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Nature Reviews. Genetics Nov 2019Over the past decade, RNA sequencing (RNA-seq) has become an indispensable tool for transcriptome-wide analysis of differential gene expression and differential splicing... (Review)
Review
Over the past decade, RNA sequencing (RNA-seq) has become an indispensable tool for transcriptome-wide analysis of differential gene expression and differential splicing of mRNAs. However, as next-generation sequencing technologies have developed, so too has RNA-seq. Now, RNA-seq methods are available for studying many different aspects of RNA biology, including single-cell gene expression, translation (the translatome) and RNA structure (the structurome). Exciting new applications are being explored, such as spatial transcriptomics (spatialomics). Together with new long-read and direct RNA-seq technologies and better computational tools for data analysis, innovations in RNA-seq are contributing to a fuller understanding of RNA biology, from questions such as when and where transcription occurs to the folding and intermolecular interactions that govern RNA function.
Topics: Alternative Splicing; Gene Expression Profiling; High-Throughput Nucleotide Sequencing; History, 21st Century; Humans; RNA, Messenger; Sequence Analysis, RNA
PubMed: 31341269
DOI: 10.1038/s41576-019-0150-2 -
Current Protocols Feb 2021Synthetic messenger RNA (mRNA)-based therapeutics are an increasingly popular approach to gene and cell therapies, genome engineering, enzyme replacement therapy, and...
Synthetic messenger RNA (mRNA)-based therapeutics are an increasingly popular approach to gene and cell therapies, genome engineering, enzyme replacement therapy, and now, during the global SARS-CoV-2 pandemic, vaccine development. mRNA for such purposes can be synthesized through an enzymatic in vitro transcription (IVT) reaction and formulated for in vivo delivery. Mature mRNA requires a 5'-cap for gene expression and mRNA stability. There are two methods to add a cap in vitro: via a two-step multi-enzymatic reaction or co-transcriptionally. Co-transcriptional methods minimize reaction steps and enzymes needed to make mRNA when compared to enzymatic capping. CleanCap AG co-transcriptional capping results in 5 mg/ml of IVT with 94% 5'-cap 1 structure. This is highly efficient compared to first-generation cap analogs, such as mCap and ARCA, that incorporate cap 0 structures at lower efficiency and reaction yield. This article describes co-transcriptional capping using TriLink Biotechnology's CleanCap AG in IVT. © 2021 Wiley Periodicals LLC. Basic Protocol 1: IVT with CleanCap Basic Protocol 2: mRNA purification and analysis.
Topics: Humans; In Vitro Techniques; Protein Biosynthesis; RNA Cap Analogs; RNA Stability; RNA, Messenger
PubMed: 33524237
DOI: 10.1002/cpz1.39 -
Tissue Engineering. Part A Jan 2019The ability to control cellular processes and precisely direct cellular reprogramming has revolutionized regenerative medicine. Recent advances in in vitro transcribed... (Review)
Review
The ability to control cellular processes and precisely direct cellular reprogramming has revolutionized regenerative medicine. Recent advances in in vitro transcribed (IVT) mRNA technology with chemical modifications have led to development of methods that control spatiotemporal gene expression. Additionally, there is a current thrust toward the development of safe, integration-free approaches to gene therapy for translational purposes. In this review, we describe strategies of synthetic IVT mRNA modifications and nonviral technologies for intracellular delivery. We provide insights into the current tissue engineering approaches that use a hydrogel scaffold with genetic material. Furthermore, we discuss the transformative potential of novel mRNA formulations that when embedded in hydrogels can trigger controlled genetic manipulation to regenerate tissues and organs in vitro and in vivo. The role of mRNA delivery in vascularization, cytoprotection, and Cas9-mediated xenotransplantation is additionally highlighted. Harmonizing mRNA delivery vehicle interactions with polymeric scaffolds can be used to present genetic cues that lead to precise command over cellular reprogramming, differentiation, and secretome activity of stem cells-an ultimate goal for tissue engineering.
Topics: Animals; Cell Differentiation; Drug Delivery Systems; Humans; RNA, Messenger; Regenerative Medicine; Stem Cells; Tissue Engineering
PubMed: 29661055
DOI: 10.1089/ten.TEA.2017.0444 -
Wiley Interdisciplinary Reviews.... Mar 2019Messenger RNA (mRNA) has become a promising class of drugs for diverse therapeutic applications in the past few years. A series of clinical trials are ongoing or will be... (Review)
Review
Messenger RNA (mRNA) has become a promising class of drugs for diverse therapeutic applications in the past few years. A series of clinical trials are ongoing or will be initiated in the near future for the treatment of a variety of diseases. Currently, mRNA-based therapeutics mainly focuses on ex vivo transfection and local administration in clinical studies. Efficient and safe delivery of therapeutically relevant mRNAs remains one of the major challenges for their broad applications in humans. Thus, effective delivery systems are urgently needed to overcome this limitation. In recent years, numerous nanoscale biomaterials have been constructed for mRNA delivery in order to protect mRNA from extracellular degradation and facilitate endosomal escape after cellular uptake. Nanoscale platforms have expanded the feasibility of mRNA-based therapeutics, and enabled its potential applications to protein replacement therapy, cancer immunotherapy, therapeutic vaccines, regenerative medicine, and genome editing. This review focuses on recent advances, challenges, and future directions in nanoscale platforms designed for mRNA delivery, including lipid and lipid-derived nanoparticles, polymer-based nanoparticles, protein derivatives mRNA complexes, and other types of nanomaterials. This article is categorized under: Nanotechnology Approaches to Biology > Nanoscale Systems in Biology Biology-Inspired Nanomaterials > Lipid-Based Structures Biology-Inspired Nanomaterials > Nucleic Acid-Based Structures.
Topics: Animals; Gene Transfer Techniques; Humans; Lipids; Nanoparticles; Nanotechnology; Proteins; RNA, Messenger
PubMed: 29726120
DOI: 10.1002/wnan.1530 -
Nature Sep 2023
Topics: RNA, Messenger; Biotechnology
PubMed: 37253980
DOI: 10.1038/d41586-023-01745-z -
Annual Review of Biochemistry 1974
Review
Topics: Adenine Nucleotides; Animals; Cell Nucleus; Centrifugation, Zonal; Crystallins; Cytoplasm; Dactinomycin; Drug Stability; Globins; Histones; Immunoglobulins; Molecular Weight; Myosins; Nucleic Acid Conformation; Ovalbumin; Polynucleotides; RNA, Messenger; Species Specificity
PubMed: 4604447
DOI: 10.1146/annurev.bi.43.070174.003201 -
Nature Jan 2024
Topics: RNA, Messenger; RNA; Proteins
PubMed: 38093050
DOI: 10.1038/d41586-023-03787-9 -
Science (New York, N.Y.) Apr 1998Fluorescence in situ hybridization (FISH) and digital imaging microscopy were modified to allow detection of single RNA molecules. Oligodeoxynucleotide probes were...
Fluorescence in situ hybridization (FISH) and digital imaging microscopy were modified to allow detection of single RNA molecules. Oligodeoxynucleotide probes were synthesized with five fluorochromes per molecule, and the light emitted by a single probe was calibrated. Points of light in exhaustively deconvolved images of hybridized cells gave fluorescent intensities and distances between probes consistent with single messenger RNA molecules. Analysis of beta-actin transcription sites after serum induction revealed synchronous and cyclical transcription from single genes. The rates of transcription initiation and termination and messenger RNA processing could be determined by positioning probes along the transcription unit. This approach extends the power of FISH to yield quantitative molecular information on a single cell.
Topics: Actins; Animals; Cell Line; Fluorescein-5-isothiocyanate; In Situ Hybridization, Fluorescence; Kinetics; Oligonucleotide Probes; RNA Processing, Post-Transcriptional; RNA, Messenger; Rats; Transcription, Genetic
PubMed: 9554849
DOI: 10.1126/science.280.5363.585 -
Molekuliarnaia Biologiia 2015Genomewide mapping of posttranscriptional modification in eukaryotic RNA allowed to reveal tens of thousands modification sites. Among modified nucleotides of eukaryotic... (Review)
Review
Genomewide mapping of posttranscriptional modification in eukaryotic RNA allowed to reveal tens of thousands modification sites. Among modified nucleotides of eukaryotic RNA 6-methyladenosine, 5-methylcytidine, pseudouridine, inosine, and others. Many modification sites are conserved, many are regulated. Function is known for a small subset of modified nucleotides, while the role of majority of them is still obscure. Global character of mRNA modifications allowed scientists to coin a new term, RNA epigenetics. The review is about posttranscriptional messenger RNA modifications in eukaryotes. Main modifications, their role in cell, their mapping techniques and proteins, that are responsible for such RNA modifications are observed.
Topics: Animals; Humans; RNA Processing, Post-Transcriptional; RNA, Messenger; RNA-Binding Proteins
PubMed: 26710771
DOI: 10.7868/S0026898415060142 -
Nature Jan 2017A growing number of nucleobase modifications in messenger RNA have been revealed through advances in detection and RNA sequencing. Although some of the biochemical... (Review)
Review
A growing number of nucleobase modifications in messenger RNA have been revealed through advances in detection and RNA sequencing. Although some of the biochemical pathways that involve modified bases have been identified, research into the world of RNA modification - the epitranscriptome - is still in an early phase. A variety of chemical tools are being used to characterize base modifications, and the structural effects of known base modifications on RNA pairing, thermodynamics and folding are being determined in relation to their putative biological roles.
Topics: Animals; Humans; Mass Spectrometry; Molecular Structure; RNA, Messenger; Sequence Analysis, RNA; Transcriptome
PubMed: 28102265
DOI: 10.1038/nature21351