Specific recognition and sensitive quantification of mRNA splice variants via one-pot ligation-dependent loop-mediated isothermal amplification DOI
Mai Zhang, Hui Wang, Jun Han

et al.

The Analyst, Journal Year: 2023, Volume and Issue: 148(22), P. 5605 - 5611

Published: Jan. 1, 2023

Specific recognition and sensitive quantification of mRNA alternative splice variants have been a necessity for exploring the regulatory mechanism RNA splicing revealing association between pre-mRNA transcriptome function, as well disease diagnosis. However, their wide abundance range high sequence homology pose enormous challenges sensitivity selectivity variants. Herein, taking advantage excellent specificity ligation powerful nucleic acid replication feature loop-mediated isothermal amplification (LAMP), we developed one-pot method (termed ligation-LAMP) specific based on two junction-specific stem-loop DNA probe subsequently initiating LAMP. The ligation-LAMP can specifically detect low 100 aM without any nonspecific signals quantify them with dynamics spanning at least six orders magnitude. We demonstrated that is versatile practical strategy accurately quantifying different in complex biological samples all one tube within 90 min, thereby providing an attractive tool variant-related studies.

Language: Английский

New insight into circRNAs: characterization, strategies, and biomedical applications DOI Creative Commons

Xin-Yi Feng,

Shun-Xin Zhu,

Ke‐Jia Pu

et al.

Experimental Hematology and Oncology, Journal Year: 2023, Volume and Issue: 12(1)

Published: Oct. 12, 2023

Abstract Circular RNAs (circRNAs) are a class of covalently closed, endogenous ncRNAs. Most circRNAs derived from exonic or intronic sequences by precursor RNA back-splicing. Advanced high-throughput sequencing and experimental technologies have enabled the extensive identification characterization circRNAs, such as novel types biogenesis, tissue-specific cell-specific expression patterns, epigenetic regulation, translation potential, localization metabolism. Increasing evidence has revealed that participate in diverse cellular processes, their dysregulation is involved pathogenesis various diseases, particularly cancer. In this review, we systematically discuss databases, challenges for circRNA discovery, new insight into strategies used studies biomedical applications. Although recent advanced understanding knowledge approaches annotation, functional applications continuously needed to provide insights circRNAs. The emergence circRNA-based protein strategy will be promising direction field biomedicine.

Language: Английский

Citations

54
circAtlas 3.0: a gateway to 3 million curated vertebrate circular RNAs based on a standardized nomenclature scheme DOI Creative Commons
Wanying Wu, Fangqing Zhao, Jinyang Zhang

et al.

Nucleic Acids Research, Journal Year: 2023, Volume and Issue: 52(D1), P. D52 - D60

Published: Sept. 22, 2023

Abstract Recent studies have demonstrated the important regulatory role of circRNAs, but an in-depth understanding comprehensive landscape circRNAs across various species still remains unexplored. The current circRNA databases are often species-restricted or based on outdated datasets. To address this challenge, we developed circAtlas 3.0 database, which contains a rich collection 2674 sequencing datasets, curated to delineate within 33 distinct tissues spanning 10 vertebrate species. Notably, represents substantial advancement over its precursor, 2.0, with number cataloged escalating from 1 007 087 3 179 560, 2 527 528 them being reconstructed into full-length isoforms. also introduces several notable enhancements, including: (i) integration both Illumina and Nanopore datasets detect extended lengths; (ii) employment standardized nomenclature scheme for providing information host gene circular exons; (iii) inclusion clinical cancer samples explore biological function context (iv) links other useful resources enable user-friendly analysis target circRNAs. updated provides platform exploring evolution implications is freely available at http://circatlas.biols.ac.cn https://ngdc.cncb.ac.cn/circatlas.

Language: Английский

Citations

46
Circular RNAs in neurological conditions – computational identification, functional validation, and potential clinical applications DOI Creative Commons

Oak Hatzimanolis,

Alex M. Sykes, Alexandre S. Cristino

et al.

Molecular Psychiatry, Journal Year: 2025, Volume and Issue: unknown

Published: Feb. 17, 2025

Abstract Non-coding RNAs (ncRNAs) have gained significant attention in recent years due to advancements biotechnology, particularly high-throughput total RNA sequencing. These developments led new understandings of non-coding biology, revealing that approximately 80% regions the genome possesses biochemical functionality. Among ncRNAs, circular (circRNAs), first identified 1976, emerged as a prominent research field. CircRNAs are abundant most human cell types, evolutionary conserved, highly stable, and formed by back-splicing events which generate covalently closed ends. Notably, circRNAs exhibit high expression levels neural tissue perform diverse functions, including acting molecular sponges for microRNAs, interacting with RNA-binding proteins regulate their availability activity, modulating transcription splicing, even translating into functional peptides some cases. Recent computational experimental methods enhanced our ability identify validate circRNAs, providing valuable insights biological roles. This review focuses on circRNA they related neuropsychiatric neurodegenerative conditions. We also explore potential applications clinical diagnostics, therapeutics, future directions. remain relatively underexplored area context neurological disorders. However, emerging evidence supports role critical players etiology mechanisms conditions such schizophrenia, bipolar disorder, major depressive Alzheimer’s disease, Parkinson’s disease. findings suggest may provide novel framework contributing dysfunctions underpinning these complex

Language: Английский

Citations

2
Triple Amplification Strategy of CHA-PER-DNAzyme for Ultrasensitive Detection of circRNA Associated with Hepatocellular Carcinoma DOI

Yanheng Yao,

Wenting Cheng, Tingting Li

et al.

Analytical Chemistry, Journal Year: 2023, Volume and Issue: 95(35), P. 13149 - 13155

Published: Aug. 22, 2023

Biological and clinical studies have indicated that aberrant expression of circMTO1 served as a crucial biomarker for the diagnosis prognosis hepatocellular carcinoma (HCC) patients well potential therapeutic target. However, detection circRNAs currently faces challenges such homologous linear RNA interference low-expression abundance certain circRNAs. Therefore, we developed triple amplification method based on catalytic hairpin assembly (CHA) activation by back-splice junction (BSJ), resulting in CHA products triggered primer exchange reaction to generate DNAzyme. Subsequently, DNAzyme cleaved fluorescent reporter chain, enabling ultrasensitive carcinoma-associated through output fluorescence signal. The opening sequence within specifically targeted BSJ circRNA, thereby avoiding false positive signals observed circRNA assays due recognition molecules. Moreover, this facilitated sensitive addressed issue low-abundance levels associated with HCC samples. Notably, our newly designed assay detecting exhibited range from 1 fM 100 nM limit 0.265 fM. Furthermore, it demonstrated excellent consistent performance even complex systems. Our proposed enabled specific various cancer cells blood samples patients, providing an innovative approach investigating role tumorigenesis development while promoting its application.

Language: Английский

Citations

27
Current advances in circular RNA detection and investigation methods: Are we running in circles? DOI
Rareş Drulă, Cornelia Braicu, Ioana Berindan‐Neagoe

et al.

Wiley Interdisciplinary Reviews - RNA, Journal Year: 2024, Volume and Issue: 15(3)

Published: May 1, 2024

Abstract Circular RNAs (circRNAs), characterized by their closed‐loop structure, have emerged as significant transcriptomic regulators, with roles spanning from microRNA sponging to modulation of gene expression and potential peptide coding. The discovery functional analysis circRNAs been propelled advancements in both experimental bioinformatics tools, yet the field grapples challenges related detection, isoform diversity, accurate quantification. This review navigates through evolution circRNA research methodologies, early detection techniques current state‐of‐the‐art approaches that offer comprehensive insights into biology. We examine limitations existing methods, particularly difficulty differentiating isoforms distinguishing linear counterparts. A critical evaluation various tools novel strategies is presented, emphasizing need for integrated enhance our understanding interpretation functions. Our underscore dynamic rapidly advancing nature research, highlighting ongoing development analytical frameworks designed address complexity facilitate assessment clinical utility. As such, this overview aims catalyze further study, fostering a deeper cellular processes implications disease. article categorized under: RNA Methods > Nanotechnology Analyses Cells In Vitro Silico

Language: Английский

Citations

10
Past, present, and future strategies for detecting and quantifying circular RNA variants DOI Creative Commons
Lin He, Vanessa M. Conn, Simon J. Conn

et al.

FEBS Journal, Journal Year: 2025, Volume and Issue: unknown

Published: Feb. 11, 2025

Circular RNAs (circRNAs) are a family of covalently closed RNA transcripts ubiquitous across the eukaryotic kingdom. CircRNAs generated by class alternative splicing called backsplicing, with resultant circularization part parental producing characteristic backsplice junction (BSJ). Because noncontiguous sequence BSJ respect to DNA genome, circRNAs remained hidden in plain sight through over decade next-generation sequencing, yet 3 million unique circRNA have been illuminated past alone. expressed cell type-specific manner, highly stable, many examples being evolutionarily conserved and/or functional specific contexts. However, can be very lowly and predictions context from BSJ-spanning reads alone confound extrapolation exact composition transcript. For these reasons, ultrasensitive detection, combined enrichment, bespoke bioinformatics pipelines and, more recently, long-read, processive sequencing is becoming critical for complete characterization all variants. Concomitantly, need targeted detection quantification has sparked numerous laboratory-based commercial approaches visualize cells quantify them biological samples, including biospecimens. This review focuses on advancements circRNAs, particular focus recent bolster variants accurately normalize between libraries.

Language: Английский

Citations

1
Lighting up three-dimensional nanolantern for circular RNA imaging and precise gene therapy DOI

Zheng Shi,

Jinping Hu,

Zichen Jiao

et al.

Biosensors and Bioelectronics, Journal Year: 2025, Volume and Issue: 276, P. 117273 - 117273

Published: Feb. 15, 2025

Language: Английский

Citations

1
Mitigating Cellular Dysfunction Through Contaminant Reduction in Synthetic circRNA for High‐Efficiency mRNA‐Based Cell Reprogramming DOI Creative Commons

Z.D. Zhang,

Lixin Tian, Xiangyu Ren

et al.

Advanced Science, Journal Year: 2025, Volume and Issue: unknown

Published: March 5, 2025

Synthetic circular RNA (circRNA) holds great promise for biomedical research and therapeutic applications, but impurities introduced during synthesis trigger innate immune responses significantly compromise its efficacy. In this study, key immunogenic byproducts, including double-stranded RNA, 5' triphosphates from uncircularized hydrolyzed fragments, are identified as impairing circRNA functionality via RNA-sensing pathways. To address this, a multi-step purification process is developed that combines enzymatic treatments cellulose-based filtration to effectively remove these contaminants. This approach reduces activation increases manufacturing yields of by over 10-fold. The purified demonstrates exceptional performance in induced pluripotent stem cells (iPSCs) reprogramming, achieving 300% reprogramming efficiency with just three OSKMLN transfection treatments. Additionally, the achieves high levels persistent expression chimeric antigen receptor (CAR) T cytotoxicity against tumor cells, making it promising candidate mRNA-based CAR-T cell therapy. These findings establish strategy scalable reliable platform producing therapeutic-grade broad applications regenerative medicine cancer immunotherapy.

Language: Английский

Citations

1
FL-circAS: an integrative resource and analysis for full-length sequences and alternative splicing of circular RNAs with nanopore sequencing DOI Creative Commons

Tai-Wei Chiang,

Song-En Jhong,

Yu‐Chen Chen

et al.

Nucleic Acids Research, Journal Year: 2023, Volume and Issue: 52(D1), P. D115 - D123

Published: Oct. 12, 2023

Abstract Circular RNAs (circRNAs) are RNA molecules with a continuous loop structure characterized by back-splice junctions (BSJs). While analyses of short-read sequencing have identified millions BSJ events, it is inherently challenging to determine exact full-length sequences and alternatively spliced (AS) isoforms circRNAs. Recent advances in nanopore long-read circRNA enrichment bring an unprecedented opportunity for investigating the issues. Here, we developed FL-circAS (https://cosbi.ee.ncku.edu.tw/FL-circAS/), which collected such data 20 cell lines/tissues thereby 884 636 BSJs 1 853 692 human 115 173 135 617 mouse. also provides multiple features. For expression, calculates expression levels each isoform, line/tissue specificity at both isoform levels, AS entropy across samples. biogenesis, identifies reverse complementary binding protein (RBP) sites residing flanking BSJs. functional patterns, potential microRNA/RBP several types evidence translation on isoform. user-friendly interfaces browsing, searching, analyzing, downloading data, serving as first resource discovering circRNAs level.

Language: Английский

Citations

15
Comprehensive analysis of m6A methylome alterations after azacytidine plus venetoclax treatment for acute myeloid leukemia by nanopore sequencing DOI Creative Commons

Zaifeng Zhang,

Lili Zhang, Jiangtao Li

et al.

Computational and Structural Biotechnology Journal, Journal Year: 2024, Volume and Issue: 23, P. 1144 - 1153

Published: March 2, 2024

Language: Английский

Citations

4