Communication on sexual and reproductive health (SRH) between caregivers and their young adolescent children plays a significant role in shaping attitudes and behaviours that are critical to laying the foundations for positive and safe SRH behaviours in later adolescence. Nevertheless, this communication is often limited, particularly in countries where adolescent sexuality is taboo. This study assessed the topics discussed (‘level’) and the comfort of caregivers with communicating with young adolescents on SRH, and their correlates. P-value < 0.05 was considered for statistical significance.A cross-sectional survey was conducted among 218 caregivers of young adolescents (10–14 years) in Mbarara district of south-western Uganda in January and February 2020. Participants were selected through consecutive sampling. A structured, pre-tested questionnaire administered by interviewers was used for data collection. The surveys were computer-assisted using Kobo Collect software. Data was exported to STATA 14 for analysis. Level of SRH communication was measured based on 10 SRH communication topics, while comfort was based on 9 SRH discussion topics. Bivariate and multivariate linear regression analyses were conducted to determine correlates of level of, and comfort with, SRH communication The mean number of topics that caregivers discussed was 3.9 (SD = 2.7) out of the 10 SRH topics explored. None of the respondents discussed all the topics; 2% reported ever discussing nine topics with their young adolescent, while 3.5% reported never discussing any of the topics. General health and bodily hygiene (89.9%) and HIV/AIDS and other sexually transmitted infections (STIs) (77.5%) were the most commonly discussed, while night emissions in boys (4.3%) and condoms (8.3%) were least discussed. The majority of caregivers (62%) reported a high level of comfort with discussing SRH. The mean comfort score was 21.9 (SD = 3.8). In general, the level of SRH communication increased with an increase in comfort with SRH communication β = 0.22 (0.04); 95% CI = . The level of comfort with SRH communication decreased with an increase in the number of YAs in a household β = -0.92 (0.38); 95%CI = .Overall, the level of SRH communication is low and varies according to the number of SRH topics. Caregivers’ comfort with SRH communication with YAs was a significant correlate of SRH communication. This justifies the need for interventions that aim to improve caregivers’ comfort with communicating with young adolescents about SRH.The online version contains supplementary material available at 10.1186/s12889-022-14561-3. Communication between parents and children about sexual and reproductive health (SRH) during the early adolescence stage (10–14 years) plays a significant role in creating positive reproductive health outcomes in later adolescence and adulthood. We conducted surveys in rural areas of Mbarara district among 218 pairs of caregivers and their children aged 10–14 years between January and February 2020. The purpose of the study was to understand whether there is communication between the two, and how comfortable they felt having discussions about SRH. We also asked caregivers and their children about their knowledge of SRH, and how they generally felt about SRH for young people. This study, however, reports only results from caregivers.We found that, on average, caregivers discussed 4 of the 10 SRH topics explored in this survey with their child, and that the majority of the caregivers reported being very comfortable discussing SRH—especially general health and bodily hygiene—with their children. On the whole, we found that caregivers’ SRH communication was largely influenced by their comfort with discussing SRH with their adolescent child. The level of comfort was influenced by the number of young adolescents living in a household. However, there is no clear justification for this finding from existing literature. In conclusion, there is a need for interventions that facilitate improvement of communication on SRH between parents/caregivers and young adolescents. These should focus on improving comfort levels by promoting communication skills for caregivers and emphasizing value clarification.The online version contains supplementary material available at 10.1186/s12889-022-14561-3. The sexual and reproductive health (SRH) of young adolescents (YAs: 10–14 years) is an emerging public health priority in developing countries. YAs comprise about half of the 1.2 billion adolescents aged 10–19 years globally [Previous research indicates that puberty accelerates risk-taking among YAs , 4. At tYAs in developing countries are disproportionately affected by SRH challenges, including coerced or forced sex, early marriage and gender-based violence . These oCaregivers play a significant role in socializing and shaping the attitudes of YAs at an early age that are critical to laying the foundations for positive and safe SRH behaviours. This is through practices such as gender socialization and communication about sexuality in general , 11. BluThere is considerable research into communication between caregivers and children about SRH in sub-Saharan Africa that emphasizes the existence of such communication despite traditional perspectives , 17. ThiCurrent research specifically into caregiver–child communication on SRH emphasizes older adolescents and barely addresses YAs . MoreoveA cross-sectional household survey was conducted in January and February 2020 among caregiver–YA dyads in six villages in Rwebishekye parish, Rwanyamahembe sub-county, Kashari county in rural Mbarara district of south-western Uganda. The study community comprised approximately 1,520 households, of which 29% headed by women, and an estimated population of 6,061 people . The comThe study sampling frame comprised all households in the study community with YAs (10–14 years) and their caregivers. A community household profiling exercise was conducted at the start of the study and established an estimate of 300 households comprising YAs. The final sample comprised 218 caregiver–YA dyads . The sample size was calculated for the effectiveness study for an intervention to improve SRH communication between caregivers and YAs. It allows a moderate change (effect size 0.2) to be measured in good caregiver–adolescent communication between pre- and post-intervention measurements with a power of 0.8 and alpha of 0.05. This required a total sample size of 277 respondents. Accounting for design effect (× 1.3) and drop-out between waves (× 0.2), the required sample size amounted to 432 participants or 216 dyads.We used consecutive sampling, based on whether a household contained a YA and whether both caregiver and YA were present simultaneously at the time of the survey. For households comprising more than one YA, we considered the oldest. Caregivers were either biological or non-biological. Within the sample, caregivers included biological parents, step-parents, foster parents or relatives, including older siblings entrusted with the greatest responsibility for the daily care and rearing of the child. Eligibility for caregivers included being 18 years or older, consenting to participate in the study, living in the community for the past six months and living with a YA in their household for whom they were the caregiver for the past six months.The survey was conducted by trained research assistants who were fluent in English and native speakers of Runyankore-Rukiga. Data were collected using a structured, pre-tested questionnaire administered by an interviewer. The surveys were computer-assisted using Kobo Collect software. The interviews with the caregiver and YA were conducted simultaneously but separately in convenient locations to avoid overhearing and to ensure open and truthful responses. The time for completion of the survey varied from one hour to one hour and 15 min. The survey team was coordinated by two team leaders and community leaders, who assisted in identifying the preselected households. There were also three monitors to check the data for consistency and completeness.Given the participatory and sensitive nature of this research project, the survey questionnaire was reviewed by a multidisciplinary team of researchers and the Community Advisory Board (CAB) in December 2019. The CAB comprised community representatives, including caregivers, young people, teachers, community leaders and influential members of the community, as well as religious leaders from the four majority faiths: Catholic, Anglican, Muslim and Pentecostal. The CAB also included representatives of different government entities, including the Ministry of Health, the Ministry of Gender, Labour and Social Development, and the Ministry of Education and Sports. These stakeholders reviewed the data collection tools and provided feedback on the pertinence and clarity of the survey questions.Caregiver–YA SRH communication was explored using 10 SRH-related topics indicated in Table Caregiver comfort with SRH communication was explored using nine SRH topics. Caregivers were asked how comfortable they were discussing any of the SRH topics with their YA children. The topics are indicated in Additional file Information on socio-demographic variables of caregivers, including age, sex, marital status and religious affiliation, was obtained . The queThis was measured using variables from the Uganda Bureau of Statistics socio-economic survey . ParametConnectedness was measured using three subscales. The parent involvement subscale comprised 10 items, and the positive parenting scale comprised 6 items. Both scales were drawn from the Alabama Parenting Questionnaire, whose target audience is caregivers of children aged 6–18 years . This quAttitudes were measured using an eight-item scale on a five-point Likert scale. Caregivers were presented with statements such as ‘You approve of your child having a boyfriend or girlfriend.’ The scores were strongly agree, moderately agree, neither agree nor disagree, moderately disagree and strongly disagree. The scale was scored based on the highest and the lowest scores, with a high score indicating a positive attitude and a low score indicating a negative attitude. The scores were reversed to allow a high score to be indicated as a positive attitude. The summated composite score for attitude was calculated with a minimum score of 8 and maximum score of 24. The scores were classified based on Bloom’s criteria [This was measured through 27 items to assess knowledge on three main sub-topics: puberty (7 questions), HIV/AIDS (13 questions) and pregnancy prevention (7 questions). A summary score was computed, with the highest score indicating a high level of knowledge and the lowest score indicating a low level of knowledge.. The summated composite score for knowledge was calculated with a minimum score of 49 and maximum score of 81. The scores were classified based on Bloom’s criteria . These wData analysis was performed using STATA 14 . Descriptive statistics were used to describe numbers and percentages for the dependent and independent variables. The prevalence of discussion for each of the 10 SRH topics was presented by dyad type. Fischer’s Exact Tests were used to test for the level of significance of the difference in SRH communication across the dyad type for each of the 10 SRH topics (a 5% level of significance was set). The mean score for the number of topics discussed across the dyads was presented. Bivariate analysis was performed between the dependent variables (level of SRH communication and caregivers’ comfort with SRH discussions) and independent variables. The dependent variables were treated as linear variables . We conducted hierarchical linear regression analyses to examine the relationship between the dependent variables (level of SRH communication and caregivers’ comfort with SRH discussions) and the independent variables . Separate linear regression models for number of SRH topics discussed and caregivers’ comfort with SRH discussions were run using a manual backward stepwise selection method. Multi-collinearity was tested using variance inflation factors; none of the variables were affected. Results from the bivariate and multivariate linear regression model for predictors of caregiver and YA communication and comfort with SRH communication are reported in Tables A total of 218 caregivers were enrolled in the study, of which 76% were women. The mean age of the caregivers was 44.9 years (SD = 12.61). Seventy-three per cent were biological caregivers, and the majority (68.3%) had attained primary education, while 11.9% had not received any formal education. The caregiver–YA dyads comprised 96 with female caregiver and daughter (44.0%), 68 male caregiver and daughter (31.2%), 30 female caregiver and son (13.8%) and 24 male caregiver and son 24 (11%). The majority (76.6%) of the households were two-caregiver households, with an average of two YAs  , followed by HIV/AIDS and other STIs (77.5%). In contrast, only 4.3% of the dyads discussed night emissions in boys. There was no significant difference in communication of SRH topics across the dyads, except for HIV/AIDS and other STIs, which were more likely to be discussed in dyads with female caregivers (5) Table .Table 2Dp > 0.05). General health and bodily hygiene was the most comfortable topic, followed by HIV/AIDS and other STIs. Having babies, birth control and night emissions in boys were the least comfortable topics  reported a high level of comfort with SRH discussions with YAs, 31.7% were moderately comfortable, and 4.9% reported a low level of comfort. There was a higher level of comfort among female caregivers (63%) than among male caregivers, but the difference was not statistically significant (p = 0.05). The median score for attitude was 11 out of a maximum of 12.The majority of the caregivers (84.8%) had a negative attitude towards SRH issues of YAs, while only about 0.7% of the caregivers had a positive attitude, and 14.5% had a moderate attitude. Female caregivers (85%) reported a significantly higher negative attitude towards SRH issues of YAs compared to male caregivers  of the caregivers reported a moderate level of SRH knowledge. Female caregivers had greater knowledge than male caregivers, though this was not statistically significant.Sixteen per cent of the caregivers reported a high level of connectedness with YAs, while 34% reported a low level of connectedness. Around half (49%) reported a medium level of connectedness. Female caregivers reported a higher level of connectedness than male caregivers. Connectedness was measured through three subscales: caregiver involvement, positive parenting, and parental expertise and accessibility. A third (33%) of the caregivers reported a high level of involvement, with the majority of these being female. Over a third (39%) of the caregivers reported a high level of positive parenting, while 32% reported a high level of parental expertise and accessibility.p < 0.001) positive linear relationship between comfort with SRH discussions and level of SRH communication. A unit of increase in comfort with SRH discussions increases SRH communication by 0.25 units (SE = 0.04). On the other hand, the level of SRH communication reduced with an increase in the number of YAs in a household .Bivariate linear regression was carried out to investigate the relationship between socio-demographic characteristics of the caregivers, comfort with SRH communication, attitudes towards SRH, level of connectedness and knowledge of SRH with SRH communication Table . The anaR2 = 0.23, F  = 7.61; p < 0.001. It was found that the level of comfort with SRH communication significantly predicted the level of SRH communication (0.22 (0.04); p > 0.001).In the multivariate linear regression analysis, we ran three models using the manual backward stepwise approach to identify the variables significantly predicting SRH communication. We considered variables that were statistically significant in the bivariate analysis (level of comfort with SRH communication and number of YAs in a household), those with a borderline p-value (caregiving structure) and those that indicate biological plausibility (based on previous findings on predictors of caregiver–child communication on SRH) Table . These iThe bivariate analysis for level of comfort with SRH communication indicated that the number of YAs in a household  significantly predicted comfort with SRH communication, although it had a negative correlation Table . The morR2 = 0.09, F  = 1.84; p = 0.06). However, it was found that the number of YAs in a household significantly predicted the level of SRH communication (p < 0.05).We ran three models using the manual backward stepwise approach. In the final model, we considered the variables that were statistically significant in the bivariate analysis and also included those that were biologically plausible, as well as borderline p-value (religion and sex) Table . The oveThis study sought to assess the current level of communication between caregivers and YAs about SRH, and caregivers’ comfort with such discussions, and identify their correlates. The study was conducted in a rural community in south-western Uganda, where an intervention to improve communication between caregivers and YAs on SRH would be tested. Unlike many studies which focus on SRH communication with older adolescents, this study focuses on YAs aged 10–14 years. This approach is driven by the notion that young adolescence is a stage of transition from childhood to adulthood where critical changes occur, especially in terms of sexual development . AddressOverall, our findings indicate that communication about SRH does take place between caregivers and their YAs. However, this was relatively rare and varied according to the topics discussed. On average, 21.6% of caregivers in the study reported ever discussing an average of 3.9 of the 10 SRH topics listed in the questionnaire. This finding falls in tandem with several other studies in similar settings—for example, a study conducted in Korogocho settlement in western Kenya indicated that communication between caregivers and very young adolescents was rare . SimilarA considerable number of caregivers reported discussing general health and bodily hygiene, and HIV/AIDS and other STIs. Indeed, on the comfort scale, caregivers reported high levels of comfort discussing HIV/AIDS and other STIs, as well as general health and bodily hygiene. Notwithstanding is the major finding of this study that the number of SRH topics discussed increased with an increasing level of comfort with SRH discussions. The probable reason for high reports of discussions on general health and bodily hygiene is that these topics can be discussed with minimal embarrassment. As far as HIV/AIDS is concerned, excess messaging around HIV/AIDS in the media, coupled with the high risk perception of HIV infection in many communities, may have triggered a lot of discussion around this topic. Topics deemed to be sensitive—such as night emissions in boys, condoms, birth control and sexual conduct—were discussed the least. Low or moderate levels of knowledge and a high proportion of caregivers reporting a negative attitude towards SRH in our findings could account for the low level of discussion of these latter topics. Additionally, evidence also shows that parents associate discussions with adolescents about condoms and birth control with being comparable to encouraging them to engage in sexual intercourse .The relationship between the SRH topics most and least commonly discussed and their perceived sensitivity strongly justifies the finding that the level of SRH communication increases with increasing level of comfort. This interrelates with the notion that open discussions about sexual issues are a taboo in many African settings, and the fact that many caregivers believed that it was too early to begin initiating discussions about sex . These fAlthough there was no significant difference in SRH communication across the dyad types for each of the SRH topics except for HIV/AIDS and other STIs, mother–daughter dyads were reported to have the highest mean number of topics discussed, while mother–son dyads were reported to have the lowest mean number of topics. Many studies implicate the influence of gender on caregiver–child communication, with mothers communicating more than fathers, and girls receiving more communication than boys. Girls are disproportionately vulnerable to SRH risks than boys, and mothers spend more time with children than fathers . MoreoveWe found that the level of comfort with SRH communication reduced with an increase in the number of YAs in a household. It is possible that caregivers may find it uncomfortable having SRH discussions with more children than it would be if they were fewer. Previous studies have particularly investigated the effect of family size on the level of SRH communication. In this study, the number of YAs in a given household could serve as an indicator for family size. Studies in Bangladesh and Ethiopia indicate that the bigger the family size, the lower the level of SRH communication , 32. AnoThe results of this study suggest that SRH communication between caregivers and YAs was low. SRH communication was also found to increase with increase in comfort with SRH communication. We also found that the more the YAs in a household, the lower the level of SRH communication. Comfort of SRH communication was found to reduce with an increase in number of YAs in a household. These findings provide a basis for interventions to improve communication between caregivers and children. First, training on value clarification and communication skills that enable caregivers to discuss SRH topics with less embarrassment and create a predisposition towards a positive attitude towards YA SRH is important. Topics focusing on general parenting skills—particularly the quality of their relationships—with the assumption that it inculcates positive caregiver–child relationships, would facilitate and increase the level of comfort with SRH discussions. There is a need for qualitative studies to gain a deeper understanding of determinants of comfort with discussing SRH with YAs. Limitations of our study include a relatively small sample size, which affects the power of the study. This affects comparison of SRH communication by gender, yet evidence highlights its important influence on SRH communication , 21. TheAdditional file 1:Appendix A. Level of comfort of SRH communication. Appendix B. SRH communication by number of topics. Appendix C. Caregiver attitudes towards SRH of Adolescents. Appendix D. Comfort of SRH Discussions by dyad type. Appendix E. Distribution of independent variables by sex of the caregiver.


Macromolecular phase separation underlies the regulated formation and dissolution of biomolecular condensates. What is unclear is how condensates of distinct and shared macromolecular compositions form and coexist within cellular milieus. Here, we use theory and computation to establish thermodynamic criteria that must be satisfied to achieve compositionally distinct condensates. We applied these criteria to an archetypal ribonucleoprotein condensate and discovered that demixing into distinct protein-RNA condensates cannot be the result of purely thermodynamic considerations. Instead, demixed, compositionally distinct condensates arise due to asynchronies in timescales that emerge from differences in long-lived protein-RNA and RNA-RNA crosslinks. This type of dynamical control is also found to be active in live cells whereby asynchronous production of molecules is required for realizing demixed protein-RNA condensates. We find that interactions that exert dynamical control provide a versatile and generalizable way to influence the compositions of coexisting condensates in live cells. A key challenge is understanding how condensates with shared and distinct macromolecular compositions can coexist as distinct functional entities6 . We used computations to ascertain the types of interactions that must be encoded to ensure that a protein can spontaneously form distinct condensates with two different types of RNA molecules. We performed lattice-based coarse-grained simulations13 of a model system in which two high valency linear polymers (designated as RNA1 and RNA2) interact with the same low valency linear polymer (a Whi3 mimic) with varying types of interactions among RNA1, RNA2, and the Whi3 mimic. Here, valency refers to the numbers of cohesive motifs (stickers) that can engage in complementary homotypic or heterotypic interactions. When the effective interactions of RNA1 and RNA2 with the Whi3 mimic are the same in strength and purely heterotypic in nature, then two coexisting phases form, with the dense phase encompassing all three macromolecules . Other interaction modes can also engender the demixing phenotype. These include repulsions between different RNAs , which we deem unlikely to be relevant since electrostatic repulsions cannot be selectively imposed among different RNA molecules. Strongly asymmetric interactions featuring heterotypic interactions of very different strengths between the RNAs and Whi3 mimics can also enable demixing .The formation of a single condensate versus sets of distinct coexisting condensates can be predicted by mean field theories where the relevant parameters are the variance of intermolecular interaction strengths and the numbers of components in multicomponent systemsolecules . In conti3 mimic . These din vitro measurements to assess the contributions of homotypic versus heterotypic interactions and determine if there are significant asymmetries in heterotypic interactions.Taken together, the simulations show that assessments of relative contributions of homotypic versus heterotypic interactions are critical for extracting the relevant thermodynamic driving forces of the phase behaviors of Whi3-RNA mixtures. Accordingly, we set out to use 12. 16. In contrast, when homotypic interactions of the RNA mimic are equivalent to heterotypic interactions with the Whi3 mimic, the RNA mimic can phase separate on its own leading to a flattening of the dilute arm of the phase boundary with decreased concentration of the Whi3 mimic . If the phase boundary is symmetrical about the diagonal, as expected in a system driven by heterotypic interactions, then a single ellipse is an optimal locus of points along the phase boundary. The addition of homotypic interactions degrades the ability to symmetrize about the diagonal leading to a poorer fit of a single ellipse  of Whi3-binding stickers on each of the RNA molecules the titratable free parameter. We vary this parameter and quantify the extent to which measured phase boundaries can be symmetrized about the diagonal .To assess if phase boundaries can be symmetrized about the diagonal, the phase boundaries were remapped in terms of sticker concentrations. Stickers can enable homotypic and heterotypic interactionsR2 values of 0.88–0.92 supporting strong fit to a single ellipse  in which the cognate Whi3-binding sites were disrupted by scrambling the consensus Whi3-binding motif. The measured phase boundary for the Whi3-BNI1mut system overlaps with that of the Whi3-BNI1 system at high Whi3 and low RNA concentrations and vice versa 20. We find that the measured phase boundaries generally show less than a 4-fold difference between Whi3-CLN3 and Whi3-BNI1 binary mixtures . This suggests that asymmetries in interaction strengths across the binary mixtures cannot account for the demixing phenotype observed in cells.Our results suggest that heterotypic interactions between Whi3 and RNA molecules are the main drivers of phase separation that give rise to Whi3-RNA condensates in binary mixtures. Heterotypic interactions involve Whi3 and its cognate RNA-binding sites, as well as additional non-cognate sites of unknown sequence specificity. Therefore, the demixing phenotypes observed in cells are unlikely to be due to the dominance of homotypic interactions over heterotypic ones. Further, for demixing to be thermodynamically driven in systems where the phase behavior is dominated by heterotypic interactions, the driving forces for phase separation between the different binary mixtures must be significantly different  and different RNA molecules (5 nM) were chosen because these are inside the two-phase regimes in binary mixtures . In contrast, in the delayed mode, we observed significant diminution of colocalization . Similar results were observed for binary mimics of ternary mixtures involving Whi3 and BNI1 as well as Whi3 and BNI1mut .We also quantified the extents of colocalization achieved in ternary mixtures of Whi3, yed mode ,h,i. ThiFigure S11a-d). The fraction of mobile proteins, which corresponds to the maximum recovery, is highest in Whi3-BNI1mut condensates, implying that the presence of cognate-binding sites decreases Whi3 mobility. In contrast, RNA molecules are essentially immobile showing undetectable recovery of fluorescence after photobleaching in all the condensates formed by binary mixtures .Next, we probed the internal molecular dynamics of Whi3 and different RNA molecules in condensates formed by four different binary mixtures. For this, we measured fluorescence recovery after photobleaching (FRAP) of the labeled components in condensates formed by binary mixtures of Whi3 and RNA molecules in aqueous buffers. In all mixtures, photobleached Whi3 shows partial recovery of its fluorescence, suggesting the presence of mobile and immobile fractions . This suggests that CLN3 has limited ability to be recruited to preexisting condensates based on RNA-RNA interactions. Additionally, while BNI1 and BNI1mut have a similar number of complementary sites, BNI1mut shows a lesser extent of demixing – a result that highlights the contribution of cognate Whi3-binding sites to dynamical demixing.FRAP measurements show that Whi3 has slightly more enhanced mobility in the presence of BNI1 and SPA2, and the presence of complementary RNA-RNA interaction sites in these molecules.The overall picture that emerges suggests a push versus pull between two distinct contributions that lead to dynamically controlled demixing of Whi3-RNA condensates. The push toward demixing appears to be caused by long-lived crosslinks between Whi3 and cognate sites on RNA molecules. This competes with a pull away from demixing that is driven by the entropy of mixing that arises from the combinatorics of interactions due to the high valence of non-cognate Whi3-binding sites in CLN3 and Whi3-BNI1 condensates in live cells of A. gossypii is likely to be under dynamical control as predicted from the in vitro assays. For this, we engineered an A. gossypii CLN3+/BNI1+ strain co-expressing the two transcripts under a common promoter. Co-expression mimics the simultaneous mode that we deployed in vitro and should enhance colocalization of the two transcripts if the timing of expression influences the extent of co-localization. A bidirectional promoter derived from the A. gossypii H2A/B histone locus was chosen to ensure robust co-expression in a wild-type background.Finally, we asked if the demixing of Whi3-7, wildtype expression showed a lack of colocalization between CLN3 and BNI1 . We also observed a spatial component to the patterns of demixing versus colocalization. Colocalized transcripts are more prominent near the nuclei, which is where the transcripts are synthesized . This suggests that the interplay between spontaneous mixing and dynamically controlled demixing will favor demixed condensates away from the nucleus, whereby longer-lasting RNAs located farther from their production sites are more likely to condense asynchronously with Whi3 thus giving rise to dynamically controlled demixed condensates.Consistent with previous resultsand BNI1 , b. In cficantly ,b. PixelCLN3 and BNI1 in condensates with Whi3 can be realized by synchronizing their expression in vivo. This mimics the simultaneous mode of adding the three components in vitro. However, in wild-type cells, the expression levels, the locations, and the timings are all different. Therefore, we propose that these dynamical factors rather than purely thermodynamic driving forces, are the primary determinants of demixing of Whi3-BNI1 and Whi3-CLN3 condensates in A. gossypii  versus the making and breaking of interactions (the contribute to elasticity) can engender phase separation on different length- and timescales. The large cytoplasm of the filamentous fungus A. gossyppi, and quite likely other mammalian cells such as axons43, are likely candidates to leverage dynamical control to enable the formation of coexisting, demixed ribonucleoprotein condensates comprising the same protein and different RNA molecules.Our findings bolster theoretical predictions


Nucleic acid nanoparticles (NANPs) are an exciting and innovative technology in the context of both basic and biomedical research. Made of DNA, RNA, or their chemical analogs, NANPs are programmed for carrying out specific functions within human cells. NANPs are at the forefront of preventing, detecting, and treating disease. Their nucleic acid composition lends them biocompatibility that provides their cargo with enhanced opportunity for coordinated delivery. Of course, the NANP system of targeting specific cells and tissues is not without its disadvantages. Accumulation of NANPs outside of the target tissue and the potential for off-target effects of NANP-mediated cargo delivery present challenges to research and medical professionals and these challenges must be effectively addressed to provide safe treatment to patients. Importantly, development of NANPs with regulated biological activities and immunorecognition becomes a promising route for developing versatile nucleic acid therapeutics. In a basic research context, NANPs can assist investigators in fine-tuning the structure-function relationship of final formulations and in this review, we explore the practical applications of NANPs in laboratory and clinical settings and discuss how we can use established nucleic acid research techniques to design effective NANPs. Nucleic Acid Nanoparticles (NANPs) are a subtype of therapeutic nucleic acids (TNAs) exclusively composed of specialized oligonucleotides designed to carry out defined architectures and functions such as delivery of therapeutic agents, biosensing, and immunostimulation . NANPs cNANPs are often used as a method of getting nucleic acids past “barriers” that exist in the body. Typically, carrier-free, naked, exogenous nucleic acids introduced without any chemical modifications would meet one of two fates within the body: rapid nuclease-mediated degradation and/or renal clearance. The engineered larger NANPs or NANPs mixed with the delivery agents assemble in such a way that the cargo they are delivering is protected from degradative nuclease activity which would otherwise stop the NANPs from achieving their therapeutic purposes. Additionally, the architectural parameters  of NANPs can be engineered in a way that is non-immunogenic and allows the payload to be shuttled to its intended target without triggering any immunological responses . Once thThere is more than one way to design NANPs and the method used to design a particular NANP depends on the material delivered as well as its action upon delivery ,10,11. TIn terms of NANP design, aptamers can be added to NANPs to enhance the specificity of NANP binding. This binding of NANPs can be to whole cells (on the cell surfaces), as well as proteins, and in some special cases, viruses . NANPs pIn the previous section, it was stated that NANP design is influenced by the cargo that is delivered as well as the intended action. The same dependence on cargo and action must be considered when selecting the nucleic acid for a NANP. Both DNA and RNA NANPs have been developed for therapeutic and research applications, but the two have several distinct qualities that make them unique from one another. In general, DNA is typically regarded as the more stable of the nucleic acids, however, that does not mean that RNA is the “lesser” nucleic acid. In fact, RNA has some properties that make it favorable for use in NANPs. A clear distinction between DNA and RNA nanoparticles is the base pairing capabilities of each nucleic acid. DNA is confined to Watson–Crick base pairing, meaning that A binds with T and C binds with G. While RNA often does form Watson–Crick base pairs, it also participates in non-canonical base pairing, such as G:U, G:A or C:A type as found in RNA structural folds . These nFor a NANP to carry out its intended function, it must be recognized by a receptor on the exterior of a target cell and endocytosed into the cell. Upon endocytosis, the NANP is inside of an endosome that functions to degrade molecules and reuse their components ,25,26. TIn summary, RNA nanoparticles are more stable thermodynamically and to pH ranges. Additionally, they have greater structural flexibility. When considered together, these features make RNAs preferred over DNAs as NANPs. Small interfering RNA (or siRNA) is a short, non-coding, regulatory RNA . Like otNANPs are particularly useful in the delivery of siRNA due to their stability . TypicalIn addition to the delivery of therapeutics to treat disease, NANPs can be used in disease prevention. RNA or DNA NANPs can be designed to deliver genetic information from pathogens in vaccines to prime the immune system for a natural exposure . This isRNAi is the phenomenon of dsRNAs knocking down or silencing the expression of endogenous mRNAs initially described by Andrew Fire and Craig Mello nearly 25 years ago ,37. RNAiCurrently, RNAi is a subject of much research and development for clinical applications . DespiteLike with all new technologies, RNAi does not come without drawbacks. Naturally, we must consider immunogenicity. RNAi therapeutics work at their best when they are delivered to the appropriate tissue without degradation. When the RNAi drug triggers an immune response, the drug may never reach its intended target, or if it does, it could be in a less effective state . In addiTo continue to develop the applications of RNAi therapeutics, researchers have gotten creative with how they have approached drawbacks. Researchers have incorporated base modifications into the RNA delivery system to get around the problem of immunogenic activation caused by RNAi therapeutics ,37,49. ITo mitigate off-target RNAi activity, researchers propose some common-sense measures to enhance safety for RNAi therapeutic recipients. The first proposed measure occurs long before the RNAi therapeutic makes it to the patient-it is extensive quality control to ensure that the drug has as few targets in the human genome as possible ,50. In tIt becomes more challenging when on-target effects in off-target tissues are considered. The drug is doing what it is supposed to do, just in a suboptimal location, which can imperil the patient’s health. Much like mitigating off-target effects in off-target tissue, researchers must design intentional, highly specific therapeutics to ensure that there are as few possible routes for the therapeutic to build up in a non-target location . In addiThe use of NANPs in humans as well as other animals carries the potential for immune activation. The immunogenic response must be thoroughly evaluated by the investigators to ensure that it is not activated in an unintended manner . Researchers do have some (though not complete) control in how immunostimulation proceeds through the design of their NANPs. A 2017 research article by Guo and colleagues examined the impact of NANP sequence as well as physical properties (size and shape) on their immunostimulatory effects . In thisThe use of adjuvants offers a robust method where immunostimulatory compounds are employed to potentiate and modulate the immune response, when used together with vaccines. Among the recent advancements of the immunomodulatory NANPs, is their ability to regulate and modulate the immune responses when encountered. The rapid clinical development of this technology is impeded by several hurdles among which is the unknown immune stimulation by NANPs. However, recent studies have reported that mammalian cells use the same patterns of recognition established to defend themselves against viral and bacterial nucleic acids to process NANPs . FurtherHuman cells have receptors for the recognition of foreign nucleic acids called pattern recognition receptors (PRRs), which can also distinguish and process NANPs. Therefore, understanding the underlying mechanisms of recognition and the NANPs structural parameters that affect their recognition process would allow the tunability of the immunostimulatory effects. This, in turn, will allow engineering the immunoquiescent NANPs intended for drug delivery, or NANPs with regulated immunological properties that could be used in immunotherapies . In the Extensive studies have been carried out to assess the immunostimulation of the representative library of NANPs introduced to freshly collected human peripheral blood mononuclear cells (PBMCs) ,59,60,61Another hurdle precluding broader clinical application of NANPs is their intracellular delivery ,66,67. OAs each set of NANPs holds a unique physicochemical and architectural profile, this creates a burden to predict the type of immune response and its magnitude. To overcome this challenge, a computational predictive tool called “artificial immune cell”, or AI-cell, was developed to guide the design of NANPs to fit the desired immunological profiles. This unprecedented computational approach is fed by physicochemical and immunological profiles for an array of various NANPs and uses innovative transformer architectures to predict the immunological activity of NANPs based on the entered oligo and their sequence compositions . This frIn 1999, P.B. Moore defined an RNA motif as a “discrete sequence or combination of base juxtapositions found in naturally occurring RNAs in unexpectedly high abundance” . Moore’sIn a 2002 review of protein domains, Ponting and Russell provided three different perspectives from which protein domains could be defined: biochemical, structural, and sequential . StructuPonting and Russell did provide a more modern definition of a domain as a structure that could adopt the necessary structural conformation for carrying out its function . While tSince the late 1980s, RNA motif research has been rapidly growing. In a 1998 review article, Conn and Draper claim that there are only a few functional RNA motifs, but when these motifs are placed together in combination, the functions that can be carried out by the structured RNA and the specificity with which these functions can be executed is enormous . NanoparRNA tectonics (or tectoRNAs) utilize naturally occurring RNA motifs to form hierarchically folded modular functional RNAs which can be used to construct RNA nanostructures ,79 and iOver the last nearly three years, the public has become increasingly aware of the use of RNA in medicine through the SARS-CoV2 vaccine. Two of the three mainstream COVID-19 vaccines contain the mRNA transcript encoding the spike protein, which facilitates viral entry into the cell . The mRNTrypanosoma brucei, the causative agent of African sleeping sickness. Telomerase RNA is a long noncoding RNA that is an integral functional subunit of a large RNA-protein complex responsible for synthesizing the G-rich strand of telomeres, the physical ends of linear chromosomes. Telomerase is critical for telomere length maintenance, thus preventing chromosome instability in eukaryotic cells .,96.Trypaa brucei  [96]. ThMore recently, a new application of selective 2′-hydroxyl acylation analyzed by primer extension emerged as juxtaposed merged pairs protocol (SHAPE-JuMP) . SHAPE-JA crucial advantage of using SHAPE-MaP techniques to predict the secondary structure of RNA is the ability to use it inside living cells in addition to outside of the cells and on deproteinized “naked” RNA . In theiSHAPE-JuMP builds upon the concept of SHAP-MaP. Its creators credit it with being better adapted at handling long range, RNA tertiary interactions that can be more prone to errors in traditional SHAPE-MaP protocols . An addiWhile quite powerful, SHAPE-MaP is not perfect. SHAPE-MaP software is unable to differentiate between multiple different isoforms of the same transcript. Different isoforms of RNAs can be predicted by several different RNA processing events which are the results of complex RNA interactions. These interactions have impacts on the RNA’s ability to perform its intended function, whether that be translation into a functional protein or a regulatory role. In addition to the inability to differentiate between structural isoforms of an RNA transcript, SHAPE-MaP requires chemical probes that can cross the plasma membrane of the cells being studied . There aThe applications of RNA structural modeling using SHAPE techniques are not limited to RNA in living systems. NANPs themselves take on discrete structures that are key to the effective delivery of their cargo to the appropriate location. The RNA-SHAPE techniques to produce structural models of RNA can be applied after NANP production in a quality control step to ensure that the nanoparticles accurately formed the intended structure. Additionally, SHAPE structural modeling techniques can be employed to evaluate how the structures of NANPs change when the cargo is delivered. Furthermore, we can use RNA-SHAPE to determine if the delivery process makes any changes to the structure of the nucleic acid cargo that may impact its ability to perform its intended function(s). These techniques can assist investigators in evaluating the structural stability and integrity of their delivery systems as well as cargo loads to enhance the efficacy of delivery and incorporation of the functional cargo.Nanotechnology has been a crucial area of research over the last decade. Nanoparticles are a growing area of academic and commercial investment, and the exploration of nucleic acid nanoparticles has opened up a wide array of research and therapeutic avenues. In the clinic, NANPs offer medical providers with potential preventative, diagnostic and therapeutic applications for patient care. Of course, as is the case with all novel technologies, there is room for nucleic acid nanoparticles to improve. Problems surrounding immunogenicity, off target activity, and unintended buildup all persist, and careful experimentation will lead researchers to optimizing specific NANPs for their unique purposes.Outside of the clinic in a basic research capacity, NANPs are of great academic value. Investigators can use NANPs to deliver nucleic acids to knock down expression of genes to clarify the purpose of the gene. Additionally, in the arena of RNA domains, we propose the use of NANPs to deliver RNA structural domains to cells that have been depleted of the same structural domains. In this planned research, one could examine the impact of RNA structural domain loss followed by complementation of the cells with the missing domain to clarify the purpose of discrete RNA structural domains. Once optimized, this system will allow us to gain a better understanding of how RNA structure influences its function.As research with nucleic acid nanoparticles progresses, investigators will need to address the challenges and disadvantages that come with working with them. Challenges such as unintended immune neutralization, NANP accumulation, and off-target effects are most common. The most powerful tool researchers have in mitigating these challenges is careful design of the NANPs so that they are non-immunogenic prior to target site delivery, do not build up in inappropriate locations, and do not act on inappropriate tissue. This can be done through robust screening of candidate NANPs to ensure that their specificity is as narrow as possible . When designing NANPs for use in the clinic, specificity is absolutely crucial for proper delivery and appropriate immune activation. Additionally, investigators must consider the stability of the NANP they wish to design. While the NANP should be stable enough to travel to the intended target tissue, once there, it must be able to deliver its cargo and then be broken down and cleared to prevent accumulation of “spent” NANPs. The structure of the NANP is a key element in its activity and special attention must be paid to how the nanostructures will react upon arrival at the target site. Advances in NANP design technology  will undoubtedly assist researchers in creating NANPs that have highly specific sequences to limit target possibilities and the proper three-dimensional structures to act on a specific target, then undergo degradation and clearance ,20,99.


Yet, the influence of chirality on the behavior of oligonucleotides in living systems, and specifically, the extent to which l-oligonucleotides interact with endogenous biomacromolecules and the resulting consequences remain unknown. In this study, we characterized the intracellular behavior of l-oligonucleotides for the first time, revealing important chirality-dependent effects on oligonucleotide cytotoxicity. We show that exogenously delivered l-oligonucleotides have the potential to be highly cytotoxic, which is dependent on backbone chemistry, sequence, and structure. Notably, for the sequences tested, we found that single-stranded G-rich l-RNAs are more cytotoxic than their d-DNA/RNA counterparts, exhibiting low nanomolar EC50 values. Importantly, RNA-seq analysis of differentially expressed genes suggests that G-rich l-RNAs stimulate an innate immune response and pro-inflammatory cytokine production. These data not only challenge the general perception that mirror image l-oligonucleotides are nontoxic and nonimmunogenic, but also reveal previously unrecognized therapeutic opportunities. Moreover, by establishing sequence/structure toxicity relationships, this work will guide how future l-oligonucleotide-based biotechnologies are designed and applied.Due to their intrinsic nuclease resistance, mirror image  l-oligonucleotides have the potential to be highly cytotoxic.We determined the influence of chirality on the behavior of oligonucleotides inside human cells, revealing that mirror-image  As such, they have enantiomers, referred to as mirror image l-DNA and l-RNA, which consist of l-(deoxy)ribose sugars  and prevents it from interacting with its intended targets.19 If l-oligonucleotides are to be routinely employed in living cells and organisms, then it is imperative that we understand how they interact with these environments and their potential consequences. Such studies may also reveal whether chirality as a design parameter can be harnessed to induce and/or modulate a desired biological response, opening the door to new applications. In this study, we conducted the first detailed characterization of how l-oligonucleotides interact with biological systems at the cellular level, revealing important chirality-dependent behaviors of l-oligonucleotides. Notably, we show that G-rich l-oligonucleotides have potent cytotoxicity and a dramatic impact on gene expression, suggesting they make extensive interactions with endogenous proteins.Given the stereospecific nature of biomolecular interactions, the behavior of l-RNAs, but not other sequences, bound tightly to PRC2 in vitro, suggesting that G-rich sequences have a propensity for interacting with native proteins. Therefore, we chose the model G-rich sequence (GGAA)8 as a starting point for our studies. We prepared both d and l stereoisomers of (GGAA)8 DNA and RNA (d/l-d(GGAA)8 and d/l-r(GGAA)8, respectively) 8 exhibited a strong concentration-dependent decrease in cell viability after 24 hours, suggesting that the l-RNA strand was uniquely cytotoxic upon transfection (d-r(GGAA)8 showed no cytotoxic effects during this timeframe, l-r(GGAA)8 killed nearly all cells after just 48 hours. The half-maximal effective concentration (EC50) of l-r(GGAA)8 was determined to be 58 ± 26 nM (d-r(GGAA)8. We extracted the various (GGAA)8 oligonucleotides from cells after 24 hours and analysed their length by gel electrophoresis. The results revealed that comparable levels of full-length material for all four versions of (GGAA)8 were present inside cells after 24 hours 8 and d-d(GGAA)8 is likely due to the Cy5 dye positioned at the 3′-end, which has been shown to greatly increase the half-life of oligonucleotides in serum-supplemented media and inside cells by protecting against 3′-exonuclease degradation.20 Importantly, the presence of full-length d-d(GGAA)8 and d-r(GGAA)8 inside cells after 24 hours indicated that their much lower cytotoxicity relative to l-r(GGAA)8 cannot be attributed to nuclease degradation. Unsurprisingly,21 the 2′-O-methyl (2′-OMe) RNA version of d-r(GGAA)8, d-Me(GGAA)8, showed no visible signs of cytotoxicity in HeLa cells 20 and l-r(G3A4)4, showed a similar toxicity profile as l-r(GGAA)8 8 series, the d-RNA versions of these RNAs, d-r(GA)20 and d-r(G3A4)4, were at least 30-fold less cytotoxic than their l-RNA counterparts (l-RNAs l-r(GGAA)8, l-r(GA)20 and l-r(G3A4)4 were cytotoxic to MCF7 cells -rich  Table S1. As an i0 see ESI. Of the sfection . We confsfection . Notably ± 26 nM , which is Fig. S2. The perLa cells , highligr(GGAA)8  and even0 values , indicatterparts . In addis Fig. S3, demonstl-RNA was selective for G-rich sequences, we also examined l-poly[A] (l-rA32) and l-poly[U] (l-rU32) (l-rU32 were mostly viable (>90%) after 96 hours, whereas cells treated with l-rA32 were ∼60% viable after the same time. The EC50 for l-rA32 was calculated to be 191 ± 42 nM, indicating that it was still much less cytotoxic than those containing both G and A (EC50 ∼ 10–50 nM) 20, and found that it was ∼4-fold less toxic than the analogous sequence containing only G and A, l-r(GA)20 10 (25% G) and l-r(GGGA)10 (75% G) to l-r(GGAA)8 and l-r(GA)20, both of which contain 50% G. Unfortunately, l-r(GGGA)10 formed aggregates under almost all conditions tested, which precluded cellular delivery and analysis. The EC50 of l-r(GAAA)10 was found to be within error of both l-r(GGAA)8 and l-r(GA)20, despite having half the number of Gs relative to As  . HeLa ce0–50 nM) . Neverth-r(GA)20 . Thus, ive to As . Thus, wl-RNAs have the potential to be highly cytotoxic to human cells, especially in the presence of adenine . Cytotoxic and antiproliferative activities have previously been reported for G-rich d-oligonucleotides.24–28 These results now indicate that such properties extend across the chiral mirror. These results also show that the stereochemistry of an oligonucleotide can influence its cytotoxicity and, in the case of G-rich sequences, we find that l-RNA is considerably more cytotoxic than its d-counterparts for those sequences tested herein. To the best of our knowledge, the first time any cytotoxicity has been assigned to l-oligonucleotides.Overall, these results demonstrate that G-rich d-DNA.28 In the case of G-rich l-RNA, potent cytotoxicity was observed for both G4-forming sequences 8) and those that cannot fold into G4s 20) 8 and l-r(GA)20 under conditions used during transfection , as well as under intracellular K+ concentrations (140 mM KCl) 8, but not l-r(GA)20, form folded G4 structures under both sets of conditions. Thus, while we can't completely rule out the involvement of G4 structures, they do not appear to be the major determinant of l-RNA toxicity.The cytotoxicity of oligonucleotides has been shown to be dependent on secondary structure and folding. For example, a positive correlation between G-quadruplex (G4) formation and cytotoxicity has been reported for G-rich r(GA)20) ,19 sugge) Fig. S4. These d29 To test this, we prepared a G-rich l-RNA, l-r(GC/GC), that contained the same number of Gs as l-(GGAA)8 but was designed to fold into a hairpin such that the majority of G residues where base paired. Cells treated with l-r(GC/GC) hairpin (200 nM) showed no cytotoxic effects after 96 hours (50 for l-r(GC/GC) hairpin was calculated to be >3 μM  and double-stranded (ds) oligonucleotides can also influence cytotoxic interactions.96 hours . The EC5be >3 μM . Thus, wl-RNA. We prepared a series of incrementally shorter versions of l-(GA)20 (Table S1l-r(GA)20, l-r(GA)15, l-r(GA)10, which contain 20, 15, and 10 Gs, respectively, were equally cytotoxic to HeLa cells (EC50 ∼ 30–50 nM) following transfection. However, when the length was further reduced to contain only five Gs (l-r(GA)5), the cytotoxicity was reduced dramatically (EC50 = 413 ± 197 nM). This suggests that a minimum of ten closely spaced Gs is sufficient for l-RNA cytotoxicity.In addition to structure, we also examined the influence of length on cytotoxicity of  Table S1. As showl-oligonucleotides with the goal determined whether these factors were correlated with cytotoxicity. We first measured the uptake of the various 3′-Cy5-labeled oligonucleotides (200 nM) into HeLa cells by flow cytometry two hours after transfection (d/l-d(GGAA)8) when transfected by Lipofectamine 3000. However, the difference in uptake between DNA and RNA series was only about 2–3-fold, whereas the EC50 values for the d/l-d(GGAA)8 were at least 10-fold higher than any of the cytotoxic l-RNA strands. Among the RNA series, little correlation was found between uptake and cytotoxicity. All d-RNA and l-RNA oligonucleotides tested, as well as d-Me(GGAA)8, had similar uptake into HeLa cells, despite l-r(GGAA)8 being substantially more cytotoxic than the others 8, d-r(GGAA)8, and d-Me(GGAA)8 formed discrete nuclear foci that were not present in cells treated with other oligonucleotides .21,23,29,32 The lack of nuclear foci for d/l-d(GGAA)8 and l-r(CG/GC) hairpin are consistent with this model, as DNA and double-stranded oligonucleotides do not interact with paraspeckle proteins.29 While future studies are needed to determine the exact nature and composition of the foci formed by these RNAs, these observations suggest that l-r(GGAA)8 can seed the formation of protein-rich nuclear structures when transfected into cells. Another major difference among the oligonucleotides examined was nucleolar localization. Whereas most of the strands were excluded from the nucleolus, l-r(GGAA)8 was significantly enriched in this compartment (l-r(GGAA)8 was also the only oligonucleotide to both form nuclear foci and accumulate in the nucleolus, suggesting that the combination of these activities underly its potent cytotoxic effects. This notion is consistent with previous studies showing that ASO-mediated sequestration of nuclear proteins, including paraspeckle proteins, into the nucleolus induces cellular toxicity through nucleolar stress.21,29Fluorescent confocal microscopy was then used to determine the subcellular localization of the different 3′-Cy5-labeled oligonucleotides post transfection  and S5†.leotides  and S5†.partment  and S5†.l-RNA form nuclear foci and accumulate in the nucleolus when transfected into HeLa cells, behaviors that are highly indicative of cytotoxic protein interactions. These data also demonstrate that in addition to sequence and structure, the subcellular localization and trafficking of oligonucleotides is dependent on its chirality.Together, these data show that G-rich l-RNA, HeLa cells were stained with annexin V and propidium iodide (PI) 24 hours post transfection and fluorescence was measured by flow cytometry. AnnexinV detects the externalization of phosphatidylserine on early apoptotic cells, while PI is used to detect necrotic or late apoptotic cell due to the loss of membrane integrity.33 Very few (<10%) apoptotic or necrotic cells were detected in untreated and Lipofectamine-only treated cells after 24 hours (l-RNAs resulted in ∼50% apoptotic cells (annexin V positive and PI negative) and 10–20% late apoptotic cells (annexin V and PI positive) following 24 hour exposure. The extent of apoptotic cells for G-rich l-RNAs exceeded that of camptothecin (CPT), a common positive control for apoptosis. None of the toxic l-RNAs induced significant necrosis (annexin V negative and PI positive)  did not trigger apoptosis or necrosis. l-rA32 also induced far less apoptosis than any of G-rich l-RNAs, consistent with its lower overall cytotoxicity. Together, these results suggest that toxic G-rich l-RNA induce apoptotic cell death.To determine the type of cell death related to cytotoxic G-rich 24 hours  and S6†.l-RNAs, and to reveal the extent of their biological interactions, we performed RNA-seq analysis of total RNA isolated from HeLa cells 12 hours after treatment with 200 nM l-r(GGAA)8 and l-r(GA)20. Cells treated with Lipofectamine only and non-toxic l-r(GC/GC) served as controls for these experiments. As shown in l-r(GGAA)8 and l-r(GA)20 compared to the controls, which were clustered together. Interestingly, gene expression profiles for l-r(GGAA)8 and l-r(GA)20 were also clustered separately, indicating that they interact differently with cells. While l-r(GGAA)8 is capable of folding into a G-quadruplex structure, l-r(GA)20 is not 8 caused significant upregulation of 507 genes (log2(ratio) ≥ 1, P-value < 0.05) and downregulation of 267 genes (log2(ratio) ≤ −1, P-value < 0.05) relative to the Lipofectamine only control (l-r(GA)20, 689 genes were upregulated and 1015 genes were downregulated relative to the Lipofectamine only control (l-r(GC/GC). Overall, these data reveal that G-rich l-RNA induce dramatic perturbations in gene expression levels, suggesting extensive engagement with cellular proteins.To better understand the source of cytotoxicity for G-rich t Fig. S4, represe control . In cell control . The exp control . No genel-r(GGAA)8 and l-r(GA)20, over-representation analyses were performed to identify biological pathways and processes enriched in differentially expressed genes. Although there were some differences, KEGG pathway analysis revealed a common set of enriched pathways for l-r(GGAA)8 and l-r(GA)20, including mitogen-activated protein kinase (MAPK) signaling pathway, cytokine–cytokine receptor interaction, (tumour necrosis factor) TNF signaling pathway, p53 signaling pathway, and transcriptional misregulation (l-r(GGAA)8 and l-r(GA)20 stimulate an innate immune response, possibly via engagement of pattern-recognition receptors such as Toll-like receptors (TLRs).34–36 Consistently, several key pro-inflammatory cytokines central to the innate immune response, including TNF, interleukin (IL)-1, IL-6, IL-12, and CXCL8, were among the most highly overexpressed genes 8 and l-r(GA)20 8, was shown to induce p53 activation and apoptotic cell death.21 Further experiments beyond the scope of this work will be required to fully identify the pathways involved in l-RNA toxicity, as well as the specific receptors  responsible for these signalling outcomes.To interpret the potential biological consequence of changes in the transcriptome caused by gulation . Collect Table S2. MoreoveS2l-RNAs , which iS2l-RNAs .37–39 A  Table S2.40,41 Ins Fig. S8. While Tl-oligonucleotides for the first time, revealing that the chirality of an oligonucleotide can have a profound effect on its behavior in living systems. Most notably, we found that single-stranded G-rich l-RNAs were highly cytotoxic to human cells, whereas d-RNA versions of the same sequence showed mild or no cytotoxicity. While the number of sequences examined herein is likely too limited to generalize that G-rich l-RNAs are more cytotoxic that their d-counterparts, our results highlight the profound influence of stereochemistry on the behavior of oligonucleotides inside cells. Furthermore, we showed that cytotoxic l-RNA induce dramatic perturbations in gene expression levels. Given that l-oligonucleotides are incapable of forming contiguous WC base pairing interactions with native DNA and RNA, we propose that these effects are the results of l-RNA–protein interactions, such has been observed previously for cytotoxic d-oligonucleotides. While future proteomics studies will be needed for confirmation and to identify key l-RNA–protein interactions responsible for the observed cytotoxic effects, an analysis of differentially expressed genes suggests that G-rich l-RNA stimulate an innate immune response and pro-inflammatory cytokine production. Thus, these data challenge the general perception that mirror image l-oligonucleotides are nontoxic and nonimmunogenic. Moreover, this work demonstrates that the antiproliferative properties of G-rich d-oligonucleotides , which are currently being pursued as anticancer agents, extends across the chiral mirror to G-rich l-RNA.23–27,48In conclusion, we characterized the intracellular behavior of l-ON are being increasingly employed in biomedical applications. However, our results emphasize that caution should be taken when employing l-oligonucleotides in cells and organisms. Moving forward, it will be important to better characterize off-target interactions underlying these cytotoxic effects and to establish detailed structure toxicity relationships. Such information could be used to better predict toxicity from sequence and potentially allow for toxic motifs to be eliminated at the design stage, which provided the key motivation for this work. Towards this goal, this study offers some preliminary design considerations for minimizing the potential toxic effects of l-oligonucleotides. For example, based on the sequences tested herein, l-DNA appears to be less cytotoxic than l-RNA, making it a better choice for most biomedical applications. However, if l-RNA must be used, long (>10 nt), single-stranded regions rich in G residues should be avoided, especially in the context of G/A-rich sequences. Furthermore, complementary interactions  appear to mitigate the cytotoxic effects of G-rich l-RNA, as evident by l-r(GC/GC) hairpin. This suggests that the cytotoxicity and potential antiproliferative activities of l-RNA could be regulated through programmable base pairing interactions, opening the door to more-targeted anticancer therapies. Finally, while most applications of l-oligonucleotides may seek to evade an immune response , l-oligonucleotides could also be highly useful as immunostimulants , especially considering their lack of off-target hybridization and superior nuclease resistance. Thus, further evaluation of the immunostimulatory effects of l-RNA, as well as the specific pathways and receptors involved, is highly warranted.Due to their favorable properties, such as nuclease resistance, l-oligonucleotides and the establishment of an l-oligonucleotide “interactome”, both of which are expected to have a broad impact on how future l-oligonucleotide-based technologies are designed and applied. Our results also reveal previously unrecognized therapeutic opportunities for l-oligonucleotides. More broadly speaking, this work should motivate similar investigations into other modified oligonucleotides, such as XNAs, that are also perceived to be highly bioorthogonal, which may reveal unforeseen biological effects.Overall, this work represents an important step in understanding the intracellular behavior of l-r(GGAA)8 and l-r(GA)20 can be accessed in the ESI File 1 document.Raw RNA-seq read and processed data are available in the NCBI GEO repository under the accession number GSE205338. The complete list of differentially expressed genes for Chen-Hsu Yu: methodology, investigation, formal analysis, visualization, writing – review & editing. Jonathan Sczepanski: conceptualization, writing – original draft, supervision, project administration, funding acquisition.The authors declare no competing financial interests.SC-014-D2SC05511B-s001SC-014-D2SC05511B-s002


P < 0.05), while age, sex, race, marital status, and liver fibrosis score were not associated with extrahepatic metastasis. After propensity score analysis, the AFP level was no longer associated with metastatic risk. The present study provided no evidence for a correlation between the clinical threshold of AFP and metastasis in small hepatocellular carcinoma.Metastasis is crucial for the prognosis of hepatocellular carcinoma (HCC). Distinguishing the potential risk factors for distant metastasis in small HCC (diameter ≤ 5 cm) is of great significance for improving the prognosis. HCC patients in the Surveillance, Epidemiology and End Results (SEER) registry with tumors ≤ 5 cm in diameter between January 2010 and December 2015 were retrieved. Demographic and clinicopathological metrics were extracted, including age, sex, race, marital status, tumor size, histological grade, T stage, N stage, M stage, alpha-fetoprotein (AFP), and liver fibrosis score. Univariate and multivariate logistic regression analyses were used to identify independent risk factors correlated with extrahepatic metastasis in small HCC. Propensity score matching (PSM) analysis was performed to balance the confounding factors in baseline characteristics. A total of 4176 eligible patients were divided into a non-metastasis group (n = 4033) and a metastasis group (n = 143) based on metastasis status. In multivariate analysis, larger tumor size, poor histological differentiation, regional lymph node metastasis, and elevated serum AFP levels were identified as independent risk factors for distant metastasis ( Hepatocellular carcinoma (HCC), as the major histological type of primary liver cancer, accounts for approximately 90% of all cases2. Although advances in therapeutic strategies, such as liver transplantation, surgical resection, radiofrequency ablation (RFA), and transarterial chemoembolization (TACE), have greatly improved the prognosis of HCC6, the 5-year survival rate remains below 20%8. Metastasis accounts for a great proportion of cancer-related deaths; approximately 90% of deaths are caused by metastatic disease rather than primary tumors, and limited treatment options can be applied for extrahepatic metastasis9.Liver cancer is currently the sixth most frequently diagnosed neoplasm and the fourth leading cause of cancer-related mortality worldwide, with approximately 841,000 new cases and 782,000 deaths in 201811. HCC diagnosed at early stages could be cured by liver transplantation and liver resection, which makes the 5-year survival over 50% possible12. Although liver transplantation is the best treatment option for those who are not suitable for surgical resection, it is limited due to donor shortage; therefore, liver resection remains the major curative treatment strategy in clinical practice10. A complete response for HCC with a diameter < 5 cm  could be achieved after surgical resection, but the recurrence rate for small HCC is still high due to various contributing factors in recurrence and metastasis13. Accordingly, identifying small HCC patients with a high risk of extrahepatic metastasis is meaningful in clinical practice.The majority of HCC cases are in middle or advanced stages at the time of diagnosis beyond the curative therapeutic regimens16. Other general characteristics of patients, including age, sex, race, etc., also indicate a certain role in the prognosis of HCC17. In addition, a growing body of literature had investigated the role of gene expression on HCC progression and metastasis. Simultaneously, gene expression was found to be associated with these tumor characteristics19. Thus, clinicopathological factors exert an important role in the estimation of HCC metastasis.Previous studies had indicated that some clinicopathological factors can were risk factors of metastasis in HCC patients. Several studies reported that tumor size, vascular invasion, histological differentiation, tumor stage, serum AFP, etc., increased the risk for distant metastasisAlthough several studies have explored the association between tumor size and extrahepatic metastatic risk in HCC, for HCC tumors less than 5 cm in diameter, as far as we know, there are limited data to fully clarify the relationship. Besides, there is no research that focused on whether AFP is the risk factor for patients with small HCC.In the present study, we retrospectively reviewed the data from the Surveillance, Epidemiology, and End Results (SEER) database to investigate the potential risk factors and AFP related to extrahepatic metastasis in small HCC (diameter ≤ 5 cm).A total of 4176 HCC patients were eligible for analysis. Of these, 143 cases (3.4%) presented with distant metastasis, while the remaining 4033 patients (96.6%) did not. The available clinicopathological metrics and their differences between the two groups are summarized in Table Upon univariate analysis, seven factors  were significantly related to extrahepatic metastasis. From the results of multivariate analysis, it was found that a larger tumor size, poorer histological differentiation, regional lymph node metastasis, and elevated AFP levels were significantly correlated with distant metastasis risk in small HCC Table .Table 2AP > 0.05), except for race and fibrosis score (P < 0.05). The distributions of propensity scores between the two groups were similar in the jitter plot  compared with AFP negativity. Thus, the AFP level may not be associated with metastatic risk estimation for small HCC.We found that the AFP level was one of four significant risk factors in the multivariate analysis. However, the value of AFP in HCC diagnosis, especially for small ones, and its ability to predict extrahepatic metastasis remain uncertain and inconsistent. Due to the imbalance in variable distributions, comparison between groups would be confounded by selection bias. To minimize the confounding impacts, PSM was performed between the AFP-negative and AFP-positive groups and resulted in 2124 patients (1062 in each group) being matched Table . After mIn the current study, we collected registry information for small HCC from the SEER database, and the multivariate analysis revealed that tumor size, histological grade, N stage, and AFP level were independent risk factors for extrahepatic metastasis in small HCC. The relationship between metastatic risk and the above variables of tumor size, histological grade, and N stage are definite, while the impact of AFP level on metastatic risk remains uncertain. To further validate the association between AFP and extrahepatic metastasis, the AFP-positive group was matched to the AFP-negative group to reduce the effects of selection bias. After PSM, the AFP level was statistically insignificant.8 reviewed the data of 33,177 HCC patients from the SEER database and indicated that a maximum tumor size over 5 cm was related to a higher risk of lung metastasis. Similarly, Yokoo et al.14 conducted a retrospective case–control study on 236 newly diagnosed HCC and found that tumor size > 5 cm was independently associated with metastasis risk but with moderate sensitivities of 75.6% (69.6–80.7%) in the screening test. In addition, an observation study that enrolled 1573 HCC patients revealed that tumor diameter > 2 cm had a significantly higher risk of extrahepatic metastasis compared to those tumors < 2 cm20. In our present study, we also observed that a larger tumor diameter within 5 cm also had a higher risk of metastasis. Consequently, HCC with tumors less than 5 cm in diameter also has a higher risk of metastasis.Tumor size was identified as an important factor for extrahepatic metastasis in several previous studies. Wu et al.21. Jia et al. reported that upregulated myeloid differentiation factor 88 (MyD88) may promote epithelial-mesenchymal transition (EMT) properties and tumor-initiating capabilities via the PI3K/Akt pathway, resulting in accelerated tumor growth and metastasis22. In addition, a large-scale study of 33,177 patients indicated that poor tumor pathological differentiation was a contributory factor to lung metastasis of HCC8, which is consistent with our results. In the present study, compared to well-differentiated HCC, poorly differentiated and undifferentiated HCC showed a significant tendency of metastasis, with the rationale being that poor tumor pathological differentiation was more likely to metastasize. Regarding lymph node metastasis, Bi et al.23 investigated the clinical relevance of MDM2 Binding Protein (MTBP) and found that MTBP was poorly expressed in HCC tissues compared to adjacent nontumor tissue, and its expression was negatively associated with lymph node metastasis and vascular invasion. Additionally, He et al.24 evaluated the clinical implications of another tumor suppressor of AT-rich interactive domain-containing protein 1A (ARID1A) and found that decreased ARID1A levels were significantly associated with regional lymph node and distant metastasis. Many similar studies indirectly suggested that lymph node metastasis was a poor prognostic factor for HCC.Histological differentiation grade and N stage (lymph node metastasis) are important characteristics of malignancy. Poorer histological differentiation and lymph node metastasis represent a higher degree of malignancy with poor prognosis. The two indicators have been extensively demonstrated to be associated with HCC metastasis. Lee et al. found that the overexpression of metastasis-associated protein MTA2 was detected in 96.2% of the 506 HCC tissue samples, which was tightly correlated with HCC size and differentiation25, especially for early stage HCC, as the sensitivity of combined serum AFP and ultrasonography only varies from 40 to 65%26. Accordingly, the value of serum AFP to predict metastasis in small HCC remains to be explored. Jin et al.27 detected the AFP mRNA level in circulating tumor cells and found that the release of AFP from HCCs released into circulation could be a significant predictor for metastasis before and after hepatic resection. Yokoo et al.14 also indicated that AFP > 400 μg/mL was strongly correlated with extrahepatic metastasis. In contrast, Giannini et al.28 found that increased AFP levels did not show significance for prognosis in well-compensated cirrhosis patients with single, small HCC. In this study, the serum AFP level was identified as a risk factor before PSM, but while adjusting for confounding factors, the AFP level was not significantly associated with HCC metastasis with a diameter < 5 cm. One reason for the opposite results may be that clinical AFP detection is generally at the protein level, rarely at the RNA level. Another reason, perhaps owing to the existence of selection bias, is that the significance of AFP for metastasis may be affected by confounding covariates. Furthermore, our studies mainly focused on small HCC, while most similar studies took all diameters into consideration.AFP has been extensively applied as a biomarker for HCC diagnosis clinically. However, recent data have shown that the low sensitivity and specificity are challenges in the diagnosis of HCC31. PSM is able to balance the variable distribution so that the baseline characteristics between groups became comparable, thus reducing the potential influence of confounders and providing a more accurate estimation on causal effect results32. Thus, the present findings were more accurate and reliable.The main strength of this study is that we focused on small HCC with diameters of less than 5 cm; a better understanding of the significant determinants of extrahepatic metastasis in small HCC can help to reasonably manage tumors. In addition, the patients’ information was retrieved from a large population-based database (SEER). Furthermore, PSM was introduced to balance confounding factors in this observational study. Although traditional regression models have been widely used to adjust for confounding factors associated with exposure and outcome, the membership bias of characteristics between the treatment and control groups may not be adequately explained. Additionally, the results estimated with regression models may vary as the parameter settings change in certain circumstances33. Second, the SEER database does not include other important risk factors, such as vascular invasion, family history, and genetic status. Therefore, more potential confounding factors should be evaluated in future research. Thirdly, the SEER database did not have the exact value of AFP, and just divided the value of AFP into positive and negative group based on the borderline. The continuous variables are converted into categorical form for analysis, which causes some information would lose34. Hence, the various cut-off value of AFP is needed to explore the relation between AFP level and metastasis of patients with small HCC.However, the present study has several limitations. First, this is a retrospective study, although PSM analysis has the advantage of eliminating the confounding effects of observed covariates, the unmeasured factors may also play roles in causal effect estimationIn summary, this study reveals that larger tumor size, poor tumor histological differentiation, and regional lymph node metastasis were associated with a higher risk of extrahepatic metastasis, but not AFP. Thus, this study provides a reference for small HCC patients with metastatic risk, and intensive attention should be paid to these patients.Data of patients with primary liver cancer (PLC) were retrieved from the Surveillance, Epidemiology, and End Results (SEER) program of the National Cancer Institute, which is publicly available and provides access to cancer-related data from cancer statistic registries 1973–2015). A total of 114,873 patients with PLC were retrieved, and their clinicopathological metrics available were extracted from the database, including age at diagnosis, sex, race, marital status, alpha-fetoprotein (AFP), liver fibrosis score, tumor size, histological grade, and TNM stage . The exclusion criteria for HCC patients are shown in Fig. –2015. A P < 0.2 in univariate analysis were used in multivariate logistic regression for further analysis35. The significance of AFP as a risk factor was further confirmed after propensity score matching (PSM) in which the following covariates were matched: age, sex, race, marital status, histological grade, T stage, N stage, and fibrosis score. The matching algorithm of nearest neighbors was used in PSM. The R (3.5.2) package “MatchIt” was used to perform PSM, distribution of propensity scores were visualized with the generic plot function, and propensity score analysis assessment plots of covariates were generated with the “cobalt” package.Univariate analysis and multivariate logistic regression analysis were applied to identify risk factors associated with extrahepatic metastasis in small HCC. Variables that were significant with P values < 0.05 were considered statistically significant in all analyses. Data were analyzed by SPSS for Windows version 22.0  except for specific data described above.In the statistical analysis, categorical variables are shown as numbers (%), and continuous variables are presented as the mean ± SD. Chi-square test or Fisher’s exact test was used to compare the differences for categorical variables, while continuous tests using Student’s t-test and Wilcoxon rank-sum test were applied when appropriate. Two-tailed


Despite the rise in morbidity and mortality associated with vascular diseases, the underlying pathophysiological molecular mechanisms are still unclear. RNA N6-methyladenosine modification, as the most common cellular mechanism of RNA regulation, participates in a variety of biological functions and plays an important role in epigenetics. A large amount of evidence shows that RNA N6-methyladenosine modifications play a key role in the morbidity caused by vascular diseases. Further research on the relationship between RNA N6-methyladenosine modifications and vascular diseases is necessary to understand disease mechanisms at the gene level and to provide new tools for diagnosis and treatment. In this study, we summarize the currently available data on RNA N6-methyladenosine modifications in vascular diseases, addressing four aspects: the cellular regulatory system of N6-methyladenosine methylation, N6-methyladenosine modifications in risk factors for vascular disease, N6-methyladenosine modifications in vascular diseases, and techniques for the detection of N6-methyladenosine-methylated RNA. RNA modification is a ubiquitous process in nature that regulates the transmission and expression of genetic information in cells. It can affect the phenotype of cells by influencing the transcription, splicing, stability, transnuclear transport, and translation of RNA –3. AccorM6A methylation, the addition of a methyl group at the nitrogen atom at position 6 of adenine, is catalysed by methyltransferases and is the most common modification of mRNA in eukaryotic cells –9. PreviPrevious studies have found that m6A is generally present in specific RNA motifs, such as DRACH , 3ʹ untranslated regions (UTRs), or around the stop codon , 18. In Writers deposit N6-methyladenosine on target RNAs. The writer complex includes the enzymes methyltransferase-like 3 (METTL3), methyltransferase-like 14 (METTL14), Wilms tumour 1-associated protein (WTAP), methyltransferase-like 16 (METTL16), and vir-like m6A methyltransferase associated (VIRMA). Two proteins, RNA-binding motif protein 15 (RBM15) and RNA-binding motif protein 15B (RBM15B), modulate the action of this complex , 31..[Previous studies have shown that METTL3 is the major catalytic subunit of the RNA methyltransferase complex. METTL3 and METTL14 form a heterodimer, in which METTL14 acts as an adaptor protein that enhances the function of METTL3 , 33. Stu. and Cham. have shoWTAP acts as a guide to direct binding of the METTL3/METTL14 heterodimer to the target RNA , 37. Pre. have suggested that METTL16 regulates the expression of S-adenosyl methionine synthase, which in turn plays a role in early embryonic development [Recent studies have shown that METTL16 is catalytically active in specific situations , 43. Stuelopment .VIRMA is another recently discovered catalytic component of the RNA methyltransferase complex that deposits N6-methyladenosine in the 3ʹUTR .2 + and α-ketoglutarate as cofactors [Erasers are demethylates that eliminate RNA m6A modifications. So far, two important erasers have been identified: the fat-mass and obesity-associated protein (FTO) and α-ketoglutarate-dependent dioxygenase AlkB homolog 5 (ALKBH5). Both are members of the α-ketoglutarate-dependent dioxygenase family of enzymes; however, while FTO is expressed in all tissues, ALKBH5 is primarily expressed in the testes –48 and tofactors , 52. FTOofactors , 52–55.Readers are proteins that can identify, bind, and link RNA N6-methyladenosine modifications to specific biological functions. Many readers have been recognized, including the YTH family  , the hetYTHDF1, YTHDF2, YTHDF3, and YTHDC2 are localized mainly in the cytoplasm. Previous studies have shown that YTHDF1 combines with eIF-3 to facilitate target mRNA translation . YTHDF2 Members of the HNRNP family process RNAs through the “m6A switch” system, where the structure of the target RNA is changed and recognized by RNA binding proteins , 63, 64.In recent years, the morbidity of vascular diseases has gradually increased; common high-risk factors of vascular diseases, including obesity, diabetes, hypertension, and atherosclerosis, have played a critical role in this rise. These factors can cause coronary heart disease (CHD), arterial occlusion, arterial dissection, aneurysms, and other common vascular diseases. Studies have shown that RNA m6A plays an independent role in the regulation of these high-risk factors. In this review, we discuss the regulatory role of RNA m6A modifications in different high-risk factors. The associations between m6A methylation and risk factors for vascular diseases are presented in Table . [Obesity is a pathological change caused by an excessive intake of exogenous lipids or a dysfunction of lipid metabolism. Mo et al.  used the. .. showed that abnormal increase in zinc-finger protein 217 (zfp217) led to abnormal increase in FTO, which in turn caused a decrease in m6A-YTHDF2, ultimately leading to obesity [., inhibition of the expression of FTO induced a decrease in total cholesterol and LDL, and suppressed the formation of atherosclerotic plaques [Several studies have shown that abnormal variations in FTO expression are closely related to obesity. A recent study carried out by Song et al obesity . In the  plaques . Increas plaques . Studies plaques , 70. The plaques . Due to T2DM often leads to intimal injury, abnormal thickening, and occlusion of peripheral blood vessels. Therefore, it is one of the high-risk factors for ischemic injury and stroke.., FTO was shown to play an important role in the development of T2DM. In that study, FTO induced an increase in the expression of forkhead box O1 (FOXO1), glucose-6-phosphatase (G6PC), and diacylglycerol O-acyltransferase 2 (DGAT2), which in turn lead to the dysfunction of lipid and glucose metabolism [In a study by Yang et altabolism . In FTO tabolism .Studies have found that RNA m6A modifications are closely related to the function and number of pancreatic β-cells. These, in turn, are key factors in glucose metabolism. β-cell arrest and insulin secretion are inhibited by decreased m6A modifications in insulin-like growth factor 1 (IGF1)-Akt-pancreatic and duodenal homeobox 1 (PDX1) transcripts . METTL14In recent years, with the fast economic growth and global improvement of living standards, the morbidity of hypertension as well as its complications, including CHD, AD, and aortic aneurysm, has increased. Previous studies have shown that RNA m6A plays an important role in the development of hypertension.. analysed data from GWAS to find a relationship between m6A-associated SNPs and hypertension. A total of 1236 SNPs, such as rs9847953 and rs197922, were found to be associated with blood pressure regulation and most of them were modified by m6A methylation [. found that, in men with hypertension, the m6A-associated SNP rs17782313 in the melanocortin 4 receptor (MC4R) had a negative association with diastolic pressure and mean blood pressure [Mo et alhylation . A recenThe eraser FTO also plays a critical role in blood pressure regulation. A decrease in FTO expression leads to protection against tachycardia, hypertension, and vascular resistance. Recent studies have also found that the pathogenesis of hypertension may be related to a change in m6A methylation levels in peripheral cells in microvessels .Studies have revealed that atherosclerosis has a close relationship with lipid metabolism and inflammation. In a study by Jian et al., ApoE knockout mice were used to investigate a relationship between RNA m6A modification and atherosclerosis. Their study revealed that FOXO1 m6A methylation was increased by METTL3 and recognized by YTHDF1. This resulted in an increase in intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1), leading to mononuclear/endothelial cell adhesion and atherosclerosis , 79. TumStudies have shown that the development of many vascular diseases is associated with RNA m6A modifications. We discuss these associations, which are further presented in Table . has shown that increased METTL14 expression leads to an increase in m6A, which in turn aggravates vascular calcification [CHD is caused by coronary luminal narrowing or occlusion due to abnormal lipid metabolism, local vascular wall inflammatory response, and other triggers. Previous studies have shown increased m6A modifications and lowered FTO expression in tissues subjected to myocardial infarction. RNA m6A modifications regulate cardiac contractility, cardiomyocyte differentiation, and metabolism. A study by Chen et alfication . Elevatefication .. has shown that the FTO rs9939609 (T > A) variant regulates the morbidity of CHD [On the other hand, genetic polymorphisms in FTO have been shown to influence the development of CHD. A study by Aijala et aly of CHD . The SNPy of CHD .. has demonstrated that m6A methylation of specific mRNAs is a candidate marker for myocardial infarction diagnosis [The detection of m6A modifications on RNA can be used as a potential diagnostic marker for myocardial infarction. A study by Saxena et aliagnosis .. have shown that many poststroke biological processes, such as inflammation, apoptosis, and transcriptional regulation, might be modulated by differential expression levels of FTO. RNA m6A modification may be a relevant indicator of poststroke pathophysiology [Stroke is caused by intracranial vascular stenosis or occlusion. Previous studies have shown that RNA m6A modification is closely related to stroke. However, research on this topic is still in its infancy. Chokkalla et alysiology .An aortic aneurysm is an enlargement of the aorta, diagnosed when its diameter exceeds 50% of the normal vascular diameter. Aortic aneurysms can lead to aortic rupture and death. Several underlying causes may exist. Previous studies have shown that RNA m6A modification has a positive relationship with abdominal aortic aneurysm formation and rupture . FTO decAortic dissection is another life-threatening vascular emergency. At present, its exact molecular mechanism is unclear. Studies have shown that the main pathological changes associated with AD are inflammation of vascular tissues, increased apoptosis of ECs and SMCs, decreased cell proliferation, decreased extracellular matrix, and congenital dysplasia of connective tissue .. has shown that macrophage differentiation and the number and function of T cells can be regulated by METTL3 through modulation of STAT1 expression and the IL-2–STAT5 signaling pathway [Previous studies have shown that METTL3 plays an important role in the development of AD. METTL3 affects hypoxia stress, which promotes the differentiation of adipose-derived stem cells into SMCs . A recen pathway .In a study performed using human umbilical vein endothelial cells, WTAP combined with Hakai, Virilizer homolog, KIAA0853, RBM15, BCLAF1, and THRAP3 to form a protein complex that functioned as an RNA processing machine and affected cell proliferation . Subsequ., HNRNPA2/B1 affected the differentiation of SMCs, which led to an increase in systolic type SMCs. Decreases in systolic type SMCs are a key factor in AD formation. Therefore, HNRNPA2/B1 maybe a potential target for the treatment of vascular degenerative disease [Readers also play an important role in the formation of AD. In a study by Wang et al disease . Additio disease .RNA m6A methylation was first identified in 1974; however, it was rediscovered in 2012 with the emergence of next-generation sequencing technology that enabled its detection through the transcriptome , 18. In Methylated RNA immunoprecipitation sequencing (MeRIP-seq and m6A-seq) is the oldest and most widely used method to detect m6A-methylated RNA. Random RNA fragmentation, m6A-specific methylated RNA immunoprecipitation, and next-generation sequencing are the basis of the MeRIP-seq and m6A-seq technique. Despite its robust technology, it presents several disadvantages, including: difficulty in accurately locating m6A, antibody bias, difficult data analysis, low reproducibility, and the requirement of large amounts of RNA , 94.Photo-crosslinking-assisted m6A sequencing (PA-m6A-seq), m6A individual-nucleoside-resolution cross-linking and immunoprecipitation (miCLIP), m6A cross-linking immunoprecipitation (m6A-CLIP), and m6A-level and isoform-characterization sequencing (m6A-LAIC-seq) were subsequently established. Their accuracy gradually improved and the required sample amounts gradually decreased; however, because the use of antibodies is still required, antibody bias cannot be avoided –99.Site-specific cleavage and radioactive-labelling followed by ligation-assisted extraction and thin-layer chromatography (SCARLET), RNA-endoribonuclease-facilitated sequencing (m6A-REF-seq), m6A-sensitive RNA digestion and sequencing (MASTER-seq), and deamination adjacent to RNA modification targets (DART-seq) are recent, quantitative, and antibody-free techniques. Their detection ability is accurate and fast, and the amount of RNA required for each detection is low. However, these technologies are still in their primary stages and cannot be used on a large scale –103.Previous studies have shown that RNA m6A modifications play a critical role in the regulation of vascular diseases. However, the research on m6A-methylated RNA is still in its infancy and many unknown fields need to be further explored. First, available studies address individual components of the m6A regulatory system; research on the interaction between different components is lacking. Second, the earlier studies were performed at the cellular and small-animal level; further large animal-level studies and clinical research need to be performed. Third, although several technologies for the detection of m6A-methylated RNA have been recently developed, many are still in their primary stage and cannot be widely used. Fourth, many RNA epigenetic modifications have already been identified. The interrelationship between RNA methylation and other RNA genetic modifications is unclear and considered a future research direction.In conclusion, RNA m6A modifications, as the most common cellular process of RNA regulation, participate in a variety of biological functions and play an important role in epigenetics. A large amount of evidence shows that RNA N6-methyladenosine modifications play a key role in the morbidity caused due to vascular diseases. Further research on the relationship between RNA N6-methyladenosine modifications and vascular diseases is necessary to understand pathophysiological mechanisms at the gene level and to provide new tools for the diagnosis and treatment of vascular diseases.