Modification Of Nucleic Acids Research Articles

Shorter Is Better: The α-(l)-Threofuranosyl Nucleic Acid Modification Improves Stability, Potency, Safety, and Ago2 Binding and Mitigates Off-Target Effects of Small Interfering RNAs.

Chemical modifications are necessary to ensure the metabolic stability and efficacy of oligonucleotide-based therapeutics. Here, we describe analyses of the α-(l)-threofuranosyl nucleic acid (TNA) modification, which has a shorter 3'-2' internucleotide linkage than the natural DNA and RNA, in the context of small interfering RNAs (siRNAs). The TNA modification enhanced nuclease resistance more than 2'-O-methyl or 2'-fluoro ribose modifications. TNA-containing siRNAs were prepared as triantennary N-acetylgalactosamine conjugates and were tested in cultured cells and mice. With the exceptions of position 2 of the antisense strand and position 11 of the sense strand, the TNA modification did not inhibit the activity of the RNA interference machinery. In a rat toxicology study, TNA placed at position 7 of the antisense strand of the siRNA mitigated off-target effects, likely due to the decrease in the thermodynamic binding affinity relative to the 2'-O-methyl residue. Analysis of the crystal structure of an RNA octamer with a single TNA on each strand showed that the tetrose sugar adopts a C4'-exo pucker. Computational models of siRNA antisense strands containing TNA bound to Argonaute 2 suggest that TNA is well accommodated in the region kinked by the enzyme. The combined data indicate that the TNA nucleotides are promising modifications expected to increase the potency, duration of action, and safety of siRNAs.

Simultaneous RNA and DNA Adductomics Using Single Data-Independent Acquisition Mass Spectrometry Analysis.

Adductomics studies are used for the detection and characterization of various chemical modifications (adducts) of nucleic acids and proteins. The advancements in liquid chromatography coupled with high-resolution tandem mass spectrometry (HRMS/MS) have resulted in efficient methods for qualitative and quantitative adductomics. We developed an HRMS-based method for the simultaneous analysis of RNA and DNA adducts in a single run and demonstrated its application using Baltic amphipods, useful sentinels of environmental disturbances, as test organisms. The novelty of this method is screening for RNA and DNA adducts by a single injection on an Orbitrap HRMS instrument using full scan and data-independent acquisition. The MS raw files were processed with an open-source program, nLossFinder, to identify and distinguish RNA and DNA adducts based on the characteristic neutral loss of ribonucleosides and 2'-deoxyribonucleosides, respectively. In the amphipods, in addition to the nearly 150 putative DNA adducts characterized earlier, we detected 60 putative RNA adducts. For the structural identification of the detected RNA adducts, the MODOMICS database was used. The identified RNA adducts included simple mono- and dimethylation and other larger functional groups on different ribonucleosides and deaminated product inosine. However, 54 of these RNA adducts are not yet structurally identified, and further work on their characterization may uncover new layers of information related to the transcriptome and help understand their biological significance. Considering the susceptibility of nucleic acids to environmental factors, including pollutants, the developed multi-adductomics methodology with further advancement has the potential to provide biomarkers for diagnostics of pollution effects in biota.

Modulation of the tetrameric I-motif folding of C-rich Tetrahymena telomeric sequences by hexitol nucleic acid (HNA) modifications

I-motifs are non-canonical DNA structures consisting of two parallel strands held together by hemiprotonated cytosine-cytosine+ base pairs, which intercalate to form a ordered column of stacked base pairs. This unique structure covers potential relevance in various fields, including gene regulation and biotechnological applications. A unique structural feature of I-motifs (iM), is the presence of sugar-sugar interactions through their extremely narrow minor grooves. Consistently, oligonucleotides containing pentose derivatives such as ribose, 2′-deoxyribose, arabinose, and 2′-deoxy-2′-fluoroarabinose highlighted a very different attitude to fold into iM. On the other hand, there is significant attention focused on exploring sugar-modifications that can increase nucleic acids resistance to nuclease degradation, a crucial requirement for therapeutic applications.An interesting example, not addressed in the iM field yet, is represented by hexitol nucleic acid (HNA), a metabolically stable six-membered ring analogue compatible with A-like double helix formation. Herein, we selected two DNA C-rich Tetrahymena telomeric sequences whose tetrameric iMs were already resolved by NMR and we investigated the iM folding of related HNA and RNA oligonucleotides by circular dichroism, differential scanning calorimetry and NMR. The comparison of their behaviours vs the DNA counterparts provided interesting insights into the influence of the sugar on iM folding. In particular, ribose and hexitol prevented iM formation. However, by clustering the hexitol-containing residues at the 3′-end, it was possible to modulate the distribution of the different topological species described for the DNA iMs. These data open new avenues for the exploitation of sugar modifications for I-motif characterization and applications.

Disease Diagnosis Based on Nucleic Acid Modifications.

Nucleic acid modifications include a wide range of epigenetic and epitranscriptomic factors and impact a wide range of nucleic acids due to their profound influence on biological inheritance, growth, and metabolism. The recently developed methods of mapping and characterizing these modifications have promoted their discovery as well as large-scale studies in eukaryotes, especially in humans. Because of these pioneering strategies, nucleic acid modifications have been shown to have a great impact on human disorders such as cancer. Therefore, whether nucleic acid modifications could become a new type of biomarker remains an open question. In this review, we briefly look back at classical nucleic acid modifications and then focus on the progress made in investigating these modifications as diagnostic biomarkers in clinical therapy and present our perspective on their development prospects.

Dynamics of nucleic acid mobility.

Advances in sequencing technologies and bioinformatic analyses are accelerating the quantity and quality of data from all domains of life. This rich resource has the potential to reveal a number of important incidences with respect to possible exchange of nucleic acids. Ancient events have impacted species evolution and adaptation to new ecological niches. However, we still lack a full picture of processes ongoing within and between somatic cells, gametes, and different organisms. We propose that events linked to acceptance of alien nucleic acids grossly could be divided into 2 main routes in plants: one, when plants are exposed to extreme challenges and, the second level, a more everyday or season-related stress incited by biotic or abiotic factors. Here, many events seem to comprise somatic cells. Are the transport and acceptance processes of alien sequences random or are there specific regulatory systems not yet fully understood? Following entrance into a new cell, a number of intracellular processes leading to chromosomal integration and function are required. Modification of nucleic acids and possibly exchange of sequences within a cell may also occur. Such fine-tune events are most likely very common. There are multiple questions that we will discuss concerning different types of vesicles and their roles in nucleic acid transport and possible intracellular sequence exchange between species.

Molecular basis for the reversible ADP-ribosylation of guanosine bases

Modification of nucleic acids by ADP-ribosylation is catalyzed by various ADP-ribosyltransferases, including the DarT enzyme. The latter is part of the bacterial toxin-antitoxin (TA) system DarTG, which was shown to provide control of DNA replication and bacterial growth as well as protection against bacteriophages. Two subfamilies have been identified, DarTG1 and DarTG2, which are distinguished by their associated antitoxins. While DarTG2 catalyzes reversible ADP-ribosylation of thymidine bases employing a macrodomain as antitoxin, the DNA ADP-ribosylation activity of DarTG1 and the biochemical function of its antitoxin, a NADAR domain, are as yet unknown. Using structural and biochemical approaches, we show that DarT1-NADAR is a TA system for reversible ADP-ribosylation of guanosine bases. DarT1 evolved the ability to link ADP-ribose to the guanine amino group, which is specifically hydrolyzed by NADAR. We show that guanine de-ADP-ribosylation is also conserved among eukaryotic and non-DarT-associated NADAR members, indicating a wide distribution of reversible guanine modifications beyond DarTG systems.

Targeting Metalloenzymes: The "Achilles' Heel" of Viruses and Parasites.

Metalloenzymes are central to the regulation of a wide range of essential viral and parasitic functions, including protein degradation, nucleic acid modification, and many others. Given the impact of infectious diseases on human health, inhibiting metalloenzymes offers an attractive approach to disease therapy. Metal-chelating agents have been expansively studied as antivirals and antiparasitics, resulting in important classes of metal-dependent enzyme inhibitors. This review provides the recent advances in targeting the metalloenzymes of viruses and parasites that impose a significant burden on global public health, including influenza A and B, hepatitis B and C, and human immunodeficiency viruses as well as Trypanosoma brucei and Trypanosoma cruzi.

Updated protein domain annotation of the PARP protein family sheds new light on biological function.

AlphaFold2 and related computational tools have greatly aided studies of structural biology through their ability to accurately predict protein structures. In the present work, we explored AF2 structural models of the 17 canonical members of the human PARP protein family and supplemented this analysis with new experiments and an overview of recent published data. PARP proteins are typically involved in the modification of proteins and nucleic acids through mono or poly(ADP-ribosyl)ation, but this function can be modulated by the presence of various auxiliary protein domains. Our analysis provides a comprehensive view of the structured domains and long intrinsically disordered regions within human PARPs, offering a revised basis for understanding the function of these proteins. Among other functional insights, the study provides a model of PARP1 domain dynamics in the DNA-free and DNA-bound states and enhances the connection between ADP-ribosylation and RNA biology and between ADP-ribosylation and ubiquitin-like modifications by predicting putative RNA-binding domains and E2-related RWD domains in certain PARPs. In line with the bioinformatic analysis, we demonstrate for the first time PARP14's RNA-binding capability and RNA ADP-ribosylation activity in vitro. While our insights align with existing experimental data and are probably accurate, they need further validation through experiments.

Design of LNA Analogues Using a Combined Density Functional Theory and Molecular Dynamics Approach for RNA Therapeutics.

Antisense therapeutics treat a wide spectrum of diseases, many of which cannot be addressed with the current drug technologies. In the quest to design better antisense oligonucleotide drugs, we propose five novel LNA analogues (A1-A5) for modifying antisense oligonucleotides and establishing each with the five standard nucleic acids: adenine (A), guanine (G), cytosine (C), thymine (T), and uracil (U). Monomer nucleotides of these modifications were considered for a detailed Density Functional Theory (DFT)-based quantum chemical analysis to determine their molecular-level structural and electronic properties. A detailed MD simulation study was done on a 14-mer ASO (5'-CTTAGCACTGGCCT-3') containing these modifications targeting PTEN mRNA. Results from both molecular- and oligomer-level analysis clearly depicted LNA-level stability of the modifications, the ASO/RNA duplexes maintaining stable Watson-Crick base pairing preferring RNA-mimicking A-form duplexes. Notably, monomer MO isosurfaces for both purines and pyrimidines were majorly distributed on the nucleobase region in modifications A1 and A2 and in the bridging unit in modifications A3, A4, and A5, suggesting that A3/RNA, A4/RNA, and A5/RNA duplexes interact more with the RNase H and solvent environment. Accordingly, solvation of A3/RNA, A4/RNA, and A5/RNA duplexes was higher compared to that of LNA/RNA, A1/RNA, and A2/RNA duplexes. This study has resulted in a successful archetype for creating advantageous nucleic acid modifications tailored for particular needs, fulfilling a useful purpose of designing novel antisense modifications, which may overcome the drawbacks and improve the pharmacokinetics of existing LNA antisense modifications.

Derivatization of Mirror-imageL-Nucleic Acids with 2'-OMe Modification for Thermal and Structural Stabilization.

To further expand the functionality and enhance the stability of mirror-image nucleic acid as advanced agent for basic research and therapeutic design, we have synthesized the 2'-deoxy2'-methoxy-L-uridine phosphoramidites and incorporate it into L-DNA and L-RNA via solid-phase synthesis quantitatively. We found that the thermostability of L-nucleic acids is dramatically improved after introducing the modifications. Moreover, we successfully crystallized both L-DNA and L-RNA duplexes containing the 2'-OMe modifications and sharing the identical sequence. The crystal structure determination and analysis reveal the overall structures of the mirrorimage nucleic acids, and for the first time interpretate the structural deviation caused by 2'-OMe and 2'-OH groups in the oligonucleotides which are highly similar. This novel chemical modification of nucleic acid has the potential to be used to design nucleic acid-based therapeutic and material in the future.

Specific prediction of mortality by oxidative stress-induced damage to RNA vs. DNA in humans.

Modifications of nucleic acids (DNA and RNA) from oxidative stress is a potential driver of aging per se and of mortality in age-associated medical disorders such as type 2 diabetes (T2D). In a human cohort, we found a strong prediction of all-cause mortality by a marker of systemic oxidation of RNA in patients with T2D (n = 2672) and in nondiabetic control subjects (n = 4079). The finding persisted after the adjustment of established modifiers of oxidative stress (including BMI, smoking, and glycated hemoglobin). In contrast, systemic levels of DNA damage from oxidation, which traditionally has been causally linked to both T2D and aging, failed to predict mortality. Strikingly, these findings were subsequently replicated in an independent general population study (n = 3649). The data demonstrate a specific importance of RNA damage from oxidation in T2D and general aging.

Widespread RNA hypoediting in schizophrenia and its relevance to mitochondrial function.

RNA editing, the endogenous modification of nucleic acids, is known to be altered in genes with important neurological function in schizophrenia (SCZ). However, the global profile and molecular functions of disease-associated RNA editing remain unclear. Here, we analyzed RNA editing in postmortem brains of four SCZ cohorts and uncovered a significant and reproducible trend of hypoediting in patients of European descent. We report a set of SCZ-associated editing sites via WGCNA analysis, shared across cohorts. Using massively parallel reporter assays and bioinformatic analyses, we observed that differential 3' untranslated region (3'UTR) editing sites affecting host gene expression were enriched for mitochondrial processes. Furthermore, we characterized the impact of two recoding sites in the mitofusin 1 (MFN1) gene and showed their functional relevance to mitochondrial fusion and cellular apoptosis. Our study reveals a global reduction of editing in SCZ and a compelling link between editing and mitochondrial function in the disease.

Abstract 215: A novel enzymatic library preparation workflow that dramatically reduces artifacts associated with damaged FFPE samples

Abstract Clinical oncology heavily relies on formalin-fixed, paraffin-embedded (FFPE) tissue samples for histology and molecular characterization. The chemical and physical modifications of nucleic acids introduced during fixation, storage and purification negatively impact molecular profiling and vary from sample-to-sample. Conventional sonication and ligation-based library preparation is considered the gold-standard approach for FFPE samples, but it is time-consuming and expensive. Importantly, these processes introduce artifacts that impact downstream analysis and interpretation (Haile et al., 2019). We have developed a novel fragmentation chemistry that virtually eliminates hairpin artifacts, achieving levels on par with non-FFPE control samples. Our fragmentation method is highly scalable, exhibits minimal sequence bias, and reduces cost and workflow inefficiencies associated with mechanical shearing. In this study, we developed a unified method for library preparation from FFPE samples which produces similar insert sizes across variable input mass and quality of FFPE samples. We carefully evaluated the performance of this method relative to a sonication-based approach employing the KAPA HyperPrep kit. Libraries were constructed from 50 to 200 ng of FFPE DNA, inputs typically used for NGS, ranging from low-to high-quality as assessed using a qPCR-based method and DNA integrity (DIN). Targeted sequencing was performed using a 37 kb custom oncology hybridization capture panel to investigate molecular complexity. Our workflow virtually eliminated hairpin artifacts that were present in up to 4.5% of reads in sonication-based libraries. Soft-clipping was also 3- to 7-fold lower in libraries prepared with the Watchmaker kit relative to sonicated DNA libraries, improving overall sequencing economy. Furthermore, the mean target coverage achieved with the Watchmaker kit was comparable to or higher than sonication libraries using the same input mass. Because input masses were normalized post-shearing, which typically results in 20-40% sample loss, coverage with our approach is significantly higher relative to sonication, if normalizing to pre-sheared DNA input. Watchmaker DNA Library Preparation with Fragmentation enables high-quality DNA library preparation from damaged FFPE samples, producing high target coverage, uniform insert size, and minimizing sequencing artifacts to improve sensitivity and specificity. This approach is highly scalable and automatable, enabling various oncology applications. Citation Format: Giulia Corbet, Philip Benson, Kailee Reed, Skyler Mishkin, Thomas Harrison, Kristin Scott, Zane Jaafar, Kristina Giorda, Josh Haimes, Martin Ranik, Brian Kudlow. A novel enzymatic library preparation workflow that dramatically reduces artifacts associated with damaged FFPE samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 215.

Oligonucleotides Containing C5-Propynyl Modified Arabinonucleic Acids: Synthesis, Biophysical and Antisense Properties.

The introduction of chemical modifications on the nucleic acid scaffold has allowed for the progress of antisense oligonucleotides (ASOs) in the clinic for the treatment of a variety of disorders. In contribution to the repertoire of gene-silencing nucleic acid modifications, herein we report the synthesis and incorporation of C5-propynyl arabinouridine (araUP ) and arabinocytidine (araCP ) into mixed-base ASOs containing a pyrimidine core. Substitution of the core with araUP and araCP resulted in stabilization of the duplex formed with RNA but not with DNA. Similar results were obtained with ASOs bearing phosphorothioate linkages or methoxyethyl (MOE) wings in a gapmer design. All modified ASOs were compatible with E. coli RNase H mediated degradation of target RNA. Substitution of DNA for araUP and araCP in the central portion of a gapmer with MOE wings demonstrated improved nuclease resistance. These results suggest C5-modified arabinonucleic acids may serve as a potential chemical modification for therapeutic ASOs.

The Long and Winding Road: On-Demand DNA Synthesis in High Demand

The Long and Winding Road: On-Demand DNA Synthesis in High Demand

Oxidative stress in the progression of Chronic Kidney Diseases in children

Current studies show that the increase in the incidence of Chronic Kidney Disease cannot be fully explained only by risk factors well elucidated in the specialty literature, as knowing that oxidative stress is prevalent in Chronic Kidney Disease patients and is considered to be an important pathogenetic mechanism. Reactive oxygen species, which appear in excess as a result of ischemic, toxic or immune mediator changes, can negatively influence various pathological processes in renal pathology. These processes mainly refer to the pathogenesis of Chronic Kidney Disease or acute kidney injury. There are at least two pathophysiological mechanisms by which oxidative stress is formed in renal pathology, and then their damaging effects can cause, maintain or aggravate the cause of the condition. Following the initiation of inflammatory processes, the progression of renal damage is accelerated, with the eventual damage to all nephron structures as well as the interstitial space. The main sources of reactive oxygen species are mitochondria, but other cellular organelles may also be involved. Oxidant radicals produce oxidation of proteins, lipids, modification of nucleic acids with destruction of cell membranes, induction of apoptosis and lysis of kidney tissue. It is shown that free radicals are normally annihilated by superoxide dismutase – the antioxidant enzyme. There are several biomarkers that can be evaluated to detect oxidative stress and antioxidant status in patients with chronic kidney disease. Their determination in the context of a complex examination represents an informational support in the elucidation of pathogenetic mechanisms with the implementation of non-invasive early diagnosis methods. We reviewed the Chronic Kidney Disease literature to see if the guidelines and data are reliable enough to justify a particular approach.

Overexpression of 18S rRNA methyltransferase CrBUD23 enhances biomass and lutein content in Chlamydomonas reinhardtii.

Post-transcriptional modification of nucleic acids including transfer RNA (tRNA), ribosomal RNA (rRNA) and messenger RNA (mRNA) is vital for fine-tunning of mRNA translation. Methylation is one of the most widespread post-transcriptional modifications in both eukaryotes and prokaryotes. HsWBSCR22 and ScBUD23 encodes a 18S rRNA methyltransferase that positively regulates cell growth by mediating ribosome maturation in human and yeast, respectively. However, presence and function of 18S rRNA methyltransferase in green algae are still elusive. Here, through bioinformatic analysis, we identified CrBUD23 as the human WBSCR22 homolog in genome of the green algae model organism Chlamydonomas reinhardtii. CrBUD23 was a conserved putative 18S rRNA methyltransferase widely exited in algae, plants, insects and mammalians. Transcription of CrBUD23 was upregulated by high light and down-regulated by low light, indicating its role in photosynthesis and energy metabolism. To characterize its biological function, coding sequence of CrBUD23 fused with a green fluorescence protein (GFP) tag was derived by 35S promoter and stably integrated into Chlamydomonas genome by glass bead-mediated transformation. Compared to C. reinhardtii wild type CC-5325, transgenic strains overexpressing CrBUD23 resulted in accelerated cell growth, thereby leading to elevated biomass, dry weight and protein content. Moreover, overexpression of CrBUD23 increased content of photosynthetic pigments but not elicit the activation of antioxidative enzymes, suggesting CrBUD23 favors growth and proliferation in the trade-off with stress responses. Bioinformatic analysis revealed the G1177 was the putative methylation site in 18S rRNA of C. reinhardtii CC-849. G1177 was conserved in other Chlamydonomas isolates, indicating the conserved methyltransferase activity of BUD23 proteins. In addition, CrTrm122, the homolog of BUD23 interactor Trm112, was found involved in responses to high light as same as CrBUD23. Taken together, our study revealed that cell growth, protein content and lutein accumulation of Chlamydomonas were positively regulated by the 18S rRNA methyltransferase CrBUD23, which could serve as a promising candidate for microalgae genetic engineering.

Acyclic (S)-glycol nucleic acid (S-GNA) modification of siRNAs improves the safety of RNAi therapeutics while maintaining potency.

Glycol nucleic acid (GNA) is an acyclic nucleic acid analog connected via phosphodiester bonds. Crystal structures of RNA-GNA chimeric duplexes indicated that nucleotides of the right-handed (S)-GNA were better accommodated in the right-handed RNA duplex than were the left-handed (R)-isomers. GNA nucleotides adopt a rotated nucleobase orientation within all duplex contexts, pairing with complementary RNA in a reverse Watson-Crick mode, which explains the inabilities of GNA C and G to form strong base pairs with complementary nucleotides. Transposition of the hydrogen bond donor and acceptor pairs using novel (S)-GNA isocytidine and isoguanosine nucleotides resulted in stable base-pairing with the complementary G and C ribonucleotides, respectively. GNA nucleotide or dinucleotide incorporation into an oligonucleotide increased resistance against 3'-exonuclease-mediated degradation. Consistent with the structural observations, small interfering RNAs (siRNAs) modified with (S)-GNA had greater in vitro potencies than identical sequences containing (R)-GNA. (S)-GNA is well tolerated in the seed regions of antisense and sense strands of a GalNAc-conjugated siRNA in vitro. The siRNAs containing a GNA base pair in the seed region had in vivo potency when subcutaneously injected into mice. Importantly, seed pairing destabilization resulting from a single GNA nucleotide at position 7 of the antisense strand mitigated RNAi-mediated off-target effects in a rodent model. Two GNA-modified siRNAs have shown an improved safety profile in humans compared with their non-GNA-modified counterparts, and several additional siRNAs containing the GNA modification are currently in clinical development.

New biochemistry in the Rhodanese-phosphatase superfamily: emerging roles in diverse metabolic processes, nucleic acid modifications, and biological conflicts.

The protein-tyrosine/dual-specificity phosphatases and rhodanese domains constitute a sprawling superfamily of Rossmannoid domains that use a conserved active site with a cysteine to catalyze a range of phosphate-transfer, thiotransfer, selenotransfer and redox activities. While these enzymes have been extensively studied in the context of protein/lipid head group dephosphorylation and various thiotransfer reactions, their overall diversity and catalytic potential remain poorly understood. Using comparative genomics and sequence/structure analysis, we comprehensively investigate and develop a natural classification for this superfamily. As a result, we identified several novel clades, both those which retain the catalytic cysteine and those where a distinct active site has emerged in the same location (e.g. diphthine synthase-like methylases and RNA 2' OH ribosyl phosphate transferases). We also present evidence that the superfamily has a wider range of catalytic capabilities than previously known, including a set of parallel activities operating on various sugar/sugar alcohol groups in the context of NAD+-derivatives and RNA termini, and potential phosphate transfer activities involving sugars and nucleotides. We show that such activities are particularly expanded in the RapZ-C-DUF488-DUF4326 clade, defined here for the first time. Some enzymes from this clade are predicted to catalyze novel DNA-end processing activities as part of nucleic-acid-modifying systems that are likely to function in biological conflicts between viruses and their hosts.

SWAMNA: a comprehensive platform for analysis of nucleic acid modifications.

The interest in MS-based analysis of modified nucleic acids is increasing due to the application of nucleic acids in therapeutics. However, there are few available integrated platforms for characterizing nucleic acid modifications. Herein, we report a general mass spectrometry-based SWATH platform to identify and quantify both RNA and DNA modifications, which we call SWATH analysis of modified nucleic acids (SWAMNA). SWAMNA incorporates the search engine, NuMo finder, enabling the analysis of modifications in native and permethylated form. SWAMNA will aid discoveries that provide new insights into nucleic acid modifications.