Nucleotide Modifications Research Articles

Expanding the epitranscriptome: Dihydrouridine in mRNA.

Nucleotide modifications can markedly influence mRNA processing and metabolism. New studies, one in PLOS Biology, show that approximately 130 yeast mRNAs contain dihydrouridine, a derivative of uridine. Functional studies show that dihydrouridine, in some cases, can affect mRNA splicing.

Ribo-DT: An automated pipeline for inferring codon dwell times from ribosome profiling data.

Protein synthesis is an energy consuming process characterised as a pivotal and highly regulated step in gene expression. The net protein output is dictated by a combination of translation initiation, elongation and termination rates that have remained difficult to measure. Recently, the development of ribosome profiling has enabled the inference of translation parameters through modelling, as this method informs on the ribosome position along the mRNA. Here, we present an automated, reproducible and portable computational pipeline to infer relative single-codon and codon-pair dwell times as well as gene flux from raw ribosome profiling sequencing data. As a case study, we applied our workflow to a publicly available yeast ribosome profiling dataset consisting of 57 independent gene knockouts related to RNA and tRNA modifications. We uncovered the effects of those modifications on translation elongation and codon selection during decoding. In particular, knocking out mod5 and trm7 increases codon-specific dwell times which indicates their potential tRNA targets, and highlights effects of nucleotide modifications on ribosome decoding rate.

Growth fitness, heme uptake and genomic variants in mutants of oxygen-tolerant Lacticaseibacillus casei and Lactiplantibacillus plantarum strains

Adaptive Laboratory Evolution (ALE) is a powerful tool to improve the fitness of industrially relevant microorganisms, because it circumvents some of the problems related to the use of genetically modified strains. In this study, we used an ALE strategy involving serial batch cultivations in aerobic and respiratory conditions to generate spontaneous mutants from the respiration-competent strain Lacticaseibacillus casei N87. Genotypic changes in selected mutants were investigated using whole genome sequencing (WGS). The O2-tolerant Lactiplantibacillus plantarum C17 and its mutant C17-m58 (obtained from a previous ALE study) were included in heme uptake experiments and in WGS and variant calling analyses. Several Lcb. casei N87 mutants cultivated under aerobic and respiratory conditions showed improved biomass production, O2 uptake and oxidative stress tolerance compared to the parental strain. Mutants of Lcb. casei and Lpb. plantarum differed from the parental strains in the ability to use heme and menaquinone. High heme concentrations (> 10 mg/L), however, were toxic for all strains. Single nucleotide modifications (SNPs) were detected in some genes encoding for proteins and transcriptional regulators involved in carbon metabolism, oxidative stress, redox balance, and cell wall properties, but their role in the evolved phenotypes needs further investigations. We conclude that prolonged adaptation to aerobic and respiratory life-style may be used as natural strategy to generate strains with improved O2-consuming ability and oxidative stress tolerance, two important features to develop robust cultures and to reduce oxidative processes in foods.

Discrimination between Different DNA Lesions by Monitoring Single-Molecule Polymerase Stalling Kinetics during Nanopore Sequencing.

O6-Carboxymethylguanosine (O6-CMG), O6-methylguanosine (O6-MeG), and abasic site (AP site) are DNA lesions induced by alkylating agents. Identification of these lesions in DNA may aid in understanding their relevance to carcinogenesis and may be used for diagnosis. Nanopore sequencing (NPS) may directly report nucleotide modifications solely from the nanopore readout. However, the conventional NPS strategy still suffers from interferences from neighboring sequences. Instead, by observation of the enzymatic stalling kinetics caused by the O6-CMG, O6-MeG, or AP site, discrimination between different DNA lesions is directly achieved. This strategy is not interfered with by the sequence context around the lesion. The lesion, which retards the movement of the DNA through the pore, efficiently prohibits misreading of the DNA lesion. These results suggest a new strategy in the identification of DNA lesions or DNA modifications. It also provides a high-resolution biophysical tool to investigate enzymatic kinetics caused by DNA lesions and the corresponding enzymes.

Development of a universal antibiotic resistance screening reporter for improving efficiency of cytosine and adenine base editing

Base editing has emerged as a revolutionary technology for single nucleotide modifications. The cytosine and adenine base editors (CBEs and ABEs) have demonstrated great potential in clinical and fundamental research. However, screening and isolating target-edited cells remains challenging. In the current study, we developed a universal Adenine and Cytosine Base-Editing Antibiotic Resistance Screening Reporter (ACBE-ARSR) for improving the editing efficiency. To develop the reporter, the CBE-ARSR was first constructed and shown to be capable of enriching cells for those that had undergone CBE editing activity. Then, the ACBE-ARSR was constructed and was further validated in the editing assays by four different CBEs and two versions of ABE at several different genomic loci. Our results demonstrated that ACBE-ARSR, compared to the reporter of transfection (RoT) screening strategy, improved the editing efficiency of CBE and ABE by 4.6- and 1.9-fold on average, respectively. We found the highest CBE and ABE editing efficiencies as enriched by ACBE-ARSR reached 90% and 88.7%. Moreover, we also demonstrated ACBE-ARSR could be employed for enhancing simultaneous multiplexed genome editing. In conclusion, both CBE and ABE activity can be improved significantly using our novel ACBE-ARSR screening strategy, which we believe will facilitate the development of base editors and their application in biomedical and fundamental research studies.

Chemical Triphosphorylation of Oligonucleotides.

The 5'-triphosphate is an essential nucleic acid modification found throughout all life and increasingly used as a functional modification of oligonucleotides in biotechnology and synthetic biology. Oligonucleotide 5'-triphosphates have historically been prepared in vitro by enzymatic methods. However, these methods are limited to natural RNA oligonucleotides, have strong sequence preferences, and tend to produce heterogeneous products. New methods of chemical triphosphorylation complement both the reduced cost of automated oligonucleotide synthesis by phosphoramidite chemistry and the diverse range of nucleotide modifications now available. Thus, the synthesis of oligonucleotide triphosphates of arbitrary sequence and length, and optionally containing various nonnatural modifications, is now accessible. This paper presents the appropriate methods and techniques for chemical triphosphorylation of oligonucleotides using salicyl phosphorochloridite and pyrophosphate. This method uses commercially available reagents, is compatible with most oligonucleotides prepared by standard solid-phase synthesis methods, and can be completed in 2 h following oligonucleotide synthesis, before deprotection and purification. Two uses of chemically triphosphorylated oligonucleotides as substrates for catalytic RNA enzymes are demonstrated, including the synthesis of a mirror-image version of the hammerhead ribozyme from nonbiological L-RNA triphosphates.

Chemical Triphosphorylation of Oligonucleotides

The 5′-triphosphate is an essential nucleic acid modification found throughout all life and increasingly used as a functional modification of oligonucleotides in biotechnology and synthetic biology. Oligonucleotide 5′-triphosphates have historically been prepared in vitro by enzymatic methods. However, these methods are limited to natural RNA oligonucleotides, have strong sequence preferences, and tend to produce heterogeneous products. New methods of chemical triphosphorylation complement both the reduced cost of automated oligonucleotide synthesis by phosphoramidite chemistry and the diverse range of nucleotide modifications now available. Thus, the synthesis of oligonucleotide triphosphates of arbitrary sequence and length, and optionally containing various nonnatural modifications, is now accessible. This paper presents the appropriate methods and techniques for chemical triphosphorylation of oligonucleotides using salicyl phosphorochloridite and pyrophosphate. This method uses commercially available reagents, is compatible with most oligonucleotides prepared by standard solid-phase synthesis methods, and can be completed in 2 h following oligonucleotide synthesis, before deprotection and purification. Two uses of chemically triphosphorylated oligonucleotides as substrates for catalytic RNA enzymes are demonstrated, including the synthesis of a mirror-image version of the hammerhead ribozyme from nonbiological L-RNA triphosphates.

Base editing the synapse: Modeling a complex neurological disorder in non-human primates

Since its discovery in 2016,1 base editing—the efficient chemical modification of single nucleotides—has enjoyed a blistering pace of advance alongside the broader field of gene editing. In this issue of Molecular Therapy, Lu and colleagues2 take an important step forward in making these technologies applicable to human disease, reporting on the successful use of a base editor to model a complex neurological disorder in non-human primates. STXBP1-related neurodevelopmental disorder (STXBP1-NDD) represents one of the most common genetic neurodevelopmental disorders and is characterized by epilepsy and cognitive, behavioral, and motor impairments3,4 Yet, a lack of adequate model systems has hindered understanding of the underlying pathophysiology.

A Test and Refinement of Folding Free Energy Nearest Neighbor Parameters for RNA Including N6-Methyladenosine

RNA folding free energy change parameters are widely used to predict RNA secondary structure and to design RNA sequences. These parameters include terms for the folding free energies of helices and loops. Although the full set of parameters has only been traditionally available for the four common bases and backbone, it is well known that covalent modifications of nucleotides are widespread in natural RNAs. Covalent modifications are also widely used in engineered sequences. We recently derived a full set of nearest neighbor terms for RNA that includes N6-methyladenosine (m6A). In this work, we test the model using 98 optical melting experiments, matching duplexes with or without N6-methylation of A. Most experiments place RRACH, the consensus site of N6-methylation, in a variety of contexts, including helices, bulge loops, internal loops, dangling ends, and terminal mismatches. For matched sets of experiments that include either A or m6A in the same context, we find that the parameters for m6A are as accurate as those for A. Across all experiments, the root mean squared deviation between estimated and experimental free energy changes is 0.67 kcal/mol. We used the new experimental data to refine the set of nearest neighbor parameter terms for m6A. These parameters enable prediction of RNA secondary structures including m6A, which can be used to model how N6-methylation of A affects RNA structure.

Self-delivering RNAi compounds as therapeutic agents in the central nervous system to enhance axonal regeneration after injury

SummaryThe therapeutic use of RNAi has grown but often faces several hurdles related to delivery systems, compound stability, immune activation, and on-target/off-tissue effects. Self-delivering RNAi (sdRNA) molecules do not require delivery agents or excipients. Here we demonstrate the ability of sdRNA to reduce the expression of PTEN (phosphatase and tensin homolog) to stimulate regenerative axon regrowth in the injured adult CNS. PTEN-targeting sdRNA compounds were tested for efficacy in vivo by intravitreal injection after adult rat optic nerve injury. We describe critical steps in lead compound generation through the optimization of nucleotide modifications, enhancements for stability in biological matrices, and screening for off-target immunostimulatory activity. The data show that PTEN expression in vivo can be reduced using sdRNA and this enhances regeneration in adult CNS neurons after injury, raising the possibility that this method could be utilized for other clinically relevant nervous system indications.

Investigation and validation of labelling loop mediated isothermal amplification (LAMP) products with different nucleotide modifications for various downstream analysis

Loop mediated isothermal amplification (LAMP) is one of the best known and most popular isothermal amplification methods. It's simplicity and speed make the method particularly suitable for point-of-care diagnostics. Nevertheless, false positive results remain a major drawback. Many (downstream) applications are known for the detection of LAMP amplicons like colorimetric assays, in-situ LAMP or CRISPR-Cas systems. Often, modifications of the LAMP products are necessary for different detection applications such as lateral flow assays. This is usually achieved with pre-modified primer. The aim of this study is to evaluate amplicon labelling with different modified nucleotides such as Cy5-dUTP, biotin-dUTP and aminoallyl-dUTP as an alternative to pre-labelled primers. To realise this, the effects on amplification and labelling efficiency were studied as a function of molecule size and nucleotide amount as well as target concentration. This research shows that diverse labelling of LAMP amplicons can be achieved using different, modified nucleotides during LAMP and that these samples can be analysed by a wide range of downstream applications such as fluorescence spectroscopy, gel electrophoresis, microarrays and lateral flow systems. Furthermore, microarray-based detection and the ability to identify and distinguish false positives were demonstrated as proof of concept.

Circular RNA: A novel and potential regulator in pathophysiology of schizophrenia.

Circular RNAs (CircRNAs) are a sub-class of non-coding RNA, which are covalently closed at the ends through a non-canonical process called, backsplicing from the precursor linear RNAs. These molecules are involved in several biological phenomena including regulation of gene expression, synaptic plasticity, and cognition. Several studies have shown that circRNA are present abundantly inside the mammalian brain and they are believed to be associated with the development of neurons and neuronal functions. It is also evident that alterations in intracellular and extracellular levels of circRNAs are linked with various neurological and neuropsychiatric disorders including schizophrenia (SZ). Detailed studies of circRNAs are required to decode the molecular mechanism behind the onset of SZ and the related biological activities during disease progression. This can help unravel their role in this neurobehavioral disorder and develop effective therapeutics against the disease. The present review mainly focuses on the expression and activities of the circRNAs in the post-mortem brain, peripheral blood, and exosomes. It also gives an insight into the role of circRNA interaction with RNA binding proteins (RBPs) and nucleotide modification and their therapeutic potential in the context of schizophrenia.

Concerted modification of nucleotides at functional centers of the ribosome revealed by single-molecule RNA modification profiling.

Nucleotides in RNA and DNA are chemically modified by numerous enzymes that alter their function. Eukaryotic ribosomal RNA (rRNA) is modified at more than 100 locations, particularly at highly conserved and functionally important nucleotides. During ribosome biogenesis, modifications are added at various stages of assembly. The existence of differently modified classes of ribosomes in normal cells is unknown because no method exists to simultaneously evaluate the modification status at all sites within a single rRNA molecule. Using a combination of yeast genetics and nanopore direct RNA sequencing, we developed a reliable method to track the modification status of single rRNA molecules at 37 sites in 18 S rRNA and 73 sites in 25 S rRNA. We use our method to characterize patterns of modification heterogeneity and identify concerted modification of nucleotides found near functional centers of the ribosome. Distinct, undermodified subpopulations of rRNAs accumulate upon loss of Dbp3 or Prp43 RNA helicases, suggesting overlapping roles in ribosome biogenesis. Modification profiles are surprisingly resistant to change in response to many genetic and acute environmental conditions that affect translation, ribosome biogenesis, and pre-mRNA splicing. The ability to capture single-molecule RNA modification profiles provides new insights into the roles of nucleotide modifications in RNA function.

Modelling aptamers with nucleic acid mimics (NAM): From sequence to three-dimensional docking.

Aptamers are single-stranded oligonucleotides, formerly evolved by Systematic Evolution of Ligands by EXponential enrichment (SELEX), that fold into functional three-dimensional structures. Such conformation is crucial for aptamers’ ability to bind to a target with high affinity and specificity. Unnatural nucleotides have been used to develop nucleic acid mimic (NAM) aptamers with increased performance, such as biological stability. Prior knowledge of aptamer-target interactions is critical for applying post-SELEX modifications with unnatural nucleotides since it can affect aptamers’ structure and performance. Here, we describe an easy-to-apply in silico workflow using free available software / web servers to predict the tertiary conformation of NAM, DNA and RNA aptamers, as well as the docking with the target molecule. Representative 2ʹ-O-methyl (2ʹOMe), locked nucleic acid (LNA), DNA and RNA aptamers, with experimental data deposited in Protein Data Bank, were selected to validate the workflow. All aptamers’ tertiary structure and docking models were successfully predicted with good structural similarity to the experimental data. Thus, this workflow will boost the development of aptamers, particularly NAM aptamers, by assisting in the rational modification of specific nucleotides and avoiding trial-and-error approaches.

Advanced preparation of fragment libraries enabled by oligonucleotide-modified 2\u2032,3\u2032-dideoxynucleotides

The ever-growing demand for inexpensive, rapid, and accurate exploration of genomes calls for refinement of existing sequencing techniques. The development of next-generation sequencing (NGS) was a revolutionary milestone in genome analysis. While modified nucleotides already were inherent tools in sequencing and imaging, further modification of nucleotides enabled the expansion into even more diverse applications. Herein we describe the design and synthesis of oligonucleotide-tethered 2′,3′-dideoxynucleotide (ddONNTP) terminators bearing universal priming sites attached to the nucleobase, as well as their enzymatic incorporation and performance in read-through assays. In the context of NGS library preparation, the incorporation of ddONNTP fulfills two requirements at once: the fragmentation step is integrated into the workflow and the obtained fragments are readily labeled by platform-specific adapters. DNA polymerases can incorporate ddONNTP nucleotides, as shown by primer extension assays. More importantly, reading through the unnatural linkage during DNA synthesis was demonstrated, with 25-30% efficiency in single-cycle extension.

Detection techniques for epitranscriptomic marks.

Similar to epigenetic DNA modification, RNA can be methylated and altered for stability and processing. RNA modifications, namely, epitranscriptomes, involve the following three functions: writing, erasing, and reading of marks. Methods for measurement and position detection are useful for the assessment of cellular function and human disease biomarkers. After pyrimidine 5-methylcytosine was reported for the first time a hundred years ago, numerous techniques have been developed for studying nucleotide modifications, including RNAs. Recent studies have focused on high-throughput and direct measurements for investigating the precise function of epitranscriptomes, including the characterization of severe acute respiratory syndrome coronavirus 2. The current study presents an overview of the development of detection techniques for epitranscriptomic marks and briefs about the recent progress in this field.

NMR assignment of non-modified tRNAIle from Escherichia coli

tRNAs are L-shaped RNA molecules of ~ 80 nucleotides that are responsible for decoding the mRNA and for the incorporation of the correct amino acid into the growing peptidyl-chain at the ribosome. They occur in all kingdoms of life and both their functions, and their structure are highly conserved. The L-shaped tertiary structure is based on a cloverleaf-like secondary structure that consists of four base paired stems connected by three to four loops. The anticodon base triplet, which is complementary to the sequence of the mRNA, resides in the anticodon loop whereas the amino acid is attached to the sequence CCA at the 3′-terminus of the molecule. tRNAs exhibit very stable secondary and tertiary structures and contain up to 10% modified nucleotides. However, their structure and function can also be maintained in the absence of nucleotide modifications. Here, we present the assignments of nucleobase resonances of the non-modified 77 nt tRNAIle from the gram-negative bacterium Escherichia coli. We obtained assignments for all imino resonances visible in the spectra of the tRNA as well as for additional exchangeable and non-exchangeable protons and for heteronuclei of the nucleobases. Based on these assignments we could determine the chemical shift differences between modified and non-modified tRNAIle as a first step towards the analysis of the effect of nucleotide modifications on tRNA’s structure and dynamics.

Bacterial DNA excision repair pathways.

Bacteria are continuously exposed to numerous endogenous and exogenous DNA-damaging agents. To maintain genome integrity and ensure cell survival, bacteria have evolved several DNA repair pathways to correct different types of DNA damage and non-canonical bases, including strand breaks, nucleotide modifications, cross-links, mismatches and ribonucleotide incorporations. Recent advances in genome-wide screens, the availability of thousands of whole-genome sequences and advances in structural biology have enabled the rapid discovery and characterization of novel bacterial DNA repair pathways and new enzymatic activities. In this Review, we discuss recent advances in our understanding of base excision repair and nucleotide excision repair, and we discuss several new repair processes including the EndoMS mismatch correction pathway and the MrfAB excision repair system.

Adaptation of the protein translational apparatus during ATDC5 chondrogenic differentiation.

IntroductionRibosome biogenesis is integrated with many cellular processes including proliferation, differentiation and oncogenic events. Chondrogenic proliferation and differentiation require a high cellular translational capacity to facilitate cartilaginous extracellular matrix production. We here investigated the expression dynamics of factors involved in ribosome biogenesis during in vitro chondrogenic differentiation and determined whether protein translation capacity adapts to different phases of chondrogenic differentiation.MaterialsSnoRNA expression during ATDC5 differentiation was analyzed by RNA sequencing of samples acquired from day 0 (progenitor stage), 7 (chondrogenic stage) and day 14 (hypertrophic stage). RT-qPCR was used to determine expression of fibrillarin, dyskerin, UBF-1, Sox9, Col2a1, Runx2, Col10a1 mRNAs and 18S, 5.8S and 28S rRNAs. Protein expression of fibrillarin, dyskerin and UBF-1 was determined by immunoblotting. Ribosomal RNA content per cell was determined by calculating rRNA RT-qPCR signals relative to DNA content (SYBR Green assay). Total protein translational activity was evaluated with a puromycilation assay and polysome profiling.ResultsAs a result of initiation of chondrogenic differentiation (Δt0-t7), 21 snoRNAs were differentially expressed (DE). Hypertrophic differentiation caused DE of 23 snoRNAs (Δt7-t14) and 43 when t0 was compared to t14. DE snoRNAs, amongst others, target nucleotide modifications in the 28S rRNA peptidyl transferase center and the 18S rRNA decoding center. UBF-1, fibrillarin and dyskerin expression increased as function of differentiation and displayed highest fold induction at day 5–6 in differentiation. Ribosomal RNA content per cell was significantly increased at day 7, but not at day 14 in differentiation. Similar dynamics in translational capacity and monosomal ribosome fraction were observed during differentiation.ConclusionThe expression of a great number of ribosome biogenesis factors is altered during chondrogenic differentiation of ATDC5 cells, which is accompanied by significant changes in cellular translational activity. This elucidation of ribosome biogenesis dynamics in chondrogenic differentiation models enables the further understanding of the role of ribosome biogenesis and activity during chondrocyte cell commitment and their roles in human skeletal development diseases.

RNA modifications stabilize the tertiary structure of tRNAfMet by locally increasing conformational dynamics.

A plethora of modified nucleotides extends the chemical and conformational space for natural occurring RNAs. tRNAs constitute the class of RNAs with the highest modification rate. The extensive modification modulates their overall stability, the fidelity and efficiency of translation. However, the impact of nucleotide modifications on the local structural dynamics is not well characterized. Here we show that the incorporation of the modified nucleotides in tRNAfMet from Escherichia coli leads to an increase in the local conformational dynamics, ultimately resulting in the stabilization of the overall tertiary structure. Through analysis of the local dynamics by NMR spectroscopic methods we find that, although the overall thermal stability of the tRNA is higher for the modified molecule, the conformational fluctuations on the local level are increased in comparison to an unmodified tRNA. In consequence, the melting of individual base pairs in the unmodified tRNA is determined by high entropic penalties compared to the modified. Further, we find that the modifications lead to a stabilization of long-range interactions harmonizing the stability of the tRNA’s secondary and tertiary structure. Our results demonstrate that the increase in chemical space through introduction of modifications enables the population of otherwise inaccessible conformational substates.