Abstract: Unlike other large biomolecules, RNA carries two main tasks: an informational coding potential governed by its sequence and a catalytic/regulatory role, determined by its secondary and tertiary structure. During the last decade, a significant improvement in biophysical and biochemical techniques has enabled researchers to initiate exploratory studies on the relationship between RNA structure and its function. Among other technological improvements, the explosion of next generation sequencing (NGS) tools has allowed the transcriptome-wide investigation of RNA folding in cells. Deeper knowledge of 2D and 3D structures is extremely important for understanding the mechanisms of RNA function as well as for designing synthetic RNAs and the development of RNA-targeted drugs. RNA molecules can adopt specific 3D motifs that are now considered druggable and offer untapped potential to therapeutically modulate numerous cellular processes, including those linked to ‘undruggable’ protein targets. In parallel to the growing interest for the RNA targetome in the pharmaceutical sector, the in-silico modelling of RNA folds is developing complementary methods. Currently, RNA structure probing methods can only capture partial structure information. The ability to directly measure intact RNA structures could facilitate investigations of the functions and regulation mechanisms as well as druggability. In this chapter, we will summarize biophysical and biochemical strategies for determining RNA structures/motifs, including latest approaches that combine molecular biology strategies with NGS readouts.

Abstract: G-quadruplexes are four-stranded nucleic acid structures, the bases of which are linked through Hoogsteen hydrogen bonding. RNA G-quadruplex is shown to take part in a wide range of cellular events such as telomere maintenance, gene expression mechanisms, etc. RNA G-quadruplex is found to be present in a significant portion of the non-coding transcriptome, thus acting as a biomarker for several diseases. RNA quadruplex is also shown to be associated with neurogenerative diseases and cancer; thus, it is a potential therapeutic target. For these reasons, it is necessary to understand the sequence, structure and functional relevance of RNA G-quadruplex. A comprehensive analysis of the structural and folding characteristics of RNA G-quadruplex structures deposited in the PDB has therefore been carried out. A total of 10 unique folds are found to be present in RNA quadruplex. Together with the existing algorithms for the transcriptome-wide prediction of G-quadruplexes, the structural features discussed here would help in the modeling of G-quadruplex structures.

Abstract: The genome is pervasively transcribed and produces various messenger RNAs and noncoding RNAs. Defective transcripts are also produced, which are cleared by RNA surveillance and quality control systems. Nuclear RNA degradation pathways play important roles in these systems and in shaping the transcriptome and preventing diseases. In this review, we summarize current knowledge of nuclear noncoding RNAs. We then discuss nuclear RNA degradation factors involved in RNA surveillance and RNA quality control systems.

Abstract: Like proteins, an RNA is only functional when it is folded into its native conformation and adopts a specific secondary and tertiary structure. Hence, the analysis of RNA structure is essential to understand the cellular roles of distinct RNA molecules. Technical approaches used to study RNA structure comprise bioinformatics tools, structural probing, and biophysical methods to integrate sequence and 3D structure information. In this review, I focus on structural probing techniques of RNA secondary structure. I discuss basic enzymatic and chemical probing techniques, and present novel approaches in combination with high-throughput sequencing. A focus is laid on SHAPE techniques and its various developments and applications. Finally, at the example of RNA G-quadruplexes, it is highlighted how an array of probing techniques can be combined to study a specific RNA structural motif in vitro and in vivo.

Abstract: Small non-coding RNAs (sRNAs) are key post-transcriptional regulators of gene expression in bacteria, with a diversity of origins, sequences, structures, and modes of action among their members. The variety within these regulatory RNAs makes it difficult to unify this remarkable heterogeneous class of RNA molecules. Structural determinants along with nucleotide sequence play important roles in defining the sRNA ability to interact with their targets. Most sRNAs bind to mRNA targets, either acting as repressors or activators. Nevertheless, the sRNAs themselves are subject to post-transcriptional regulation, either by ribonucleases, RNA chaperones, and other RNA-binding proteins, as well as RNA sponges. In this chapter we summarize the information on structure and function of bacterial sRNAs and provide several examples to better illustrate the broad diversity of this class of regulatory RNAs.

Abstract: G-quadruplexes (G4s) are four-stranded nucleic acid secondary structures that are formed by the stacking of square planar guanine arrangements and stabilized by favorable cations. Potential G4-forming sequences are distributed in the regulatory regions of the genome and transcriptome. G4s are proposed to modulate various physiological and pathophysiological cellular processes. As such RNA G4s (rG4s) have been implicated in several key processes of gene regulation such as RNA maturation, mRNA translation, and RNA transport. rG4s often impact cellular biology by associating different RNA binding proteins, both of which could act as crucial therapeutic targets in the fight for developing novel therapeutics for the diseases associated with rG4-containing transcripts.

Abstract: The development of high-throughput total RNA sequencing technologies and novel computational tools have discovered the ubiquitous expression of circular RNAs (circRNAs) in eukaryotes across yeast to humans. Hundreds of studies demonstrated that circRNAs are abundant, conserved, stable, and expressed in a tissue-specific manner. Although a huge number of circRNAs are known to be expressed in various organisms, the cellular functions of most circRNAs remain to be explored. Several computational pipelines were developed for the systematic analysis of circRNA functions. This chapter describes an easy-to-use workflow for computational analysis of circRNA functions prior to experimental analysis. Here, we illustrate the main steps of circRNA functional analysis, including prediction of mature circRNA sequence, circRNA-RBP interaction, circRNA-miRNA-mRNA regulatory network, and protein-coding ability.

Abstract: More than 50 years ago, viroids were firstly described as the smallest RNA molecules capable to infect certain plants and to autonomously self-replicate in host plants. Viroids are covalently closed circular single-stranded RNAs that are non-coding and depend for most of their infection cycle on host proteins. Today, viroids are subdivided into the two families Avsunviroidae and Pospiviroidae. Members of Avsunviroidae replicate in the chloroplast and have a highly bifurcated structure including hammerhead ribozymes, which cleave oligomeric replication intermediates into monomers and ligate them to mature circles. Members of Pospiviroidae accumulate in the nucleus, have a rod-like structure and depend on host proteins for cleavage and ligation. We will describe our present knowledge on sequence and structural elements of viroids in connection to their replication and trafficking.

Abstract: Prostate cancer (CaP) is associated with considerably reduced overall survival with high mortality rates due to the development of resistance to both standard and novel therapies. This acquired resistance is a multifactorial problem involving the interplay of several genetic and epigenetic alterations that contribute significantly to the growth, invasiveness, and metastasis of CaP. A deeper understanding may lead to novel therapeutic interventions and ultimately prolong patient survival. The recent progress in high-throughput RNA sequencing (RNA-seq) techniques has identified several non-coding RNAs (ncRNAs) that play important roles in the progression of different diseases including cancer. ncRNAs are accumulated at higher concentrations in serum, plasma, and/or urine samples of CaP patients. Thus, differential involvement of ncRNA from normal patients to localised and metastatic cancers, makes it an acceptable biomarker for early diagnosis of metastatic disease. In this chapter, we highlight the emerging impacts and the translational applications of non-coding RNA in CaP progression for developing noble therapeutic strategies.

Abstract: The great potential of nucleic acids as therapeutics has been recognized for a while but has experienced a tremendous attention with the recent development of RNA vaccines. In contrast to protein-targeting strategies, nucleic acid-based approaches often have the advantage that the required target selectivity is not realized via matching a specific structure, but plainly via the primary sequence of the applied RNA or DNA construct. This sequence is then either directly processed or comprises an additional unit capable of processing a target molecule. The latter is true for a number of DNA sequences, called DNAzymes, that are capable of both binding and processing a target with a high selectivity. While the mRNA technology has the inherent strength of bringing something into the system, RNA-processing DNA catalysts such as RNA-cleaving DNAzymes have the inherent strength of taking something out of the system. Consequently, the DNAzyme technology has the potential to emerge as counterpart to the mRNA technology. However, and in line with the endeavors that were required for the success of the mRNA technology, specific improvements need to be realized to unravel the full potential of RNA-processing DNAzymes. This review provides an overview of recent findings and remaining limitations.

Abstract: RNAs can form complex 3D structures to influence biology and disease. Small self-cleaving ribozymes and riboswitches are some well-characterized examples of structured RNAs, functional RNA sequences with 3D structures. In this chapter, we will discuss the structural features of hammerhead, hairpin, glmS, and twister small self-cleaving ribozymes that are relevant for their biological function, and specific biochemical studies that help elucidate the mechanisms of their self-cleavage reactions. We will also discuss the structural elements of the bacterial purine and thiamine pyrophosphate riboswitches responsible for recognizing specific ligands. Binding to their cognate ligands is an essential step in the regulation of gene expression by the riboswitches. Structured RNAs have also been targeted for developing drugs such as Ribocil and Risdiplam/Branaplam. These drugs are notable examples of approved therapies for bacterial infections and spinal muscular atrophy, respectively, that target the RNA structures. In this chapter, we will discuss the targeting potential of riboswitches for developing antibacterial therapy and the mechanism of Ribocil recognition by the FMN riboswitch.

Abstract: RNA modifications contribute to the various functions of RNAs in all living organisms. Some of these modifications are dynamic and contribute to the regulation of gene expression. In bacteria, their roles in stress, environmental adaptation, and in infections caused by pathogens have been recently fully recognized. In this review, we describe several methodologies including mass spectrometry, next-generation RNA sequencing methods, biochemical approaches, and cryo-EM structural analysis that are used to detect and localize the modifications in tRNAs and rRNAs. We illustrate how the combination of methods was necessary to avoid technical biases for a successful mapping of the modifications in tRNAs and rRNAs in Staphylococcus aureus.

Abstract: More than 100 modified nucleosides with different structures and functions are known as components of nucleic acids. Carbohydrate-modified (disaccharide) nucleosides are components of tRNA and poly(ADP-ribose) and also participate as second messengers in plants and animals. Base-modified nucleosides contain methylations, acylations, hydroxylations, amino acid and hydrocarbon functionalities, cyclic structure, sulfur or selenium. These modifications are represented in various types of RNA (transport—t, ribosomal—r, matrix—m, small-interfering—si, non-coding—nc RNAs) among all domains of life. They have many important biological implications: RNA splicing, protein biosynthesis, altering RNA structure and functional organization of ribosomes. Many reviews and books were devoted to this theme, but the problems accompanied by the structural diversity of ribonucleosides and their participation in the regulation of macromolecules’ biosynthesis are updated with novel, complex data. In this chapter, general aspects of the structure and functions of modified nucleosides as minor RNA components are given in considering novel scientific achievements. This work highlights essential structural features of various general classes of naturally modified nucleosides and their biosynthetic formation and biological functions. A significant part of this work is devoted to medicinal chemistry. Here we consider the mechanism of action of synthetic nucleosides and drugs on their basis, changing properties of viral RNAs and thus leading to inhibition of viral reproduction and application of nucleoside stable isotope labeled internal standards (SILIS) for analysis of RNA probes.

Abstract: RNAs form complex structures in vivo to perform diverse functions. These RNA structures dynamically change in response to internal cellular regulatory factors. Latest technological innovations have allowed us to detect RNA structures on a large scale with recent analyses of RNA structure revealing how dynamic RNA structures mediate RNA regulatory functions. In this chapter, we review the latest technological advances in RNA structure probing and analysis. We also outline the technical challenges of RNA structure probing methods and discuss future directions.

Abstract: From initially overlooked as peculiarities of uncertain biological importance or rare isoforms generated as a result of splicing errors to commonly regarded as relevant regulatory molecules, circular RNAs (circRNAs) now represent an extensively studied subgroup of non-coding RNAs (ncRNAs). The structural uniqueness of these endogenous biomolecules confers resistance to exonuclease activity, resulting in higher stability than observed in their linear counterparts, which alongside their reported function of gene expression regulators, predisposes them to potential use as robust biomarkers and/or powerful therapeutic targets. During the last decade, we have witnessed a boom in circRNA research deciphering various mechanisms of their biogenesis and an in-depth description of their biological functions, including miRNA and protein sponging, yet regulation of these events remains unclear. Despite recent advances in high-throughput genomic technology and novel bioinformatic approaches allowing circRNAs characterization in detail, analyzing these molecules continues to be challenging, leaving multiple biological knowledge gaps unsolved.

Abstract: Cell death is attributed to the unrecoverable disturbance of cells exposed to intracellular or extracellular stimulus. Regulated cell death (RCD), also known as programmed cell death (PCD), involves signaling cascades in which effector molecules participate in and play important roles for maintaining organism homeostasis. As a basic biological phenomenon of cells, RCD displays exceptional influence on the occurrence and development of many pathophysiological processes. N6-methyladenosine (m6A) is an important epigenetic modification that regulates RNA fate and subsequent bioprocesses. It occurs extensively on multiple RNA species and is the most abundant internal modification of eukaryotic messenger RNA (mRNA). The abundance of m6A modification is regulated by its methyltransferases and demethylases, and the diverse biological functions of m6A are executed by distinct m6A binding proteins. Accruing evidence shows that m6A modification participates in eukaryotic cell fate transition and determination. In recent years, the disorder of m6A modification status has been observed during the initiation and progression of various RCDs, including classical apoptosis, necroptosis, pyroptosis, and the newly described ferroptosis. Due to the importance of m6A and RCD in cellular and organismal homeostasis, it is worthwhile to recapitulate and organize the current knowledge of the crosstalk between m6A status and RCDs with distinct biochemical and morphological features, as well as immune consequences. In this chapter, we systematically summarize the regulatory networks of m6A modification on RCDs to provide the profiling of m6A-RCD regulatory axis as comprehensively as possible.

Abstract: RNA nanotechnology is the bottom-up self-assembly of nanoscale RNA structures, the main framework of which is mainly composed of RNA. Scaffolds, ligands, therapeutics, and modulators can all be composed of RNA. Classical RNA research has focused on interactions and 2D/3D structures within RNA. RNA nanotechnology can elucidate and exploit RNA-RNA interactions and quaternary (4D) structures. RNA technology is like “Lego bricks”. Ideal materials for building blocks should have the following attributes: 1. Diversity. 2. Capable of constructing structures with various shapes, sizes, and modifiable stoichiometry. 3. Mix together to self-assemble. 4. Thermodynamically, chemically, and enzymatically stable, with long shelf life. The potential of RNA NPs (nanoparticles) in various drug delivery applications including anticancer drug delivery is enormous. Therefore, RNA NPs can be developed in cancer research for chemotherapy and immunotherapy. The next decade is expected to witness many clinical trials in this field. RNA instability is no longer an issue and can be overcome through various chemical modifications. If we look at the big picture, the therapeutic potential of RNA, well-defined RNA NPs, and recent successes in stabilizing them not only support that RNA nanotechnology at the beginning of a new era but also suggest that future therapeutic prospects may predominate. Only 1.5% of the human genome encodes proteins. A significant portion of the remaining 98.5% of so-called “junk DNA” codes for important small or long non-coding RNAs. All substances that are toxic to cells are known to stop cancer growth. Currently, most cancer chemotherapies are highly toxic to normal cells and viral organoids. RNA nanoparticles have been shown to be a motile, dynamic, and deformable material that spontaneously runs enriched with cancer vasculature and is rapidly excreted into the urine via the glomerulus with little accumulation in vital organs. Therefore, RNA nanoparticles can efficiently and specifically target tumor tissues while being rapidly cleared from the body. Thus, RNA technology can convert toxic drugs in chemotherapy into non-toxic drugs. The promise of RNA as the third milestone in drug development has come true. Importantly, RNA itself can be used as a drug, such as siRNA, miRNA, aptamer, ribozyme, anti-miRNA, etc. Another area of interest is the use of small chemical drugs as ligands targeting non-coding RNA or mRNA. This field has been attractive and emerging but progress has been slow. This segment is expected to witness significant growth in the coming years.

Abstract: RNA molecules are highly versatile and dynamic components within cells that orchestrate fundamental molecular biological processes, including information transfer and decoding, splicing, transcriptional regulation and translation. These vital processes are in large part determined by the complex structures and modular folding adopted by RNA. Nanopore technology has emerged as a powerful technique to interrogate native RNA directly at the single molecule level. In this chapter we will highlight and discuss applications of nanopore technology to investigate the conformation of RNA and its interaction partners. First, we will feature RNA translocation experiments which can extract key signatures of RNA size and global secondary or tertiary structure using nanopore electrical and temporal measurements. Second, we will focus on applications of nanopore technology to decipher RNA–ligand and RNA–protein interactions. Finally, we will summarize recent approaches using direct RNA nanopore sequencing in conjunction with chemical probing to obtain RNA structural profiles at high-throughput. Nanopore technology is well positioned to address the needs for single-molecule, high-throughput RNA detection and structural characterization.

Abstract: We highlight the role played by molecular modeling and simulation to unravel, at an atomistic and even electronic scale, the complex structural dynamic equilibrium assumed by RNA sequences, either cellular or viral. After pointing out the role played by specific RNA structures in regulating key biological functions, or in assuring either viral replication or immune system activation, we will show how computationally efficient multiscale approaches lead to the understanding of the fundamental biological processes, in terms of nucleic acid structural dynamic and their interaction with protein partners, and hence may be successfully used to rationally develop novel therapeutic approaches. By a selection of examples involving cellular and viral RNA, we will show that molecular modeling and simulation is nowadays assuming its role of a virtual microscope complementing and deepening the insight gained by structural and cellular biology.

Abstract: Piwi-interacting RNAs (piRNAs) are single-stranded, small noncoding RNAs that are 24–30 nucleotides long and have a 5′-U end and a 2′-O-methylation at the 3′ end. piRNAs associate with Piwi proteins that cleave them then enters the nucleus to induce epigenetic modification, transcriptional and post-transcriptional gene silencing. The piRNA and PIWI proteins mediate various epigenetic changes, such as DNA methylation, histone modification via methylation, gene regulation via transposon silencing, and chromatin remodelling. Various DNA methyltransferases, such as DNMT1, DNMT3A, and DNMT3B, catalyse the cytosine methylation of DNA molecules. Histone methylation on lysine and arginine residues of H3 and H4 histone proteins is done by various methyl transferases. Suv39H1 and SETDB1 methyltransferase enzymes are involved in lysine methylation on histone H3 and H4, and the protein arginine methyltransferase-5 (PRMT-5) enzyme causes the methylation of arginine residues on histone H3. Upregulation and downregulation of Piwi proteins and piRNAs may have the ability to suppress cancer growth and metastasis. Various studies have shown that piRNA-mediated modifications can be used as biomarkers for the diagnosis, prognosis of various diseases, such as different types of human malignancies, type-II diabetes mellitus, and Alzheimer’s disease. In this chapter, the current findings and functions of piRNAs in cancer epigenetics are discussed.

Abstract: A recent focus on the regulatory roles of non-coding RNAs has widened our knowledge of their biological functions. These regulatory roles inherently exist owing to their unique structures. Cancer research is increasingly based on the roles of multiple non-coding RNAs. Crucial to explore are the major cancer hallmarks that combine to bring about an aggressive phenotype in cancer cells. Epithelial-to-mesenchymal transition (EMT) contributes towards an increasing metastatic potential in cancer cells, additionally leading to the domination of cancer stem cells (CSCs), which have a higher propensity to seed secondary tumors. An ever aggressively growing tumor gains a survival advantage by reprogramming its metabolism and becomes therapy resistant. Tumor hypoxia is centric to an aggressive cancer phenotype along with other physiological factors. Literature has shown how hypoxia could contribute towards EMT in cancer. This chapter focuses on the crucial role of several non-coding RNAs that mediate hypoxia-driven EMT in multiple cancers. We aim to discuss the structural and functional aspects of these regulatory non-coding RNAs. Importantly, we focus on the cell signaling pathway crosstalk, mediated by ncRNAs in multiple cancers, and describe how different pathways that are crucial to both EMT and hypoxia converge onto some important molecular players. Interestingly, several ncRNAs have been implicated as novel biomarkers and therapeutic targets. A detailed understanding of the ncRNAs regulating hypoxia-driven EMT would help enable future studies of these phenomena.

Abstract: The occurrence, development and prognosis of cardiovascular disease is a multi-factor and multi-path pathological process. In addition to environmental factors, epigenetic regulation mechanisms also play an important role in the occurrence and development of cardiovascular disease. The most common and abundant internal modification of mRNA is m6A. Together with RNA editing, which is an alternative RNA modification, both play important roles in regulating gene expression and affect the fate of RNA molecules. In addition, with the advances in next-generation sequencing technology, non-coding RNAs such as microRNA, long non-coding RNA, and circular RNA which are usually not involved in protein synthesis, but can participate in cardiac homeostasis, cardiomyocyte growth, proliferation and apoptosis, endothelial cell function, cardiac remodeling and repair, and inflammatory response through various mechanisms. Grasping the cognition of RNA modifications and non-coding RNAs in cardiovascular disease may help us to better understand mechanisms and develop new biomarkers or therapeutic strategies in cardiovascular disease. This chapter summarizes the roles of long non-coding RNA, microRNA, circular RNA, and RNA modification in cardiovascular diseases.

Abstract: Many studies have shown RNA modifications including deamination of cytosine to uracil (C-to-U), deamination of adenosine to inosine (A-to-I), N1-methyladenosine (m1A), 5-methylcytosine (m5C), N6-methyladenosine (m6A), and pseudouridine (ψ), and ribose 2′-O-methyl to have key roles in regulating RNA stability, processing and gene expression. The manipulation and regulation of RNA modifications can be seen to create a trickling effect on biological processes. Here, we review the knowledge of RNA modifications of some of the most detrimental sexually transmitted infections and relate our current understanding of the regulatory roles behind such modifications in infections and their association with other diseases and cancer. With the increase in techniques to identify RNA modification, we anticipate further breakthrough of the RNA modifications’ influence on bacterial biological processes and their link to various diseases.

Abstract: Post-transcriptional modifications are crucial for RNA function, with roles ranging from the stabilization of functional RNA structures to modulation of RNA–protein interactions. Additionally, artificially modified RNAs have been suggested as optimal oligonucleotides for therapeutic purposes. The impact of chemical modifications on secondary structure has been rationalized for some of the most common modifications. However, the characterization of how the modifications affect the three-dimensional RNA structure and dynamics and its capability to bind proteins is still highly challenging. Molecular dynamics simulations, coupled with enhanced sampling methods and integration of experimental data, provide direct access to RNA structural dynamics. In the context of RNA chemical modifications, alchemical simulations where a wild type nucleotide is converted to a modified one are particularly common. In this Chapter, we review recent molecular dynamics studies of modified ribonucleotides. We discuss the technical aspects of the reviewed works, including the employed force fields, enhanced sampling methods, and alchemical methods, in a way that is accessible to experimentalists. Finally, we provide our perspective on this quickly growing field of research. The goal of this Chapter is to provide a guide for experimentalists to understand molecular dynamics works and, at the same time, give molecular dynamics experts a solid review of published articles that will be a useful starting point for new research.

Abstract: Mammalian genomes encode large amounts of noncoding RNAs (ncRNAs), which tend to form intricate structures and interact with their target RNA molecules through complementary base pairing with the help of proteins. Mapping of intra- and inter-molecular RNA–RNA interactions (RRIs) is required to unravel the structure and targets of ncRNAs which are two essential aspects for understanding the molecular mechanisms of ncRNAs in various biological processes. At this frontiers, we recently invented RNA in situ conformation sequencing (RIC-seq) technology to profile protein-mediated RNA–RNA spatial interactions at single-nucleotide resolution in an unbiased manner. We have demonstrated that RIC-seq-identified RRIs are helpful for simultaneously deducing ncRNA structures and targets. Here, we summarize methods for probing RRIs and describe a step-by-step protocol for generating a successful RIC-seq library.

Abstract: The global deployment of mRNA vaccines against SARS-CoV-2 and the projected expansion of therapeutic applications of synthetic mRNA call for robust and high-precision analytical methods to evaluate attributes that are crucial to the safety and efficacy of the mRNA drug substances. Liquid chromatography–mass spectrometry (LC–MS) is one of the few techniques that can provide a direct and high-confidence readout of the identity and incorporation efficiency of the 5′ cap, length of the poly(A) tail, nucleotide sequence, and modification profile of synthetic mRNA molecules. Prior to LC–MS analysis, the RNA molecules are partially digested by specific endoribonucleases into oligonucleotides that are suitable for charge state-dependent fragmentation and mass deconvolution. The most commonly used endoribonuclease for RNA sequence mapping is the guanosine-specific RNase T1. RNase T1 has been employed for analysis of mRNA, rRNA, and tRNA as well as for mRNA poly(A) tail length verification. For mRNA 5′ cap analysis, selective excisions using probe-restrained RNase H or (deoxy)ribozymes are typically required. In this chapter, we will review the application of endoribonucleases for mRNA analysis, with emphasis on a recently characterized endoribonuclease derived from human RNase 4. We will also discuss the latest methods to assess 5′ cap and poly(A) tail incorporation in synthetic mRNA. Finally, we will highlight why more enzymatic tools are needed and how they can contribute to improving the quality of synthetic RNA analysis, and to help understand the biology of RNA modifications in the cell.

Abstract: We review the basic concepts and tools for mechanically unzipping RNA hairpins using force spectroscopy. By pulling apart the ends of an RNA molecule using optical tweezers, it is possible to measure the folding free energy at varying experimental conditions. Energy measurements permit us to characterize the thermodynamics of RNA hybridization (base pairing and stacking), the dynamics of the formation of native and kinetic (intermediates and misfolded) molecular states, and interactions with metallic ions. This paper introduces basic concepts and reviews recent developments related to RNA force thermodynamics, native and barrier RNA energy landscapes, and RNA folding dynamics. We emphasize the implications of mechanical unzipping experiments to understand non-coding RNAs and RNAs in extreme environments.

Abstract: Modification of RNA molecules has a significant effect on their structure and function. Many different examples of RNA modifications have been observed and each contributes in various ways to ensure proper biological activity. One of the most common modifications is pseudouridine which occurs in key dynamic locations in many RNAs. Despite its prevalence in natural RNA sequences, organic synthesis of pseudouridine has been challenging because of the stereochemistry requirement and the sensitivity of reaction steps to moisture. Herein, we describe the semi-enzymatic synthetic route for the synthesis of pseudouridine using adenosine-5’-monophosphate and uracil as the starting materials and a reverse reaction catalyzed by a pseudouridine monophosphate glycosidase. Moreover, we describe the conversion from nucleoside (pseudouridine) to nucleotide triphosphate (pseudouridine-5’-triphosphate) to incorporate into RNA via in vitro transcription for biochemical and biophysical studies.