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The YTH N 6 -methyladenosine RNA binding proteins (YTHDFs) mediate the functional effects of N 6 -methyladenosine (m 6 A) on RNA. Recently, a report proposed that all YTHDFs work redundantly to facilitate RNA decay, raising questions about the exact functions of individual YTHDFs, especially YTHDF1 and YTHDF2. We show that YTHDF1 and YTHDF2 differ in their low-complexity domains (LCDs) and exhibit different behaviors in condensate formation and subsequent physiological functions. Biologically, we also find that the global stabilization of RNA after depletion of all YTHDFs is driven by increased P-body formation and is not strictly m 6 A dependent., (© 2023. The Author(s).)

This review covers research findings reported over the past decade concerning the ability of low complexity (LC) domains to self-associate in a manner leading to their phase separation from aqueous solution. We focus our message upon the reductionist use of two forms of phase separation as biochemical assays to study how LC domains might function in living cells. Cells and their varied compartments represent extreme examples of material condensates. Over the past half century, biochemists, structural biologists, and molecular biologists have resolved the mechanisms driving innumerable forms of macromolecular condensation. In contrast, we remain largely ignorant as to how 10%-20% of our proteins actually work to assist in cell organization. This enigmatic 10%-20% of the proteome corresponds to gibberish-like LC sequences. We contend that many of these LC sequences move in and out of a structurally ordered, self-associated state as a means of offering a combination of organizational specificity and dynamic pliability to living cells. Finally, we speculate that ancient proteins may have behaved similarly, helping to condense, organize, and protect RNA early during evolution., (© 2022 Kato et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Existing methods for RNA diagnostics, such as reverse transcription PCR (RT-PCR), mainly rely on nucleic acid amplification (NAA) and RT processes, which are known to introduce substantial issues, including amplification bias, cross-contamination, and sample loss. To address these problems, we introduce a confinement effect-inspired Cas13a assay for single-molecule RNA diagnostics, eliminating the need for NAA and RT. This assay involves confining the RNA-triggered Cas13a catalysis system in cell-like-sized reactors to enhance local concentrations of target and reporter simultaneously, via droplet microfluidics. It achieves >10 000-fold enhancement in sensitivity when compared to the bulk Cas13a assay and enables absolute digital single-molecule RNA quantitation. We experimentally demonstrate its broad applicability for precisely counting microRNAs, 16S rRNAs, and SARS-CoV-2 RNA from synthetic sequences to clinical samples with excellent accuracy. Notably, this direct RNA diagnostic technology enables detecting a wide range of RNA molecules at the single-molecule level. Moreover, its simplicity, universality, and excellent quantification capability might render it to be a dominant rival to RT-qPCR.

MicroRNAs (miRNAs) have been regarded as potential biomarkers in early diagnosis of cancer. Since the high sequence similarity among miRNA family members, biosensing miRNAs with single-base resolution is still a challenge, particularly when the different base is located at the terminal of miRNA. Herein, we developed two real-time fluorescence monitoring methods for miRNA detection utilizing efficient PBCV-1 DNA ligase mediated target miRNA dependent DNA ligation, followed by rolling circle signal amplification. Compared to duplex-specific nuclease (DSN) enhanced rolling circle transcription (RCT) system, nicking endonuclease (NEase) assisted rolling circle amplification (PRCA-NESA) can provide higher amplification efficiency, and achieve a limit-of-detection of 0.5 amol for miR-17 in 10 μL sample. More importantly, benefiting from the unique characteristics of PBCV-1 DNA ligase, we designed an asymmetric PRCA-NESA method, which can greatly discriminate the single-base difference at either 5'- or 3'-terminals of miRNAs. MiR-17 from various tumor cells also can be reliably detected. In conclusion, our strategy exploited the application potential of PBCV-1 DNA ligase in biosensing, and provided a new idea to highly specific miRNA detection, thereby would possess a promising potential for further application in biomedical research and early cancer diagnosis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

It is well known that graphene oxide (GO), a planar nanomaterial, is endowed with the capacity to immobilize short ssRNA via π-π stacking, thus enhancing its stability. However, whether large RNA molecules, such as total RNA, extracted from biological tissues can be protected using GO has not been investigated. It is usually believed that the protection of total RNA by GO is not effective because the lengths of total RNA, which range from a few to thousands of bases, are inclined to undergo desorption due to their complicated structure. Herein, the nanobiological effects of total RNA/GO are first investigated and demonstrate that the total RNA can be harbored on the surface of GO, thus resulting in a shield effect. This shield effect allows total RNA to highly resist RNase degradation and maintain RNA stability at room temperature up to 4 days, enabling the discovery of GO as the potential next-generation RNase nanoinhibitor. Furthermore, GO can be conjugated to nanomagnetic beads, defined as magnetic graphene oxide, enabling the rapid purification and protection of RNA from animal cells and tissues, whole blood, bacteria, and plant tissue.

Few methods for the detection of SARS‐CoV‐2 currently have the capability to simultaneously detect two genes in a single test, which is a key measure to improve detection accuracy, as adopted by the gold standard RT‐qPCR method. Developed here is a CRISPR/Cas9‐mediated triple‐line lateral flow assay (TL‐LFA) combined with multiplex reverse transcription‐recombinase polymerase amplification (RT‐RPA) for rapid and simultaneous dual‐gene detection of SARS‐CoV‐2 in a single strip test. This assay is characterized by the detection of envelope (E) and open reading frame 1ab (Orf1ab) genes from cell‐cultured SARS‐CoV‐2 and SARS‐CoV‐2 viral RNA standards, showing a sensitivity of 100 RNA copies per reaction (25 μL). Furthermore, dual‐gene analysis of 64 nasopharyngeal swab samples showed 100 % negative predictive agreement and 97.14 % positive predictive agreement. This platform will provide a more accurate and convenient pathway for diagnosis of COVID‐19 or other infectious diseases in low‐resource regions. [ABSTRACT FROM AUTHOR]

Increases of the activity of mitochondrial electron transport chain generally lead to increases of production of ATP and reactive oxygen species (ROS) as by-products. MnSOD is the first line of defense against the stress induced by mitochondrial ROS. Our previous studies demonstrated that EAE progression increased Na,K-ATPase activity in the mouse kidney cortex. Since mitochondria are the major source of ATP, our present studies were sought to determine whether EAE progression increased mitochondrial activity. We found that severe EAE increased mitochondrial complex II and IV activities without significantly affecting complex I activity with corresponding increases of ROS in the isolated mitochondria and native kidney cortex. Severe EAE augmented both cytosolic and mitochondrial MnSOD protein levels and activities and decreased the specific activity of mitochondrial MnSOD when the total mitochondrial MnSOD activity was normalized to the protein level. Using HEK293 cells as a model free of interference from immune reactions, we found that activation of Na,K-ATPase by monensin for 24 hours increased complex II activity, mitochondrial ROS and MnSOD protein abundance, and decreased the specific activity of the mitochondrial MnSOD. Inhibition of Na,K-ATPase by ouabain or catalase attenuated the effects of monensin on the mitochondrial complex II activity, ROS, MnSOD protein level and specific activity. Kockdown of MnSOD by RNAi reduced the mitochondrial ability to generate ATP. In conclusion, EAE increases mitochondrial activity possibly to meet the energy demand from increased Na,K-ATPase activity. EAE increases mitochondrial MnSOD protein abundance to compensate for the loss of the specific activity of the enzyme, thus minimizing the harmful effects of ROS. [ABSTRACT FROM AUTHOR]

Catalytic transfer of genetic information from DNA to RNA is very important in life activities. The unconventional RNA transcription level may be related to the source of genetic diseases. At present, conventional methods for detection of RNA transcripts usually involve cumbersome preparative steps, or require sophisticated laboratory equipments. In this study, we presented a rapid, sensitive nano-detection platform for monitoring of RNA transcript levels. T7 RNA polymerase transcription reaction is employed as the example to test the feasibility of this method. In this design, in vitro synthesized RNA products can be hybridized to the FAM labeled single strand DNA (ssDNA) probes which can be adsorbed onto the graphene oxide (GO) surface. Using GO as the fluorescence switch, excellent capacity of the signal-on fluorescence platform for detection of RNA transcripts level is demonstrated. Transcription levels sensing with this nano-platform achieved a sensitivity of 5 pmol/L transcription template. It is anticipated that current developed RNA transcript nano-detection mode has the potential to be an alternative to the conventional RNA transcript detection methods. [ABSTRACT FROM AUTHOR]

A label-free approach with multiple enhancement of the signal for microRNA detection has been introduced. The key idea of this strategy is achieved by taking advantage of a novel graphene oxide (GO)/intercalating dye based fluorescent hairpin probe (HP) and an isothermal polymerization reaction. In this paper, we used microRNA-21 (mir-21) as the target to examine the desirable properties of this assay. When the target, as a 'trigger', was hybridized with the HP and caused a conformation change, an efficient isothermal polymerization reaction was activated to achieve the first step of the 'signal' amplification. After incubation with the platform of GO/intercalating dye, the formed complex of DNA interacted with the high-affinity dye and then detached from the surface of the GO, a process that was accompanied by distinguishable fluorescence recovery. Further signal enhancement has been accomplished by a mass of intercalating dye inserting into the minor groove of the long duplex replication product. Due to the efficient and multiple amplification steps, this approach exerted a substantial enhancement in sensitivity and could be used for rapid and selective detection of Mir-21 at attomole levels. Proof-of-concept evidence has been provided for the proposed cost-effective strategy; thus, this strategy could expand the application of GO-material-based bioanalysis for nucleic acid studies. [ABSTRACT FROM AUTHOR]

Development of rapid and accurate detection of miRNAs in complex samples is of great significance for potential early diagnosis of disease. Herein, we report a magnetic relaxation switching (MRS)-based strategy for direct detection of miRNAs in complex samples via the assistance of signal amplification system of CRISPR/Cas13a which has the ability to specifically recognize target RNA. In the designed strategy, 30 nm-magnetic nanoparticles (MB 30) and 1000 nm-magnetic particles (MM 1000) linked by single-strand RNA 1 complexes (MB 30 -RNA 1 - MM 1000) were employed as signal probe. After the target miRNAs (taking miR-21 as model) recognition by CRISPR/Cas13a system, the resulted trans -cleavage degrades the MB 30 -RNA 1 -MM 1000 , releasing MB 30 which caused transverse relaxation time (T 2) signal change. The combination of CRISPR/Cas13a assisted signal amplification and the MRS assay achieved direct detection of miR-21 in the serum sample without extracting within 60min, with a detection limit of 0.22 pM. Moreover, the detection accuracy is confirmed by performing the detection of miR-21 using qRT-PCR. The CRISPR/Cas13a system assisted MRS assay successfully achieved accurate, simple, and rapid detection of miRNAs in complex samples, showing great potential for detection miRNAs in potential clinical applications. [Display omitted] • The proposed strategy here enables the direct detection of miR-21 in serum samples without pre-extraction of miR-21. • The aid of the CRISPR/Cas13a signal amplification system increases detection sensitivity. • This platform can realize the detection of different kinds of miRNAs by changing the crRNA sequence. [ABSTRACT FROM AUTHOR]