as well as performance power. Since the preventive care market is the most promising application sector, we also investigated whether the developments predominantly occur in the context of early disease detection or surveillance of therapy success. In addition, we provided a careful view on the current biotechnology investment activities in this sector to indicate the most attractive strategies for future economic success. Taken together, our review shall serve as a current reference, at the interplay of technology, clinical use and economic potential, to guide the interested readers in this rapid developing sector of precision medicine. Liquid biopsy is a simple and minimal invasive procedure alternative to surgical biopsies. It has gained relevance first as a method of analysing circulating tumour cells (CTCs) contained in blood and has since expanded to include the analysis of the materials secreted by the cells, which include circulating free nucleic acids  as well as exosomes . The intLiquid biopsy therefore is a groundbreaking new alternative to the current decision making for targeted therapy in patients . In addiIn liquid biopsy, the most commonly investigated sources of biomarkers are CTC, circulating free nucleic acids and exosomes (cell-free) . For claCTCs were first described in 1869 and are defined as cancer cells released by primary and/or secondary (metastatic) places that can be identified in the circulating blood , therefoCirculating free DNA (cf-DNA) is formed of short fragments of around 160 base pairs. The portion that is originated from tumour cells is named circulating tumour DNA (ct-DNA) . BecauseCirculating free RNA are degraded and released RNA fragments to the bloodstream. Depending on the length, they can be divided into small RNAs (20–200 nucleotides) and long RNAs (>200 nt). Despite the fact that CTCs and ct-DNA have mainly led the research field of liquid biopsy, the focus has expanded to the analysis of cell-released RNA. Several cf-RNA types are highly abundant in the circulation, and since the majority of cf-DNA is released by necrotic or apoptotic cells, cf-RNA can give both information from apoptotic/necrotic tumour cells and from active tumour cells, such as exosome-mediated signalling, providing real-time information on the cancer . HoweverDue to their higher stability and abundance, there is nowadays a great increase in the research done in non-coding RNAs (ncRNAs), particularly in small RNAs that could serve as diagnostic and prognostic biomarkers for disease . HoweverIn the majority of human cancers, miRNA levels are altered, and its expression is tissue-specific. Consequently, they are promising biomarkers under study, and many cancer types can therefore be classified considering their miRNA profile . In 2019Extracellular vesicles are released from both eukaryotes and prokaryotes and are classified into three groups: exosomes, microvesicles and apoptotic bodies . Each grDue to heterogenous cargo and reflection of parent cells, exosomes are rapidly emerging as a powerful source of biomarkers for cancer diagnosis, prognosis and therapeutic response . ExosomeWhen analysing cf-RNA from blood, centrifugation steps are necessary to obtain the plasma or serum fraction from which the miRNAs are isolated. The main steps are described in different studies. Since cf-RNA is a source of multiple RNA types as described above, some, such as cf-miRNA, are more stable than others. Cf-miRNA interacts with other biomolecules or packaging membrane-coated or membrane-free particles which protect them from the RNases present in the circulation .Exosomes as a source of biomarkers must first be reliably and successfully isolated from the blood sample. Several factors should be addressed, including the patient’s age, gender, ethnicity, body mass index, disease, medication, and food intake. The more detailed information regarding factors influencing the exosome release and content is described in . AdditioThe majority of the most well-studied body fluids for both circulating free and exosome fractions are, beside blood, plasma and serum ,49,50, sThe review from Bryzgunova et al., 2021 outlines the variety of methods of cf-miRNA isolation from different body fluids . As in bRNAs in body fluids can be released from cells as free RNAs associated with proteins (mainly argonaute-2), but also with high-density lipoproteins (HDLs), or are present inside extracellular vesicles . AnalysiExtraction methods of cf-RNA vary depending on the manufacturer and can be based on the use of the TRIzol reagent, silica membranes  or magnetic beads  ,65. PuriTRIzol™ is a ready-to-use reagent designed to isolate high-quality total RNA. Different types of commercial kits, such as Trizol LS , nowadays use this phenol-based method for the isolation of cf-miRNA because of its main advantages: it is easy and fast to use and the whole procedure is low-cost. In addition, since one of its main disadvantages is the presence of ethanol-based contaminants, many protocols try to improve the isolation yield by combining the TRIzol reagent procedure with the use of columns for purification and miRNA enrichment .Silica matrices have revolutionized the procedures for RNA isolation both for research and commercial purposes. The affinity between negatively charged RNA and positively charged silica membranes (gel or glass) results in selective binding of the RNA while the rest of the components and chemicals of the sample are washed away . On the In 2015, Brunet-Vega et al. performed a study acknowledging the problem of cf-RNA isolation, particularly in the case of miRNAs . They anIn 2014, Hantzsch et al. performed a research study comparing different RNA isolation protocols regarding miRNA and mRNA quantity and quality recovered using PAXgene  or Tempus  tubes  . RNA yieIn a study conducted by Pezzi et al. in 2018, the potential impact of both DNA and RNA nucleic acid isolation based on the magnetic bead technology was evaluated. The two kits used were Dynabeads mRNA DIRECT  for magnetic bead-based isolation and RNeasy Mini Kit  for silica membrane isolation, detecting for both methods no significant loss compared to the control. Both techniques were effective in the isolation of RNA . There aFrom small non-coding RNAs as a valuable source of RNA biomarkers, circulating free miRNAs are evaluated as the most promising biomarkers. However, the majority of the isolation protocols nowadays exist for total cf-RNA isolation, while several recent protocols from companies such as Qiagen or Norgen Biotek include the option to specifically isolate small RNAs as cf-miRNAs, providing that total RNA isolation is without loss of miRNAs or that there is selective purification of miRNAs in the pool . Extractg for 10 min at RT conditions. Afterwards, miRNA was isolated from the plasma. Synthetic miRNA from Caenorhabditis elegans was added to all the samples as an exogenous spiked-in control for sample-to-sample variation normalization. Once the plasma was obtained, it was subjected to a second centrifugation at 12,000× g at 4 °C for 15 min to remove all cellular debris. The four protocols for cf-miRNA extraction were as follows: standard extraction protocol using TRIzol LS ; miRNeasy mini kit ; mirVana PARIS kit ; and miRNA purification kit . The researchers concluded that the TRIzol protocol gave the worst results in the percentage of miRNA recovery compared to the mirVANA PARIS kit. Among the commercially available kits evaluated in the study, the mirVANA PARIS kit featured the highest recovery yield and the most reproducible results.In the study conducted by Sourvinou et al. in 2013, they evaluated four different extraction protocols for circulating free miRNAs from blood . After cIn 2012, Li and Kowdley evaluated three different commercial miRNA isolation kits comparing performance and recovery yield miRNeasy kit , the miRVana PARIS kit  and the total RNA isolation kit  following the manufacturer’s protocols and improvements. The miRNA fraction from total RNA was present in all the three kits . The resA brief summary of the methods for isolating exosomes described below is presented in g [Ultracentrifugation is the most commonly employed method for exosome isolation and is used in 81% of all cases . It consg . Ultraceg ,45. The g ,81. Anotg .A variation of ultracentrifugation is density gradient ultracentrifugation, which separates vesicles based on their density (exosome density being 1.08–1.22 g/mL) ,79. AfteWith size-exclusion chromatography (SEC), vesicles are separated by their size, as pores capture smaller molecules and vesicles elute first . It is p® Exosome RNA Column Purification Kit  or Total Exosome RNA & Protein Isolation Kit  [Immunoaffinity-based methods exploit the presence of specific proteins on the surface of exosomes that can be captured with the corresponding antibody. Several commercial kits have been developed for this purpose, such as ExoMir (TM) Kit , ExoQuickMA, USA) ,84. The MA, USA) . CompareMA, USA) ,85. On tMA, USA) . The maiMA, USA) . MoreoveIn 2018, Stranska et al. compared the membrane affinity-based method  and SEC  for exosome isolation from human plasma samples . The resExosome precipitation is based on applying water-excluding polymers, such as polyethylene glycol (PEG). PEG attracts water molecules and forces less soluble components out of the sample. Usually, the mixture is incubated overnight at 4 °C and the exosomes are isolated with centrifugation or ultrafiltration . CurrentIn 2015, Lobb et al. compared ExoQuick , ExoSpin ; the method is based on the combination of precipitation and SEC) and SEC for plasma exosome isolation. The yield of exosomes was the lowest using SEC, but the purity of samples was the highest. Furthermore, the amount of albumin contaminants was considerably lower compared to ExoQuick and ExoSpin .In 2017, Helwa et al. compared ultracentrifugation and three different precipitation commercial kits for exosome isolation from serum: miRCURY RNA Isolation Kit , ExoQuick  and Total Exosome Isolation Reagent . The number of exosomes isolated by differential centrifugation was considerably lower  compared to commercial kits. All the commercial kits had a similar yield; only TEIR produced slightly higher yield compared to miRCURY. When RNA was isolated from exosomes, interestingly, there were no differences in the amounts of RNA between commercial kits and differential centrifugation .In 2019, Macías et al. compared six commercial kits for serum exosome isolation: ExoEasy  (membrane affinity-based method), ExoQuick , ExoSpin , ME (peptide binding method), ExoQuick Plus  (polymer precipitation followed by immunoaffinity capturing—negative selection) and Exo-Flow  (polymer precipitation followed by immunoaffinity capturing the anti-CD63 antibody). ExoQuick, ExoQuick Plus and ExoSpin had the highest yield while the yield of ME was around 400 times smaller compared to those three methods. In exosome samples isolated by Exo-Flow and ME, no apolipoprotein B was detected, and among the exosomes isolated with ExoQuick Plus, only one sample out of five was positive in apoB. On the other hand, exosomes isolated by ExoQuick only had large amount of contaminants .Exosomal miRNAs are attractive cancer biomarkers as they are protected from RNAse degradation, are stable for a longer period of time and resistant to freeze–thaw cycles . The disMicroRNA may be isolated from exosomes using different commercially available kits. Interestingly, using TRIzol, the most popular method for RNA isolation, might not be suitable for exosome isolation, as certain miRNAs, especially poor in the GC content, may be lost during the procedure . In 2014Prendergast analysed the exosomal RNA amount isolated from archival sera using different commercial kits and adapted protocols . ExosomeIn 2017, Tang et al. analysed RNA isolated from serum exosomes . ExosomeIn 2018, Buschmann et al. isolated exosomal RNA from patients’ serums using different methods for exosome and RNA isolation: (1) exoRNeasy , (2) SEC and miRCURY , (3) Exo-spin  and miRCURY RNA isolation kit , (4) miRCURY Exosome Isolation kit  and miRCURY Exosome RNA isolation kit , (5) Ultracentrifugation and exoRNeasy  . The resA brief summary of all the detection and quantification methods described below is presented in Quantification of miRNA after the initial isolation is a crucial step for all further downstream applications. The indicated assays demonstrating a high flexibility and adaptability to all downstream applications such as the ddPCR, NGS or qPCR or can be used as stand-alone detection method.® 200 PRO Nanoquant Spectrophotometer  or Nanodrop Spectrophotometers . One major disadvantage of the spectrometer-based methods is that they cannot discriminate between the different types of RNAs, therefore revealing false yield of miRNA. Specifically, below the concentration of 1 ng/µL Nanoquant and the Nanodrop Spectrophotometers revealed high variability in single measurements while other platforms remained in the reproducible range [Common methods to quantify RNA directly for further downstream applications are fluorescence-based detection using a Qubit Fluorometer (Technologies) or spectrophotometer-based technologies Bioanalyzer , Tecan Infinitele range . Similarle range .Unlike these techniques, Qubit  employs specific fluorescent dyes selective for small RNA or miRNA over other forms of RNA, and therefore the concentration values obtained with this platform should correspond mainly to the portion of small RNA . A broadNext-generation sequencing (NGS) describes a number of different modern sequencing technologies. All of these technologies allow the sequencing of short or long reads of DNA or RNA in a high-throughput manner. The advantages of these systems are that they are much faster, display a high accuracy and are usually cheaper when analysing a large amount of samples than the previously used methods.Over the past few decades, RNA sequencing has significantly progressed and mostly employs NGS technology. Using this technology, it is now possible to detect even miRNAs in individual cells by using single-cell RNA-seq ,123,124;Quantitative PCR  is based on the PCR cycle analysis allowing for quantification of the desired sample; qPCR-based analyses combine “traditional” endpoint detection PCR with fluorescent detection technologies based on different fluorescent dyes, such as SYBR Green or TaqMan of FRET probes, to gain specificity. The basic mechanism consists in the multiple amplification cycles in which cDNA (reverse-transcribed from the RNA sample) follows cycles of denaturation and renaturation followed by the extension of the complementary strand by a thermostable DNA polymerase, resulting in an exponential increase of the sample.The length of miRNA is usually around 21–23 base pairs, so it is challenging to design conventional PCR primers which are typically 20 base pairs long. In general, two different groups of reverse transcriptions are known, universal and specific reverse transcription. During the reverse transcription step, the length of miRNA is extended by adding the poly A tail to the 3’ end of miRNA with poly A polymerase for universal reverse transcription or using miRNA-specific stem loop primer for specific reverse transcription ,126. TheIn the specific reverse transcription method, linear primers, pincer probes and stem loop primers are used. The development of stem loop RT-qPCR techniques provides a novel method for accurate and sensitive detection of miRNA . Stem loProgress has been made over the last decade in the development of more sensitive PCR-based methods. Digital PCR and digital droplet PCR methods are highly sensitive and low-cost methods and the workflow is similar to TaqMan probe real-time PCR. They are able to target the amounts in samples that are below the limit of conventional qPCR methods . In ddPCThere is an increasing research effort on the ddPCR technology for assessing and quantifying the presence of various miRNA from different body fluids to get accurate absolute quantification of cf-miRNAs ,128.Microarray technology is a powerful high-throughput method where known high-density immobilized DNA probes are hybridized to detect target miRNA . Anyway,In a study conducted by Callari et al. in 2013, it was researched if microarrays can be accurately used for circulating miRNAs from plasma . The relLiquid biopsy of circulating biomarkers has the potential to be developed as a minimally invasive diagnostic methodology for precision medicine, and its potential values have received increasing attention over the last decades ,132,133.In addition, liquid biopsy techniques can overlap the existing diagnostic methodologies  but can also substitute some, like invasive tissue biopsy. High-risk population groups can benefit from liquid biopsy as it can be used as a method for early cancer detection. This way, patients with predisposition for cancer can be tested for assessing their risk of disease development. In addition, patients can also be pharmacologically evaluated in order to assign the most efficient treatment regimens . It alsoFrom an economic point of view, early and accurate cancer diagnosis can significantly reduce costs of cancer care. For instance, in 2017, the average cost for solid biopsy for lung cancer was $8869, while the total cost for a patient reached $37,745 because of 20% complications from needle biopsies ,143. MorRNA-based technologies for prognostic, diagnostic and predictive therapies will be developing in the following years. A report published by Allied Market Research showed that by 2020, around 1.2 billion dollars had been spent on RNA diagnostics, and 6.8 billion dollars will be invested by 2028. The main targets would be diseases such as cancer, AIDS and different genetic disorders .An indicator of the attractiveness of this field is acquisition of RNA-based companies by pharmaceutic giants, for example, of Santaris Pharma by Roche (2014) and of SiRNA Therapeutics by Merck (2007), followed by the acquisition of this division by Alnylam Pharmaceuticals (2014) .The FDA-approved DNA-based liquid biopsy tests highly outnumber the RNA-based liquid biopsy assays. The assay that entered clinical practice detects the BCR–ABL1 fusion gene and was developed by several manufacturers . The tesCancer is the primary driver of the miRNA research field. This is supported by the data that today miRNA studies in cancer present 50% of the total field . miRNAs This steady growth, estimated to be 18 to 20% within the next five years, is an indirect result of the increasing number of prevalence of chronic diseases including cancer. Moreover, the competition among companies to develop such tests will likely speed up the demand for their services in clinical settings. The biggest market is USA (45.8%), while Europe is the second largest holding 29.2% of global miRNA market share. Still, the miRNA market is growing fastest in Asia Pacific with a compound annual growth rate of 6% . However®/ThyGENX®) for Thyroid and pancreatic cancer risk classifier, Rosetta Genomics/Precision Therapeutics (miRview mets) and Genoptix  are companies for detecting the origin of cancer and TAmiRNA (OsteomiR and ThrombomiR) detecting markers for osteoporosis or cardiovascular diseases, respectively. Furthermore, several other companies reaching pre-clinical and phase one testing. DiamiR is reaching the market using miRNA panel analysis for Alzheimer and brain disease diagnostics [Smaller already established companies working and active promoting miRNA-based diagnostics for various disease types including test detecting single specific miRNAs are Interpace Diagnostics , as well as robust workflows for probe extraction, quantification  and validation for molecular analysis of liquid biopsy samples must be guaranteed prior to market release ,163,164.For clinical approaches, the most important criteria for RNA, particularly miRNA, as diagnostic and prognostic biomarker is high sensitivity and specificity to avoid false positive or negative diagnostic results. Furthermore, the appropriate biomarker for a specific cancer type needs to be both significantly differentially expressed and in correlation with the outcome of patients. It has to be considered that miRNAs in some cancer types are up regulated but the same miRNA can be downregulated in patients suffering from another cancer type ,166. BesThe last point to be considered for the transfer from basic research into routine laboratory diagnostics are costs. At the moment, liquid biopsy is too expensive when summarizing the costs for equipment, reagents, and the salary for professionals such as technical personnel, biochemists, bioinformatics specialists, nurses and physicians. Taken this factor into account, the current costs for liquid biopsies are much higher than those for comparable conventional biopsies.In the latest decade, the potential role of RNA as cancer biomarker has revolutionized the development of clinic diagnostics. It has especially shown advantages in non-invasive methodology, high precision and sensitivity, detecting disease at early stage, therapy follow-up, real-time monitoring and learning of the heterogenicity of the tumour profile. The detection of circulating free RNA, particularly miRNAs in liquid biopsies can be used as prognostic, diagnostic and predictive biomarkers. Moreover, it could help to assist clinicians to make decisions on patients with cancer types difficult to diagnose. In spite of these important advantages, the main challenges nowadays in cf-RNA field include the low concentration and stability of some cf-RNAs. However, miRNAs are intrinsically more stable for clinical applications. Isolating miRNAs and also other RNAs from exosomes may be more valuable resource. However, currently there is no universally accepted gold standard method for cf-miRNA or exosome isolation. A difficult impediment in both analysis is namely co-isolation of contaminants, especially lipoproteins, which contain RNAs themselves and can interfere with further analysis.Liquid biopsy will unlikely substitute current established diagnosing methods completely but can be very useful for early diagnosis or in cases where tissue is inaccessible or unavailable. Although they have the potential to be applied for personalized medicine, the commercialization process of liquid biopsy methods (based on the detection of ctRNA or exosomes) requires numerous time-consuming laboratory studies and rigorous clinical trials. The current gold standard method for miRNA quantification is qPCR. Novel methods as biosensors (known from blood glucose measurements or pregnancy tests) suppose a rapid, low-cost and easy-to-handle point of care testing for cf-RNA diagnostics. Once the protocols for circulating biomarkers are optimized, all validation and safety tests are passed, and approvals are obtained, they can have a significant impact on reducing the diagnostic costs. At the moment, the implementation of cf-RNA diagnostics in the clinic is rapidly increasing worldwide and billions of dollars are being invested in the field, and the amount is expected to rise in the following years.


Appropriate gene delivery systems are essential for successful gene therapy in clinical medicine. Lipid-mediated nucleic acid delivery is an alternative to viral vector-mediated gene delivery and has the following advantages. Lipid-mediated delivery of DNA or mRNA is usually more rapid than viral-mediated delivery, offers a larger payload, and has a nearly zero risk of incorporation. Lipid-mediated delivery of DNA or RNA is therefore preferable to viral DNA delivery in those clinical applications that do not require long-term expression for chronic conditions. Delivery of RNA may be preferable to non-viral DNA delivery in some clinical applications, since transit across the nuclear membrane is not necessary, and onset of expression with RNA is therefore even faster than with DNA, although both are faster than most viral vectors. Delivery of RNA to target organ(s) has previously been challenging due to RNA’s rapid degradation in biological systems, but cationic lipids complexed with RNA, as well as lipid nanoparticles (LNPs), have allowed for delivery and expression of the complexed RNA both in vitro and in vivo. This review will focus on the non-viral lipid-mediated delivery of RNAs, including mRNA, siRNA, shRNA, and microRNA, to the central nervous system (CNS), an organ with at least two unique challenges. The CNS contains a large number of slowly dividing or non-dividing cell types and is protected by the blood brain barrier (BBB). In non-dividing cells, RNA-lipid complexes demonstrated increased transfection efficiency relative to DNA transfection. The efficiency, timing of the onset, and duration of expression after transfection may determine which nucleic acid is best for which proposed therapy. Expression can be seen as soon as 1 h after RNA delivery, but duration of expression has been limited to 5–7 h. In contrast, transfection with a DNA lipoplex demonstrates protein expression within 5 h and lasts as long as several weeks after transfection. Gene therapy has the potential to significantly advance clinical medicine, but the risks and duration of gene delivery should be closely matched to the proposed clinical application . Long-teCationic lipid- or polymer-mediated gene transfer is particularly suited for transient gene expression, both in basic research and in selected clinical applications. Cationic lipids are commonly comprised of a polar headgroup and non-polar symmetric or dissymmetric carbon-based tail and may also include nuclear localization signals (NLS), antibodies, polymers, or other targeting moieties. Negatively charged nucleic acids condense and self-assemble into heterogeneous complexes when mixed with cationic lipids . The strBarriers to lipid or polymer-mediated nucleic acid transfection include: (1) transport of the nucleic acid/lipid complex through the extracellular environment to target tissue(s); (2) association and uptake of the nucleic acid/lipid complex by the target cell ; and (3)Currently, more than 70% of clinical trials investigating nucleic acids as therapy use some form of viral vector, including retroviruses, lentiviruses, adenoviruses, and adeno-associated viruses . These vThe potential hazards of viral vectors have led many investigators to consider the use of non-viral vectors for delivery of therapeutic nucleic acids, including for treatment of genetic diseases. Patisiran, a non-viral vector in the form of a lipid nanoparticle carrying an siRNA targeted to inhibit transthyretin protein for the treatment of hereditary transthyretin amyloidosis, recently received FDA approval . PatientCationic lipids and polymers have different advantages. Cationic polymers tend to complex with nucleic acids into smaller and more uniform nanoparticles, enhancing transfection efficiency. Their high cationic charge also favors efficient endosomal uptake, but this charge also carries some cytotoxicity . In addiBoth DNA and RNA, recognized by the immune system as foreign genetic material, are rapidly degraded in biologic systems. Both nucleic acids therefore present challenges when considered as therapeutics and encapsulating into a viral or non-viral vector is critical. The use of DNA (mostly as cDNA) has been seen as preferable for therapy, requiring long-term expression, such as replacement therapy for genetic diseases. Incorporation of DNA into the genome using non-viral vector delivery remains a theoretical if extremely remote possibility, and even this rare chance is avoided with mRNAs, which are transcribed in the cytoplasm and do not need to reach the nucleus. This is not true of some small RNAs that target gene promoters or for CRISPR applications. Delivery of DNA has the additional barriers of transport through the cytoplasm, avoiding degradative pathways and transit across the nuclear membrane. Episomal DNA residing in the cytoplasm is likely not permanent, for a variety of reasons ,41. EpisAlthough there are numerous successes in vitro and in animal models using both DNA and RNA, RNA has the benefit of being more efficiently transfected into non-dividing cells . One of Cationic lipids have been used to protect nucleic acids and increase the efficiency in the process of transfection for decades. Felgner et al defined three functional properties needed for transfection using cationic lipids: spontaneous capture of the nucleic acid by the cationic lipid to form lipoplexes, increased cellular uptake, due to interaction of the positively charged complexes with biological surfaces, and fusion with the plasmalemma or endosome . The lipLipid nanoparticles are another means to carry nucleic acid into cells/tissues/organisms and have been used in the pharmaceutical industry for the delivery of poorly soluble drugs. Their safety is well known for dermal, ocular, and oral administration, but the use of surfactants for stabilization may induce inflammatory reactions in other routes of administration . InvestiCentral nervous system diseases, such as Parkinson’s disease, Alzheimer’s disease, and brain tumors, have been resistant to traditional therapies, at least in part due to the nearly impenetrable blood brain barrier (BBB). Gene therapy represents a new avenue for treating these diseases and a great deal of research has been focused on such approaches. Several studies have shown therapeutic benefits of cationic lipid: siRNA or LNP:siRNA transfection in the reduction of glioblastoma cells either in vitro or when tumor cells have been seeded subcutaneously in mice (ex vivo), but the BBB is not a factor in these studies +++ . DeliverIntracranial injection of nucleic acids complexes was one of the first approaches to bypassing the BBB. Intracortical injection in mice of pVEGF-GFP complexed with cationic lipid showed angiogenesis in the regions surrounding the injection/transfection sites . This apTransfection of the CNS with nucleic acids in lipoplexes can also be achieved by delivery of the lipoplex at a time when the BBB is disrupted. Intranasal delivery of a cationic lipid: GFP-mRNA was successful  in demonAnother avenue for crossing the BBB is to use the endogenous transport mechanisms within the BBB. For small molecule delivery, there are carrier-mediated transporters (CMT). These transporters take vitamins and other nutrients into the brain. CMT systems include, for example, GLUT1, LAT1 , and CAT1 (cationic amino acid transporter). LAT1 is the transport system that brings L-DOPA, gabapentin, and AAV9 into the brain . Other mThe BBB can be bypassed by direct injection. Thakker et al. used intrathecal injection of an siRNA to demonstrate widespread inhibition of protein expression . In thisIn our laboratory, we have demonstrated repeatedly in a variety of species and over many years the rapid and widespread distribution and expression of a variety of reporter and neuroprotective gene sequences by intrathecal injection of the nucleic acid complex. Anderson et al. demonstrated the stability of cationic lipid:mRNA complexes in both human and rat cerebral spinal fluid (CSF) . The catDetection of light by IVIS imaging from luciferin breakdown in the transfected rats required optimization of several experimental parameters. Gene therapy has become an active research avenue for many diseases of the CNS. Ongoing research programs, using model systems for AD, PD, MS, stroke, and brain tumors, are actively searching for cures and treatments of these and other CNS diseases. Once candidate treatments are found, delivery must contend with the BBB and transfection into a largely nondividing cell population. Both viral and non-viral vectors have been used to deliver gene therapy to the CNS, but non-viral vectors have several advantages, including lower toxicity, lower immunogenicity, and the ability to package a large nucleic acid load. Non-viral vectors include physical transfection methods, but lipid-based non-viral vectors, such as cationic lipid, nucleic acid complexes, and lipid nanoparticles encasing nucleic acid, have greater potential for whole organ transfection or even full systemic transfection and have been a greater focus of research efforts. Decades ago, cationic lipids were first complexed with nucleic acids to protect them and also to neutralize the strongly negative charge of these macromolecules. Since that time, chemical manipulation of cationic lipids, as well as their incorporation as part of LNPs, have increased transfection efficiency and decreased toxicity and immunogenicity. More recently, there has been an increased interest in the use of RNA for genetic therapy to the CNS. This is at least in part due to the fact that transfection of nondividing cells with RNA is more efficient than transfection with DNA. While RNA-based gene therapy is shorter lived than DNA therapy, we have shown effects lasting longer than might be expected. Although there have been only a few examples of FDA approved RNA drugs, Patisiran is an siRNA treatment for a genetic disease that is given intravenously once every 3 weeks, a reasonable treatment regimen.Intrathecal injection is the means we have used to successfully transit the BBB and transfect the CNS with single CSF injections. We have shown that transfection with both pDNA and mRNA is possible, efficient, and can be followed by a time course using a reporter gene and in vivo imaging. There has been tremendous progress in overcoming the road blocks to gene therapy for the CNS, and a focus on RNA-based therapies, as well as delivery methods that cross the BBB, will likely increase the speed of this progress in the years to come.


PLOS Biology finds that interferon (IFN)-induced adenosine deaminase acting on RNA 1 (ADAR1) mRNA is N6-methyladenosine (m6A) modified to promote its translation, enabling ADAR1 to modify self-double-stranded RNAs (dsRNAs) generated during the IFN response and preventing activation of the melanoma differentiation–associated protein 5 (MDA5)-mediated host antiviral response.A new study in  This Primer explores a new study in PLOS Biology which finds that interferon-induced ADAR1 mRNA is m6A-modified to promote its translation, enabling ADAR1 to modify self-dsRNAs generated during the interferon response; this prevents recognition of these self RNAs and erroneous activation of the MDA5-mediated host antiviral response. The discrimination between self- and foreign-derived nucleic acids, such as from viruses, is critical to allow for induction of type I interferon (IFN) while preventing autoinflammation. IFN is induced when cytosolic RNA pattern recognition receptors (PRRs) retinoic acid–inducible gene I (RIG-I) and melanoma differentiation–associated protein 5 (MDA5) sense specific patterns in RNA, which are commonly found in viruses, as nonself or foreign. The RNA patterns that mark mRNA as foreign include lack of a 7-methylguanosine cap, absence of 2′O-methylation on the first nucleotide, or double-stranded RNA (dsRNA) structures. As such, proper mRNA capping and conversion of dsRNA to single-stranded RNA (ssRNA) are required for self-mRNA to escape immune detection. The cellular RNA deaminase adenosine deaminase acting on RNA 1 (ADAR1) catalyzes the conversion of adenosine to inosine (A-to-I) in dsRNA structures, resulting in ssRNA that is largely protected from immune sensing .6A) has emerged as another RNA pattern that marks RNA as self. m6A can shield mRNA from sensing by RIG-I, a mechanism that has been co-opted by RNA viruses in order to escape immune surveillance [6A on viral RNA is not limited to immune evasion, and it can have both positive and negative impacts on viral infection due to either direct effects on viral RNA or indirect effects on cellular mRNAs that encode factors important for the immune response [6A on IFNB1, which encodes the cytokine IFN-β, decreases its mRNA half-life, and m6A on antiviral mRNAs can influence their expression by increasing translation or decreasing stability. Thus, modifications to RNA, including m6A and A-to-I editing, can play important and diverse roles in viral infection and the host innate immune response. Despite this, the full interplay of how these and other RNA modifications orchestrate antiviral signaling remains unclear.Recently, the RNA modification N6-methyladenosine , which promotes translation of m6A-containing mRNAs, binds both isoforms of ADAR1 to varying degrees, with strong enrichment seen for the IFN-induced p150 isoform and less enrichment for the basally expressed p110 isoform (6A and A-to-I editing, the expression of the IFN-induced ADAR1 p150 protein was significantly impaired following the depletion of either the m6A writer enzymes METTL3 or METTL14 or the m6A-binding protein YTHDF1. This indicates that m6A deposition and its recognition by YTHDF1 are important for the translation of the ADAR1 p150 isoform. Consequently, this resulted in a global reduction of A-to-I editing events of cellular mRNA, leading to increased levels of cellular dsRNA and subsequent recognition by the dsRNA sensor MDA5 to induce IFN. These results support the work of others, indicating that MDA5 is activated by self-RNA upon ADAR1 deficiency [6A-binding protein YTHDF1 enhances ADAR1 p150 expression in glioblastoma cells to facilitate A-to-I editing of self-RNA and prevent activation of dsRNA sensors.In a new study published in  isoform . Contrarficiency ,7,8. Thu6A and its regulatory proteins could have important roles in preventing autoinflammation. Interestingly, in the cell types tested, this regulation was unique to glioblastoma cells as other cell types did not induce IFN in response to YTHDF1 depletion, suggesting that this might be a mechanism shared in human cells of glial origin. As other studies of YTHDF1 in RNA virus infection have not found that loss of its expression results in broad induction of IFN [This study by Terajima and colleagues broadens our understanding of how RNA modifications can directly control gene expression and indirectly regulate innate immune signaling, all of which could contribute to autoimmune disorders, leaving the field with much to consider for future study. For example, the depletion of YTHDF1 resulted in increased dsRNA and IFN induction, suggesting that mn of IFN , there aInterestingly, the source of dsRNA produced in response to IFN treatment of glioblastoma cells is also unknown. IFN does not globally alter the frequency of Alu elements, a common source of dsRNA ,8, altho