Complementary RNA Research Articles

Synthesis of LNA gapmers that replace a phosphorothioate linkage with a sulfonamide in the gap region, and their ability to form duplexes with complementary RNA targets.

Antisense oligodeoxynucleotides can bind to target RNAs and cleave them using RNase H. Despite the high activity of antisense oligodeoxynucleotides modified with locked nucleic acids (LNA) at several bases at both the 5' and 3' ends (LNA gapmer), toxicity has been reported, necessitating additional backbone modifications to reduce toxicity. In this study, we introduced a sulfonamide linkage into the LNA gapmer to elucidate its fundamental properties such as hybridization, base recognition, and induction of RNase H activity. A new chemically stable sulfonyltriazole was used as a synthetic intermediate to introduce a sulfonamide linkage between the two nucleosides. We studied the properties of the duplex of the sulfonamide-linked gapmer and target RNAs, such as melting temperature, circular dichroism, and cleavage of RNA strands by RNase H. We found that the gapmers had a lower but tolerable duplex stability with base-pair specificity and the ability to induce RNase H activity.

Influenza A virus transcription generates capped cRNAs that activate RIG-I.

During influenza A virus (IAV) infection, host pathogen receptor retinoic acid-inducible gene I (RIG-I) detects the partially complementary, 5'-triphosphorylated ends of the viral genome segments and non-canonical replication products. However, it has also been suggested that innate immune responses may be triggered by viral transcription. In this study, we investigated whether an immunostimulatory RNA is produced during IAV transcription. We show that the IAV RNA polymerase can read though the polyadenylation signal during transcription termination, generating a capped complementary RNA (ccRNA), which contains the 5' cap of an IAV mRNA and the 3' terminus of a cRNA instead of a poly(A) tail. ccRNAs are detectable in vitro and in both ribonucleoprotein reconstitution assays and IAV infections. Mutations that disrupt polyadenylation enhance ccRNA synthesis and increase RIG-I-dependent innate immune activation. Notably, while ccRNA itself is not immunostimulatory, it forms a RIG-I agonist by hybridizing with a complementary negative-sense viral RNA. These findings thus identify a novel non-canonical IAV RNA species and suggest an alternative mechanism for RIG-I activation during IAV infection.

Mouse Pachytene piRNAs Cleave Hundreds of Transcripts, But Alter the Steady-State Abundance of Only a Minority of Targets.

In animals, 18-35-nt piRNAs guide PIWI proteins to regulate complementary RNAs. During male meiosis, mammals produce an exceptionally abundant class of piRNAs called pachytene piRNAs. Pachytene piRNAs are required for spermatogenesis and have been proposed to control gene expression by various mechanisms. Here, we show that pachytene piRNAs regulate targets predominantly, if not exclusively, by endonucleolytic cleavage. Remarkably, pachytene piRNAs slice hundreds of RNAs, yet a change in steady-state level is detectable for a small fraction of transcripts. Our data suggest that cleavage of the few targets whose abundance is reduced significantly by piRNAs is essential for male fertility. Other pachytene piRNA targets are enriched for highly transcribed genes, which may explain why piRNA cleavage is often inconsequential for the steady-state abundance of targets. We propose that the retention of pachytene piRNAs throughout mammalian evolution is driven by the selective advantage conferred by a tiny minority of piRNAs.

Bifunctional SnO2/NiO/rGO nanocomposite electrode for hydrogen evolution reaction and detection of winter wheat cold-resistant RNA.

Aconvenient, non-toxic, and low-cost tin dioxide (SnO2)/nickel oxide (NiO)/reduced graphene oxide (rGO) nanocatalytic electrode was investigated, which can effectively promote the hydrogen evolution reaction and can also be used to construct a sensitive winter wheat cold-tolerant RNA sensor. Due to the good synergy and photocurrent generation between SnO2 and NiO, which accelerates the electron transfer and catalyzes the hydrolysis reaction, the resultant data for the hydrogen evolution reaction in alkaline environment of this material are very impressive, with cathodic current densities of up to 10mAcm-2. The marvelous heterostructures and photovoltaic properties form a signal amplification system, which enables the nanocomposites to have an excellent linear sensitivity in low-concentration RNA solutions. The single-stranded complementary RNA of the target RNA was immobilized on the surface of the nanocomposites, which enabled the materials to recognize the target RNA under enzyme-free conditions. The test results showed that the modified electrode materials had good single detection performance in a wide linear range from 0.1nM to 2mM, with the limit of detection (LOD) of 0.078nM. The developed nanocomposite electrode has good performance in hydrogen evolution and winter wheat cold-resistant RNA detection, and is a bifunctional device with superior performance.

Two amino acid residues in the N-terminal region of the polymerase acidic protein determine the virulence of Eurasian avian-like H1N1 swine influenza viruses in mice.

Reassortant Eurasian avian-like H1N1 (rEA H1N1) viruses carrying the internal genes of H1N1/2009 virus have been circulating in pigs for more than 10 years and have caused sporadic human infections. The enhanced virulence phenotype of the rEA H1N1 viruses highlights potential risks to public health. However, the molecular mechanism underlying the viral pathogenicity of the currently circulating rEA H1N1 viruses remains unclear. In this study, we found that two naturally isolated rEA H1N1 swine influenza viruses, A/swine/Liaoning/FX38/2017 (FX38) and A/swine/Liaoning/SY72/2018 (SY72), possessed similar genetic characteristics but exhibited significantly different pathogenicity in a mouse model. Using reverse genetics, we demonstrated that amino acid mutations at positions 100 and 122 in the polymerase acidic (PA) protein had individual and synergistic effects on the polymerase activity and viral replication capacity in vitro, as well as the viral pathogenicity in mice. Furthermore, we revealed that amino acid residue 100 in PA influenced the transcription of viral RNA (vRNA) by altering the endonuclease activity, and amino acid residue 122 affected the synthesis of complementary RNA and messenger RNA by altering the RNA-binding ability and endonuclease activity of the PA protein. Taken together, we identified that two naturally occurring amino acid mutations in PA derived from H1N1/2009 virus are crucial determinants of the virulence of rEA H1N1 viruses and revealed the differential mechanism by which these two mutations affect the transcription and replication of vRNA. These findings will extend our understanding of the roles of PA in the virulence of influenza A viruses.IMPORTANCEMultiple genetic determinants are involved in the virulence of influenza A viruses. In this study, we identified two naturally occurring amino acid mutations, located at residues 100 and 122 in the polymerase acidic (PA) protein, which are associated with viral polymerase activity, replication competence, and pathogenicity in mice. In particular, we clarified the specific mechanism by which the two residues play an important role in viral transcription and replication. These findings will help to improve understanding the functions of amino acid residues in the N-terminal region of the PA protein involved in the pathogenicity of influenza A viruses.

Synthesis, Biophysical and Biological Evaluation of Splice-Switching Oligonucleotides with Multiple LNA-Phosphothiotriester Backbones.

Polyanionic antisense oligonucleotides hold great promise as RNA targeting drugs but issues with bioavailability hinder their development. Uncharged phosphorus-based backbones are promising alternatives but robust methods to produce them are limited. We report the synthesis and properties of oligonucleotides containing charge-neutral LNA alkyl phosphothiotriester backbones combined with 2'-O-methyl phosphorothioate nucleotides for therapeutic applications. The nature of the triester alkyl group dictates the success of solid-phase synthesis; tertiary alkyl groups are lost during the P(III) oxidation step, whereas primary alkyl groups are partially cleaved during deprotection. In contrast, oligonucleotides containing secondary phosphothiotriester linkages are stable, and large numbers of triesters can be incorporated. The modified oligonucleotides have excellent duplex stability with complementary RNA and exhibit strong nuclease resistance. To expand synthetic flexibility, oligonucleotides containing multiple internal alkynyl phosphothiotriesters can be conjugated to lipids, carbohydrates, or small molecules through CuAAC click chemistry. Oligonucleotides containing LNA-THP phosphothiotriesters exhibit high levels of pre-mRNA splice switching in eukaryotic cells.

An Electrochemical Bioplatform Based on Acrylamide‐Induced Inhibition of DNA‐RNA Hybridization for the Determination of Acrylamide

AbstractThis study reports the development of an innovative electrochemical bioplatform for the indirect determination of acrylamide (AM) based on its inhibitory effect on the hybridization of nucleic acids through the formation of AM‐guanine single‐stranded (ssDNA) adducts. The method combines the advantages of the formation and selective immunorecognition of DNA/RNA heteroduplex, with the use of MBs and amperometric transduction using disposable SPCEs. Increasing concentrations of acrylamide prevented the efficient hybridization of a synthetic biotinylated DNA probe immobilized on streptavidin‐coated magnetic beads with its complementary RNA target and the as‐formed heteroduplex recognition with a specific antibody and a secondary HRP‐labeled antibody used for electrochemical readout. The resulting magnetic bioconjugates were magnetically trapped on the surface of the working electrode of a SPCE and the amperometric transduction was carried out in the presence of the H2O2/HQ system, recording a cathodic response inversely proportional to the AM concentration. The developed bioplatform is more competitive with other reported methods in terms of simplicity and assay time, allowing the selective detection of 1 nM AM in 60 min. In addition, the bioplatform was applied to the analysis of potato crisp water extracts.

A proteomics approach to study mouse long bones: examining baseline differences and mechanical loading-induced bone formation in young-adult and old mice.

With aging, bone mass declines and the anabolic effects of skeletal loading diminish. While much research has focused on gene transcription, how bone ages and loses its mechanoresponsiveness at the protein level remains unclear. We developed a novel proteomics approach and performed a paired mass spectrometry and RNA-seq analysis on tibias from young-adult (5-month) and old (22-month) mice. We report the first correlation estimate between the bone proteome and transcriptome (Spearman ρ = 0.40), which is in line with other tissues but indicates that a relatively low amount of variation in protein levels is explained by the variation in transcript levels. Of 71 shared targets that differed with age, eight were associated with bone mineral density in previous GWAS, including understudied targets Asrgl1 and Timp2. We used complementary RNA in situ hybridization to confirm that Asrgl1 and Timp2 had reduced expression in osteoblasts/osteocytes in old bones. We also found evidence for reduced TGF-beta signaling with aging, in particular Tgfb2. Next, we defined proteomic changes following mechanical loading. At the protein level, bone differed more with age than with loading, and aged bone had fewer loading-induced changes. Overall, our findings underscore the need for complementary protein-level assays in skeletal biology research.

Inhibitory Effects on RNA Binding and RNase H Induction Activity of Prodrug-Type Oligodeoxynucleotides Modified with a Galactosylated Self-Immolative Linker Cleavable by β-Galactosidase.

Prodrug-type oligonucleotides (prodrug-ONs) are a class of oligonucleotide designed for activation under specific intracellular conditions or external stimuli. Prodrug-ONs can be activated in the target tissues or cells, thereby reducing the risk of adverse effects. In this study, we synthesized prodrug-type oligodeoxynucleotides activated by β-galactosidase, an enzyme that is overexpressed in cancer and senescent cells. These oligodeoxynucleotides (ODNs) contain a modified thymidine conjugated with galactose via a self-immolative linker at the O4-position. UV-melting analysis revealed that the modifications decreased the melting temperature (Tm) compared with that of the unmodified ODN when hybridized with complementary RNA. Furthermore, cleavage of the glycosidic bond by β-galactosidase resulted in the spontaneous removal of the linker from the nucleobase moiety, generating unmodified ODNs. Additionally, the introduction of multiple modified thymidines into ODNs completely inhibited the RNase H-mediated cleavage of complementary RNA. These findings suggest the possibility of developing prodrug-ONs, which are specifically activated in cancer cells or senescent cells with high β-galactosidase expression.

A heterozygous SPRY4 variant identified in female infertility characterized by reduced oocyte potential and early embryonic arrest.

Can novel genetic factors contributing to early embryonic arrest in infertile patients be identified, along with the underlying mechanisms of the pathogenic variant? We identified a heterozygous variant in the SPRY4 (sprouty RTK signaling antagonist 4) in infertile patients and conducted in vitro and in vivo studies to investigate the effects of the variant/deletion, highlighting its critical role in female reproductive health. SPRY4 acts as a negative regulator of receptor tyrosine kinases (RTKs) and functions as a tumor suppressor. Its abnormal expression can lead to recurrent miscarriage by affecting trophoblast function. In mice, Spry4 knockout (KO) leads to craniofacial anomalies and growth defects. A human study links the SPRY4 variant to a male patient with isolated hypogonadotropic hypogonadism (IHH), hypothetically impacting gonadotropin-releasing hormone (GnRH) neurons, and causing reproductive dysfunctions. SPRY4 is thus potentially integral in regulating endocrine homeostasis and reproductive function. To date, no study has reported SPRY4 variants associated with female fertility, and a causal relationship has not been established with functional evidence. Whole-exome sequencing (WES) was performed in 392 infertile women who suffered from primary infertility of unknown reason, and the heterozygous SPRY4 variant were identified in one independent family. The infertile patients presenting were recruited from July 2017 to November 2023. Women diagnosed with primary infertility were recruited from the Reproduction Center of Zhongshan Hospital, Fudan University. Genomic DNA was extracted from peripheral blood for WES analysis. The SPRY4 variant were identified through WES, in silico analysis, and variant screening. All variants were confirmed by Sanger sequencing. The effects of the variants were investigated in human embryonic kidney (HEK) 293T (HEK293T) cells via western blotting, and in mouse oocytes and embryos through complementary RNA (cRNA) injection, RNA sequencing, fluorescence, absorbance, and RT-qPCR assays. Gene function was further examined in Spry4 KO mice via histology, western blotting, ELISA, and RT-qPCR assays. We identified a missense heterozygous pathogenic variant in SPRY4 (GRCh38, GenBank: NM_030964.5, c.157C>T p.(Arg53Trp), rs200531302) that reduces SPRY4 protein levels in HEK293T cells and disrupts the redox system and mitochondrial function in mouse oocyte, and perturbs developmental potential in mouse embryos. These phenotypes could be partially reversed by the exogenous addition of Nrf1 cRNA. Additionally, Spry4-/- mice exhibit ovarian oxidative stress and decreased ovarian function. Due to the limited WES data and population, we identified only one family with a SPRY4 mutation. The deeper mechanism and therapeutic strategy should be further investigated through mutant mice and recovery experiment. Our study has identified a pathogenic variant in SPRY4 associated with early embryonic arrest in humans. These findings enhance our understanding of the role of SPRY4 in early embryonic development and present a new genetic marker for female infertility. This work was supported by the National Natural Science Foundation of China (82071643 and 82171655) and Natural Science Foundation of Shanghai (22ZR1456200). None of the authors have any competing interests. N/A.

Identification of leader-trailer helices of precursor ribosomal RNA in all phyla of bacteria and archaea.

Ribosomal RNAs are transcribed as part of larger precursor molecules. In Escherichia coli, complementary RNA segments flank each rRNA and form long leader-trailer (LT) helices, which are crucial for subunit biogenesis in the cell. A previous study of 15 representative species suggested that most but not all prokaryotes contain LT helices. Here, we use a combination of in silico folding and covariation methods to identify and characterize LT helices in 4464 bacterial and 260 archaeal organisms. Our results suggest that LT helices are present in all phyla, including Deinococcota, which had previously been suspected to lack LT helices. In very few organisms, our pipeline failed to detect LT helices for both 16S and 23S rRNA. However, a closer case-by-case look revealed that LT helices are indeed present but escaped initial detection. Over 3600 secondary structure models, many well supported by nucleotide covariation, were generated. These structures show a high degree of diversity. Yet, all exhibit extensive base-pairing between the leader and trailer strands, in line with a common and essential function.

Inhibition of SARS-CoV-2 growth in the lungs of mice by a peptide-conjugated morpholino oligomer targeting viral RNA

Further development of direct-acting antiviral agents against human SARS-CoV-2 infections remains a public health priority. Here, we report that an antisense peptide-conjugated morpholino oligomer (PPMO), named 5’END-2, targeting a highly conserved sequence in the 5’UTR of SARS-CoV-2 genomic RNA, potently suppressed SARS-CoV-2 growth in vitro and in vivo. In Hela-ACE 2 cells, 5’END-2 produced IC50 values of between 40 nM and 1.15 μM in challenges using six genetically disparate strains of SARS-CoV-2, including JN.1. In vivo, using K18-hACE2 mice and the WA-1/2020 virus isolate, two doses of 5’END-2 at 10 mg/kg, administered intranasally on the day before and the day after infection, produced approximately 1.4 log10 virus titer reduction in lung tissue at 3 days post-infection. Under a similar dosing schedule, intratracheal administration of 1.0 to 2.0 mg/kg 5’END-2 produced over 3.5 log10 virus growth suppression in mouse lungs. Electrophoretic mobility shift assays characterized specific binding of 5’END-2 to its complementary target RNA. Further, using reporter constructs containing SARS-CoV-2 5’UTR leader sequence, in an in-cell system, we observed that 5’END-2 could interfere with translation in a sequence-specific manner. The results demonstrate that direct pulmonary delivery of 5’END-2 PPMO is a promising antiviral strategy against SARS-CoV-2 infections and warrants further development.

Development of a Gold Nanoparticle-Based Sensor for Authentication of Organic Milk Based on Differential Levels of miRNA.

Dairy production systems significantly impact environmental sustainability, animal welfare, and human health. Intensive farming maximizes output through high-input practices, raising concerns about environmental degradation, animal welfare, and health risks from antibiotic residues. Conversely, organic farming emphasizes sustainable practices, animal welfare, and minimal synthetic inputs, potentially enhancing biodiversity, soil health, and milk quality. MicroRNAs (miRNAs), non-coding RNAs regulating gene expression, are promising biomarkers due to their response to various conditions. In this study, miRNAs bta-miR-103 and bta-miR-155, which are abundant in milk from pasture-fed cows, were selected. Additionally, bta-miR-215, which is abundant in milk fat from intensive systems, was also studied, in order to differentiate dairy production systems. A novel, cost-effective gold nanoparticle (AuNP)-based sensor was developed for miRNA detection, leveraging the unique plasmonic properties of AuNPs for visual detection. The method involves functionalizing AuNPs with complementary RNA probes and detecting miRNA-induced aggregation through colorimetric changes. This rapid, results in 30 min, and sensitive, visual limit of detection of 200 nM, assay requires minimal instrumentation and can be easily interpreted, offering significant advantages for field implementation in characterizing dairy production systems. This study demonstrates the successful application of this sensor in detecting miRNAs in 350 nM miRNA spiked raw milk, highlighting its potential for in situ dairy industry applications.

Light‐Guided Genetic Scissors Based on Phosphorene Quantum Dot

AbstractGenetic engineering faces persistent challenges in achieving precise Deoxyribonucleic acid (DNA) cleavage, especially with the limitations associated with current enzyme‐based methods, exemplified by issues in CRISPR technologies. This study introduces a groundbreaking approach: utilizing reactive oxygen species (ROS) generated by Multiphoton Absorption (MPA)‐excited Black Phosphorus Quantum Dots (BPQDs) under femto‐second laser irradiation. This innovative method not only allows excitation with lower energy but also enhances overall efficiency. The integration of complementary RNA sequences facilitates high‐efficiency, site‐selective DNA cleavage in the system, named “TADPOLE” (Targeted DNA Precision Oriented Laser Excision). Beyond its precision in targeting arbitrary DNA sequences using quantum dots, TADPOLE harnesses the unique multiphoton absorption property of BPQDs, enabling excitation with lower‐energy light sources suitable for in vivo applications in the future. Moreover, the approach integrates guiding RNA and ultrafast laser technology to provide precise control over local ROS generation and minimal heat production. This guarantees site‐specific DNA cleavage while mitigating the risk of damage to non‐targeted sequences. In summary, this study catalyzes advancements in enzyme‐free DNA cleavage technologies, with transformative implications for genetic engineering, biotechnology, and medicine. The holistic precision, versatility, and endurance presented by TADPOLE open new avenues for targeted gene therapies and transformative applications in related fields.

CD163+ macrophages in mantle cell lymphoma induce activation of prosurvival pathways and immune suppression

AbstractMantle cell lymphoma (MCL) is dependent on a supportive tumor immune microenvironment (TIME) in which infiltration of CD163+ macrophages has a negative prognostic impact. This study explores how abundance and spatial localization of CD163+ cells are associated with the biology of MCL, using spatial multiomic investigations of tumor and infiltrating CD163+ and CD3+ cells. A total of 63 proteins were measured using GeoMx digital spatial profiling in tissue microarrays from 100 diagnostic MCL tissues. Regions of interest were selected in tumor-rich and tumor-sparse tissue regions. Molecular profiling of CD163+ macrophages, CD20+ MCL cells, and CD3+ T-cells was performed. To validate protein profiles, 1811 messenger RNAs were measured in CD20+ cells and 2 subsets of T cells. Image analysis was used to extract the phenotype and position of each targeted cell, thereby allowing the exploration of cell frequencies and cellular neighborhoods. Proteomic investigations revealed that CD163+ cells modulate their immune profile depending on their localization and that the immune inhibitory molecules, V-domain immunoglobulin suppressor of T-cell activation and B7 homolog 3, have higher expression in tumor-sparse than in tumor-rich tissue regions and that targeting should be explored. We showed that MCL tissues with more abundant infiltration of CD163+ cells have a higher proteomic and transcriptional expression of key components of the MAPK pathway. Thus, the MAPK pathway may be a feasible therapeutic target in patients with MCL with CD163+ cell infiltration. We further showed the independent and combined prognostic values of CD11c and CD163 beyond established risk factors.

Divergent Biochemical Properties and Disparate Impact of Arrhythmogenic Calmodulin Mutations on Zebrafish Cardiac Function.

Calmodulin (CaM) is a ubiquitous, small cytosolic calcium (Ca2+)-binding sensor that plays a vital role in many cellular processes by binding and regulating the activity of over 300 protein targets. In cardiac muscle, CaM modulates directly or indirectly the activity of several proteins that play a key role in excitation-contraction coupling (ECC), such as ryanodine receptor type 2 (RyR2),l-type Ca2+ (Cav1.2), sodium (NaV1.5) and potassium (KV7.1) channels. Many recent clinical and genetic studies have reported a series of CaM mutations in patients with life-threatening arrhythmogenic syndromes, such as long QT syndrome (LQTS) and catecholaminergic polymorphic ventricular tachycardia (CPVT). We recently showed that four arrhythmogenic CaM mutations (N98I, D132E, D134H, and Q136P) significantly reduce the binding of CaM to RyR2. Herein, we investigate in vivo functional effects of these CaM mutations on the normal zebrafish embryonic heart function by microinjecting complementary RNA corresponding to CaMN98I, CaMD132E, CaMD134H, and CaMQ136P mutants. Expression of CaMD132E and CaMD134H mutants results in significant reduction of the zebrafish heart rate, mimicking a severe form of human bradycardia, whereas expression of CaMQ136P results in an increased heart rate mimicking human ventricular tachycardia. Moreover, analysis of cardiac ventricular rhythm revealed that the CaMD132E and CaMN98I zebrafish groups display an irregular pattern of heart beating and increased amplitude in comparison to the control groups. Furthermore, circular dichroism spectroscopy experiments using recombinant CaM proteins reveals a decreased structural stability of the four mutants compared to the wild-type CaM protein in the presence of Ca2+. Finally, Ca2+-binding studies indicates that all CaM mutations display reduced CaM Ca2+-binding affinities, with CaMD132E exhibiting the most prominent change. Our data suggest that CaM mutations can trigger different arrhythmogenic phenotypes through multiple and complex molecular mechanisms.

РОЛЬ МИКРОРНК В РАЗВИТИИ УСТОЙЧИВОСТИ РАСТЕНИЙ К ХОЛОДУ

Plants, as sessile organisms, are forced to adapt to changes in the environment, including temperature changes that limit growth, development and productivity). Abiotic stresses have become a serious problem due to their widespread nature and destructive effects on plants. However, plants have developed complex ways to perceive and respond to environmental changes. The mechanisms of plant resistance to hypothermia have been studied for a long time and transcriptional control of expression of genes sensitive to cold has been studied in some detail. To activate protective mechanisms, plants trigger a network of genetic regulators, including changing the expression of a significant part of genes using transcriptional and/or translational regulators. microRNAs can be considered important participants in the response of plants to cold stress. The key to understanding the involvement of microRNAs in plant stress reactions was studies that showed the regulation of their expression under stress. microRNAs are small non-coding RNAs that manifest themselves as important regulatory components in plants. microRNA expression includes transcription of microRNA genes by RNA polymerase II, multistage processing of primary transcripts using the enzyme DICER-LIKE1 (DCL1) and the formation of an effector complex consisting of miRNAs and proteins of the ARGONAUTE (AGO) family. Such complexes interact with complementary RNA targets, suppressing their expression at the transcriptional and post-transcriptional levels. Thus, microRNAs regulate a variety of biological processes, including responses to changes in the environment. Currently, microRNAs are considered as an important tool for regulating the work of genes. Such significant processes in plants as maintaining homeostasis, growth and development, transition from the vegetative to the reproductive phase, signaling and response to various stresses are regulated by microRNAs. The article describes the role of microRNAs in the response of plants to cold stress, analyzes microRNA targets, and discusses the prospects for using microRNAs in the practice of improving plant resistance to external influences.

Systematic assessment of long-read RNA-seq methods for transcript identification and quantification

The Long-read RNA-Seq Genome Annotation Assessment Project Consortium was formed to evaluate the effectiveness of long-read approaches for transcriptome analysis. Using different protocols and sequencing platforms, the consortium generated over 427 million long-read sequences from complementary DNA and direct RNA datasets, encompassing human, mouse and manatee species. Developers utilized these data to address challenges in transcript isoform detection, quantification and de novo transcript detection. The study revealed that libraries with longer, more accurate sequences produce more accurate transcripts than those with increased read depth, whereas greater read depth improved quantification accuracy. In well-annotated genomes, tools based on reference sequences demonstrated the best performance. Incorporating additional orthogonal data and replicate samples is advised when aiming to detect rare and novel transcripts or using reference-free approaches. This collaborative study offers a benchmark for current practices and provides direction for future method development in transcriptome analysis.

Impedimetric monitoring of microRNA-200c-3p using hydroxyapatite nanoparticles modified pencil graphite electrodes

It is important to monitor miRNA-200c-3p as it can potentially serve as an important biomarker for respiratory diseases including COVID-19 and cancer. Despite the importance of microRNA monitoring, there are few previous studies for miRNA-200c-3p monitoring, and the application of hydroxyapatite nanoparticles (HaNP) in miRNA biosensors is quite limited. This study aims to fill this gap by utilizing the advantageous properties of HaNPs to develop a powerful strategy to detect microRNA-200c-3p. First, HaNPs were modified on the surface of pencil graphite electrodes (PGEs). Subsequently, hybridization between a phosphate-labeled miRNA-200c-3p-specific DNA probe and its complementary RNA target was carried out in the solution phase. The DNA-RNA hybrid forms were immobilized on the surface of the HaNP-PGEs and the impedimetric measurements were performed. The changes at the charge transfer resistance value (Rct) were evaluated in terms of the hybridization and optimization of the experimental conditions. The detection limits (DLs) were calculated as 0.12 µg/mL (16.19 nM) in phosphate buffer solution (PBS, pH 7.40) and 0.31 µg/mL (41.82 nM) in synthetic plasma. The selectivity of the developed biosensor was tested against miRNA-200c-5p and miRNA-141-3p. The results promise a significant improvement in public health in terms of a leap forward in the early diagnosis of many serious diseases.

Detecting the FLJ22447 lncRNA in Ovarian Cancer with Cyclopentane-Modified FIT-PNAs (cpFIT-PNAs).

Ovarian cancer (OC) is one of the most lethal gynecologic cancers that is typically diagnosed at the very late stage of disease progression. Thus, there is an unmet need to develop diagnostic probes for early detection of OC. One approach may rely on RNA as a molecular biomarker. In this regard, FLJ22447 lncRNA is an RNA biomarker that is over-expressed in ovarian cancer (OC) and in cancer-associated fibroblasts (CAFs). CAFs appear early on in OC as they provide a metastatic niche for OC progression. FIT-PNAs (forced intercalation-peptide nucleic acids) are DNA analogs that are designed to fluoresce upon hybridization to their complementary RNA target sequence. In recent studies, we have shown that the introduction of cyclopentane PNAs into FIT-PNAs (cpFIT-PNA) results in superior RNA sensors. Herein, we report the design and synthesis of cpFIT-PNAs for the detection of this RNA biomarker in living OC cells (OVCAR8) and in CAFs. cpFIT-PNA was compared to FIT-PNA and the cell-penetrating peptide (CPP) of choice was either a simple one (four L-lysines) or a CPP with enhanced cellular uptake (CLIP6). The combination of CLIP6 with cpFIT-PNA resulted in a superior sensing of FLJ22447 lncRNA in OVCAR8 cells as well as in CAFs. Moreover, incubation of CLIP6-cpFIT-PNA in OVCAR8 cells leads to a significant decrease (ca. 60%) in FLJ22447 lncRNA levels and in cell viability, highlighting the potential theranostic use of such molecules.