Base-resolution m5C profiling across the mammalian transcriptome by bisulfite-free enzyme-assisted chemical labeling approach.

5-methylcytosine (m5C) is a prevalent RNA modification crucial for gene expression regulation. However, accurate and sensitive m5C sites identification remains challenging due to severe RNA degradation and reduced sequence complexity during bisulfite sequencing (BS-seq). Here, we report m5C-TAC-seq, a bisulfite-free approach combining TET-assisted m5C-to-f5C oxidation with selective chemical labeling, therefore enabling direct base-resolution m5C detection through pre-enrichment and C-to-T transitions at m5C sites. With m5C-TAC-seq, we comprehensively profiled the m5C methylomes in human and mouse cells, identifying a substantially larger number of confident m5C sites. Through perturbing potential m5C methyltransferases, we deciphered the responsible enzymes for most m5C sites, including the characterization of NSUN5's involvement in mRNA m5C deposition. Additionally, we characterized m5C dynamics during mESC differentiation. Notably, the mild reaction conditions and preservation of nucleotide composition in m5C-TAC-seq allow m5C detection in chromatin-associated RNAs. The accurate and robust m5C-TAC-seq will advance research into m5C methylation functional investigation.

RNA modifications identification based on chemical reactions.

RNA modification identification is an emerging field in epigenetics due to its indispensable regulatory role in the cell life cycle. With advancements in identification methods, an increasing number of RNA modifications has been discovered, thereby driving the development of more efficient and accurate techniques for localizing modified RNAs and elucidating their functions. High-throughput sequencing approaches for modified RNA detection can be categorized into antibody-based, enzymatic-based, and chemical-labeling-based methods. Given the intrinsic chemical reactions involved in all biochemical processes, we provide a comprehensive review of recent advancements in artificial chemical labeling and transformations of ten distinct RNA modifications and their applications in sequencing. Our aim is to contribute to a deeper understanding of the mechanisms underlying these modifications. We focus on the chemical reactions associated with RNA modifications and briefly compare the advantages and disadvantages of detection methods based on these reactions. Additionally, we introduce several approaches that identify multiple modifications through chemical labeling. As the field of RNA modification research continues to expand, we anticipate that the techniques and insights presented in this review will serve as a valuable resource for future studies aimed at further elucidating the functional roles of RNA modifications in biological processes.

Ultrabright Acrylic Polymers with Tunable Fluorescence Enabled by Imprisoning Single TICT Probe.

Bright and colorful fluorescent polymers are ideal materials for a variety of applications. Although polymers could be made fluorescent by physical doping or chemical binding of fluorescent units, it is a great challenge to get colorful and highly emissive polymers with a single fluorophore. Here the development of a general and facile method to synthesize ultrabright and colorful polymers using a single twisted intramolecular charge transfer (TICT) probe is reported. By incorporating polymerizable, highly fluorescent, and environmental sensitive TICT probe, a series of colorful acrylic polymers (emission from 481 to 543 nm) with almost 100% fluorescence quantum yields are prepared. Like the solvatochromic effect, functional groups within side chains of acrylic polymers (including alkyl chain, tetrahydrofurfuryl group, and hydroxyl group) provide varied environmental polarity for the incorporated fluorophore, resulting in a series of colorful polymeric materials. Benefiting from the excellent photophysical properties, the polymers show great potential in encryption, cultural relics protection, white light-emitting diode bulb making, and fingerprint identification.

Recent Advances in Analytical Approaches for Glycan and Glycopeptide Quantitation.

Growing implications of glycosylation in physiological occurrences and human disease have prompted intensive focus on revealing glycomic perturbations through absolute and relative quantification. Empowered by seminal methodologies and increasing capacity for detection, identification, and characterization, the past decade has provided a significant increase in the number of suitable strategies for glycan and glycopeptide quantification. Mass-spectrometry-based strategies for glycomic quantitation have grown to include metabolic incorporation of stable isotopes, deposition of mass difference and mass defect isotopic labels, and isobaric chemical labeling, providing researchers with ample tools for accurate and robust quantitation. Beyond this, workflows have been designed to harness instrument capability for label-free quantification, and numerous software packages have been developed to facilitate reliable spectrum scoring. In this review, we present and highlight the most recent advances in chemical labeling and associated techniques for glycan and glycopeptide quantification.

Switching of Photocatalytic Tyrosine/Histidine Labeling and Application to Photocatalytic Proximity Labeling.

Weak and transient protein interactions are involved in dynamic biological responses and are an important research subject; however, methods to elucidate such interactions are lacking. Proximity labeling is a promising technique for labeling transient ligand-binding proteins and protein-protein interaction partners of analytes via an irreversible covalent bond. Expanding chemical tools for proximity labeling is required to analyze the interactome. We developed several photocatalytic proximity-labeling reactions mediated by two different mechanisms. We found that numerous dye molecules can function as catalysts for protein labeling. We also identified catalysts that selectively modify tyrosine and histidine residues and evaluated their mechanisms. Model experiments using HaloTag were performed to demonstrate photocatalytic proximity labeling. We found that both ATTO465, which catalyzes labeling by a single electron transfer, and BODIPY, which catalyzes labeling by singlet oxygen, catalyze proximity labeling in cells.

Ultrasensitive Simultaneous Detection of Multiple Rare Modified Nucleosides as Promising Biomarkers in Low-Put Breast Cancer DNA Samples for Clinical Multi-Dimensional Diagnosis.

Early cancer diagnosis is essential for successful treatment and prognosis, and modified nucleosides have attracted widespread attention as a promising group of cancer biomarkers. However, analyzing these modified nucleosides with an extremely low abundance is a great challenge, especially analyzing multiple modified nucleosides with a different abundance simultaneously. In this work, an ultrasensitive quantification method based on chemical labeling, coupled with LC-MS/MS analysis, was established for the simultaneous quantification of 5hmdC, 5fdC, 5hmdU and 5fdU. Additionally, the contents of 5mdC and canonical nucleosides could be obtained at the same time. Upon derivatization, the detection sensitivities of 5hmdC, 5fdC, 5hmdU and 5fdU were dramatically enhanced by several hundred times. The established method was further applied to the simultaneous detection of nine nucleosides with different abundances in about 2 µg genomic DNA of breast tissues from 20 breast cancer patients. The DNA consumption was less than other overall reported quantification methods, thereby providing an opportunity to monitor rare, modified nucleosides in precious samples and biology processes that could not be investigated before. The contents of 5hmdC, 5hmdU and 5fdU in tumor tissues and normal tissues adjacent to the tumor were significantly changed, indicating that these three modified nucleosides may play certain roles in the formation and development of tumors and be potential cancer biomarkers. While the detection rates of 5hmdC, 5hmdU and 5fdU alone as a biomarker for breast cancer samples were 95%, 75% and 85%, respectively, by detecting these three cancer biomarkers simultaneously, two of the three were 100% consistent with the overall trend. Therefore, simultaneous detection of multiple cancer biomarkers in clinical samples greatly improved the accuracy of cancer diagnosis, indicating that our method has great application potential in clinical multidimensional diagnosis.

A Mutation-Based Method for Pinpointing a DNA N6 -Methyladenine Methyltransferase Modification Site at Single Base Resolution.

DNA N6 -methyladenine (6mA) has recently received notable attention due to an increased finding of its functional roles in higher eukaryotes. Here we report an enzyme-assisted chemical labeling method to pinpoint the DNA 6mA methyltransferase (MTase) substrate modification site at single base resolution. A designed allyl-substituted MTase cofactor was applied in the catalytic transfer reaction, and the allyl group was installed to the N6 -position of adenine within a specific DNA sequence to form N6 -allyladenine (6aA). The iodination of 6aA allyl group induced the formation of 1, N6 -cyclized adenine which caused mutations during DNA replication by a polymerase. Thus the modification site could be precisely detected by a mutation signal. We synthesized 6aA deoxynucleoside and deoxynucleotide model compounds and a 6aA-containing DNA probe, and screened nine DNA polymerases to define an optimal system capable of detecting the substrate modification site of a DNA 6mA MTase at single-base resolution.

Insights into Dynamic Mechanism of Ligand Binding to Peroxisome Proliferator-Activated Receptor γ toward Potential Pharmacological Applications.

Peroxisome proliferator-activated receptor γ (PPARγ) is a member of the nuclear receptor superfamily, which regulates the transcription of a variety of genes involved in lipid and glucose metabolism, inflammation, and cell proliferation. These functions correlate with the onset of type-2 diabetes, obesity, and immune disorders, which makes PPARγ a promising target for drug development. The majority of PPARγ functions are regulated by binding of small molecule ligands, which cause conformational changes of PPARγ followed by coregulator recruitment. The ligand-binding domain (LBD) of PPARγ contains a large Y-shaped cavity that can be occupied by various classes of compounds such as full agonists, partial agonists, natural lipids, and in some cases, a combination of multiple molecules. Several crystal structure studies have revealed the binding modes of these compounds in the LBD and insight into the resulting conformational changes. Notably, the apo form of the PPARγ LBD contains a highly mobile region that can be stabilized by ligand binding. Furthermore, recent biophysical investigations have shed light on the dynamic mechanism of how ligands induce conformational changes in PPARγ and result in functional output. This information may be useful for the design of new and repurposed structures of ligands that serve a different function from original compounds and more potent pharmacological effects with less undesirable clinical outcomes. This review provides an overview of the peculiar characteristics of the PPARγ LBD by examining a series of structural studies focused on the dynamic mechanism of binding and the potential applications of strategies for ligand screening and chemical labeling.

Chemical-Labeling-Assisted Detection of Nucleobase Modifications by Quantum-Tunneling-Based Single-Molecule Sensing.

Quantum-tunneling-based DNA sensing is a single-molecule technique that promises direct mapping of nucleobase modifications. However, its applicability is seriously limited because of the small difference in conductivity between modified and unmodified nucleobases. Herein, a chemical labeling strategy is presented that facilitates the detection of modified nucleotides by quantum tunneling. We used 5-Formyl-2'-deoxyuridine as a model compound and demonstrated that chemical labeling dramatically alters its molecular conductance compared with that of canonical nucleotides; thus, facilitating statistical discrimination, which is impeded in the unlabeled state. This work introduces a chemical strategy that overcomes the intrinsic difficulty in quantum-tunneling-based modification analysis-the similarity of the molecular conductance of the nucleobases of interest.

Molecules and elements for quantitative bioanalysis: The allure of using electrospray, MALDI, and ICP mass spectrometry side-by-side.

To understand biological processes, not only reliable identification, but quantification of constituents in biological processes play a pivotal role. This is especially true for the proteome: protein quantification must follow protein identification, since sometimes minute changes in abundance tell the real tale. To obtain quantitative data, many sophisticated strategies using electrospray and MALDI mass spectrometry (MS) have been developed in recent years. All of them have advantages and limitations. Several years ago, we started to work on strategies, which are principally capable to overcome some of these limits. The fundamental idea is to use elemental signals as a measure for quantities. We began by replacing the radioactive 32 P with the "cold" natural 31 P to quantify modified nucleotides and phosphorylated peptides and proteins and later used tagging strategies for quantification of proteins more generally. To do this, we introduced Inductively Coupled Plasma Mass Spectrometry (ICP-MS) into the bioanalytical workflows, allowing not only reliable and sensitive detection but also quantification based on isotope dilution absolute measurements using poly-isotopic elements. The detection capability of ICP-MS becomes particularly attractive with heavy metals. The covalently bound proteins tags developed in our group are based on the well-known DOTA chelate complex (1,4,7,10-tetraazacyclododecane-N,N',N″,N‴-tetraacetic acid) carrying ions of lanthanoides as metal core. In this review, I will outline the development of this mutual assistance between molecular and elemental mass spectrometry and discuss the scope and limitations particularly of peptide and protein quantification. The lanthanoide tags provide low detection limits, but offer multiplexing capabilities due to the number of very similar lanthanoides and their isotopes. With isotope dilution comes previously unknown accuracy. Separation techniques such as electrophoresis and HPLC were used and just slightly adapted workflows, already in use for quantification in bioanalysis. Imaging mass spectrometry (MSI) with MALDI and laser ablation ICP-MS complemented the range of application in recent years.

Compilation of Modern Technologies To Map Genome-Wide Cytosine Modifications in DNA.

Over the past few decades, various DNA modification detection methods have been developed; many of the high-resolution methods are based on bisulfite treatment, which leads to DNA degradation, to a degree. Thus, novel bisulfite-free approaches have been developed in recent years and shown to be useful for epigenome analysis in otherwise difficult-to-handle, but important, DNA samples, such as hmC-seal and hmC-CATCH. Herein, an overview of advances in the development of epigenome sequencing methods for these important DNA modifications is provided.

Metabolic analysis of the melatonin biosynthesis pathway using chemical labeling coupled with liquid chromatography-mass spectrometry.

Characterization of the melatonin (MLT) biosynthesis pathway in plants is still limited. Additionally, a metabolomic analysis of MLT biosynthesis in plants is still a challenge due to analyte structural and chemical diversity, low analyte abundances, and plant matrix complexities. Herein, a sensitive liquid chromatography-mass spectrometry (LC-MS) method enabling the simultaneous determination of seven plant MLT biosynthetic metabolites was developed. In the proposed strategy, the targeted metabolites, which included tryptophan (Trp), tryptamine (TAM), 5-hydroxytryptophan (5HTP), serotonin (5HT), N-acetylserotonin (NAS), 5-methoxytryptamine (5MT), and MLT, were purified from plant extracts using a one-step dispersive solid-phase extraction (DSPE). The samples were then chemically labeled with dansyl chloride (DNS-Cl), followed by analysis using LC-MS. The limit of detection (LOD) values ranged from 0.03 to 1.36 pg/mL and presented a 22- to 469-fold decrease when compared to the unlabeled metabolites. Due to the high sensitivity of the proposed method, the consumption of plant materials was reduced to 10 mg FW. Ultimately, the established method was utilized to examine the distributions of MLT and its intermediates in rice shoots and roots with or without cadmium (Cd) stress. The results suggested that under normal condition, MLT may also be generated via a Trp/TAM/5HT/5MT/MLT path (Pathway II) in addition to the previously reported Trp/TAM/5HT/NAS/MLT path (Pathway I), although Pathway I was shown to be dominant. During Cd stress, MLT was also shown to be produced through these two pathways, with Pathway II shown to be dominant in rice shoots and roots.

A new boronic acid reagent for the simultaneous determination of C27-, C28-, and C29-brassinosteroids in plant tissues by chemical labeling-assisted liquid chromatography-mass spectrometry.

Brassinosteroids (BRs) are endogenous plant growth-promoting hormones affecting growth and development during the entire life cycle of plants. Naturally occurring BRs can be classified into C27-, C28-, or C29-BRs based on the nature of the alkyl groups occupying the C-24 position in the side chain of the 5a-cholestane carbon skeleton. However, while C27-BRs exhibit similar bioactivities to C28- and C29-BRs, the biosynthetic pathways of C27-BRs in plants have not yet been clearly characterized. In addition to a lack of biochemical and enzymatic evidence regarding the biosynthetic pathways of C27-BRs, even most of the intermediate compounds on their pathways have not been explored and identified due to the lower endogenous levels of C27-BRs. Therefore, the development of highly sensitive analytical methods is essential for studying the biosynthetic pathways and physiological functions of C27-BRs. Accordingly, this study establishes qualitative and quantitative methods for identifying and detecting C27-, C28-, and C29-BRs using a newly synthesized boronic acid reagent denoted as 2-methyl-4-phenylaminomethylphenylboronic acid (2-methyl-4-PAMBA) in conjunction with liquid chromatography-mass spectrometry (LC-MS). Labeling with 2-methyl-4-PAMBA provides derivatives with excellent stability, and the detection sensitivities of BRs, particularly for C27-BRs, are dramatically improved. The limits of detection (with a signal-to-noise ratio of 3) for six BRs, including 2 C27-BRs (28-norCS and 28-norBL), 3 C28-BRs (CS, BL, and TY), and a single C29-BR (28-homoBL), are found to be 0.10-1.68 pg/mL after labeling with 2-methyl-4-PAMBA. Finally, the proposed analytical method is successfully applied for the detection of endogenous BRs in small mass samples of Oryza sativa seedlings, Rape flowers, Arabidopsis shoots, and Arabidopsis flowers. In addition, a method for profiling potential BRs in plants is also developed using LC-MS in multiple reaction monitoring scan mode assisted by 2-methyl-4-PAMBA and 2-methyl-4-PAMBA-d5 labeling. The developed method is able to identify 10 potential BRs in a Rape flower extract. The proposed quantitative and qualitative methods established by 2-methyl-4-PAMBA labeling are helpful for facilitating an understanding of the physiological functions and biosynthetic pathways of BRs, particularly for C27-BRs. Graphical abstract.

A boronic acid modified binary matrix consisting of boron nitride and α-cyano-4-hydroxycinnamic acid for determination of cis-diols by MALDI-TOF MS.

A MALDI-TOF mass spectrometric method is described for the determination of small molecule compounds with cis-diol. It is based on the use of a binary matrix consisting of boron nitride (BN) and α-cyano-4-hydroxycinnamic acid that was modified with the derivatization reagent of (3-(acridin-9-ylamino)phenyl)boronic acid which can recognize cis-diols. The binary matrix is used for desorption/ionization (DI) in the positive ion mode. The mechanism leading to DI enhancement was investigated. The results imply that BN is beneficial for the DI because it induces an enhancement in the positive ion mode. The boronic acid-functionalized binary matrix was successfully applied to capture the glucose, shikimic acid and quinic acid. The method was applied to the determination of 3-chloro-1,2-propanediol in plant oil. Graphical abstract Schematic representation of a method for detecting the cis-diol compounds on matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) using the binary matrix of boron nitride (BN)/α-cyano-4-hydroxycinnamic acid (CHCA) that was modified with (3-(acridin-9-ylamino)phenyl) boronic acid (AYPBA).

A tool to assure the geographical origin of local food products (glasshouse tomatoes) using labeling with rare earth elements.

BACKGROUND: Trace element fingerprinting has been widely used for identification of provenance of regional food. In the case of products from conventional agriculture, it is expected that the elemental composition will comply with that of the commercially available substrate of the plants. Therefore, for products without a direct relationship with the regional soil the region-specific differences in elemental composition are no longer recognizable. The idea of this work is the labeling of tomatoes with rare earth elements (REE) in the ultra-trace range for food authentication. RESULTS: Labeling of tomatoes was carried out either by watering the soil with Nd- and Er-spiked water or by adding these elements as solid oxides to the soil. In both cases enrichment of Nd and Er relative to the control group was detected in tomato fruits and leaves using inductively coupled plasma-mass spectrometry. Tomato plants rapidly absorb the dissolved REE from the irrigation water, and watering for a short period just before ripeness is sufficient to induce REE labels. CONCLUSION: Labeling with trace amounts of REE could potentially be used to assure the provenance of tomatoes of local origin and separate these from products of foreign origin. © 2018 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Detection of nucleic acid modifications by chemical reagents.

Nucleic acids, especially RNA, naturally contain a diversity of chemically modified nucleosides. To understand the biological role of these modified nucleosides, nucleic acid scientists need tools to specifically label, detect and enrich modified nucleic acids. These tools comprise a diverse set of chemical reagents which have been established in the early years of nucleic acid research. Recent developments in high-throughput sequencing and mass spectrometry utilize these chemical labeling strategies to efficiently detect and localize modifications in nucleic acids. As a consequence the transcriptome-wide distribution of modified nucleosides, especially 5-methylcytosine and pseudouridine, in all domains of life could be analyzed. With the help of these techniques and the gained knowledge, it becomes possible to understand the functions of modifications and even study their connections to human health and disease. Here, the differential chemical reactivity of modified nucleosides and their canonical counterpart is reviewed and discussed.

Lysine conjugation properties in human IgGs studied by integrating high-resolution native mass spectrometry and bottom-up proteomics.

Antibody-drug conjugates (ADCs) are a novel class of biopharmaceuticals several of which are now being investigated in clinical studies. In ADCs, potent cytotoxic drugs are coupled via a linker to reactive residues in IgG monoclonal antibodies. Linkage to lysine residues in the IgGs, using N-hydroxysuccinimide ester based chemistry, is one of the possible options. To control drug load and specificity, proper knowledge is required about which lysine residues are most accessible and reactive. Here, we combine native MS and bottom-up proteomics to monitor the overall drug load and site-specific lysine reactivity, using N-hydroxysuccinimide-based tandem mass tags. High-resolution Orbitrap native MS enables us to monitor and quantify, due to the achieved baseline resolution, the sequential incorporation of up to 69 tandem mass tag molecules into human IgGs. Complementary, bottom-up proteomics facilitates the identification of some very reactive "hot-spot" conjugation sites. However, we also identify lysine residues that are highly resistant to chemical labeling. Our integrated approach gives insight into the conjugation properties of IgGs at both the intact protein and residue levels, providing fundamental information for controlling drug load and specificity in lysine-linked ADCs.

Low-pH Solid-Phase Amino Labeling of Complex Peptide Digests with TMTs Improves Peptide Identification Rates for Multiplexed Global Phosphopeptide Analysis.

We present a novel tandem mass tag solid-phase amino labeling (TMT-SPAL) protocol using reversible immobilization of peptides onto octadecyl-derivatized (C18) solid supports. This method can reduce the number of steps required in complex protocols, saving time and potentially reducing sample loss. In our global phosphopeptide profiling workflow (SysQuant), we can cut 24 h from the protocol while increasing peptide identifications (20%) and reducing side reactions. Solid-phase labeling with TMTs does require some modification to typical labeling conditions, particularly pH. It has been found that complete labeling equivalent to standard basic pH solution-phase labeling for small and large samples can be achieved on C18 resins under slightly acidic buffer conditions. Improved labeling behavior on C18 compared to that with standard basic pH solution-phase labeling is demonstrated. We analyzed our samples for histidine, serine, threonine, and tyrosine labeling to determine the degree of overlabeling and observed higher than expected levels (25% of all peptide spectral matches (PSMs)) of overlabeling at all of these amino acids (predominantly at tyrosine and serine) in our standard solution-phase labeling protocol. Overlabeling at all of these sites is greatly reduced (4-fold, to 7% of all PSMs) by the low-pH conditions used in the TMT-SPAL protocol. Overlabeling seems to represent a so-far overlooked mechanism causing reductions in peptide identification rates with NHS-activated TMT labeling compared to that with label-free methods. Our results also highlight the importance of searching data for overlabeling when labeling methods are used.

Overcoming the detrimental O-acylation in TMTpro labeling improves the proteome depth and quantification precision.

BACKGROUND: With the advent of proline-based reporter isobaric Tandem Mass Tag (TMTpro) reagents, the sample multiplexing capacity of tandem mass tags (TMTs) has been expanded, and up to 18 samples can be quantified in a multiplexed manner. Like classic TMT reagents, TMTpro reagents contain a tertiary amine group, which markedly enhances their reactivity toward hydroxyl groups and results in O-acylation of serine, threonine and tyrosine residues. This overlabeling significantly compromises proteome analysis in terms of depth and precision. In particular, the reactivity of hydroxyl-containing residues can be dramatically enhanced when coexisting with a histidine in the same peptides, leading to a severe systematic bias against the analysis of these peptides. Although some protocols using a reduced molar excess of TMT under alkaline conditions can alleviate overlabeling of histidine-free peptides to some extent, they have a limited effect on histidyl- and hydroxyl-containing peptides. RESULTS: Here, we report a novel TMTpro labeling method that overcomes detrimental overlabeling while providing high labeling efficiency of amines. Additionally, our method is cost-effective, as it requires only half the amount of TMTpro reagents recommended by the reagent manufacturer. In a deep-scale analysis of a yeast/human two-proteome model sample, we compared our method with a typical alkaline labeling method using a reduced molar excess of TMTpro. Even at a depth of over 10,000 proteins, our method detected 23.7% more unique peptides and 8.7% more protein groups compared to the alkaline labeling method. Moreover, our method significantly improved the quantitative precision due to the reduced variability in labeling and increased protein sequence coverage. This substantially enhanced the statistical power of our method for detecting differentially abundant proteins, providing an average of 13% more yeast proteins that reached statistical significance. SIGNIFCANCE: We presented a novel TMTpro labeling method that overcomes the detrimental O-acylation and thus significantly improves the depth and quantitative precision for proteome analysis.

ICLAMP: a novel technique to explore adenosine deamination via inosine chemical labeling and affinity molecular purification.

Recent developments in sequencing and bioinformatics have advanced our understanding of adenosine-to-inosine (A-to-I) RNA editing. Surprisingly, recent analyses have revealed the capability of adenosine deaminase acting on RNA (ADAR) to edit DNA:RNA hybrid strands. However, edited inosines in DNA remain largely unexplored. A precise biochemical method could help uncover these potentially rare DNA editing sites. We explore maleimide as a scaffold for inosine labeling. With fluorophore-conjugated maleimide, we were able to label inosine in RNA or DNA. Moreover, with biotin-conjugated maleimide, we purified RNA and DNA containing inosine. Our novel technique of inosine chemical labeling and affinity molecular purification offers substantial advantages and provides a versatile platform for further discovery of A-to-I editing sites in RNA and DNA.