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PubMed1990

Electrophoretic analysis of DNA modifications induced by cis- and trans-diamminedichloroplatinum (II) during heat-denaturation of conformation isomers of pBR322 DNA.

The use of conformation isomers of pBR322 DNA in the study of interactions of Pt complexes with DNA provided for a good monitoring of induced changes in the structure of DNA by gel electrophoresis. On the basis of characteristic changes in the gel electrophoretic mobility of platinated isomers of pBR322 DNA we detected the presence of Pt-DNA adducts representing both intra- and interstrand bifunctional binding of Pt complexes to DNA. Also, this method made it possible to distinguish between DNA modifications induced by the therapeutically active cis-diamminedichloroplatinum (II) (cis-DDP) alone, and those induced by its therapeutically inactive trans-isomer (trans-DDP). The electrophoretically detected DNA modifications were more effective if the interaction of the Pt complex took place with heat-denatured DNA. This process, as compared to that performed with native DNA, ran 100 times faster.

Cisplatin↗
PubMed1995

Rapid decreases in indigenous covalent DNA modifications (I-compounds) of male Fischer-344 rat liver DNA by diquat treatment.

I-compounds are indigenously appearing covalent DNA modifications that can be detected by 32P-postlabeling assay in tissues of normal animals without known exposure to any carcinogens or toxins. Although these compounds have not been structurally identified, indirect evidence from earlier work suggested the possibility of involvement of molecular fragments derived from lipid peroxides. Diquat is a herbicide that stimulates lipid peroxidation and massive intrahepatic oxidant stress through redox cycling-mediated generation of reactive oxygen species. In the present study, we examined the effects of diquat on hepatic I-compounds of male Fischer-344 rats. Two groups of rats, approximately 14 weeks and 8 weeks old, were given a hepatotoxic dose (0.1 mmol/kg) of diquat or equal volumes of saline, i.p. Two and 6 h later plasma alanine aminotransferase (ALT) activities were measured and hepatic DNA I-compound levels were examined by nuclease P1-enhanced 32P-postlabeling. Elevated ALT activities were observed in some animals in both groups, at both time points, but considerable inter-animal variation was seen. A total of 15-16 I-compound fractions were measured in control and in diquat-treated animals, but no extra spots indicative of treatment-induced adducts were detected. Despite the qualitative similarities, the quantities of individual I-compounds were markedly decreased at 2 h in diquat-treated animals of both age groups. In 14 week old rats the hepatic I-compound contents were decreased at 2 h by 22-59%, which was statistically significant (ANOVA, P < 0.05) for all of the 9 polar I-compound fractions and none of the non-polar fractions. Eleven I-spots from this group showed significant negative linear correlations (P < 0.05) with ALT values. In 8 week old rats treated with diquat a 22-43% depletion in I-compound contents was statistically significant for 4 of the 7 nonpolar and 2 of the 8 polar adduct fractions, but there was no significant correlation of I-compound contents with ALT values at the 2 h time point. By 6 h most of the I-spot levels had returned to normal or above normal values in both groups of animals. While most I-spots from 14 week old rats did not correlate with ALT levels at 6 h, two I-spots displayed positive correlations in the 8 week group. Overall, the susceptibility to diquat-associated DNA alterations appeared to differ with age.(ABSTRACT TRUNCATED AT 400 WORDS)

Alanine Transaminase↗
PubMedDate not supplied

Exogenous and endogenous DNA modifications as monitored by 32P-postlabeling: relationships to cancer and aging.

32P-postlabeling analysis, a highly sensitive method for the detection and measurement of covalent carcinogen-DNA adducts and other DNA modifications, does not require radioactive test substances and, therefore, can be applied to DNA of mammals, including humans exposed to low doses of environmental or occupational genotoxicants. The basic procedure entails the enzymatic incorporation of 32P-label into hydrolysis products of DNA, followed by chromatographic mapping and autoradiography of the 32P-labeled digestion products and quantitative scintillation spectrometry. Microgram amounts of DNA are analyzed: Thus the assay is suited for limited amounts of cells or tissues. Various versions of the assay afford different sensitivities of adduct detection. A single aromatic or bulky/hydrophobic adduct in 10(8)-10(10) nucleotides can be detected and measured (corresponding to 0.3-30 amol adduct/micrograms DNA or 0.1-10 nmol adduct/mol DNA-P). In animal models, the assay has been successfully applied to a variety of mutagenic (genotoxic) as well as nonmutagenic carcinogens. In humans, DNA specimens from cigarette smokers, iron foundry workers, and coke oven workers whose total aromatic adduct levels ranged from 1 adduct in 10(6)-10(8) DNA nucleotides have been examined by 32P-postlabeling. The assay also detects DNA modifications--Indigenous (I)-compounds--that increase with age in untreated animals. I-compound profiles and levels are highly species-, strain-, sex-, and tissue-specific, and also depend on diet composition. Caloric restriction, a highly efficient method for improving resistance to carcinogenesis and extending life span, increased rather than decreased I-compound levels in various tissues of male rats. Nonmutagenic hepatocarcinogens reduced levels of I-compounds in the target organ. Because of the specificity of this effect, reduction of I-compound levels appears to represent a novel biomarker for the action of nonmutagenic carcinogens. DNA from various hepatomas was found largely devoid of I-compounds. The results support a possible antineoplastic and antiaging role of these DNA modifications.

Aging↗
PubMed1991

Measurement of the sequence specificity of covalent DNA modification by antineoplastic agents using Taq DNA polymerase.

A polymerase stop assay has been developed to determine the DNA nucleotide sequence specificity of covalent modification by antineoplastic agents using the thermostable DNA polymerase from Thermus aquaticus and synthetic labelled primers. The products of linear amplification are run on sequencing gels to reveal the sites of covalent drug binding. The method has been studied in detail for a number of agents including nitrogen mustards, platinum analogues and mitomycin C, and the sequence specificities obtained accord with those obtained by other procedures. The assay is advantageous in that it is not limited to a single type of DNA lesion (as in the piperidine cleavage assay for guanine-N7 alkylation), does not require a strand breakage step, and is more sensitive than other primer extension procedures which have only one cycle of polymerization. In particular the method has considerable potential for examining the sequence selectivity of damage and repair in single copy gene sequences in genomic DNA from cells.

Antineoplastic Agents↗
PubMed1999

Bulky endogenous DNA modifications (I-compounds) -possible structural origins and functional implications.

I-compounds are bulky covalent DNA modifications which increase with age in tissues of unexposed laboratory animals and are derived from endogenous DNA-reactive intermediates of nutrient and oxygen metabolism. They have been classified into 2 major groups, i.e., type I and type II. Profiles and levels of type I I-compounds show considerable variation depending on species, strain, tissue, and gender, but are also affected by diet and chemical and hormonal exposures, indicating their formation to be determined by genetic and environmental factors. For example, sex hormones, dietary oat lipids, and isoprenoids affect their profiles and/or levels in tissue DNA. A gradual depletion of many type I I-compounds occurs during carcinogenesis, as many carcinogens/tumor promoters significantly reduce their levels, and neoplasms display very low levels, apparently independent of growth rate, indicating a loss of the ability to form these modified nucleotides. Conversely, dietary restriction, the most effective method to retard carcinogenesis and aging, significantly elevates type I I-compound levels, as compared to age-matched ad libitum-fed animals. Levels of many liver and kidney I-compounds exhibit genotype- and diet-dependent positive linear correlations with median life span. Formation of high levels of oat-related type I I-compounds has been associated with reduced formation of carcinogen-induced preneoplastic hepatic foci. These results suggest that such DNA modifications may not represent DNA lesions but may rather be functionally important. This view is supported by circadian rhythms displayed by some I-compounds. Thus, certain type I I-compounds may play a protective role against carcinogenesis and age-associated degenerative processes. Type II I-compounds, on the other hand, represent DNA damage and include several bulky lesions, which are enhanced by pro-oxidant carcinogens such as ferric nitrilotri- acetate (Fe-NTA) in target organ (kidney) DNA of rodents and are identical to products generated by oxidizing DNA or oligonucleotides under Fenton reaction conditions in vitro. Some of these products appear to be base-base or base-sugar intrastrand crosslinks. Notably, Fe-NTA reduces the levels of type I I-compounds in renal DNA. Type II I-compound levels are increased in tissue DNA of normal newborn rats. The formation of oxidative DNA lesions in neonates is most likely caused by oxidative stress associated with the sudden increase of partial oxygen pressure in arterial blood and tissues at birth. In view of the rapid cell replication at this developmental stage, endogenous oxidative DNA lesions sustained early in life may contribute to the development of cancer and degenerative diseases later in life.

Animals↗
PubMed1999

Dietary oat lipids-induced novel DNA modifications and suppression of altered hepatic foci formation.

Previous studies have shown that the presence of several hepatic I-compounds, i.e., age-dependent covalent DNA modifications, is related to the presence of a natural ingredient, i.e., oats, in the diet. To demonstrate the biological significance of these novel DNA modifications, the effect of oat lipids on tumor initiation and promotion was examined in a rat liver tumor model. Female Sprague-Dawley rats were treated with a single dose of diethylnitrosamine, a hepatic carcinogen, 24 hours after a 70% partial hepatectomy, then subjected to dietary phenobarbital promotion. Diets containing 10% oat lipids or corn oil were given during the initiation or the promotion stage of the tumorigenesis. At the end of the feeding, hepatic I-compounds were measured by 32P postlabeling, and the number and volume of enzyme-altered hepatic foci, which served as preneoplastic markers, were measured in serial sections of liver by the method of quantitative stereology. Rats receiving oat lipids-supplemented diets had five- to sixfold higher levels of I-compounds in their liver DNA than those receiving control diets. Meanwhile, rats receiving diets containing oat lipids during promotion had significantly smaller numbers and reduced volume of altered hepatic foci compared with those fed the control diet containing corn oil. These observations support the hypothesis that some I-compounds, e.g., the oats-specific I-compounds, are novel DNA modifications related to nutrient metabolism. The diet containing oat lipids may have chemopreventive activities, as demonstrated in this model system.

Analysis of Variance↗
PubMed1992

Oat lipids-induced covalent DNA modifications (I-compounds) in female Sprague-Dawley rats, as determined by 32P-postlabeling.

Previous studies have shown that the presence of oats in the diet contributes to formation of I-compounds (age-dependent covalent DNA modifications detected by 32P-postlabeling assay) in female Sprague-Dawley rat liver DNA. The current study explored the possible ingredients in oats responsible for the observed effects on DNA. Feeding AIN-76A diet containing 5% oat lipids (obtained by methanol extraction and dissolved in trioctanoin) in place of corn oil for 2 months successfully induced the formation of 3 oats-specific (spots 2-4) and 4 natural ingredient diet-specific I-compounds (spots 6-9) in liver DNA. Barley, an oatlike cereal, induced 3 of these spots at very low intensities but not the 3 oats-specific I-spots. Oral administration of oat lipids to weanling rats of both sexes for 7 days elicited trace amounts of the oats-specific spots and spot 9 in liver DNA. However, when oat lipids were given at 6 or 9 weeks of age, the oats-specific spots were detected at high levels in female but not in male rats. These oats-related DNA modifications were also present in 6-week-old female rats which had received oat lipids p.o. for 2 or 3 days or i.p. for 4 days. Rats given trioctanoin or extracts from natural ingredient Wayne diet (lacking oats) did not show any of these spots. On the other hand, rats treated with extracts from an oats-containing Teklad diet displayed a trace amount of one of these I-compounds. Oat lipids did not induce any extra spots in rat kidney DNA. Feeding of AIN diet supplemented with oats to female Syrian hamsters did not elicit any renal or hepatic DNA alterations, as detected by 32P-postlabeling. Rats fed oat lipids-supplemented AIN diet or Purina diet showed the highest levels of I-compounds overall in liver among all dietary groups and these two groups also had significantly higher hepatic DNA synthesis rates. Oat lipids enhanced kidney DNA synthesis also. The total hepatic or renal cytochrome P-450 contents were not significantly affected by different diets. These results demonstrate a novel link between a natural dietary ingredient and covalent DNA modifications and shed light on the origins of certain I-compounds.

Animals↗
PubMed2000

Engineered zinc finger proteins that respond to DNA modification by HaeIII and HhaI methyltransferase enzymes.

Zinc finger modules are capable of specifically interacting with DNA that contains 5-methylcytosine (5-mC) in place of cytosine, suggesting that zinc finger-DNA binding could be regulated by extrinsic methylation of DNA. Here, we have used phage display to engineer zinc finger proteins that detect and discriminate DNA methylation by the prokaryotic enzymes HaeIII and HhaI. In these systems, zinc finger-DNA complexes are induced by DNA modification using the appropriate enzyme, which can therefore act as a switch. To further develop the specificity of the switch, zinc finger discrimination between 5-mC and thymine in DNA sequences is demonstrated despite the presence of the characteristic major groove methyl group that is common to both bases. Specificity was achieved using a DNA-binding strategy involving synergy between adjacent zinc fingers. We propose that engineered zinc fingers that recognise particular DNA modifications, such as sequence-specific DNA methylation, could be integrated into artificial regulatory circuits for the control of gene expression and other biological processes.

Bacteriophages↗
PubMed1982

Evidence for DNA modification in the maintenance of X-chromosome inactivation of adult mouse tissues.

The role of DNA modification in the maintenance of mammalian X-chromosome inactivation was investigated by using the technique of DNA transformation in mammalian cells. The ability of inactive X-chromosome DNA from adult mouse tissues to act in transformation for the X-linked hypoxanthine phosphoribosyltransferase gene (Hprt) could be ascertained by utilizing a recently discovered electrophoretic variant form of the hypoxanthine phosphoribosyltransferase enzyme and a previously available X:autosome translocation. Our findings indicate that inactive X-chromosome DNA from several tissues of adult female mice is strikingly inefficient, in comparison to active X-chromosome DNA, in eliciting genetic transformation for hypoxanthine phosphoribosyltransferase. These results provide in vivo evidence that is consistent with DNA modification playing an important role in the maintenance of X-chromosome inactivation.

Animals↗
PubMed1990

Acute and long-term effects of carbon tetrachloride on DNA modifications (I-compounds) in male mouse liver.

I-compounds are recently discovered species and tissue dependent covalent DNA modifications which are detectable by the 32P-postlabeling assay for DNA adducts and tend to increase with the animal's age. The effects of the hepatocarcinogen carbon tetrachloride (CCl4) on hepatic I-compounds were studied in 10-12-month-old male ICR mice using the 32P-postlabeling assay. CCl4 was dissolved in corn oil (20%, v/v) and intraperitoneally (i.p.) injected in doses of 0.75 ml/kg (0.375 ml/100 g body weight, 20% CCl4 in corn oil) while control mice received corn oil only (0.375 ml/100 g body wt). Twenty-four h after a single injection of CCl4, the intensity of non-polar I-spots in liver DNA was significantly increased as compared with corn oil treated controls, while the level of one polar I-compound was reduced at 24 h. DNA synthesis (as indicated by [3H]thymidine incorporation) was not significantly affected at 24 h after a single dose of CCl4. To study the long-term effects of CCl4, five groups of mice were given two consecutive weekly injections of 0.75 ml/kg CCl4 (as above) and were sacrificed 1, 4, 8, 12 and 22 weeks after the second treatment. In these groups the total liver I-compound levels were reduced to 17.3-49.0% compared with corresponding controls. The maximum decline was observed at 4 weeks (17.3% of control). Comparison of thymidine incorporation showed no significant increase between control and treated liver DNAs at 1, 4 and 8 weeks after CCl4, suggesting that the decrease in I-compound levels was probably not a secondary effect of increased DNA synthesis during postnecrotic proliferation. Even though there was a trend of recovery between 8 and 22 weeks, I-compound levels still remained significantly lower at 22 weeks (49.0%). Since I-compounds appear to be normal DNA modifications, the results suggest that persistent reduction of I-compound levels contributes to the hepatocarcinogenic effect of CCl4.

Age Factors↗
PubMed1994

Characterization of a Streptomyces-lividans-type site-specific DNA modification system in the avermectin-producer Streptomyces avermitilis permits investigation of two novel giant linear plasmids, pSA1 and pSA2.

The degradation of Streptomyces avermitilis DNA samples analysed by conventional pulsed-field gel electrophoresis was shown to be due to Tris-dependent, double-strand cleavage. Using alternative electrophoretic conditions, separation of intact DNA molecules was achieved, permitting the identification of two novel giant linear plasmids: the 100 kb pSA1 and 250 kb pSA2. Use of pSA2 DNA as a probe showed that pSA1 does not cross-hybridize, indicating that the plasmids are not closely related. The site-specificity of the DNA modifications, which render the DNA susceptible to Tris-dependent cleavage, was found to be essentially identical to that of similar modifications found in the DNA of S. lividans.

Bacterial Proteins↗
PubMed1988

A comparison between different types of covalent DNA modifications (I-compounds, persistent carcinogen adducts and 5-methylcytosine) in regenerating rat liver.

I-Compounds have been recently identified as adduct-like nonpolar covalent DNA modifications that are detectable by 32P-postlabeling assay in tissues of untreated experimental animals and increase with age. Additional I-compounds have now been observed in liver DNA of male Sprague-Dawley rats when the chromatographic conditions were modified to allow for the detection of more polar adducts exhibiting low affinity to polyethyleneimine (PEI)--cellulose anion-exchange thin-layer material. The total I-compound level in 10-month-old animals was as high as one modification in approximately 10(7) nucleotides. This represented a minimum estimate since 100% recovery of all rat liver I-compounds in 32P-labeled form presumably was not achieved by the procedures used. The I-compound pattern was reproducible and variation of I-compound levels among individual animals of the same age was small. We have used regenerating rat liver herein as a model to compare the properties of I-compounds with those of persistent 2-acetylaminofluorene (AAF)-induced DNA adducts and of 5-methylcytosine (m5C), a normal enzymatic DNA modification. Eight- to 10-month-old male Sprague-Dawley rats were given 2-AAF (50 mg/kg in DMSO) or vehicle (DMSO) by i.p. injection. Partial hepatectomy was performed 6 weeks later (i.e. after AAF adduct levels had stabilized) and regenerating liver samples were taken 1 week after the operation for DNA analysis. Consistent with the restoration of cell and tissue loss, the overall levels of I-compounds and 2-AAF adducts were reduced to approximately 47% and approximately 45% respectively of control in regenerating liver by dilution with newly synthesized DNA, while the m5C level was not affected. Thus, in regenerating liver, I-compounds resembled carcinogen--DNA adducts and not m5C. This supports our hypothesis that the formation of these DNA modifications may be due to the binding to DNA of small amounts of reactive electrophilic by-products of normal metabolic activities, leading to the slow accumulation of I-compounds in tissue DNA with ageing.

2-Acetylaminofluorene↗
PubMed1998

Distribution of Xanthomonas oryzae pv. oryzae DNA modification systems in Asia.

The presence or absence of two DNA modification systems, XorI and XorII, in 195 strains of Xanthomonas oryzae pv. oryzae collected from different major rice-growing countries of Asia was assessed. All four possible phenotypes (XorI+ XorII+, XorI+ XorII-, XorI- XorII+ and XorI- XorII-) were detected in the population at a ratio of approximately 1:2:2:2. The XorI+ XorII+ and XorI- XorII+ phenotypes were observed predominantly in strains from southeast Asia (Philippines, Malaysia, and Indonesia), whereas strains with the phenotypes XorI- XorII- and XorI+ XorII- were distributed in south Asia (India and Nepal) and northeast Asia (China, Korea, and Japan), respectively. Based on the prevalence and geographic distribution of the XorI and XorII systems, we suggest that the XorI modification system originated in northeast Asia and was later introduced to southeast Asia, while the XorII system originated in southeast Asia and moved to northeast Asia and south Asia. Genomic DNA from all tested strains of X. oryzae pv. oryzae that were resistant to digestion by endonuclease XorII or its isoschizomer PvuI also hybridized with a 7.0-kb clone that contained the XorII modification system, whereas strains that were digested by XorII or PvuI lacked DNA that hybridized with the clone. Size polymorphisms were observed in fragments that hybridized with the 7.0-kb clone. However, a single hybridization pattern generally was found in XorII+ strains within a country, indicating clonal maintenance of the XorII methyl-transferase gene locus. The locus was monomorphic for X. oryzae pv. oryzae strains from the Philippines and all strains from Indonesia and Korea.

DNA Restriction Enzymes↗
PubMed1993

Quantification of oxidative DNA modifications in mitochondria.

Specific repair endonucleases were used to quantify oxidative modifications in mitochondrial DNA (mtDNA) from rat liver and from porcine liver and kidney by means of a relaxation assay. In rat liver mitochondria the number of modifications sensitive to formamidopyrimidine--DNA glycosylase (FPG protein), which include 8-hydroxyguanine (8-oxo-7,8-dihydroguanine) residues, was only 0.8 +/- 0.2 per 10(5) base pairs (bp). Even lower values were observed in porcine kidney (0.5 +/- 0.3 per 10(5) bp) and liver (0.4 +/- 0.2 per 10(5) bp). The numbers of sites of base loss (AP sites) sensitive to T4 endonuclease V and of 5,6-dihydropyrimidines sensitive to endonuclease III were less than 0.2 per 10(5) bp in all cases. The data provide evidence that the steady-state levels of oxidative mtDNA modifications are low under physiological conditions, either because reactive oxygen species generated in the mitochondria are instantly inactivated or because of efficient DNA repair processes inside mitochondria.

Animals↗
PubMed1994

DNA modification induced by 6-mercaptopurine riboside in murine embryos.

The cytostatic drug 6-mercaptopurine riboside (6-MPr) was investigated in mice in order to test the hypothesis that the teratogenicity of this antimetabolite is paralleled by an incorporation into the DNA of the embryos during organogenesis. DNA modification in the embryos was analysed 4 h following s.c. administration of [35S]-labelled 6-MPr to the dams on day 11 of pregnancy. The DNA of the embryos was isolated and hydrolysed to the bases by formic acid. Following separation by cation-exchange HPLC 6-thioguanine was found in the hydrolysate. Quantitation was performed by liquid scintillation counting. Evaluations of 6 doses in the range of 8-25 mg/kg were performed. An incorporation rate of 6-thioguanine from 32-56 pmol per mumol guanine was found in the DNA of the embryos. These findings suggest that, similar to the previously studied alkylating agents, the teratogenicity of 6-MPr may be, at least in part, induced via DNA modification of the embryos.

Animals↗
PubMed1998

Changes in expression site control and DNA modification in Trypanosoma brucei during differentiation of the bloodstream form to the procyclic form.

We have adapted a system for in vitro differentiation of a monomorphic trypanosome strain to monitor changes in transcription and DNA modification in expression sites during the transition of the bloodstream-form to the procyclic trypanosome. We have used trypanosomes that have a gene for drug resistance integrated in an expression site, just downstream of either an expression site promoter, or a ribosomal promoter replacing the endogenous promoter. During the transition from bloodstream-form to procyclic, the promoters in an active expression site behave as expected on the basis of previous work on these promoters in procyclics, i.e. the ribosomal replacement promoter remains fully active, whereas the expression site promoter is (incompletely) down-regulated. A silent bloodstream-form expression site promoter does not remain tightly silenced, however. There is a transient increase of transcription of the marker gene during the transition from bloodstream-form to procyclic, indicating that the control of silent expression sites differs between the bloodstream-form and the procyclic trypanosome, and that a short time is required to reset the silencing mechanisms. One of the differences between bloodstream-form and procyclic trypanosomes is the presence of the modified base beta-D-glucosyl-hydroxymethyluracil (J) in and around bloodstream-form expression sites. We have studied loss of this DNA modification and find that the change in expression site control from bloodstream-form to procyclic does not require active removal of J. Base J is lost by synthesis of new, unmodified DNA, which happens after the major changes in expression site transcription have occurred.

Animals↗
PubMed2025

Write and Read: Harnessing Synthetic DNA Modifications for Nanopore Sequencing.

An exciting feature of nanopore sequencing is its ability to record multi-omic information on the same sequenced DNA molecule. Well-trained models allow the detection of nucleotide-specific molecular signatures through changes in ionic current as DNA molecules translocate through the nanopore. Thus, naturally occurring DNA modifications, such as DNA methylation and hydroxymethylation, may be recorded simultaneously with the genetic sequence. Additional genomic information, such as chromatin state or the locations of bound transcription factors, may also be recorded if their locations are chemically encoded into the DNA. Here, we present a versatile "write-and-read" framework, where chemo-enzymatic DNA labeling with unnatural synthetic tags results in predictable electrical fingerprints in nanopore sequencing. As a proof-of-concept, we explore a DNA glucosylation approach that selectively modifies 5-hydroxymethylcytosine (5hmC) with glucose or glucose-azide adducts. We demonstrate that these modifications generate distinct and reproducible electrical shifts, enabling the direct detection of chemically altered nucleotides. We further demonstrate that enzymatic alkylation, such as the enzymatic transfer of azide residues to the N6 position of adenines, also produces characteristic nanopore signal shifts relative to the native adenine and 6-methyladenine. Beyond direct nucleotide detection, this approach introduces new possibilities for bio-orthogonal DNA labeling, enabling an extended alphabet of sequence-specific detectable moieties. The future use of programmable chemical modifications for simultaneous analysis of multiple omics features on individual molecules opens new avenues for genetic research and discovery.

5-hydroxymethylcytosine (5hmC)↗