Canonical Nucleosides Research Articles

Robuste Bisulfit‐freie Einzelmolekül‐Echtzeitsequenzierung von Methyldesoxycytidin auf der Grundlage eines neuartigen hpTet3‐Enzyms

AbstractNeben den vier kanonischen Nukleosiden dA, dG, dC und T enthält die genomische DNA die zusätzliche Base 5‐Methyldesoxycytidin (mdC). Die Anwesenheit dieses methylierten Cytidin‐Nukleosids in Promotorregionen oder Genkörpern beeinflusst die Transkriptionsaktivität des entsprechenden Gens erheblich. Folglich sind die Methylierungsmuster von Genen entscheidend dafür, ob Gene stillgelegt oder aktiviert werden. Die Sequenzierung der mdC‐Positionen im Genom ist daher für die Krebsfrüherkennung von größter Bedeutung, da sie hilft, eine fehlerhafte Genexpression zu erkennen. Derzeit ist die Bisulfit‐Methode der Goldstandard für die mdC‐Sequenzierung. Diese Methode hat jedoch den Nachteil, dass der größte Teil der Ausgangs‐DNA während der Bisulfit‐Behandlung zerstört wird. Außerdem ist die Bisulfit‐Sequenzierung anfällig für Fehler. Wir berichten hier über eine schonende, bisulfitfreie mdC‐Sequenzierungsmethode, die wir als EMox‐seq bezeichnen und auf der Einzelmolekül‐SMRT‐Sequenzierung der dritten Generation beruht. Die Grundlage unserer Technologie ist ein neues Tet3‐Enzym, das mdC effizient zu 5‐Carboxycytidin (cadC) oxidiert. CadC wiederum liefert mit speziell trainierten KI‐basierten Algorithmen ein hervorragendes Signal bei der SMRT‐Sequenzierung.

Robust Bisulfite-Free Single-Molecule Real-Time Sequencing of Methyldeoxycytidine Based on a Novel hpTet3 Enzyme.

In addition to the four canonical nucleosides dA, dG, dC and T, genomic DNA contains the additional base 5-methyldeoxycytidine (mdC). The presence of this methylated cytidine nucleoside in promoter regions or gene bodies significantly affects the transcriptional activity of the corresponding gene. Consequently, the methylation patterns of genes are crucial for either silencing or activating genes. Sequencing the positions of mdC in the genome is therefore of paramount importance for early cancer diagnostics as it helps determine incorrect gene expression. Currently, the bisulfite method is the gold standard for mdC-sequencing. However, this method has the drawback that the majority of the input DNA is degraded during the bisulfite treatment. Additionally, bisulfite sequencing is prone to errors. Here, we report a benign, bisulfite-free mdC sequencing method termed EMox-seq, which is based on third-generation single-molecule SMRT sequencing. The foundation of this technology is a new Tet3 enzyme that efficiently oxidizes mdCs to 5-carboxycytidine (cadC). In turn, cadC provides an excellent readout by SMRT sequencing using specially trained AI-based algorithms.

Influence of 5-fluorination on the structure and glycosidic bond stability of the protonated canonical DNA and RNA cytidine nucleosides: Oh, what a difference the 2′-hydroxy substituent makes?

Fluorinated nucleosides are well-established as anticancer and antiviral medications. As with many pharmaceuticals, the effects of fluorine modifications are often only partially understood. In this work, the effects of 5-fluorination of the cytosine nucleobase on the structures and glycosidic bond stabilities of the protonated canonical DNA and RNA cytidine nucleosides (dCyd and Cyd) are examined. Infrared multiple photon dissociation action spectroscopy experiments and electronic structure calculations are employed to probe the structural influences of 5-fluorination. Spectral signatures in the IR fingerprint and hydrogen-stretching regions indicate that 5-fluorination heavily directs for and solely produces O2 protonation para to the 5-fluoro substituent. This differs from the canonical cytidine nucleosides where roughly equal populations of O2 and N3 protonated structures were observed. Energy-resolved collision-induced dissociation experiments combined with survival yield analyses are performed to probe the influence of 5-fluorination on glycosidic bond stability. Trends in the energy-dependence of the survival yield curves indicate that 5-fluorination weakens the glycosidic bond, and that the influence of 5-fluorination on glycosidic bond stability is much greater for dCyd than Cyd. Theory also finds that 5-fluorination weakens the glycosidic bond but predicts that the influence of 5-fluorination on N-glycosidic bond stability is roughly the same for dCyd and Cyd. Oh, what a difference the 2′-hydroxy substituent makes?

The Excited State Dynamics of a Mutagenic Guanosine Etheno Adduct Investigated by Femtosecond Fluorescence Spectroscopy and Quantum Mechanical Calculations.

Femtosecond fluorescence upconversion experiments were combined with CASPT2 and time dependent DFT calculations to characterize the excited state dynamics of the mutagenic etheno adduct 1,N2-etheno-2'-deoxyguanosine (ϵdG). This endogenously formed lesion is attracting great interest because of its ubiquity in human tissues and its highly mutagenic properties. The ϵdG fluorescence is strongly modified with respect to that of the canonical nucleoside dG, notably by an about 6-fold increase in fluorescence lifetime and quantum yield at neutral pH. In addition, femtosecond fluorescence upconversion experiments reveal the presence of two emission bands with maxima at 335 nm for the shorter-lived and 425 nm for the longer-lived. Quantum mechanical calculations rationalize these findings and provide absorption and fluorescence spectral shapes similar to the experimental ones. Two different bright minima are located on the potential energy surface of the lowest energy singlet excited state. One planar minimum, slightly more stable, is associated with the emission at 335 nm, whereas the other one, with a bent etheno ring, is associated with the red-shifted emission.

The Influence of 2'-Deoxyguanosine Lesions on the Electronic Properties of OXOG:::C Base Pairs in Ds-DNA: A Comparative Analysis of Theoretical Studies.

DNA is continuously exposed to a variety of harmful factors, which, on the one hand, can force undesirable processes such as ageing, carcinogenesis and mutagenesis, while on the other hand, can accelerate evolutionary changes. Of all the canonical nucleosides, 2'-deoxyguanosine (dG) exhibits the lowest ionization potential, making it particularly prone to the one-electron oxidizing process. The most abundant type of nucleobase damage is constituted by 7,8-dihydro-8-oxo-2'-deoxyguanosine (OXOdG), with an oxidation potential that is 0.56 V lower than that of canonical dG. All this has led to OXOdG, as an isolated lesion, being perceived as a sink for radical cations in the genome. In this paper, a comparative analysis of the electronic properties of an OXOGC base pair within the context of a clustered DNA lesion (CDL) has been conducted. It is based on previous DFT studies that were carried out at the M06-2x/6-31++G** level of theory in non-equilibrated and equilibrated condensed phases. The results of the comparative analysis presented here reveal the following: (A) The ionization potentials of OXOG4C2 were largely unaffected by a second lesion. (B) The positive charge and spin were found predominantly on the OXOG4C2 moiety. (C) The electron-hole transfers A3T3→G4C2 and G4C2←A5T1 were found in the Marcus inverted region and were resistant to the presence of a second DNA lesion in close proximity. It can therefore be reasonably postulated that OXOGC becomes the sink for a radical cation migrating through the double helix, irrespective of the presence of other 2'-deoxyguanosine lesions in the CDL structure.

Synthesis, Detection, and Metabolism of Pyridone Ribosides, Products of NAD Overoxidation.

Pyridone-containing adenine dinucleotides, ox-NAD, are formed by overoxidation of nicotinamide adenine dinucleotide (NAD+) and exist in three distinct isomeric forms. Like the canonical nucleosides, the corresponding pyridone-containing nucleosides (PYR) are chemically stable, biochemically versatile, and easily converted to nucleotides, di- and triphosphates, and dinucleotides. The 4-PYR isomer is often reported with its abundance increasing with the progression of metabolic diseases, age, cancer, and oxidative stress. Yet, the pyridone-derived nucleotides are largely under-represented in the literature. Here, we report the efficient synthesis of the series of ox-NAD and pyridone nucleotides and measure the abundance of ox-NAD in biological specimens using liquid chromatography coupled with mass spectrometry (LC-MS). Overall, we demonstrate that all three forms of PYR and ox-NAD are found in biospecimens at concentrations ranging from nanomolar to midmicromolar and that their presence affects the measurements of NAD(H) concentrations when standard biochemical redox-based assays are applied. Furthermore, we used liver extracts and 1H NMR spectrometry to demonstrate that each ox-NAD isomer can be metabolized to its respective PYR isomer. Together, these results suggest a need for a better understanding of ox-NAD in the context of human physiology since these species are endogenous mimics of NAD+, the key redox cofactor in metabolism and bioenergetics maintenance.

Prebiotic synthesis of dihydrouridine by photoreduction of uridine in formamide.

In this report, we show that a very common modification (especially in tRNA), dihydrouridine, was efficiently produced by photoreduction of the canonical pyrimidine ribonucleoside, uridine in formamide. Formamide not only acts as a solvent in this reaction, but also as the reductant. The other three components of the canonical alphabet (C, A, G) remained intact under the same conditions, suggesting that dihydrouridine might have coexisted with all four canonical RNA nucleosides (C, U, A, G) at the dawn of life.

Contiguous Silver(I)-Mediated Base Pairs of Imidazophenanthroline and Canonical Nucleobases in DNA Duplexes: Formation of Classical Duplexes versus Homodimer Formation.

Metal-mediated base pairs represent a topical alternative to canonical hydrogen-bonded base pairs. In this context, the ligand 1H-imidazo[4,5-f][1,10]phenanthroline (P) was introduced as an artificial nucleobase in a glycol nucleic acid-based nucleoside analogue into a DNA oligonucleotide in a way that the oligonucleotide contains a central block of six contiguous P residues. The ability to engage in Ag+-mediated base pairing was evaluated with respect to the four canonical nucleosides in positions complementary to P. Highly stabilizing Ag+-mediated base pairs were formed with cytosine and guanine (i.e., P-Ag+-C and P-Ag+-G base pairs), whereas the analogous base pairs with thymine and adenine were much less stable and hence formed incompletely. Surprisingly, the intermediate formation of a homodimeric duplex of the P-containing oligonucleotide was observed in all cases, albeit to a different extent. The homodimer is composed of P-Ag+-P base pairs and 18 overhanging mismatched canonical nucleobases. It demonstrates the obstacles present when designing metal-mediated base pairs as metal complexation may take place irrespective of the surrounding natural base pairs. Homodimer formation was found to be particularly prominent when the designated metal-mediated base pairs are of low stability, suggesting that homodimers and regular duplexes are formed in a competing manner.

Photochemical Stability of 5-Methylcytidine Relative to Cytidine: Photophysical Insight for mRNA Therapeutic Applications.

5-Methylcytidine (5mCyd) has recently been investigated with renewed interest in its utilization in mRNA therapeutics. However, its photostability following exposure to electromagnetic radiation has been overlooked. This Letter compares the photostability and excited-state dynamics of 5mCyd with those of the canonical RNA nucleoside, cytidine (Cyd), using steady-state and femtosecond transient absorption spectroscopy under physiologic conditions. 5mCyd is shown to have a 5-fold higher fluorescence yield and a 5-fold longer 1ππ* excited-state decay lifetime. Importantly, however, the excited-state population in 5mCyd decays primarily by internal conversion, with a photodegradation rate 3 times smaller than that in Cyd. In Cyd, the population of a 1nπ* state with a lifetime of ca. 45 ps is implicated in the formation 6-hydroxycytidine and other photoproducts.

Wavelength dependent excited state dynamics observed in canonical pyrimidine nucleosides

Epidemiological evidence indicates that damage to DNA/RNA initialized by ultraviolet (UV) radiation is associated with skin cancer. Wavelength dependence of DNA photodamage was proposed as early as 1990s and demonstrated later on. Unraveling the photo-activated dynamics involved in related reactions is essential. However, studies aimed at uncovering the wavelength dependent excited state dynamics in canonical pyrimidine nucleosides have not received enough attention. In this work, excitation wavelength dependent excited state dynamics of 2′-deoxy-thymidine (dThd) and oxy-uridine (Urd) are investigated in acetonitrile solutions by femtosecond broadband transient absorption spectroscopy. Varying the excitation wavelength leads to a significant difference in the branching of the excited state population at the Franck-Condon (FC) region, resulting higher fluorescence quantum yield with 285 nm pump but higher triplet state quantum yield under 267 nm excitation. Based on our results, a vibronic coupling regulated excited state relaxation mechanism is proposed. This mechanism information is important for understanding the formation of harmful photoproducts for DNA/RNA with different wavelength UV excitations.

Aminodealkenylation: Ozonolysis and copper catalysis convert C(sp3)-C(sp2) bonds to C(sp3)-N bonds.

Great efforts have been directed toward alkene π bond amination. In contrast, analogous functionalization of the adjacent C(sp3)-C(sp2) σ bonds is much rarer. Here we report how ozonolysis and copper catalysis under mild reaction conditions enable alkene C(sp3)-C(sp2) σ bond-rupturing cross-coupling reactions for the construction of new C(sp3)-N bonds. We have used this unconventional transformation for late-stage modification of hormones, pharmaceutical reagents, peptides, and nucleosides. Furthermore, we have coupled abundantly available terpenes and terpenoids with nitrogen nucleophiles to access artificial terpenoid alkaloids and complex chiral amines. In addition, we applied a commodity chemical, α-methylstyrene, as a methylation reagent to prepare methylated nucleosides directly from canonical nucleosides in one synthetic step. Our mechanistic investigation implicates an unusual copper ion pair cooperative process.

Influence of 2'-Modifications (O-Methylation, Fluorination, and Stereochemical Inversion) on the Base Pairing Energies of Protonated Cytidine Nucleoside Analogue Base Pairs: Implications for the Stabilities of i-Motif Structures.

Naturally occurring and chemically engineered modifications are among the most powerful strategies explored for fine-tuning the conformational characteristics and intrinsic stability of nucleic acids topologies. Modifications at the 2'-position of the ribose or 2'-deoxyribose moieties differentiate nucleic acid structures and have a significant impact on their electronic properties and base-pairing interactions. 2'-O-Methylation, a common post-transcriptional modification of tRNA, is directly involved in modulating specific anticodon-codon base-pairing interactions. 2'-Fluorinated and arabino nucleosides possess novel and beneficial medicinal properties and find use as therapeutics for treating viral diseases and cancer. However, the potential to deploy 2'-modified cytidine chemistries for tuning i-motif stability is largely unknown. To address this knowledge gap, the effects of 2'-modifications including O-methylation, fluorination, and stereochemical inversion on the base-pairing interactions of protonated cytidine nucleoside analogue base pairs, the core stabilizing interactions of i-motif structures, are examined using complementary threshold collision-induced dissociation techniques and computational methods. The 2'-modified cytidine nucleoside analogues investigated here include 2'-O-methylcytidine, 2'-fluoro-2'-deoxycytidine, arabinofuranosylcytosine, 2'-fluoro-arabinofuranosylcytosine, and 2',2'-difluoro-2'-deoxycytidine. All five 2'-modifications examined here are found to enhance the base-pairing interactions relative to the canonical DNA and RNA cytidine nucleosides with the greatest enhancements arising from 2'-O-methylation and 2',2'-difluorination, suggesting that these modifications should well be tolerated in the narrow grooves of i-motif conformations.

Chemical Diversification of Carbocyclic Fluorinated Pyrimidine Nucleosides: Introducing 2′-Arabino Analogues and Ring Unsaturation

AbstractAnalogues of the canonical nucleosides have a longstanding presence and proven capability within medicinal chemistry and drug-discovery research. Herein, we report chemical diversification of carbocyclic pyrimidine nucleosides containing CF2 and CHF in place of the furanose oxygen to introduce ring unsaturation and 2′-epimers. Utilizing gram-scale access to 6′-(R)-monofluoro- and 6′-gem-difluorouridine, we explore the provision of 2′,3′-didehydro-2′,3′-dideoxy, and 1′,2′-didehydro-2′-deoxy analogues, alongside the first example of a 6′-(R)-fluoro arabino-carbauridine. Key stereochemistries and the presence of unsaturation are confirmed using X-ray crystallography and NMR, and an indicative conformational preference for a monofluoro 2′,3′-didehydro-2′,3′-dideoxy system is presented. This synthetic blueprint offers a potential to explore biological activity for these hitherto unavailable materials, including a direct comparison to established nucleoside analogue drugs.

Replacement of the phosphodiester backbone between canonical nucleosides with a dirhenium carbonyl "click" linker-a new class of luminescent organometallic dinucleoside phosphate mimics.

The first-in-class luminescent dinucleoside phosphate analogs with a [Re2(μ-Cl)2(CO)6(μ-pyridazine)] "click" linker as a replacement for the natural phosphate group are reported together with the synthesis of luminescent adenosine and thymidine derivatives having the [Re2(μ-Cl)2(CO)6(μ-pyridazine)] entity attached to positions 5' and 3', respectively. These compounds were synthesized by applying inverse-electron-demand Diels-Alder and copper(I)-catalyzed azide-alkyne 1,3-dipolar cycloaddition reactions in three or four steps. The obtained compounds exhibited orange emission (λPL ≈ 600 nm, ΦPL ≈ 0.10, and τ = 0.33-0.61 μs) and no toxicity (except for one nucleoside) to human HeLa cervical epithelioid and Ishikawa endometrial adenocarcinoma cancer cells in vitro. Furthermore, the compounds' ability to inhibit the growth of Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli bacterial strains was moderate and only observed at a high concentration of 100 μM. Confocal microscopy imaging revealed that the "dirhenium carbonyl" dinucleosides and nucleosides localized mainly in the membranous structures of HeLa cells and uniformly inside S. aureus and E. coli bacterial cells. An interesting finding was that some of the tested compounds were also found in the nuclei of HeLa cells.

5-Halogenation of Uridine Suppresses Protonation-Induced Tautomerization and Enhances Glycosidic Bond Stability of Protonated Uridine: Investigations via IRMPD Action Spectroscopy, ER-CID Experiments, and Theoretical Calculations.

Uridine (Urd), a canonical nucleoside of RNA, is the most commonly modified nucleoside among those that occur naturally. Uridine has also been an important target for the development of modified nucleoside analogues for pharmaceutical applications. In this work, the effects of 5-halogenation of uracil on the structures and glycosidic bond stabilities of protonated uridine nucleoside analogues are examined using tandem mass spectrometry and computational methods. Infrared multiple photon dissociation (IRMPD) action spectroscopy experiments and theoretical calculations are performed to probe the structural influences of these modifications. Energy-resolved collision-induced dissociation experiments along with survival yield analyses are performed to probe glycosidic bond stability. The measured IRMPD spectra are compared to linear IR spectra predicted for the stable low-energy conformations of these species computed at the B3LYP/6-311+G(d,p) level of theory to determine the conformations experimentally populated. Spectral signatures in the IR fingerprint and hydrogen-stretching regions allow the 2,4-dihydroxy protonated tautomers (T) and O4- and O2-protonated conformers to be readily differentiated. Comparisons between the measured and predicted spectra indicate that parallel to findings for uridine, both T and O4-protonated conformers of the 5-halouridine nucleoside analogues are populated, whereas O2-protonated conformers are not. Variations in yields of the spectral signatures characteristic of the T and O4-protonated conformers indicate that the extent of protonation-induced tautomerization is suppressed as the size of the halogen substituent increases. Trends in the energy-dependence of the survival yield curves find that 5-halogenation strengthens the glycosidic bond and that the enhancement in stability increases with the size of the halogen substituent.

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.

Influence of 5-Halogenation on the Base-Pairing Energies of Protonated Cytidine Nucleoside Analogue Base Pairs: Implications for the Stabilities of Synthetic i-Motif Structures for DNA Nanotechnology Applications.

DNA nanotechnology has been employed to develop devices based on i-motif structures. The protonated cytosine-cytosine base pairs that stabilize i-motif conformations are favored under slightly acidic conditions. This unique property has enabled development of the first DNA molecular motor driven by pH changes. The ability to alter the stability and pH transition range of such DNA molecular motors is desirable. Understanding how i-motif structures are influenced by modifications, and which modifications enhance stability and/or affect the pH characteristics, are therefore of great interest. Here, the influence of 5-halogenation of the cytosine nucleobases on the base pairing of protonated cytidine nucleoside analogue base pairs is examined using complementary threshold collision-induced dissociation techniques and computational methods. The nucleoside analogues examined here include the 5-halogenated forms of the canonical DNA and RNA cytidine nucleosides. Comparisons among these systems and to the analogous canonical base pairs previously examined enable the influence of 5-halogenation and the 2'-hydroxy substituent on the base pairing to be elucidated. 5-Halogenation of the cytosine nucleobases is found to enhance the strength of base pairing of DNA base pairs and generally weakens the base pairing for RNA base pairs. Trends in the strength of base pairing indicate that both inductive and polarizability effects influence the strength of base pairing. Overall, the present results suggest that 5-halogenation, and in particular, 5-fluorination and 5-iodination, provide effective means of stabilizing DNA i-motif conformations for applications in nanotechnology, whereas only 5-iodination is effective for stabilizing RNA i-motif conformations but the enhancement in stability is less significant.

Identification of nucleoside monophosphates and their epigenetic modifications using an engineered nanopore.

RNA modifications play critical roles in the regulation of various biological processes and are associated with many human diseases. Direct identification of RNA modifications by sequencing remains challenging, however. Nanopore sequencing is promising, but the current strategy is complicated by sequence decoding. Sequential nanopore identification of enzymatically cleaved nucleoside monophosphates may simultaneously provide accurate sequence and modification information. Here we show a phenylboronic acid-modified hetero-octameric Mycobacterium smegmatis porin A nanopore, with which direct distinguishing between monophosphates of canonical nucleosides, 5-methylcytidine, N6-methyladenosine, N7-methylguanosine, N1-methyladenosine, inosine, pseudouridine and dihydrouridine was achieved. A custom machine learning algorithm, which reports an accuracy of 0.996, was also applied to the quantitative analysis of modifications in microRNA and natural transfer RNA. It is generally suitable for sensing of a variety of other nucleoside or nucleotide derivatives and may bring new insights to epigenetic RNA sequencing.

Prebiotic synthesis and triphosphorylation of 3'-amino-TNA nucleosides.

Nucleosides are essential to the emergence of life, and so their synthesis is a key challenge for prebiotic chemistry. Although amino-nucleosides have enhanced reactivity in water compared with ribonucleosides, they are assumed to be prebiotically irrelevant due to perceived difficulties with their selective formation. Here we demonstrate that 3'-amino-TNA nucleosides (TNA, threose nucleic acid) are formed diastereoselectively and regiospecifically from prebiotic feedstocks in four high-yielding steps. Phosphate provides an unexpected resolution, leading to spontaneous purification of the genetically relevant threo-isomer. Furthermore, 3'-amino-TNA nucleosides are shown to be phosphorylated directly in water, under mild conditions with cyclic trimetaphosphate, forming a nucleoside triphosphate (NTP) in a manner not feasible for canonical nucleosides. Our results suggest 3'-amino-TNA nucleosides may have been present on the early Earth, and the ease with which these NTPs form, alongside the inherent selectivity for the Watson-Crick base-pairing threo-monomer, warrants further study of the role they could play during the emergence of life.

Abstract 2257: DNA adductome analysis in human colorectal tissues

Abstract Due to recent advancement of DNA sequencing technologies, cancer genome analysis has shown that somatic mutations have different trends in various tissues and individuals. Some of trends has been associated with exposure of chemically reactive compounds derived from exogenous or endogenous sources and genetic deficiencies in DNA repair and DNA replication as mutational signatures. Accumulation of these information will ultimately expect to identify carcinogenic etiologies in an individual case. However, what types of DNA damage caused the mutational signatures and how extent they contribute to human carcinogenesis has not yet fully explained.To directly evaluate the chemical types of DNA damage in human colorectal tissues, we performed comprehensive identification of DNA adducts by a liquid chromatography coupled with mass spectrometry. In this DNA adductome methodology, genome DNA was enzymatically hydrolyzed into mononucleoside and separated using a reversed-phase chromatography. Chemical structure of DNA adducts including products of epigenetic modification in genome DNA was identified using column retention time and m/z of chemical standard library of DNA adducts. Peak areas of DNA adducts were normalized by that of naturally-occurring isotopologues of canonical DNA nucleoside and compared with clinicopathological information. We identified several DNA adducts in human colorectal tissue. These included DNA adducts of alkylation, oxidation, and lipid peroxidation. C5-methyl-2’-deoxycytidine was most abundant atypical DNA in human colorectal tissue and prevalent in all cases. C5-hydroxymethyl-2’-deoxycytdine was decreased in colorectal cancer cases compared with non-colorectal cancer cases. Other DNA adducts like 1,N6-etheno-2’-deoxyadenosine were present with group- or individual-specificity. Compared with our previous results of DNA adductome analysis in human stomach mucosae and kidney tissues, DNA adducts observed in human colorectal tissue showed some tissue specificity. We will discuss whether profiles of DNA adducts are based on similar contexts of chemical exposure among individuals. DNA adducts observed in our study potentially indicated chemical causes of DNA mutation in human colorectal tissue. We propose that the integration of DNA adductomics using mass spectrometric profiling with other genetic analysis such as mutational signature analysis leverage the exploration of chemical and genetic etiology in an individual carcinogenic context and evaluate gene-environment interaction in human carcinogenesis. Citation Format: Yuji Iwashita, Shunsuke Ohtsuka, Ippei Ohnishi, Yuto Matsushita, Takashi Yamashita, Keisuke Inaba, Atsuko Fukazawa, Hideto Ochiai, Keigo Matsumoto, Nobuhito Kurono, Yoshitaka Matsushima, Hiroki Mori, Shioto Suzuki, Shohachi Suzuki, Fumihiko Tanioka, Haruhiko Sugimura. DNA adductome analysis in human colorectal tissues [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2257.