Bioorthogonal labeling and profiling of N6-isopentenyladenosine (i6A) modified RNA.

Chemical modifications in RNAs play crucial roles in diversifying their structures and regulating numerous biochemical processes. Since the 1990s, several hydrophobic prenyl-modifications have been discovered in various RNAs. Prenyl groups serve as precursors for terpenes and many other biological molecules. The processes of prenylation in different macromolecules have been extensively studied. We introduce here a novel chemical biology toolkit that not only labels i6A, a prenyl-modified RNA residue, by leveraging the unique reactivity of the prenyl group, but also provides a general strategy to incorporate fluorescence functionalities into RNAs for molecular tracking purposes. Our findings revealed that iodine-mediated cyclization reactions of the prenyl group occur rapidly, transforming i6A from a hydrogen-bond acceptor to a donor. Based on this reactivity, we developed an Iodine-Mediated Cyclization and Reverse Transcription (IMCRT) tRNA-seq method, which can profile all nine endogenous tRNAs containing i6A residues in Saccharomyces cerevisiae with single-base resolution. Furthermore, under stress conditions, we observed a decline in i6A levels in budding yeast, accompanied by significant decrease of mutation rate at A37 position. Thus, the IMCRT tRNA-seq method not only permits semi-quantification of i6A levels in tRNAs but also holds potential for transcriptome-wide detection and analysis of various RNA species containing i6A modifications.

Structural effects of inosine substitution in telomeric DNA quadruplex.

The telomeric DNA, a distal region of eukaryotic chromosome containing guanine-rich repetitive sequence of (TTAGGG)n, has been shown to adopt higher-order structures, specifically G-quadruplexes (G4s). Previous studies have demonstrated the implication of G4 in tumor inhibition through chromosome maintenance and manipulation of oncogene expression featuring their G-rich promoter regions. Besides higher order structures, several regulatory roles are attributed to DNA epigenetic markers. In this work, we investigated how the structural dynamics of a G-quadruplex, formed by the telomeric sequence, is affected by inosine, a prevalent modified nucleotide. We used the standard (TTAGGG)n telomere repeats with guanosine mutated to inosine at each G position. Sequences (GGG)4, (IGG)4, (GIG)4, (GGI)4, (IGI)4, (IIG)4, (GII)4, and (III)4, bridged by TTA linker, are studied using biophysical experiments and molecular modeling. The effects of metal cations in quadruplex folding were explored in both Na+ and K+ containing buffers using CD and UV-melting studies. Our results show that antiparallel quadruplex topology forms with the native sequence (GGG)4 and the terminal modified DNAs (IGG)4 and (GGI)4 in both Na+ and K+ containing buffers. Specifically, quadruplex hybrid was observed for (GGG)4 in K+ buffer. Among the other modified sequences, (GIG)4, (IGI)4 and (GII)4 show parallel features, while (IIG)4 and (III)4 show no detectable conformation in the presence of either Na+ or K+. Our studies indicate that terminal lesions (IGG)4 and (GGI)4 may induce certain unknown conformations. The folding dynamics become undetectable in the presence of more than one inosine substitution except (IGI)4 in both buffer ions. In addition, both UV melting and CD melting studies implied that in most cases the K+ cation confers more thermodynamic stability compared to Na+. Collectively, our conformational studies revealed the diverse structural polymorphisms of G4 with position dependent G-to-I mutations in different ion conditions.

Inosine-Induced Base Pairing Diversity during Reverse Transcription.

A-to-I editing catalyzed by adenosine deaminase acting on RNAs impacts numerous physiological and biochemical processes that are essential for cellular functions and is a big contributor to the infectivity of certain RNA viruses. The outcome of this deamination leads to changes in the eukaryotic transcriptome functionally resembling A-G transitions since inosine preferentially pairs with cytosine. Moreover, hyper-editing or multiple A to G transitions in clusters were detected in measles virus. Inosine modifications either directly on viral RNA or on cellular RNA can have antiviral or pro-viral repercussions. While many of the significant roles of inosine in cellular RNAs are well understood, the effects of hyper-editing of A to I on viral polymerase activity during RNA replication remain elusive. Moreover, biological strategies such as molecular cloning and RNA-seq for transcriptomic interrogation rely on RT-polymerase chain reaction with little to no emphasis placed on the first step, reverse transcription, which may reshape the sequencing results when hypermodification is present. In this study, we systematically explore the influence of inosine modification, varying the number and position of inosines, on decoding outcomes using three different reverse transcriptases (RTs) followed by standard Sanger sequencing. We find that inosine alone or in clusters can differentially affect the RT activity. To gain structural insights into the accommodation of inosine in the polymerase site of HIV-1 reverse transcriptase (HIV-1-RT) and how this structural context affects the base pairing rules for inosine, we performed molecular dynamics simulations of the HIV-1-RT. The simulations highlight the importance of the protein-nucleotide interaction as a critical factor in deciphering the base pairing behavior of inosine clusters. This effort sets the groundwork for decrypting the physiological significance of inosine and linking the fidelity of reverse transcriptase and the possible diverse transcription outcomes of cellular RNAs and/or viral RNAs where hyper-edited inosines are present in the transcripts.

Bio-Orthogonal Chemistry Conjugation Strategy Facilitates Investigation of N-methyladenosine and Thiouridine Guide RNA Modifications on CRISPR Activity.

The CRISPR-Cas9 system is an important genome editing tool that holds enormous potential toward the treatment of human genetic diseases. Clinical success of CRISPR technology is dependent on the incorporation of modifications into the single-guide RNA (sgRNA). However, chemical synthesis of modified sgRNAs, which are over 100 nucleotides in length, is difficult and low-yielding. We developed a conjugation strategy that utilized bio-orthogonal chemistry to efficiently assemble functional sgRNAs containing nucleobase modifications. The described approach entails the chemical synthesis of two shorter RNA oligonucleotides: a 31-mer containing tetrazine (Tz) group and a 70-mer modified with a trans-cyclooctene (TCO) moiety. The two oligonucleotides were conjugated to form functional sgRNAs. The two-component conjugation methodology was utilized to synthesize a library of sgRNAs containing nucleobase modifications such as N1-methyladenosine (m1A), N6-methyladenosine (m6A), 2-thiouridine (s2U), and 4-thiouridine (s4U). The impact of these RNA modifications on overall CRISPR activity were investigated in vitro and in Cas9-expressing HEK293T cells.

Probing the Substrate Requirements of the In†Vitro Geranylation Activity of Selenouridine Synthase (SelU).

Natural RNA modifications diversify the structures and functions of existing nucleic acid building blocks. Geranyl is one of the most hydrophobic groups recently identified in bacterial tRNAs. Selenouridine synthase (SelU, also called mnmH) is an enzyme with a dual activity which catalyzes selenation and geranylation in tRNAs containing 2-thiouridine using selenophosphate or geranyl-pyrophosphate as cofactors. In this study, we explored the in vitro geranylation process of tRNA anticodon stem loops (ASL) mediated by SelU and showed that the geranylation activity was abolished when U35 was mutated to A35 (ASL-tRNALys (s2U)UU to ASL-tRNAIle (s2U)AU ). By examining the SelU cofactor geranyl-pyrophosphate (gePP) and its analogues, we found that only the geranyl group, but not dimethylallyl- and farnesyl-pyrophosphate with either shorter or longer terpene chains, could be incorporated into ASL. The degree of tRNA geranylation in the end-point analysis for SelU follows the order of ASLLys (s2UUU) ≃ ASLGln (s2UUG) >ASLGlu (s2UUC) . These findings suggest a putative mechanism for substrate discrimination by SelU and reveal key factors that might influence its enzymatic activity. Given that SelU plays an important role in bacterial translation systems, inhibiting this enzyme and targeting its geranylation and selenation pathways could be exploited as a promising strategy to develop SelU-based antibiotics.

Fluorescence-Based Binding Characterization of Small Molecule Ligands Targeting CUG RNA Repeats.

Pathogenic CUG and CCUG RNA repeats have been associated with myotonic dystrophy type 1 and 2 (DM1 and DM2), respectively. Identifying small molecules that can bind these RNA repeats is of great significance to develop potential therapeutics to treat these neurodegenerative diseases. Some studies have shown that aminoglycosides and their derivatives could work as potential lead compounds targeting these RNA repeats. In this work, sisomicin, previously known to bind HIV-1 TAR, is investigated as a possible ligand for CUG RNA repeats. We designed a novel fluorescence-labeled RNA sequence of r(CUG)10 to mimic cellular RNA repeats and improve the detecting sensitivity. The interaction of sisomicin with CUG RNA repeats is characterized by the change of fluorescent signal, which is initially minimized by covalently incorporating the fluorescein into the RNA bases and later increased upon ligand binding. The results show that sisomicin can bind and stabilize the folded RNA structure. We demonstrate that this new fluorescence-based binding characterization assay is consistent with the classic UV Tm technique, indicating its feasibility for high-throughput screening of ligand-RNA binding interactions and wide applications to measure the thermodynamic parameters in addition to binding constants and kinetics when probing such interactions.

Synthesis of Novel MicroRNA-30c Analogs to Reduce Apolipoprotein B Secretion in Human Hepatoma Cells.

Atherosclerosis, a condition characterized by thickening of the arteries due to lipid deposition, is the major contributor to and hallmark of cardiovascular disease. Although great progress has been made in lowering the lipid plaques in patients, the conventional therapies fail to address the needs of those that are intolerant or non-responsive to the treatment. Therefore, additional novel therapeutic approaches are warranted. We have previously shown that increasing the cellular amounts of microRNA-30c (miR-30c) with the aid of viral vectors or liposomes can successfully reduce plasma cholesterol and atherosclerosis in mice. To avoid the use of viruses and liposomes, we have developed new methods to synthesize novel miR-30c analogs with increasing potency and efficacy, including 2'-O-methyl (2'OMe), 2'-fluoro (2'F), pseudouridine (á´ª), phosphorothioate (PS), and N-acetylgalactosamine (GalNAc). The discovery of these modifications has profoundly impacted the modern RNA therapeutics, as evidenced by their increased nuclease stability and reduction in immune responses. We show that modifications on the passenger strand of miR-30c not only stabilize the duplex but also aid in a more readily uptake by the cells without the aid of viral vectors or lipid emulsions. After uptake, the analogs with PS linkages and GalNAc-modified ribonucleotides significantly reduce the secretion of apolipoprotein B (ApoB) without affecting apolipoprotein A1 (ApoA1) in human hepatoma Huh-7 cells. We envision an enormous potential for these modified miR-30c analogs in therapeutic intervention for treating cardiovascular diseases. This protocol was validated in: J Biol Chem (2021), DOI: 10.1016/j.jbc.2022.101813.

Synthesis and Functionality Study of Photoswitchable Hydrazone Oligodeoxynucleotides.

This article provides a detailed procedure for the chemical synthesis and characterization of photoswitchable hydrazone phosphoramidite and its incorporation into oligodeoxynucleotides. The synthesis starts with commercially available deoxyuridine, followed by conversion of the 4-oxo into a 4-chloro moiety via Appel reaction to install the key hydrazone group in the absence of base. The hydrazone phosphoramidite building block is compatible with the conventional amidite chemistry protocols for solid-phase synthesis of oligodeoxynucleotides. Our method expands the current nucleotide pool by adding a novel, functional DNA building block that is suitable for a broad spectrum of applications, including the regulation of DNA-enzyme interactions and DNA synthesis by irradiation with cell-friendly blue light. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Preparation of photoswitchable hydrazone phosphoramidite Basic Protocol 2: Synthesis and purification of oligodeoxynucleotides containing the hydrazone photoswitch Basic Protocol 3: Primer extension assay for functionality studies of hydrazone cytidine.

Synthesis and Purification of N3 -Methylcytidine (m3 C) Modified RNA Oligonucleotides.

This protocol describes a step-by-step chemical synthesis approach to prepare N3 -methylcytidine (m3 C) and its phosphoramidite. The method for synthesizing m3 C starts from commercially available cytidine, and proceeds via N3 -methylation in the presence of MeI, which generates the N3 -methylcytidine (m3 C) nucleoside, followed by the installation of several protecting groups at sites that include the 5'-hydroxyl group (4,4'-dimethoxytrityl protection), the 4-amino group (benzoyl protection), and the 2'-hydroxyl group (tert-butyldimethylsilyl, TBDMS, protection). Standard phosphoramidite chemistry is applied to prepare the final m3 C phosphoramidite for solid-phase synthesis of a series of RNA oligonucleotides. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Synthesis of N3 -methylcytidine (m3 C) and its phosphoramidite Basic Protocol 2: Automated synthesis of m3 C modified RNA oligonucleotides.

Synthesis of N4 -Methylcytidine (m4 C) and N4 ,N4 -Dimethylcytidine (m4 2 C) Modified RNA.

This article summarizes the protocols for phosphoramidite chemistry and solid phase synthesis of RNA oligonucleotides containing N4 -methylcytidine (m4 C) and N4 ,N4 -dimethylcytidine (m4 2 C) residues for base-pairing, structural, and enzymatic activity studies. The two key m4 C and m4 2 C phosphoramidite building blocks can be synthesized starting from the partially protected cytidine nucleosides, followed by solid-phase synthesis and HPLC purification of the modified target RNA oligonucleotides. These modified RNA strands are then prepared for base pairing stability, specificity, and structural studies using UV-melting temperature (Tm ) measurements and X-ray crystallography. Functional studies are performed by reverse transcription assays in primer extension reactions employing different enzymes. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Chemical synthesis of m4 C phosphoramidite Basic Protocol 2: Synthesis of m4 2 C phosphoramidite Basic Protocol 3: Synthesis and purification of m4 C and m4 2 C containing RNA oligonucleotides.

Sulfur modification in natural RNA and therapeutic oligonucleotides.

Sulfur modifications have been discovered on both DNA and RNA. Sulfur substitution of oxygen atoms at nucleobase or backbone locations in the nucleic acid framework led to a wide variety of sulfur-modified nucleosides and nucleotides. While the discovery, regulation and functions of DNA phosphorothioate (PS) modification, where one of the non-bridging oxygen atoms is replaced by sulfur on the DNA backbone, are important topics, this review focuses on the sulfur modification in natural cellular RNAs and therapeutic nucleic acids. The sulfur modifications on RNAs exhibit diversity in terms of modification location and cellular function, but the various sulfur modifications share common biosynthetic strategies across RNA species, cell types and domains of life. The first section reviews the post-transcriptional sulfur modifications on nucleobases with an emphasis on thiouridine on tRNA and phosphorothioate modification on RNA backbones, as well as the functions of the sulfur modifications on different species of cellular RNAs. The second section reviews the biosynthesis of different types of sulfur modifications and summarizes the general strategy for the biosynthesis of sulfur-containing RNA residues. One of the main goals of investigating sulfur modifications is to aid the genomic drug development pipeline and enhance our understandings of the rapidly growing nucleic acid-based gene therapies. The last section of the review focuses on the current drug development strategies employing sulfur substitution of oxygen atoms in therapeutic RNAs.

Structural Insights Into the 5'UG/3'GU Wobble Tandem in Complex With Ba2+ Cation.

G•U wobble base pair frequently occurs in RNA structures. The unique chemical, thermodynamic, and structural properties of the G•U pair are widely exploited in RNA biology. In several RNA molecules, the G•U pair plays key roles in folding, ribozyme catalysis, and interactions with proteins. G•U may occur as a single pair or in tandem motifs with different geometries, electrostatics, and thermodynamics, further extending its biological functions. The metal binding affinity, which is essential for RNA folding, catalysis, and other interactions, differs with respect to the tandem motif type due to the different electrostatic potentials of the major grooves. In this work, we present the crystal structure of an RNA 8-mer duplex r[UCGUGCGA]2, providing detailed structural insights into the tandem motif I (5'UG/3'GU) complexed with Ba2+ cation. We compare the electrostatic potential of the presented motif I major groove with previously published structures of tandem motifs I, II (5'GU/3'UG), and III (5'GG/3'UU). A local patch of a strongly negative electrostatic potential in the major groove of the presented structure forms the metal binding site with the contributions of three oxygen atoms from the tandem. These results give us a better understanding of the G•U tandem motif I as a divalent metal binder, a feature essential for RNA functions.

Detection and Quantification of RNA Phosphorothioate Modifications Using Mass Spectrometry.

This article describes a protocol for detecting and quantifying RNA phosphorothioate modifications in cellular RNA samples. Starting from solid-phase synthesis of phosphorothioate RNA dinucleotides, followed by purification with reversed-phase HPLC, phosphorothioate RNA dinucleotide standards are prepared for UPLC-MS and LC-MS/MS methods. RNA samples are extracted from cells using TRIzol reagent, then digested with a nuclease mixture and analyzed by mass spectrometry. UPLC-MS is employed first to identify RNA phosphorothioate modifications. An optimized LC-MS/MS method is then employed to quantify the frequency of RNA phosphorothioate modifications in a series of model cells. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Synthesis, purification, and characterization of RNA phosphorothioate dinucleotides Basic Protocol 2: Digestion of RNA samples extracted from cells Basic Protocol 3: Detection and quantification of RNA phosphorothioate modifications by mass spectrometry.

RNA Phosphorothioate Modification in Prokaryotes and Eukaryotes.

RNA modifications play important roles in RNA structures and regulation of gene expression and translation. We report the first RNA modification on the phosphate, the RNA phosphorothioate (PS) modification, discovered in both prokaryotes and eukaryotes. The PS modification is also first reported on nucleic acids of eukaryotes. The GpsG modification exists in the Rp configuration and was quantified with liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). By knocking out the DndA gene in E. coli, we show the Dnd clusters that regulate DNA PS modification may also play roles in RNA PS modification. We also show that the GpsG modification locates on rRNA in E. coli, L. lactis, and HeLa cells, and it is not detected in rRNA-depleted total RNAs from these cells.

RNA modifications and cancer.

RNA plays essential roles in not only translating nucleic acids into proteins, but also in gene regulation, environmental interactions and many human diseases. Nature uses over 150 chemical modifications to decorate RNA and diversify its functions. With the fast-growing RNA research in the burgeoning field of 'epitranscriptome', a term describes post-transcriptional RNA modifications that can dynamically change the transcriptome, it becomes clear that these modifications participate in modulating gene expression and controlling the cell fate, thereby igniting the new interests in RNA-based drug discovery. The dynamics of these RNA chemical modifications is orchestrated by coordinated actions of an array of writer, reader and eraser proteins. Deregulated expression of these RNA modifying proteins can lead to many human diseases including cancer. In this review, we highlight several critical modifications, namely m6A, m1A, m5C, inosine and pseudouridine, in both coding and non-coding RNAs. In parallel, we present a few other cancer-related tRNA and rRNA modifications. We further discuss their roles in cancer promotion or tumour suppression. Understanding the molecular mechanisms underlying the biogenesis and turnover of these RNA modifications will be of great significance in the design and development of novel anticancer drugs.

Dietary agents in cancer prevention: an immunological perspective.

Skin cancer is the most common form of cancer diagnosed in the United States. Exposure to solar ultraviolet (UV) radiations is believed to be the primary cause for skin cancer. Excessive UV radiation can lead to genetic mutations and damage in the skin's cellular DNA that in turn can lead to skin cancer. Lately, chemoprevention by administering naturally occurring non-toxic dietary compounds has proven to be a potential strategy to prevent the occurrence of tumors. Attention has been drawn toward several natural dietary agents such as resveratrol, one of the major components found in grapes, red wines, berries and peanuts, proanthocyanidins from grape seeds, (-)-epigallocatechin-3-gallate from green tea, etc. However, the effect these dietary agents have on the immune system and the immunological mechanisms involved therein are still being explored. In this review, we shall focus on the role of key chemopreventive agents on various immune cells and discuss their potential as antitumor agents with an immunological perspective.