Nucleoside Modifications Research Articles

The impact of nucleoside base modification in mRNA vaccine is influenced by the chemistry of its lipid nanoparticle delivery system

The use of modified nucleosides is an important approach to mitigate the intrinsic immunostimulatory activity of exogenous mRNA and to increase its translation for mRNA therapeutic applications. However, for vaccine applications, the intrinsic immunostimulatory nature of unmodified mRNA could help induce productive immunity. Additionally, the ionizable lipid nanoparticles (LNPs) used to deliver mRNA vaccines can possess immunostimulatory properties that may influence the impact of nucleoside modification. Here we show that uridine replacement with N1-methylpseudouridine in an mRNA vaccine encoding influenza hemagglutinin had a significant impact on the induction of innate chemokines/cytokines and a positive impact on the induction of functional antibody titers in mice and macaques when MC3 or KC2 LNPs were used as delivery systems, while it impacted only minimally the titers obtained with L319 LNPs, indicating that the impact of nucleoside modification on mRNA vaccine efficacy varies with LNP composition. In line with previous observations, we noticed an inverse correlation between the induction of high innate IFN-α titers in the macaques and antigen-specific immune responses. Furthermore, and consistent with the species specificity of pathogen recognition receptors, we found that the effect of uridine replacement did not strictly translate from mice to non-human primates.

DLC-ac4C: A Prediction Model for N4-acetylcytidine Sites in Human mRNA Based on DenseNet and Bidirectional LSTM Methods.

N4 acetylcytidine (ac4C) is a highly conserved nucleoside modification that is essential for the regulation of immune functions in organisms. Currently, the identification of ac4C is primarily achieved using biological methods, which can be time-consuming and labor-intensive. In contrast, accurate identification of ac4C by computational methods has become a more effective method for classification and prediction. To the best of our knowledge, although there are several computational methods for ac4C locus prediction, the performance of the models they constructed is poor, and the network structure they used is relatively simple and suffers from the disadvantage of network degradation. This study aims to improve these limitations by proposing a predictive model based on integrated deep learning to better help identify ac4C sites. In this study, we propose a new integrated deep learning prediction framework, DLC-ac4C. First, we encode RNA sequences based on three feature encoding schemes, namely C2 encoding, nucleotide chemical property (NCP) encoding, and nucleotide density (ND) encoding. Second, one-dimensional convolutional layers and densely connected convolutional networks (DenseNet) are used to learn local features, and bi-directional long short-term memory networks (Bi-LSTM) are used to learn global features. Third, a channel attention mechanism is introduced to determine the importance of sequence characteristics. Finally, a homomorphic integration strategy is used to limit the generalization error of the model, which further improves the performance of the model. The DLC-ac4C model performed well in terms of sensitivity (Sn), specificity (Sp), accuracy (Acc), Mathews correlation coefficient (MCC), and area under the curve (AUC) for the independent test data with 86.23%, 79.71%, 82.97%, 66.08%, and 90.42%, respectively, which was significantly better than the prediction accuracy of the existing methods. Our model not only combines DenseNet and Bi-LSTM, but also uses the channel attention mechanism to better capture hidden information features from a sequence perspective, and can identify ac4C sites more effectively.

RNase2 is a possible trigger of acute-on-chronic inflammation leading to mRNA vaccine-associated cardiac complication

Post-mRNA vaccination-associated cardiac complication is a rare but life-threatening adverse event. Its risk has been well balanced by the benefit of vaccination-induced protection against severe COVID-19. As the rate of severe COVID-19 has consequently declined, future booster vaccination to sustain immunity, especially against infection with new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants, may encounter benefit-risk ratios that are less favorable than at the start of the COVID-19 vaccination campaign. Understanding the pathogenesis of rare but severe vaccine-associated adverse events to minimize its risk is thus urgent. Here, we report a serendipitous finding of a case of cardiac complication following a third shot of COVID-19 mRNA vaccine. As this case was enrolled in a cohort study, pre-vaccination and pre-symptomatic blood samples were available for genomic and multiplex cytokine analyses. These analyses revealed the presence of subclinical chronic inflammation, with an elevated expression of RNASE2 at pre-booster baseline as a possible trigger of an acute-on-chronic inflammation that resulted in the cardiac complication. RNASE2 encodes for the ribonuclease RNase2, which cleaves RNA at the 3' side of uridine, which may thus remove the only Toll-like receptor (TLR)-avoidance safety feature of current mRNA vaccines. These pre-booster and pre-symptomatic gene and cytokine expression data provide unique insights into the possible pathogenesis of vaccine-associated cardiac complication and suggest the incorporation of additional nucleoside modification for an added safety margin. This work was funded by the NMRC Open Fund-Large Collaborative Grant on Integrated Innovations on Infectious Diseases (OFLCG19May-0034).

Research progress of the Elongator complex in plant

The Elongator complex consists of six subunits (ELP1-ELP6), where ELP1-ELP3 forms the core subcomplex and ELP4-ELP6 forms the auxiliary subcomplex. Deletion of any of the six subunits results in an almost identical phenotype, suggesting that all six subunits are essential for cellular function. All six subunits are evolutionarily conserved in terms of sequence and their interactions with other subunits. The most striking features are the structural conservation of the protein complexes and the phenotypic similarity caused by loss-of-function mutations in any protein subunit. Similar to elongation factors in translation, there is a strong interaction between the Elongator complex and RNA polymerase II during transcription. The Elongator complex is also involved in a variety of cellular pathways, including histone modification/acetylation, DNA methylation, tRNA nucleoside modification, etc. Here, we summarized the functions and mechanisms of the Elongator complex in plant growth and development, molecular pathways, and gene regulation. In this way, we aimed to provide a reference for an in-depth study of the Elongator complex.

Synthesis of oligonucleotides containing 5′-homo-4′-selenouridine derivative and its increased resistance against nuclease

As technologies using RNA or DNA have been developed, various chemical modifications of nucleosides have been attempted to increase the stability of oligonucleotides. Since it is known that 2′-OMe-modification greatly contributes to increasing the stability of oligonucleotides, we added 2′-OMe to our previously developed 4′-selenonucleoside and 5′-homo-4′-selenonucleoside as the modified monomers for oligonucleotide: 2′-methoxy-4′-selenouridine (2′-OMe-4′-Se-U) and 5′-homo-2′-methoxy-4′-selenouridine (5′-homo-2′-OMe-4′-Se-U). We synthesized oligonucleotides containing the chemically modified 4′-selenouridine and evaluated their thermal stability and nuclease resistance. In conclusion, the nuclease stability of the oligonucleotide containing 5′-homo-2′-OMe-4′-selenouridine increased while its thermal stability decreased.

Methylated guanosine and uridine modifications in S. cerevisiae mRNAs modulate translation elongation.

Chemical modifications to protein encoding messenger RNAs (mRNAs) influence their localization, translation, and stability within cells. Over 15 different types of mRNA modifications have been observed by sequencing and liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) approaches. While LC-MS/MS is arguably the most essential tool available for studying analogous protein post-translational modifications, the high-throughput discovery and quantitative characterization of mRNA modifications by LC-MS/MS has been hampered by the difficulty of obtaining sufficient quantities of pure mRNA and limited sensitivities for modified nucleosides. We have overcome these challenges by improving the mRNA purification and LC-MS/MS pipelines. The methodologies we developed result in no detectable non-coding RNA modifications signals in our purified mRNA samples, quantify 50 ribonucleosides in a single analysis, and provide the lowest limit of detection reported for ribonucleoside modification LC-MS/MS analyses. These advancements enabled the detection and quantification of 13 S. cerevisiae mRNA ribonucleoside modifications and reveal the presence of four new S. cerevisiae mRNA modifications at low to moderate levels (1-methyguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, and 5-methyluridine). We identified four enzymes that incorporate these modifications into S. cerevisiae mRNAs (Trm10, Trm11, Trm1, and Trm2, respectively), though our results suggest that guanosine and uridine nucleobases are also non-enzymatically methylated at low levels. Regardless of whether they are incorporated in a programmed manner or as the result of RNA damage, we reasoned that the ribosome will encounter the modifications that we detect in cells. To evaluate this possibility, we used a reconstituted translation system to investigate the consequences of modifications on translation elongation. Our findings demonstrate that the introduction of 1-methyguanosine, N2-methylguanosine and 5-methyluridine into mRNA codons impedes amino acid addition in a position dependent manner. This work expands the repertoire of nucleoside modifications that the ribosome must decode in S. cerevisiae. Additionally, it highlights the challenge of predicting the effect of discrete modified mRNA sites on translation de novo because individual modifications influence translation differently depending on mRNA sequence context.

Abstract 2286: Molecular characterization of the effect of RNA nucleoside modification on Flaviviral RNA dependent RNA polymerase in vitro transcription

Abstract 2286: Molecular characterization of the effect of RNA nucleoside modification on Flaviviral RNA dependent RNA polymerase in vitro transcription

Base Modifications of Nucleosides via the Use of Peptide-Coupling Agents, and Beyond.

Several naturally occurring purine and pyrimidine nucleosides contain an amide linkage as part of the heterocyclic aglycone. Enolization of the amide and conversion to leaving groups at the amide carbon atom permits base modification by addition-elimination types of processes. Although a number of methods have been developed over the years for accomplishing such conversions, the present Personal Account describes efforts from the Lakshman laboratories. Facile activation of the amido groups in nucleobases can be achieved with peptide-coupling agents. Subsequent reaction with nucleophiles then accomplishes the base modifications. In many cases, the activation and displacement steps can be done as two-step, one-pot processes, whereas in other cases, discrete storable activated nucleosides can be isolated for subsequent displacement reactions. Using such an approach a wide range of nucleoside base modifications is readily achievable. In many instances, mechanistic investigations have been conducted so as to understand the activation process.

Water-Soluble Pd-Imidate Complexes as Versatile Catalysts for the Modification of Unprotected Halonucleosides.

Modification of unprotected nucleosides has been attracting continuous interest, since these building blocks themselves and their phosphate-upgraded corresponding nucleotides have shown a plethora of uses in fields like biochemistry or pharmacy. Pd-catalyzed cross-coupling reactions, conducted in water or its mixtures with polar organic solvents, have frequently been the researchers' choice for the functionalization of the purine/pyrimidine base of the unprotected nucleosides. In this scenario, the availability of hydrophilic ligands and its water-soluble palladium complexes has markedly set the pace of the advances. The approach of our group to the synthesis of such complexes, Pd-imidates specifically, has faced critical stages, namely the jump to synthesize water soluble complexes from our experience working in conventional solvents, the preparation of phosphine free complexes and the overall goal of getting catalytic systems able to work close to room temperature. The continuous feedback with Kapdi's group, experienced in the chemistry of nucleosides, has produced over the last decade the interesting results in both fields presented here.

Probing Nascent RNA with Metabolic Incorporation of Modified Nucleosides.

The discovery of previously unknown functional roles of RNA in biological systems has led to increased interest in revealing novel RNA molecules as therapeutic targets and the development of tools to better understand the role of RNA in cells. RNA metabolic labeling broadens the scope of studying RNA by incorporating of unnatural nucleobases and nucleosides with bioorthogonal handles that can be utilized for chemical modification of newly synthesized cellular RNA. Such labeling of RNA provides access to applications including measurement of the rates of synthesis and decay of RNA, cellular imaging for RNA localization, and selective enrichment of nascent RNA from the total RNA pool. Several unnatural nucleosides and nucleobases have been shown to be incorporated into RNA by endogenous RNA synthesis machinery of the cells. RNA metabolic labeling can also be performed in a cell-specific manner, where only cells expressing an essential enzyme incorporate the unnatural nucleobase into their RNA. Although several discoveries have been enabled by the current RNA metabolic labeling methods, some key challenges still exist: (i) toxicity of unnatural analogues, (ii) lack of RNA-compatible conjugation chemistries, and (iii) background incorporation of modified analogues in cell-specific RNA metabolic labeling. In this Account, we showcase work done in our laboratory to overcome these challenges faced by RNA metabolic labeling.To begin, we discuss the cellular pathways that have been utilized to perform RNA metabolic labeling and study the interaction between nucleosides and nucleoside kinases. Then we discuss the use of vinyl nucleosides for metabolic labeling and demonstrate the low toxicity of 5-vinyluridine (5-VUrd) compared to other widely used nucleosides. Next, we discuss cell-specific RNA metabolic labeling with unnatural nucleobases, which requires the expression of a specific phosphoribosyl transferase (PRT) enzyme for incorporation of the nucleobase into RNA. In the course of this work, we discovered the enzyme uridine monophosphate synthase (UMPS), which is responsible for nonspecific labeling with modified uracil nucleobases. We were able to overcome this background labeling by discovering a mutant uracil PRT (UPRT) that demonstrates highly specific RNA metabolic labeling with 5-vinyluracil (5-VU). Furthermore, we discuss the optimization of inverse-electron-demand Diels-Alder (IEDDA) reactions for performing chemical modification of vinyl nucleosides to achieve covalent conjugation of RNA without transcript degradation. Finally, we highlight our latest endeavor: the development of mutually orthogonal chemical reactions for selective labeling of 5-VUrd and 2-vinyladenosine (2-VAdo), which allows for potential use of multiple vinyl nucleosides for simultaneous investigation of multiple cellular processes involving RNA. We hope that our methods and discoveries encourage scientists studying biological systems to include RNA metabolic labeling in their toolkit for studying RNA and its role in biological systems.

Suzuki-Miyaura Coupling, Heck Alkenylation, and Amidation of DMTr-Protected 5-Iodo-2'-Deoxyuridine via Palladium-catalyzed Reactions.

Modification of nucleosides via cross-coupling processes has been carried out extensively on unprotected halonucleosides to produce functionalized nucleosides that are often developed for incorporation into oligonucleotides or used as fluorescent probes. This approach requires protection of the 5'-OH with the 4,4'-dimethoxytrityl (DMTr) group, which is complicated and a common cause of reaction failure. Here we report a method for direct functionalization of 5'-O-DMTr-5-iodo-2'-deoxyuridine via Suzuki-Miyaura cross-coupling, Heck alkenylation, and carboamidation. This approach facilitates rapid synthesis of a variety of C5-substituted 5'-O-DMTr-2'-deoxyuridine derivatives. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Synthesis of the SerrKap palladacycle complex Basic Protocol 2: Suzuki-Miyaura coupling of 5'-O-DMTr-5-iodo-2'-deoxyuridine using SerrKap palladacycle Basic Protocol 3: Heck coupling of 5'-O-DMTr-5-iodo-2'-deoxyuridine using SerrKap palladacycle Basic Protocol 4: Heck coupling of 5'-O-DMTr-5-iodo-2'-deoxyuridine with Ruth linker using Pd(OAc)2 /PTABS Basic Protocol 5: Carbonylative amidation of 5'-O-DMTr-5-iodo-2'-deoxyuridine using Pd(OAc)2 /PTABS.

Synthesis of Sugar and Nucleoside Analogs and Evaluation of Their Anticancer and Analgesic Potentials.

Chemical modification of sugars and nucleosides has a long history of producing compounds with improved selectivity and efficacy. In this study, several modified sugars (2–3) and ribonucleoside analogs (4–8) have been synthesized from α-d-glucose in a total of 21 steps. The compounds were tested for peripheral anti-nociceptive characteristics in the acetic acid-induced writhing assay in mice, where compounds 2, 7, and 8 showed a significant reduction in the number of writhes by 56%, 62%, and 63%, respectively. The compounds were also tested for their cytotoxic potential against human HeLa cell line via trypan blue dye exclusion test followed by cell counting kit-8 (CCK-8) assay. Compound 6 demonstrated significant cytotoxic activity with an IC50 value of 54 µg/mL. Molecular docking simulations revealed that compounds 2, 7, and 8 had a comparable binding affinity to cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) enzymes. Additionally, the bridged nucleoside analogs 7 and 8 potently inhibited adenosine kinase enzyme as well, which indicates an alternate mechanistic pathway behind their anti-nociceptive action. Cytotoxic compound 6 demonstrated strong docking with cancer drug targets human cytidine deaminase, proto-oncogene tyrosine-protein kinase Src, human thymidine kinase 1, human thymidylate synthase, and human adenosine deaminase 2. This is the first ever reporting of the synthesis and analgesic property of compound 8 and the cytotoxic potential of compound 6.

Bacterial tRNA landscape revisited

The updated Wobble Hypothesis reasonably explains why some 40 tRNA species are sufficient to decode the 61 amino acid codons of the Universal Genetic Code. However, we still have no clue why eubacteria lack tRNA isoacceptors with ANN anticodons, whereas eukaryotes universally lack eight GNN anticodons, only one of which is also absent in bacteria. Direct tRNA sequencing could resolve the patterns of nucleoside modification that had been driving the divergent evolution in prokaryotes and eukaryotes, but this task will require the development of AI-supported base-callers that can recognize modified nucleosides without any subsequent analytical verification. Our knowledge of the bacterial tRNA landscape is moreover broadened by the recent discovery of antisense tRNAs and tRNA-derived fragments that should be examined in their roles for gene expression, translation, bacterial physiology or metabolism.

Combinatorial optimization of mRNA structure, stability, and translation for RNA-based therapeutics

Therapeutic mRNAs and vaccines are being developed for a broad range of human diseases, including COVID-19. However, their optimization is hindered by mRNA instability and inefficient protein expression. Here, we describe design principles that overcome these barriers. We develop an RNA sequencing-based platform called PERSIST-seq to systematically delineate in-cell mRNA stability, ribosome load, as well as in-solution stability of a library of diverse mRNAs. We find that, surprisingly, in-cell stability is a greater driver of protein output than high ribosome load. We further introduce a method called In-line-seq, applied to thousands of diverse RNAs, that reveals sequence and structure-based rules for mitigating hydrolytic degradation. Our findings show that highly structured “superfolder” mRNAs can be designed to improve both stability and expression with further enhancement through pseudouridine nucleoside modification. Together, our study demonstrates simultaneous improvement of mRNA stability and protein expression and provides a computational-experimental platform for the enhancement of mRNA medicines.

Identification and mapping of post-transcriptional modifications on the HIV-1 antisense transcript Ast in human cells.

The human immunodeficiency virus type 1 (HIV-1) encodes multiple RNA molecules. Transcripts that originate from the proviral 5' long terminal repeat (LTR) function as messenger RNAs for the expression of 16 different mature viral proteins. In addition, HIV-1 expresses an antisense transcript (Ast) from the 3'LTR, which has both protein-coding and noncoding properties. While the mechanisms that regulate the coding and noncoding activities of Ast remain unknown, post-transcriptional modifications are known to influence RNA stability, interaction with protein partners, and translation capacity. Here, we report the nucleoside modification profile of Ast obtained through liquid chromatography coupled with mass spectrometry (LC-MS) analysis. The epitranscriptome includes a limited set of modified nucleosides but predominantly ribose methylations. A number of these modifications were mapped to specific positions of the sequence through RNA modification mapping procedures. The presence of modifications on Ast is consistent with the RNA-modifying enzymes interacting with Ast The identification and mapping of Ast post-transcriptional modifications is expected to elucidate the mechanisms through which this versatile molecule can carry out diverse activities in different cell compartments. Manipulation of post-transcriptional modifications on the Ast RNA may have therapeutic implications.

Ribozyme Assays to Quantify the Capping Efficiency of In Vitro-Transcribed mRNA.

The presence of the cap structure on the 5′-end of in vitro-transcribed (IVT) mRNA determines its translation and stability, underpinning its use in therapeutics. Both enzymatic and co-transcriptional capping may lead to incomplete positioning of the cap on newly synthesized RNA molecules. IVT mRNAs are rapidly emerging as novel biologics, including recent vaccines against COVID-19 and vaccine candidates against other infectious diseases, as well as for cancer immunotherapies and protein replacement therapies. Quality control methods necessary for the preclinical and clinical stages of development of these therapeutics are under ongoing development. Here, we described a method to assess the presence of the cap structure of IVT mRNAs. We designed a set of ribozyme assays to specifically cleave IVT mRNAs at a unique position and release 5′-end capped or uncapped cleavage products up to 30 nt long. We purified these products using silica-based columns and visualized/quantified them using denaturing polyacrylamide gel electrophoresis (PAGE) or liquid chromatography and mass spectrometry (LC–MS). Using this technology, we determined the capping efficiencies of IVT mRNAs with different features, which include: Different cap structures, diverse 5′ untranslated regions, different nucleoside modifications, and diverse lengths. Taken together, the ribozyme cleavage assays we developed are fast and reliable for the analysis of capping efficiency for research and development purposes, as well as a general quality control for mRNA-based therapeutics.

Phytophthora capsici infection causes dynamic alterations in tRNA modifications and their associated gene candidates in black pepper

The efforts to signify the relevance of tRNA modifications were always within the limits of prokaryotes, humans, and some fewer model plant systems. The story of tRNA modifications in higher plants is still overlooked, especially in non-model spice crops. Stress causes alterations in tRNA modifications to facilitate the downstream functions of tRNAs. The present study was done to identify and better understand the fate of tRNA nucleoside modifications during biotic stress response in a widely used spice crop called Black pepper. We have uncovered the various tRNA nucleoside modifications present in black pepper. Methylations were the predominant nucleoside modifications in black pepper tRNAs.Furthermore, the different methyltransferase gene candidates implicated in catalyzing tRNA nucleoside methylations in black pepper were also identified. The LC-MS profile showed that certain tRNA nucleoside modifications showed varied abundance upon P. capsici infection. The N4-acetylcytidine (ac4C) nucleoside modification has shown a constant hike at 24 and 48 hpi. At the same time, some nucleoside modifications have exhibited a time-dependent abundance. Altogether our study suggests that tRNA modifications and the expression of associated enzymes are altered during biotic stress regulation.

Advances in mRNA vaccines.

mRNA vaccines have been increasingly recognized as a powerful vaccine platform since the FDA approval of two COVID-19 mRNA vaccines, which demonstrated outstanding prevention efficacy as well as great safety profile. Notably, nucleoside modification and lipid nanoparticle-facilitated delivery has greatly improved the immunogenicity, stability, and translation efficiency of mRNA molecule. Here we review the recent progress in mRNA vaccine development, including nucleoside modification, in vitro synthesis and product purification, and lipid nanoparticle vectors for in vivo delivery and efficient translation. We also briefly introduce the clinical application of mRNA vaccine in preventing infectious diseases and treating inflammatory diseases including cancer.

Mutation enrichment in human DNA samples via UV-mediated cross-linking.

Detection of low-level DNA mutations can reveal recurrent, hotspot genetic changes of clinical relevance to cancer, prenatal diagnostics, organ transplantation or infectious diseases. However, the high excess of wild-type (WT) alleles, which are concurrently present, often hinders identification of salient genetic changes. Here, we introduce UV-mediated cross-linking minor allele enrichment (UVME), a novel approach that incorporates ultraviolet irradiation (∼365 nm UV) DNA cross-linking either before or during PCR amplification. Oligonucleotide probes matching the WT target sequence and incorporating a UV-sensitive 3-cyanovinylcarbazole nucleoside modification are employed for cross-linking WT DNA. Mismatches formed with mutated alleles reduce DNA binding and UV-mediated cross-linking and favor mutated DNA amplification. UV can be applied before PCR and/or at any stage during PCR to selectively block WT DNA amplification and enable identification of traces of mutated alleles. This enables a single-tube PCR reaction directly from genomic DNA combining optimal pre-amplification of mutated alleles, which then switches to UV-mediated mutation enrichment-based DNA target amplification. UVME cross-linking enables enrichment of mutated KRAS and p53 alleles, which can be screened directly via Sanger sequencing, high-resolution melting, TaqMan genotyping or digital PCR, resulting in the detection of mutation allelic frequencies of 0.001–0.1% depending on the endpoint detection method. UV-mediated mutation enrichment provides new potential for mutation enrichment in diverse clinical samples.

An Update on mRNA-Based Viral Vaccines.

With the success of COVID-19 vaccines, newly created mRNA vaccines against other infectious diseases are beginning to emerge. Here, we review the structural elements required for designing mRNA vaccine constructs for effective in vitro synthetic transcription reactions. The unprecedently speedy development of mRNA vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was enabled with previous innovations in nucleoside modifications during in vitro transcription and lipid nanoparticle delivery materials of mRNA. Recent updates are briefly described in the status of mRNA vaccines against SARS-CoV-2, influenza virus, and other viral pathogens. Unique features of mRNA vaccine platforms and future perspectives are discussed.