CHEMMEDCHEM REVIEWS DOI: 10.1002/cmdc.201402114
DNA–Osmium Complexes: Recent Developments in the Operative Chemical Analysis of DNA Epigenetic Modifications Akimitsu Okamoto*[a]
2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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CHEMMEDCHEM REVIEWS The development of a reaction for the detection of one epigenetic modification in a long DNA strand is a chemically and biologically challenging research subject. Herein, we report and discuss the formation of 5-methylcytosine–osmium complexes that are used as the basis for a bisulfite-free chemical assay for DNA methylation analysis. Osmium in the oxidized state reacts with C5-methylated pyrimidines in the presence of a bipyridine ligand to give a stable ternary complex. On the basis of this reaction, an adenine derivative with a tethered bipyridine moiety has been designed for sequence-specific
www.chemmedchem.org osmium complex formation. Osmium complexation is then achieved by hybridization of a short DNA molecule containing this functional nucleotide to a target DNA sequence and results in the formation of a cross-linked structure. This novel concept of methylation-specific reaction, based on a straightforward chemical process, expands the range of methods available for the analysis of epigenetic modifications. Advantages of the described method include amplification-insensitive detection, 5-hydroxymethylcytosine complexation, and visualization through methylation-specific in situ hybridization.
Introduction Gene expression is regulated by the epigenetic modification of biopolymers, such as DNA and histone proteins, independent of their primary sequences. In particular, methylation and demethylation of cytosine (C) in DNA is an important epigenetic modification of the genome in many animals and plants, and this process plays a crucial role in the regulation of developmental gene expression, chromatin remodeling, genomic imprinting, X-chromosome inactivation, and genome stability.[1–4] Aberrant DNA methylation is an early and fundamental event in the pathogenesis of many human diseases, including cancer.[5] The mechanism of DNA methylation, that is, the formation of 5-methylcytosine (5mC) has been studied very well, and recent studies on DNA demethylation have revealed that 5-hydroxymethylcytosine (5hmC) is an important intermediate for replication-dependent and/or -independent demethylation (Figure 1).[6, 7] In addition, erroneous DNA methylation may contribute to the etiopathogenesis of tumorigenesis and aging.[5, 8] The methylation and demethylation of C are the most important epigenetic events occurring in DNA, and their analysis is very significant. Much effort has gone into developing a simple reaction that can be used for the detection of 5mC and 5hmC. However, the ability to distinguish 5mC and 5hmC from C, that is, to detect the existence of only one methyl group in a long DNA strand, is still a chemically and biologically challenging subject. A variety of methods have been developed to detect DNA methylation.[9] For example, recent advances in high-throughput DNA sequencing technology, along with the use of immunoprecipitation,[10] affinity-based pull-down,[11] and bisulfite conversion,[12–14] has now made it possible to map 5mC in the genome at base resolution. At the cellular level, global DNA methylation patterns can be microscopically visualized by using either anti-5mC antibodies[15, 16] or methylated DNA-binding domain fusion proteins.[17, 18] Bisulfite methods have now been widely applied for creating whole methylome maps of the human genome in addition to others. Sodium bisulfite causes C deamination in a single-strand DNA through the for[a] Dr. A. Okamoto Research Center for Advanced Science & Technology The University of Tokyo 4-6-1 Komaba, Meguro-ku, Tokyo 153-8904 (Japan) E-mail: [email protected]
2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Figure 1. Methyl groups in DNA structure. a) Methylation and demethylation of cytosine. Two epigenetically modified cytosine residues, 5mC and 5hmC, generated in the methylation and demethylation processes. b) Two methyl groups in a fully methylated DNA. The methylation is a very small change in the DNA duplex structure.
mation of a 5,6-dihydrocytosine-6-sulfonate intermediate at acidic pH values. The deaminated bisulfite adduct is converted into a uracil residue through elimination of bisulfite at alkaline pH values. 5mC also yields thymine (T) with sodium bisulfite, but the reaction rate for bisulfite adduct formation is much slower.[19, 20] Thus, the bisulfite assay requires long reaction times (5–24 h, normally an overnight reaction) to avoid false positives for methylcytosine. Methylation-specific polymerase chain reaction (PCR) was developed on the basis of the modification of DNA by sodium bisulfite to rapidly assess the methylation status within a C–phosphate–G island.[21, 22] The biggest problem in bisulfite assays is DNA degradation during the long reaction times.[23] By treating a 100-mer DNA with bisulfite over 16 h under typical bisulfite assay conditions, the sigmoid curves of quantitative PCR show that of the 1014 copies of DNA ChemMedChem 2014, 9, 1958 – 1965
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CHEMMEDCHEM REVIEWS in the sample before the bisulfite assay, only approximately 1011 copies remain after bisulfite treatment for 16 h. This is 0.1 % of the original DNA amount, and a critical level of degradation is caused by depyrimidination during bisulfite treatment. Several radical scavengers and standardized kits have been developed for routine bisulfite treatment nowadays. However, a DNA methylation detection method with a quite different chemical mechanism is still required for further extension of epigenetic studies. A rapid and selective chemical reaction capable of detecting 5mC and 5hmC in DNA would clearly be very useful for efficiently analyzing the status of epigenetic modifications at a specific site in a gene. The five key points required for a new chemical assay for methylation detection are as follows. 1) Sequence selectivity: Nowadays, the focus of methylation studies is the elucidation of the role of each methylation in cell functions. The development of a conceptually new chemical approach to site-specific detection is thus very desirable. 2) Modification-positive assay: Reactions selective to epigenetic modification sites should be developed. 3) Simple detection process: The most facile method to detect methylation is to label the methylation sites directly with signal-sending units. Apart from DNA microarray analysis of sequence-converted DNA through bisulfite treatment, there are very few fluorescent assays available for the detection of C methylation.[24, 25] 4) No strand damage: DNA samples are damaged by strand scission during bisulfite treatment.[23] Nonspecific cleavage complicates the detection process and decreases quantification precision. 5) Short assay time: To obtain reliable results, the bisulfite assay usually requires approximately half a day for complete modification. A shorter reaction time is clearly desirable.
Akimitsu Okamoto obtained his PhD in synthetic chemistry and biological chemistry from Kyoto University in 1998, where he was appointed Assistant Professor in 1999 after a year as a Postdoctoral Fellow at Massachusetts Institute of Technology. He moved to RIKEN as an Initiative Research Scientist (unit leader) in 2006. Akimitsu Okamoto joined the faculty at the University of Tokyo in 2012 as a Professor. His research focuses on the design, synthesis, and physical properties of new, manmade biopolymers with various functionalities and on the design of unique organic chemical systems for recognizing, transforming, and visualizing a single component or atom in biopolymers of interest.
2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemmedchem.org In this review, we introduce newly designed chemical assays for the detection of DNA epigenetic modifications. The chemistry-based assay includes many advantages that are quite different from conventional assays. The new concept of sequencespecific rapid methylation analysis based on a straightforward chemical reaction will be the starting point for a novel methylation-type assay.
5mC Forms a Stable Complex with Osmium A rapid, target-selective, DNA-compatible chemical reaction capable of distinguishing between C and 5mC would be useful to analyze efficiently the status of C methylation at a specific site in a gene. The oxidation of pyrimidine bases is a practical approach that could be used to determine the presence or absence of a methyl group at the C5 position of C, because the number of substituents on the C5=C6 double bond is different in these two species. The C5=C6 double bonds of T and 5mC bases are known to be oxidized by osmium tetroxide, and these bases are converted into the corresponding glycols.[26, 27] In single-stranded DNA molecules, T is also oxidized by osmium tetroxide, and this reaction is sometimes used for T sequencing.[28–32] We developed the selective oxidation of 5mC through exquisite control of a reaction that distinguishes between C and 5mC (Scheme 1).[33, 34] One example of the reaction conditions
Scheme 1. Oxidative osmium complex formation at 5mC.
is as follows: 5 mm potassium osmate (an oxidant much less intractable than osmium tetroxide), 100 mm potassium hexacyanoferrate(III) (an activator), and 100 mm 2,2’-bipyridine (a reaction-accelerating ligand) in 100 mm tris(hydroxymethyl)aminomethane (Tris)·HCl buffer (pH 7.7), 1 mm ethylenediaminetetraacetic acid (EDTA), and 10 % acetonitrile (for dissolution of 2,2’-bipyridine). The reaction mixture is incubated with the DNA of interest at 0 8C for 5 min, whereupon the oxidized strand is cleaved at a damaged pyrimidine base with hot piperidine. Cleaved DNA strands can then be identified as shortened DNA fragments by using polyacrylamide gel electrophoresis. Strand cleavage at C is negligible, whereas DNA strands containing 5mC are cleaved at the site of modification. The product of the osmium-mediated oxidation reaction is a 5mC glycol–dioxidoosmium–bipyridine ternary complex.[35] The major form of the glycol is in the (5R,6S) configuration. The osmium complex formed selectively at 5mC is stable in aqueous solutions under weakly acidic, weakly basic, and elevated temperature conditions. ChemMedChem 2014, 9, 1958 – 1965
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The tendency of the reaction to react preferentially with C or 5mC is determined by the ligand. The reactivity and selectivity was investigated by using a variety of pyridyl ligands including pyridine, 2,2’-bipyridine, 4,4’-diamino-2,2’-bipyridine, 4,4’-dinitro-2,2’-bipyridine, and phenanthroline (Table 1).[36] The
Table 1. Effect of the ligand on the selectivity of osmium-mediated oxidation.[a] Ligand
Conc. [mm]
5mC [%]
C [%]
2,2’-bipyridine pyridine 4,4’-diamino-2,2’-bipyridine 4,4’-dinitrobipyridine 1,10-phenanthroline
100 250 10 10 10
54 9 > 98 0 96
2 0 > 98 0 17
[a] The radiolabeled DNA 5’-32P-d(AAAAAAG[C/5mC]GAAAAAA)-3’ (1 mm) was added to 5 mm potassium osmate, 100 mm potassium hexacyanoferrate(III), ligand, 1 mm EDTA, and 10 % acetonitrile in 100 mm Tris·HCl (pH 7.7), and then the mixture was incubated at 0 8C for 5 min. After hot piperidine treatment (90 8C, 20 min), the yield at the target base was calculated from DNA cleavage bands in polyacrylamide gel electrophoresis.
reaction employing pyridine can distinguish between C and 5mC as a result of the difference in reaction rates; however, the reaction rate for 5mC is much slower than that observed in the reaction employing 2,2’-bipyridine. 4,4’-Diamino-2,2’-bipyridine, which has electron-donating groups, accelerates the reactions with both C and 5mC, whereas 4,4’-dinitro-2,2’-bipyridine, which has electron-withdrawing groups, exhibits a low reaction rate with both C and 5mC. Conducting the reaction with phenanthroline gives higher reaction rates, but it does not suppress the reaction for C. Therefore, the inclusion of 2,2’-bipyridine in the assay allows the difference in reaction rates with C and 5mC to be maximized while keeping the overall reaction time to a minimum. The 5mC in single-stranded DNA efficiently forms a metal complex, whereas complex formation at the 5mC in a duplex is suppressed.[33] The weak reactivity in a duplex is attributed to inhibition of the attack of an osmium on the p orbital of a C5=C6 double bond by base stacking in the duplex structure. The use of steric control, such as the formation of a bulge structure or a mismatched base pair, is effective for sequencespecific osmium complex formation. Efficient strand cleavage is observed at 5mC in the bulged structure, similar to that found in the reaction with single-stranded DNA. The calculated rate constants for 5mC-bulged and C-bulged duplexes are 1.11 10 2 and 2.51 10 5 s 1, respectively. The use of a bulge-inducing DNA fixed on polystyrene beads make 5mC analysis straightforward. After hybridization of the target DNA with bulge-inducing DNA, the duplex is incubated in a reaction mixture containing potassium osmate. After washing and treatment with hot piperidine, the DNA amplification is monitored with quantitative PCR. A curve with a retarded start and a small curvature is displayed for a 5mC-containing sequence, because complex formation at 5mC results in DNA cleavage at the complexation site and inhibition of DNA amplification. 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chemical Labeling of 5mC The ability to label 5mC sites directly with signal-sending units would lead to a simple detection assay. To this end, functionalization of the bipyridine ligand with a signaling unit would allow tagging of the 5mC residues in the DNA of interest through coordination to the osmate. Substitution at C4 of bipyridine does not hinder complex formation with 5mC.[36] Indeed, tag-attachable bipyridine ligands for direct 5mC labeling have been synthesized and used in fluorescent and electrochemical assays.[37] As an implementation of the above approach in a fluorescent assay, a new ligand, 4-[6-(4-aminobutylamino)-6-oxohexyl]-4’methyl-2,2’-bipyridine, has been developed for the attachment of fluorescent dyes to 5mC sites. After formation of the osmium complex, a fluorescent dye is attached to the amino end of the linker extended from the ligand (5mC–Os–HEX, (HEX = hexachlorofluorescein), Figure 2 a). Direct fluorescent labeling to 5mC residues through osmium complex formation thus allows the presence or absence of 5mC to be judged visually and quantified through the use of Fçrster resonance energy transfer (FRET) from a hybridization probe labeled with fluorescein to 5mC-Os-HEX.
Figure 2. Fluorescence detection of 5mC. a) Fluorescence labeling of 5mC by tethering bipyridine to a fluorescent dye. b) Fluorescence quenching by osmium complex formation.
A DNA probe with a microenvironment-sensitive fluorescent dye, 6-dimethylamino-2-acylnaphthalene (DAN), has also been designed.[38–44] The fluorescence of the duplex of the osmiumcomplexed DNA and the DAN-labeled probe can be significantly quenched (Figure 2 b). Photoinduced electron transfer occurring from the excited DAN to the osmium complex contributes to the quenching process.
ICON Probe for 5mC Detection A new “ligand” has been developed for sequence-specific 5 mC detection.[45] Thus, an adenine base forming a misChemMedChem 2014, 9, 1958 – 1965
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www.chemmedchem.org the ICON probes. After osmium-mediated oxidation, the wells are rinsed and then coated with bovine serum albumin. The fixed DNA is detected by using PCR amplification of a part of the cross-linked target DNA. The 5mC-containing DNA shows exponential growth of the PCR product. On-chip capture of methylated DNA by the ICON probe at 5mC makes the sequence-specific detection of 5mC through PCR amplification possible.
Fluorescence in situ Hybridization To Visualize 5mC in Chromosomes
Scheme 2. Interstrand crosslinking between 5mC in the target DNA and the complementary ICON probe.
matched pair with 5mC is tethered to the bipyridine ligand required for osmium-centered complex formation (bipyridinetethered adenine, B; Scheme 2) to form interstrand complexes at the target 5mC residue. The formation of a 5mC/A mismatched pair causes partial disruption of the p stacking of the DNA duplex and facilitates oxidation at the 5mC C5=C6 double bond, which is forced out of the duplex major groove. The combination of the formation of a mismatched base pair together with an appropriately located bipyridine should result in complex formation at a specific 5mC, regardless of the presence of other reactive bases in a long DNA strand. Nucleic acids containing B probes can form interstrand crosslinks through osmium complex formation, which allows sequenceselective labeling and obstruction of PCR amplification at the target 5mC residue. The sequence-specific osmium complexation accelerating nucleic acid probe is now described as an interstrand complexation formed by osmium and nucleic acid (ICON) probe. Blocking of PCR by interstrand crosslinking with an ICON probe at 5mC makes the sequence-specific quantification of methylation possible.[46] The DNA of interest is incubated with ICON probes in an osmium reaction mixture. The quantitative PCR assay exhibits a linear relationship between the amplification starting point and the logarithm of the proportion of methylation. Interstrand crosslinking with ICON probes designed for each target 5mC is formed easily, independent of the number of other 5mC sites. For example, sequence-specific ICON probes have been used to quantify the methylation of mouse genome collected from different tissues.[45] Samples from different tissues exhibit characteristic methylation levels for the two methylation sites, which is in close agreement with the degree of methylation determined by mass spectrometric analysis of fragmented and bisulfite-treated genome samples. Interstrand crosslinking between 5mC in the target DNA and the complementary ICON probe fixed to the bottom of microwells has assisted the on-chip detection of a specific 5mC in the target DNA.[47] After ICON DNA is fixed through covalent bonds to the bottom of each well of a series of microwell strips, the target DNA is put into the wells and hybridized with 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ICON technology is useful to identify 5mC residues in target sequences at both the cellular and chromosomal levels, because ICON probes can label the target 5mC with fluorescent dyes without causing serious DNA damage, as observed with bisulfite methods, and without problems associated with nonsequence-specific 5mC binding, as observed with anti-5mC antibodies. The Sasaki group have applied ICON technology to develop a novel method, named methylation-specific fluorescence in situ hybridization (MeFISH), to visualize the DNA methylation status at specific sequences in individual nuclei or chromosomes (Figure 3).[48] MeFISH could be used to detect DNA methylation at centromeric and pericentromeric repeat sequences in both mouse and human cells. The overall MeFISH protocol involves conventional FISH technology for sequencespecific target detection and ICON technology for 5mC detection.[45] Tissue sections, cells, or chromosome spreads are first subjected to FISH with the fluorescence-labeled ICON probes,
Figure 3. Outline of the MeFISH protocol.
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CHEMMEDCHEM REVIEWS and the location of fluorescent signals is then recorded. Next, the specimens are treated with osmium to induce 5mC-dependent crosslinking, and non-cross-linked probes are removed in a denaturation step. Subsequent comparison of the FISH and MeFISH images allows the methylation status to be estimated. For example, ICON probes have been designed against major and minor satellite repeats of wild-type and Dnmt-TKO (Dnmt1, Dnmt3a, and Dnmt3b triple knock out) mouse embryonic stem cells,[49] and two-color FISH has been performed by using these probes. On metaphase chromosomes, the major satellite probe displays a strong signal near the centromeric end, and the minor satellite probe gives a doublet on the sister chromatids at the centromeric end. In interphase nuclei, whereas the major satellite probe gives strong signals that are co-localized with the 4’,6-diamidino-2-phenylindole (DAPI)dense regions, the minor satellite probe gives smaller spots at the periphery of the major satellite signals (Figure 4). The specimens are then treated with osmium and denatured to remove non-cross-linked probes. The specific retention of ICON signals in wild-type stem cells, but not in Dnmt-TKO embryonic stem cells, is clear.
www.chemmedchem.org bryonic tissues by the Abe group.[52] The whole-mount MeFISH approach enables the observation of the DNA methylation status of satellite repeats in developing mouse primordial germ cells. The combination of whole-mount MeFISH with immunostaining or RNA-FISH facilitates the simultaneous visualization of DNA methylation and protein or RNA expression at single-cell resolution without destroying embryonic and nuclear structures.
Reactions Controlled at Thymine Osmium complex formation occurs at T as well as at 5mC residues.[26, 27] Indeed, the reactivity at T is almost twice that at 5mC. However, in the ICON probe, T does not form any osmium complex even if located opposite B, because the B base forms a matched base pair with T in the duplex. However, if the location of a B nucleotide within the ICON probe is varied, the reactivity and base selectivity of the osmium crosslinking change. Osmium-mediated oxidation by using the ICON probe containing B at the end of the strand has shown high crosslink yields not only for T opposite B, but also for T located at the unhybridized region of the target DNA (Figure 5 a).[53] An ICON strand end has higher flexibility of its basestacking structure; thus, the oxidants have better access to the target C=C bond of T than to the T/B base pair located inside the target–probe duplex. The lack of specificity with respect to the reaction site on the target DNA opposite B is attributable to the structural flexibility of both the unhybridized region of the target DNA and the alkyl linker tethering the bipyridine to the purine ring in B.
Figure 4. The MeFISH signal from major satellite regions in mouse embryonic stem cells observed by our group. a) The MeFISH signal after osmium treatment and denaturation (green, ICON probe labeled with Cy3; blue, DAPI). b) The MeFISH signal after denaturation without osmium treatment.
MeFISH has also made it possible to detect 5mC at human satellite repeat sequences. Human classical satellites 2 and 3 and a satellites are generally highly methylated; however, they are hypomethylated in cells from patients with immunodeficiency, centromeric instability, and facial abnormalities (ICF) syndrome.[50] Whereas type 1 ICF shows normal methylation at a satellites, type 2 ICF shows hypomethylation at a satellites.[51] Both types of ICF syndrome show hypomethylation at classical satellites 2 and 3 on chromosomes 1, 9, and 16. Upon using MeFISH assays to test lymphoblast cell lines derived from type 1 and type 2 ICF patients, the control cells clearly show both classical- and a-satellite signals, whereas weaker signals are detected in type 2 ICF cells under the same photographic exposure conditions. In type 1 ICF cells, although a-satellite MeFISH signals are detectable, classical-satellite signals are very weak. Quantitative measurement of the MeFISH signals confirms the observed differences among the three groups. The utility of MeFISH technology has very recently been extended to the imaging of intact early embryos and small em 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Figure 5. Complex formation at T with B-end-labeled ICON. a) Reaction sites cross-linked by a 5’-B-labeled ICON. b) Application of B-end-labeled ICON to fluorimetric 5mC detection.
The reaction of a B-end-labeled ICON probe can also be used as a control in conjunction with a B-internal-labeled probe. For example, an ICON probe containing a Cy5 label at the 5’-end and a B base inside the sequence (discrimination ICON), and an ICON probe containing a Cy3 label near the 3’end and a B base at the 5’-end (control ICON), which can recChemMedChem 2014, 9, 1958 – 1965
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CHEMMEDCHEM REVIEWS ognize the 3’-neighbor of the sequence to which the discrimination ICON binds, have been used to investigate test HoxA5 fragments that had been attached to beads (Figure 5 b). Upon methylation of the target C in the fragment, the beads show Cy5 fluorescence at 655 nm through FRET upon Cy3 excitation. In contrast, no Cy5 fluorescence is observed from beads to which unmethylated fragments are attached. A combination of 5mC-specific crosslinking from B-internal ICON probes and nonspecific crosslinking from B-terminal ICON probes is therefore effective for fluorimetric detection of 5mC. In the reaction with ICON probes, T is usually inactive against osmate because B and T can form a Watson–Crick base-like pair, and T is correctly stacked by flanking base pairs, as described above. Clearly, the T of an A/T base pair is also inactive. However, osmium complex formation with the use of the ICON probe results in oxidation of T in the T5mCG–CBA duplex.[54] Interestingly, whereas oxidation is observed at both T and 5mC for a T5mCG sequence, oxidation is negligible at both T and C in a TCG sequence. The reason behind this difference in reactivity is not clear. Evidently, the T base flanking a B/5mC mismatched base pair may be more reactive than T in the normal stacking duplex, because it is easier for the oxidant to access the T in the mismatched pair. Therefore, oxidation of T in the T5mCG/CBA duplex is understandable. However, the suppression of oxidation at T in a TCG sequence is still difficult to explain. It may be due to the small steric and electronic effect imparted by the 3’-neighboring C. As a result, although complex formation at the T of T5mCG occurs, the function of the ICON probes, which discriminate 5mC from C by using complexation efficiency, is maintained completely, and quantification of 5mC through interstrand crosslinking between the target methylated DNA and the ICON probe is straightforward. Why does the reactivity of T change depending on the 3’neighboring base? Upon investigating T oxidation by osmate in detail, interesting T reactivity has been observed: osmium oxidation progresses more rapidly for sequences in which two T residues are consecutive (TT) than for sequences containing an isolated T.[55] The half-lives of isolated T and TT sequences are 86 and 13 s in the osmium oxidation mixture, respectively. In addition, in a TT sequence, the ratio of reaction rates at 5’T and 3’T (k1/k2) is 2.2:1. This difference in the oxidation efficiency could be attributable to the accessibility of the oxidants to T. The predominant formation of (5R,6S)-T glycol, as determined by X-ray crystallographic analysis, suggests that the accessibility of oxidants to T is influential, and this results in a difference in oxidation efficiency at each T.
www.chemmedchem.org group extending from the nucleobase does not coordinate to an osmium complex core. A high level of 5hmC at the centromeric repeats has been discovered by MeFISH analysis of developing male germ cells. Sasaki and co-workers[48] report that 5hmC signals are observed in Dnmt3L-KO germ cells by using ICON probes. The results suggest that the formation of 5hmC in mitotic chromosomes at postnatal day 2 of germ cells is independent of de novo methylation in prospermatogonia and that 5hmC residues in the centromeres are produced from preexisting 5mC residues in prospermatogonia. A variety of 5hmC detection methods have recently been reported.[57–64] The combination of the osmium-mediated oxidation and the 5hmC detection technologies with different chemical approaches will extend the range of applications in epigenetics research.
Summary The chemistry of specific osmium complex formation at methylated C in DNA for the analyses of 5mC has been discussed. This method of detecting 5mC is bisulfite free and significantly expands the range of technologies available for analysis of epigenetic modifications. Detailed investigations into osmium complexation efficiency and selectivity have contributed to the development of a quantitative, sequence-selective 5mC detection method based on the site of incorporation of B in ICON probes. Osmium-mediated oxidation is effective not only for the detection of 5mC but also for the detection of 5hmC. Extending the range of 5hmC detection methods is expected to be very useful for the next generation of epigenetic studies. The chemical reactions developed herein to target 5mC offer a promising prospect for several new technologies for 5mC analysis that are quite different from conventional approaches. For example, a new, high-value aspect of on-chip methylation analysis has been developed through fixation of an ICON probe that can form an osmium complex with the target 5mC onto a microwell and subsequent PCR amplification. Methods for the microscopic visualization of 5mC in specific DNA sequences in individual cells or chromosomes have also been developed. MeFISH analysis is suitable for interrogating repeat sequences for which a priori knowledge of the DNA methylation/hydroxymethylation profile is known. In principle, the method is applicable to interspersed repeats and singlecopy sequences.
Outlook Reactions of ICON Probes with 5hmC in Demethylation Processes The 5hmC residue in DNA also shows high reactivity upon osmium-mediated oxidation.[56] The reactivity of 5hmC in DNA is almost the same as that observed for 5mC in DNA. A stable 5hmC glycol–dioxidoosmium–bipyridine ternary complex is formed, and the configuration of glycol in the complex has been determined to be (5R,6S). In addition, the hydroxymethyl 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Further aspects remain to be examined to establish a more straightforward analysis of epigenetic modification. A higher signal-to-noise ratio may also be required for higher sensitivity of methylation detection, which may be achieved by the use of multiple probes, multiple labeling, signal amplification, as well as suppression of nonspecific signals. However, the osmium-based 5mC detection system described herein is applicable to the efficient analysis of 5mC in DNA by using conventional fluorescence detection methods familiar to biologists, ChemMedChem 2014, 9, 1958 – 1965
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CHEMMEDCHEM REVIEWS such as microscopy and flow cytometry. Such an approach may be particularly useful for studying cells that are only available in small numbers, such as early embryonic cells, tissue stem cells, developing germ cells, and clinical specimens. This new chemical-based concept of effective 5mC/5hmC analysis equips researchers in this field with new approaches for epoch-making epigenotyping assay. Keywords: DNA methylation · epigenetics · fluorescent probes · genomics · osmium [1] [2] [3] [4] [5] [6]
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Received: April 9, 2014 Published online on July 2, 2014
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DNA–Osmium Complexes: Recent Developments in the Operative Chemical Analysis of DNA Epigenetic Modifications Akimitsu Okamoto*[a]
2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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CHEMMEDCHEM REVIEWS The development of a reaction for the detection of one epigenetic modification in a long DNA strand is a chemically and biologically challenging research subject. Herein, we report and discuss the formation of 5-methylcytosine–osmium complexes that are used as the basis for a bisulfite-free chemical assay for DNA methylation analysis. Osmium in the oxidized state reacts with C5-methylated pyrimidines in the presence of a bipyridine ligand to give a stable ternary complex. On the basis of this reaction, an adenine derivative with a tethered bipyridine moiety has been designed for sequence-specific
www.chemmedchem.org osmium complex formation. Osmium complexation is then achieved by hybridization of a short DNA molecule containing this functional nucleotide to a target DNA sequence and results in the formation of a cross-linked structure. This novel concept of methylation-specific reaction, based on a straightforward chemical process, expands the range of methods available for the analysis of epigenetic modifications. Advantages of the described method include amplification-insensitive detection, 5-hydroxymethylcytosine complexation, and visualization through methylation-specific in situ hybridization.
Introduction Gene expression is regulated by the epigenetic modification of biopolymers, such as DNA and histone proteins, independent of their primary sequences. In particular, methylation and demethylation of cytosine (C) in DNA is an important epigenetic modification of the genome in many animals and plants, and this process plays a crucial role in the regulation of developmental gene expression, chromatin remodeling, genomic imprinting, X-chromosome inactivation, and genome stability.[1–4] Aberrant DNA methylation is an early and fundamental event in the pathogenesis of many human diseases, including cancer.[5] The mechanism of DNA methylation, that is, the formation of 5-methylcytosine (5mC) has been studied very well, and recent studies on DNA demethylation have revealed that 5-hydroxymethylcytosine (5hmC) is an important intermediate for replication-dependent and/or -independent demethylation (Figure 1).[6, 7] In addition, erroneous DNA methylation may contribute to the etiopathogenesis of tumorigenesis and aging.[5, 8] The methylation and demethylation of C are the most important epigenetic events occurring in DNA, and their analysis is very significant. Much effort has gone into developing a simple reaction that can be used for the detection of 5mC and 5hmC. However, the ability to distinguish 5mC and 5hmC from C, that is, to detect the existence of only one methyl group in a long DNA strand, is still a chemically and biologically challenging subject. A variety of methods have been developed to detect DNA methylation.[9] For example, recent advances in high-throughput DNA sequencing technology, along with the use of immunoprecipitation,[10] affinity-based pull-down,[11] and bisulfite conversion,[12–14] has now made it possible to map 5mC in the genome at base resolution. At the cellular level, global DNA methylation patterns can be microscopically visualized by using either anti-5mC antibodies[15, 16] or methylated DNA-binding domain fusion proteins.[17, 18] Bisulfite methods have now been widely applied for creating whole methylome maps of the human genome in addition to others. Sodium bisulfite causes C deamination in a single-strand DNA through the for[a] Dr. A. Okamoto Research Center for Advanced Science & Technology The University of Tokyo 4-6-1 Komaba, Meguro-ku, Tokyo 153-8904 (Japan) E-mail: [email protected]
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Figure 1. Methyl groups in DNA structure. a) Methylation and demethylation of cytosine. Two epigenetically modified cytosine residues, 5mC and 5hmC, generated in the methylation and demethylation processes. b) Two methyl groups in a fully methylated DNA. The methylation is a very small change in the DNA duplex structure.
mation of a 5,6-dihydrocytosine-6-sulfonate intermediate at acidic pH values. The deaminated bisulfite adduct is converted into a uracil residue through elimination of bisulfite at alkaline pH values. 5mC also yields thymine (T) with sodium bisulfite, but the reaction rate for bisulfite adduct formation is much slower.[19, 20] Thus, the bisulfite assay requires long reaction times (5–24 h, normally an overnight reaction) to avoid false positives for methylcytosine. Methylation-specific polymerase chain reaction (PCR) was developed on the basis of the modification of DNA by sodium bisulfite to rapidly assess the methylation status within a C–phosphate–G island.[21, 22] The biggest problem in bisulfite assays is DNA degradation during the long reaction times.[23] By treating a 100-mer DNA with bisulfite over 16 h under typical bisulfite assay conditions, the sigmoid curves of quantitative PCR show that of the 1014 copies of DNA ChemMedChem 2014, 9, 1958 – 1965
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CHEMMEDCHEM REVIEWS in the sample before the bisulfite assay, only approximately 1011 copies remain after bisulfite treatment for 16 h. This is 0.1 % of the original DNA amount, and a critical level of degradation is caused by depyrimidination during bisulfite treatment. Several radical scavengers and standardized kits have been developed for routine bisulfite treatment nowadays. However, a DNA methylation detection method with a quite different chemical mechanism is still required for further extension of epigenetic studies. A rapid and selective chemical reaction capable of detecting 5mC and 5hmC in DNA would clearly be very useful for efficiently analyzing the status of epigenetic modifications at a specific site in a gene. The five key points required for a new chemical assay for methylation detection are as follows. 1) Sequence selectivity: Nowadays, the focus of methylation studies is the elucidation of the role of each methylation in cell functions. The development of a conceptually new chemical approach to site-specific detection is thus very desirable. 2) Modification-positive assay: Reactions selective to epigenetic modification sites should be developed. 3) Simple detection process: The most facile method to detect methylation is to label the methylation sites directly with signal-sending units. Apart from DNA microarray analysis of sequence-converted DNA through bisulfite treatment, there are very few fluorescent assays available for the detection of C methylation.[24, 25] 4) No strand damage: DNA samples are damaged by strand scission during bisulfite treatment.[23] Nonspecific cleavage complicates the detection process and decreases quantification precision. 5) Short assay time: To obtain reliable results, the bisulfite assay usually requires approximately half a day for complete modification. A shorter reaction time is clearly desirable.
Akimitsu Okamoto obtained his PhD in synthetic chemistry and biological chemistry from Kyoto University in 1998, where he was appointed Assistant Professor in 1999 after a year as a Postdoctoral Fellow at Massachusetts Institute of Technology. He moved to RIKEN as an Initiative Research Scientist (unit leader) in 2006. Akimitsu Okamoto joined the faculty at the University of Tokyo in 2012 as a Professor. His research focuses on the design, synthesis, and physical properties of new, manmade biopolymers with various functionalities and on the design of unique organic chemical systems for recognizing, transforming, and visualizing a single component or atom in biopolymers of interest.
2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemmedchem.org In this review, we introduce newly designed chemical assays for the detection of DNA epigenetic modifications. The chemistry-based assay includes many advantages that are quite different from conventional assays. The new concept of sequencespecific rapid methylation analysis based on a straightforward chemical reaction will be the starting point for a novel methylation-type assay.
5mC Forms a Stable Complex with Osmium A rapid, target-selective, DNA-compatible chemical reaction capable of distinguishing between C and 5mC would be useful to analyze efficiently the status of C methylation at a specific site in a gene. The oxidation of pyrimidine bases is a practical approach that could be used to determine the presence or absence of a methyl group at the C5 position of C, because the number of substituents on the C5=C6 double bond is different in these two species. The C5=C6 double bonds of T and 5mC bases are known to be oxidized by osmium tetroxide, and these bases are converted into the corresponding glycols.[26, 27] In single-stranded DNA molecules, T is also oxidized by osmium tetroxide, and this reaction is sometimes used for T sequencing.[28–32] We developed the selective oxidation of 5mC through exquisite control of a reaction that distinguishes between C and 5mC (Scheme 1).[33, 34] One example of the reaction conditions
Scheme 1. Oxidative osmium complex formation at 5mC.
is as follows: 5 mm potassium osmate (an oxidant much less intractable than osmium tetroxide), 100 mm potassium hexacyanoferrate(III) (an activator), and 100 mm 2,2’-bipyridine (a reaction-accelerating ligand) in 100 mm tris(hydroxymethyl)aminomethane (Tris)·HCl buffer (pH 7.7), 1 mm ethylenediaminetetraacetic acid (EDTA), and 10 % acetonitrile (for dissolution of 2,2’-bipyridine). The reaction mixture is incubated with the DNA of interest at 0 8C for 5 min, whereupon the oxidized strand is cleaved at a damaged pyrimidine base with hot piperidine. Cleaved DNA strands can then be identified as shortened DNA fragments by using polyacrylamide gel electrophoresis. Strand cleavage at C is negligible, whereas DNA strands containing 5mC are cleaved at the site of modification. The product of the osmium-mediated oxidation reaction is a 5mC glycol–dioxidoosmium–bipyridine ternary complex.[35] The major form of the glycol is in the (5R,6S) configuration. The osmium complex formed selectively at 5mC is stable in aqueous solutions under weakly acidic, weakly basic, and elevated temperature conditions. ChemMedChem 2014, 9, 1958 – 1965
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The tendency of the reaction to react preferentially with C or 5mC is determined by the ligand. The reactivity and selectivity was investigated by using a variety of pyridyl ligands including pyridine, 2,2’-bipyridine, 4,4’-diamino-2,2’-bipyridine, 4,4’-dinitro-2,2’-bipyridine, and phenanthroline (Table 1).[36] The
Table 1. Effect of the ligand on the selectivity of osmium-mediated oxidation.[a] Ligand
Conc. [mm]
5mC [%]
C [%]
2,2’-bipyridine pyridine 4,4’-diamino-2,2’-bipyridine 4,4’-dinitrobipyridine 1,10-phenanthroline
100 250 10 10 10
54 9 > 98 0 96
2 0 > 98 0 17
[a] The radiolabeled DNA 5’-32P-d(AAAAAAG[C/5mC]GAAAAAA)-3’ (1 mm) was added to 5 mm potassium osmate, 100 mm potassium hexacyanoferrate(III), ligand, 1 mm EDTA, and 10 % acetonitrile in 100 mm Tris·HCl (pH 7.7), and then the mixture was incubated at 0 8C for 5 min. After hot piperidine treatment (90 8C, 20 min), the yield at the target base was calculated from DNA cleavage bands in polyacrylamide gel electrophoresis.
reaction employing pyridine can distinguish between C and 5mC as a result of the difference in reaction rates; however, the reaction rate for 5mC is much slower than that observed in the reaction employing 2,2’-bipyridine. 4,4’-Diamino-2,2’-bipyridine, which has electron-donating groups, accelerates the reactions with both C and 5mC, whereas 4,4’-dinitro-2,2’-bipyridine, which has electron-withdrawing groups, exhibits a low reaction rate with both C and 5mC. Conducting the reaction with phenanthroline gives higher reaction rates, but it does not suppress the reaction for C. Therefore, the inclusion of 2,2’-bipyridine in the assay allows the difference in reaction rates with C and 5mC to be maximized while keeping the overall reaction time to a minimum. The 5mC in single-stranded DNA efficiently forms a metal complex, whereas complex formation at the 5mC in a duplex is suppressed.[33] The weak reactivity in a duplex is attributed to inhibition of the attack of an osmium on the p orbital of a C5=C6 double bond by base stacking in the duplex structure. The use of steric control, such as the formation of a bulge structure or a mismatched base pair, is effective for sequencespecific osmium complex formation. Efficient strand cleavage is observed at 5mC in the bulged structure, similar to that found in the reaction with single-stranded DNA. The calculated rate constants for 5mC-bulged and C-bulged duplexes are 1.11 10 2 and 2.51 10 5 s 1, respectively. The use of a bulge-inducing DNA fixed on polystyrene beads make 5mC analysis straightforward. After hybridization of the target DNA with bulge-inducing DNA, the duplex is incubated in a reaction mixture containing potassium osmate. After washing and treatment with hot piperidine, the DNA amplification is monitored with quantitative PCR. A curve with a retarded start and a small curvature is displayed for a 5mC-containing sequence, because complex formation at 5mC results in DNA cleavage at the complexation site and inhibition of DNA amplification. 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chemical Labeling of 5mC The ability to label 5mC sites directly with signal-sending units would lead to a simple detection assay. To this end, functionalization of the bipyridine ligand with a signaling unit would allow tagging of the 5mC residues in the DNA of interest through coordination to the osmate. Substitution at C4 of bipyridine does not hinder complex formation with 5mC.[36] Indeed, tag-attachable bipyridine ligands for direct 5mC labeling have been synthesized and used in fluorescent and electrochemical assays.[37] As an implementation of the above approach in a fluorescent assay, a new ligand, 4-[6-(4-aminobutylamino)-6-oxohexyl]-4’methyl-2,2’-bipyridine, has been developed for the attachment of fluorescent dyes to 5mC sites. After formation of the osmium complex, a fluorescent dye is attached to the amino end of the linker extended from the ligand (5mC–Os–HEX, (HEX = hexachlorofluorescein), Figure 2 a). Direct fluorescent labeling to 5mC residues through osmium complex formation thus allows the presence or absence of 5mC to be judged visually and quantified through the use of Fçrster resonance energy transfer (FRET) from a hybridization probe labeled with fluorescein to 5mC-Os-HEX.
Figure 2. Fluorescence detection of 5mC. a) Fluorescence labeling of 5mC by tethering bipyridine to a fluorescent dye. b) Fluorescence quenching by osmium complex formation.
A DNA probe with a microenvironment-sensitive fluorescent dye, 6-dimethylamino-2-acylnaphthalene (DAN), has also been designed.[38–44] The fluorescence of the duplex of the osmiumcomplexed DNA and the DAN-labeled probe can be significantly quenched (Figure 2 b). Photoinduced electron transfer occurring from the excited DAN to the osmium complex contributes to the quenching process.
ICON Probe for 5mC Detection A new “ligand” has been developed for sequence-specific 5 mC detection.[45] Thus, an adenine base forming a misChemMedChem 2014, 9, 1958 – 1965
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www.chemmedchem.org the ICON probes. After osmium-mediated oxidation, the wells are rinsed and then coated with bovine serum albumin. The fixed DNA is detected by using PCR amplification of a part of the cross-linked target DNA. The 5mC-containing DNA shows exponential growth of the PCR product. On-chip capture of methylated DNA by the ICON probe at 5mC makes the sequence-specific detection of 5mC through PCR amplification possible.
Fluorescence in situ Hybridization To Visualize 5mC in Chromosomes
Scheme 2. Interstrand crosslinking between 5mC in the target DNA and the complementary ICON probe.
matched pair with 5mC is tethered to the bipyridine ligand required for osmium-centered complex formation (bipyridinetethered adenine, B; Scheme 2) to form interstrand complexes at the target 5mC residue. The formation of a 5mC/A mismatched pair causes partial disruption of the p stacking of the DNA duplex and facilitates oxidation at the 5mC C5=C6 double bond, which is forced out of the duplex major groove. The combination of the formation of a mismatched base pair together with an appropriately located bipyridine should result in complex formation at a specific 5mC, regardless of the presence of other reactive bases in a long DNA strand. Nucleic acids containing B probes can form interstrand crosslinks through osmium complex formation, which allows sequenceselective labeling and obstruction of PCR amplification at the target 5mC residue. The sequence-specific osmium complexation accelerating nucleic acid probe is now described as an interstrand complexation formed by osmium and nucleic acid (ICON) probe. Blocking of PCR by interstrand crosslinking with an ICON probe at 5mC makes the sequence-specific quantification of methylation possible.[46] The DNA of interest is incubated with ICON probes in an osmium reaction mixture. The quantitative PCR assay exhibits a linear relationship between the amplification starting point and the logarithm of the proportion of methylation. Interstrand crosslinking with ICON probes designed for each target 5mC is formed easily, independent of the number of other 5mC sites. For example, sequence-specific ICON probes have been used to quantify the methylation of mouse genome collected from different tissues.[45] Samples from different tissues exhibit characteristic methylation levels for the two methylation sites, which is in close agreement with the degree of methylation determined by mass spectrometric analysis of fragmented and bisulfite-treated genome samples. Interstrand crosslinking between 5mC in the target DNA and the complementary ICON probe fixed to the bottom of microwells has assisted the on-chip detection of a specific 5mC in the target DNA.[47] After ICON DNA is fixed through covalent bonds to the bottom of each well of a series of microwell strips, the target DNA is put into the wells and hybridized with 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ICON technology is useful to identify 5mC residues in target sequences at both the cellular and chromosomal levels, because ICON probes can label the target 5mC with fluorescent dyes without causing serious DNA damage, as observed with bisulfite methods, and without problems associated with nonsequence-specific 5mC binding, as observed with anti-5mC antibodies. The Sasaki group have applied ICON technology to develop a novel method, named methylation-specific fluorescence in situ hybridization (MeFISH), to visualize the DNA methylation status at specific sequences in individual nuclei or chromosomes (Figure 3).[48] MeFISH could be used to detect DNA methylation at centromeric and pericentromeric repeat sequences in both mouse and human cells. The overall MeFISH protocol involves conventional FISH technology for sequencespecific target detection and ICON technology for 5mC detection.[45] Tissue sections, cells, or chromosome spreads are first subjected to FISH with the fluorescence-labeled ICON probes,
Figure 3. Outline of the MeFISH protocol.
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CHEMMEDCHEM REVIEWS and the location of fluorescent signals is then recorded. Next, the specimens are treated with osmium to induce 5mC-dependent crosslinking, and non-cross-linked probes are removed in a denaturation step. Subsequent comparison of the FISH and MeFISH images allows the methylation status to be estimated. For example, ICON probes have been designed against major and minor satellite repeats of wild-type and Dnmt-TKO (Dnmt1, Dnmt3a, and Dnmt3b triple knock out) mouse embryonic stem cells,[49] and two-color FISH has been performed by using these probes. On metaphase chromosomes, the major satellite probe displays a strong signal near the centromeric end, and the minor satellite probe gives a doublet on the sister chromatids at the centromeric end. In interphase nuclei, whereas the major satellite probe gives strong signals that are co-localized with the 4’,6-diamidino-2-phenylindole (DAPI)dense regions, the minor satellite probe gives smaller spots at the periphery of the major satellite signals (Figure 4). The specimens are then treated with osmium and denatured to remove non-cross-linked probes. The specific retention of ICON signals in wild-type stem cells, but not in Dnmt-TKO embryonic stem cells, is clear.
www.chemmedchem.org bryonic tissues by the Abe group.[52] The whole-mount MeFISH approach enables the observation of the DNA methylation status of satellite repeats in developing mouse primordial germ cells. The combination of whole-mount MeFISH with immunostaining or RNA-FISH facilitates the simultaneous visualization of DNA methylation and protein or RNA expression at single-cell resolution without destroying embryonic and nuclear structures.
Reactions Controlled at Thymine Osmium complex formation occurs at T as well as at 5mC residues.[26, 27] Indeed, the reactivity at T is almost twice that at 5mC. However, in the ICON probe, T does not form any osmium complex even if located opposite B, because the B base forms a matched base pair with T in the duplex. However, if the location of a B nucleotide within the ICON probe is varied, the reactivity and base selectivity of the osmium crosslinking change. Osmium-mediated oxidation by using the ICON probe containing B at the end of the strand has shown high crosslink yields not only for T opposite B, but also for T located at the unhybridized region of the target DNA (Figure 5 a).[53] An ICON strand end has higher flexibility of its basestacking structure; thus, the oxidants have better access to the target C=C bond of T than to the T/B base pair located inside the target–probe duplex. The lack of specificity with respect to the reaction site on the target DNA opposite B is attributable to the structural flexibility of both the unhybridized region of the target DNA and the alkyl linker tethering the bipyridine to the purine ring in B.
Figure 4. The MeFISH signal from major satellite regions in mouse embryonic stem cells observed by our group. a) The MeFISH signal after osmium treatment and denaturation (green, ICON probe labeled with Cy3; blue, DAPI). b) The MeFISH signal after denaturation without osmium treatment.
MeFISH has also made it possible to detect 5mC at human satellite repeat sequences. Human classical satellites 2 and 3 and a satellites are generally highly methylated; however, they are hypomethylated in cells from patients with immunodeficiency, centromeric instability, and facial abnormalities (ICF) syndrome.[50] Whereas type 1 ICF shows normal methylation at a satellites, type 2 ICF shows hypomethylation at a satellites.[51] Both types of ICF syndrome show hypomethylation at classical satellites 2 and 3 on chromosomes 1, 9, and 16. Upon using MeFISH assays to test lymphoblast cell lines derived from type 1 and type 2 ICF patients, the control cells clearly show both classical- and a-satellite signals, whereas weaker signals are detected in type 2 ICF cells under the same photographic exposure conditions. In type 1 ICF cells, although a-satellite MeFISH signals are detectable, classical-satellite signals are very weak. Quantitative measurement of the MeFISH signals confirms the observed differences among the three groups. The utility of MeFISH technology has very recently been extended to the imaging of intact early embryos and small em 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Figure 5. Complex formation at T with B-end-labeled ICON. a) Reaction sites cross-linked by a 5’-B-labeled ICON. b) Application of B-end-labeled ICON to fluorimetric 5mC detection.
The reaction of a B-end-labeled ICON probe can also be used as a control in conjunction with a B-internal-labeled probe. For example, an ICON probe containing a Cy5 label at the 5’-end and a B base inside the sequence (discrimination ICON), and an ICON probe containing a Cy3 label near the 3’end and a B base at the 5’-end (control ICON), which can recChemMedChem 2014, 9, 1958 – 1965
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CHEMMEDCHEM REVIEWS ognize the 3’-neighbor of the sequence to which the discrimination ICON binds, have been used to investigate test HoxA5 fragments that had been attached to beads (Figure 5 b). Upon methylation of the target C in the fragment, the beads show Cy5 fluorescence at 655 nm through FRET upon Cy3 excitation. In contrast, no Cy5 fluorescence is observed from beads to which unmethylated fragments are attached. A combination of 5mC-specific crosslinking from B-internal ICON probes and nonspecific crosslinking from B-terminal ICON probes is therefore effective for fluorimetric detection of 5mC. In the reaction with ICON probes, T is usually inactive against osmate because B and T can form a Watson–Crick base-like pair, and T is correctly stacked by flanking base pairs, as described above. Clearly, the T of an A/T base pair is also inactive. However, osmium complex formation with the use of the ICON probe results in oxidation of T in the T5mCG–CBA duplex.[54] Interestingly, whereas oxidation is observed at both T and 5mC for a T5mCG sequence, oxidation is negligible at both T and C in a TCG sequence. The reason behind this difference in reactivity is not clear. Evidently, the T base flanking a B/5mC mismatched base pair may be more reactive than T in the normal stacking duplex, because it is easier for the oxidant to access the T in the mismatched pair. Therefore, oxidation of T in the T5mCG/CBA duplex is understandable. However, the suppression of oxidation at T in a TCG sequence is still difficult to explain. It may be due to the small steric and electronic effect imparted by the 3’-neighboring C. As a result, although complex formation at the T of T5mCG occurs, the function of the ICON probes, which discriminate 5mC from C by using complexation efficiency, is maintained completely, and quantification of 5mC through interstrand crosslinking between the target methylated DNA and the ICON probe is straightforward. Why does the reactivity of T change depending on the 3’neighboring base? Upon investigating T oxidation by osmate in detail, interesting T reactivity has been observed: osmium oxidation progresses more rapidly for sequences in which two T residues are consecutive (TT) than for sequences containing an isolated T.[55] The half-lives of isolated T and TT sequences are 86 and 13 s in the osmium oxidation mixture, respectively. In addition, in a TT sequence, the ratio of reaction rates at 5’T and 3’T (k1/k2) is 2.2:1. This difference in the oxidation efficiency could be attributable to the accessibility of the oxidants to T. The predominant formation of (5R,6S)-T glycol, as determined by X-ray crystallographic analysis, suggests that the accessibility of oxidants to T is influential, and this results in a difference in oxidation efficiency at each T.
www.chemmedchem.org group extending from the nucleobase does not coordinate to an osmium complex core. A high level of 5hmC at the centromeric repeats has been discovered by MeFISH analysis of developing male germ cells. Sasaki and co-workers[48] report that 5hmC signals are observed in Dnmt3L-KO germ cells by using ICON probes. The results suggest that the formation of 5hmC in mitotic chromosomes at postnatal day 2 of germ cells is independent of de novo methylation in prospermatogonia and that 5hmC residues in the centromeres are produced from preexisting 5mC residues in prospermatogonia. A variety of 5hmC detection methods have recently been reported.[57–64] The combination of the osmium-mediated oxidation and the 5hmC detection technologies with different chemical approaches will extend the range of applications in epigenetics research.
Summary The chemistry of specific osmium complex formation at methylated C in DNA for the analyses of 5mC has been discussed. This method of detecting 5mC is bisulfite free and significantly expands the range of technologies available for analysis of epigenetic modifications. Detailed investigations into osmium complexation efficiency and selectivity have contributed to the development of a quantitative, sequence-selective 5mC detection method based on the site of incorporation of B in ICON probes. Osmium-mediated oxidation is effective not only for the detection of 5mC but also for the detection of 5hmC. Extending the range of 5hmC detection methods is expected to be very useful for the next generation of epigenetic studies. The chemical reactions developed herein to target 5mC offer a promising prospect for several new technologies for 5mC analysis that are quite different from conventional approaches. For example, a new, high-value aspect of on-chip methylation analysis has been developed through fixation of an ICON probe that can form an osmium complex with the target 5mC onto a microwell and subsequent PCR amplification. Methods for the microscopic visualization of 5mC in specific DNA sequences in individual cells or chromosomes have also been developed. MeFISH analysis is suitable for interrogating repeat sequences for which a priori knowledge of the DNA methylation/hydroxymethylation profile is known. In principle, the method is applicable to interspersed repeats and singlecopy sequences.
Outlook Reactions of ICON Probes with 5hmC in Demethylation Processes The 5hmC residue in DNA also shows high reactivity upon osmium-mediated oxidation.[56] The reactivity of 5hmC in DNA is almost the same as that observed for 5mC in DNA. A stable 5hmC glycol–dioxidoosmium–bipyridine ternary complex is formed, and the configuration of glycol in the complex has been determined to be (5R,6S). In addition, the hydroxymethyl 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Further aspects remain to be examined to establish a more straightforward analysis of epigenetic modification. A higher signal-to-noise ratio may also be required for higher sensitivity of methylation detection, which may be achieved by the use of multiple probes, multiple labeling, signal amplification, as well as suppression of nonspecific signals. However, the osmium-based 5mC detection system described herein is applicable to the efficient analysis of 5mC in DNA by using conventional fluorescence detection methods familiar to biologists, ChemMedChem 2014, 9, 1958 – 1965
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CHEMMEDCHEM REVIEWS such as microscopy and flow cytometry. Such an approach may be particularly useful for studying cells that are only available in small numbers, such as early embryonic cells, tissue stem cells, developing germ cells, and clinical specimens. This new chemical-based concept of effective 5mC/5hmC analysis equips researchers in this field with new approaches for epoch-making epigenotyping assay. Keywords: DNA methylation · epigenetics · fluorescent probes · genomics · osmium [1] [2] [3] [4] [5] [6]
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Received: April 9, 2014 Published online on July 2, 2014
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