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Osmium Complexation of Mismatched DNA: Effect of the Bases Adjacent to Mismatched 5-Methylcytosine Akiko Nomura, Kazuki Tainaka, and Akimitsu Okamoto* Advanced Science Institute, RIKEN (The Institute of Physical and Chemical Research), Wako, Saitama 351-1098, Japan. Received December 10, 2008; Revised Manuscript Received January 12, 2009
The efficiency of osmium complex formation at 5-methylcytosine in mismatched DNA duplexes is a key point for the design of sequence-specific detection of DNA methylation. Osmium complexation was not observed in fully matched duplexes, whereas the complexation site and efficiency in mismatched duplexes changed depending on the type of 5′-neighboring base of the 5-methylcytosine forming a mismatched base pair. In particular, when the base adjacent to the 5′ side of the mismatched base pair was thymine, a unique “side reaction” was observed. However, the nature of the mismatched base pairs in the reaction site did not influence the selectivity of osmium complex formation with methylated DNA.
INTRODUCTION A number of reactions for pyrimidine bases have been reported, such as photodimerization (1, 2), Michael addition (3-5), and oxidation (6-9). Osmium oxidation is one of the known pyrimidine-targeting sequencing methods, and in this case, the C5-C6 double bonds of thymine and 5-methylcytosine are oxidized (10-15). A highly stable pyrimidine glycol-osmatebipyridine triad is formed on reaction with osmium tetroxide (10, 11) or potassium osmate activated by potassium hexacyanoferrate(III) in the presence of bipyridine as a ligand (Figure 1a) (12-15). On the other hand, the oxidation of cytosine is much slower than that of 5-methylcytosine (10, 12). Cytosine methylation, in which the C5 position of the cytosine base is methylated enzymatically, is an epigenetic modification that can play an important role in the control of gene expression in mammalian cells, and about 70% to 80% of cytosines are methylated in the CpG sequence. The sequence-selective distinction between methylated and unmethylated cytosines is very important for studies of gene deactivation (16, 17). In addition, erroneous DNA methylation may contribute to the etiopathogenesis of tumorigenesis (18-20). Cytosine methylation is one of the most important epigenetic events in cells, and its detection is very significant. The oxidation of pyrimidine bases may be useful for the detection of the presence/absence of a methyl group at cytosine C5. A 5-methylcytosine-adenine mismatched base pair made possible the sequence-selective formation of an osmium complex at 5-methylcytosine (21). This is different from the negligible reactivity of the fully matched duplex, because the formation of a cytosine-adenine mismatched pair causes partial disruption of π-stacking of the DNA duplex and facilitates oxidation at the C5-C6 double bond of the cytosine forced out of the DNA major groove. This system was applied to a new DNA probe for methylation detection, the so-called ICON probe (Figure 1b). The formation of a mismatched base pair by hybridization with the ICON probe and the location of bipyridine attached to the probe DNA results in complexation at a specific 5-methylcytosine. Toward development of more effective, sequenceselective 5-methylcytosine detection based on the formation of mismatched base pairs, a more detailed investigation of the * To whom correspondence may be addressed. Phone: (+81) 48467-9238. Fax: (+81) 48-467-9205. E-mail:
[email protected].
Figure 1. Osmium complex formation at 5-methylcytosine. (a) Formation of a ternary complex with 5-methylcytosine, osmate, and bipyridine. (b) Interstrand cross-link by osmium complexation with the ICON probe.
osmium complexation efficiency and selectivity depending on the nature of the mismatched base pairs and neighboring sequences is required. This knowledge facilitates prior estimation of the reaction abilities of probes and helps in the design of highly methylation sensitive probes. In this paper, we describe the osmium oxidation efficiency at methylated CpG (MpG) in a series of duplexes with a single base mismatch. The oxidation efficiency was investigated through alkaline strand cleavage for a variety of duplexes with different complementary bases of 5-methylcytosine and different 5′-flanking base pairs of MpG. Sequence-selective osmium complexation at the target 5-methylcytosine was detected for mismatched duplexes, and the unique reactivity of 5′-neighboring thymine was also observed.
EXPERIMENTAL PROCEDURES General. The reagents for 5-methylcytosine oxidation were purchased from Wako, and the reagents for the DNA synthe-
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sizer, such as A, G, C, T, and M-β-cyanoethyl phosphoramidite, were purchased from Glen Research. Oligodeoxynucleotides (ODNs) were purchased from Gene Design. HPLC was performed on a Cosmosil 5C-18AR or Chemcobond 5-ODS-H column (10 × 150 mm) with a Gilson Chromatography model 305 using a UV detector model 118 at 254 nm. Micro BioSpin chromatography columns used for desalting were purchased from Bio-Rad. ICON probes were prepared as in our previous report (21). Preparation of 32P-5′-End-Labeled DNA. The ODNs (400 pmol-strand) were 5′-end-labeled by phosphorylation with 4 µL of [γ-32P]ATP (GE Healthcare) and T4 polynucleotide kinase using a standard procedure. The 5′-end-labeled ODN was recovered by ethanol precipitation and further purified by 15% denaturing polyacrylamide gel electrophoresis (PAGE). Osmium Oxidation of 32P-5′-End-Labeled DNA. The 5′end-labeled ODN (10 pmol-strand) to be examined was incubated in a solution of 1 µM mismatch-forming ODN or ICON probe, 5 mM potassium osmate, 100 mM potassium hexacyanoferrate(III), 100 mM bipyridine, and 1 mM EDTA in 100 mM Tris-HCl buffer (pH 7.7) and 10% acetonitrile at 0 °C for 5 min. The reaction mixture was ethanol precipitated with the addition of 15 µL of 3 M sodium acetate (pH 5.0), 10 µL of salmon sperm DNA (1 mg/mL), and 1 mL of cold ethanol. The precipitate was washed with 150 µL of 80% cold ethanol and dried in vacuo. The residue was redissolved in 50 µL of 10% piperidine (v/v), heated at 90 °C for 20 min, and then evaporated to dryness by vacuum rotary evaporation. The dried reaction sample was resuspended in 5-20 µL of 80% formamide loading buffer (a solution of 80% formamide (v/v), 1 mM EDTA, 0.1% xylenecyanol, and 0.1% bromophenol blue). The samples (1 µL, 3-10 kcpm) were loaded onto 15% denaturing 19:1 acrylamide-bisacrylamide gel containing 7 M urea, electrophoresed at 1900 V for approximately 1 h, transferred to a cassette, and stored at -80 °C with X-ray film. Melting Temperature (Tm) Measurement. All Tm values of the ODN duplexes (2.5 µM, duplex concentration) were taken in a buffer containing 1 mM EDTA, 100 mM Tris-HCl buffer, and 100 mM sodium chloride, pH 7.7. Absorbance vs temperature profiles were measured at 260 nm using a Shimadzu UV2550 UV/vis spectrometer connected to a Shimadzu TMSPC-8 temperature controller. The absorbance of the samples was monitored at 260 nm from 5 to 90 °C at a heating rate of 1 °C/min. From these profiles, first derivatives were calculated to determine Tm values.
RESULTS AND DISCUSSION Osmium Oxidation at Mismatched 5-Methylcytosine. We prepared 32P-labeled DNA containing an MpG or CpG dinucleotide with different 5′-flanking bases, or ODN(XNG), 5′-32Pd(AAAAGAXNGAGAAAA)-3′ (N ) M or C; X ) C, G, A, or T), which were hybridized with ODN′(CN′X′) 5′d(TTTTCTCN′X′TCTTTT)-3′ (N′ ) G, A, C, or T; X′ ) G, C, T, or A), to examine the reactivity on osmium oxidation (Figure 2a and Supporting Information Figure S1). The duplex was incubated in a solution of 5 mM potassium osmate, 100 mM potassium hexacyanoferrate(III), 100 mM bipyridine, 1 mM EDTA in 100 mM Tris-HCl (pH ) 7.7) and 10% acetonitrile at 0 °C for 5 min. The oxidized strand was cleaved at the damaged pyrimidine base with hot piperidine (90 °C, 20 min), and the products were analyzed as bands representing the shortened strands using PAGE. Typical results of PAGE are shown in Figure 2b. No cleavage band was observed for the fully Watson-Crick base-paired duplex (N′ ) G). The strong suppression of oxidation of bases was due to the protection of the C5-C6 double bond by the stacking of the flanking base pairs. On the other hand, the osmium oxidation specifically
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Figure 2. Osmium oxidation of mismatched duplexes. (a) The sequence of the duplex 32P-ODN(XNG)/ODN′(CN′X′). (b) Two typical examples of gel images after PAGE of the oxidation products. The duplexes were incubated in a solution of 5 mM potassium osmate, 100 mM potassium hexacyanoferrate(III), 1 mM EDTA, and 100 mM bipyridine in 100 mM Tris-HCl (pH ) 7.7), and 10% acetonitrile at 0 °C for 5 min. Recovered DNA was treated with piperidine (10% v/v, 90 °C, 20 min) to cleave the oxidized strand. (Left) Oxidation of 32P-ODN(GMG)/ ODN′(CN′C). (Right) Oxidation of 32P-ODN(TMG)/ODN′(CN′A).
proceeded at the mismatched 5-methylcytosine of ODN(GMG), ODN(AMG), and ODN(CMG), although some difference in strand cleavage efficiency between the types of bases opposite 5-methylcytosine appeared (Figure 3). The small difference in the reactivity can be explained by duplex stability or Tm values. Higher duplex stability resulted in the appearance of the weaker strand cleavage band at 5-methylcytosine (Figure 4). For example, the Tm value of ODN(CMG)/ODN′(CAG) was slightly higher (34 °C) among those of the mismatched duplexes. This mismatched duplex showed relatively low strand cleavage intensity (the relative band intensity was 9%). It is more difficult for oxidants to access the 5-methylcytosine of stable mismatched duplexes compared with 5-methylcytosines in less stable mismatched duplexes, as well as strong suppression of oxidation because of low accessibility in the fully matched duplex. Cleavage of unmethylated ODNs, namely, ODN(GCG), ODN(ACG), and ODN(CCG), was negligible regardless of the nature of the base opposite cytosine. Unique Reaction Site Selectivity in TMG Sequence. The site selectivity of the strand cleavage observed for ODN(TMG) was quite different from that of the sequences described above (Figures 2 and 3). The ODN(TMG) strand was oxidized at 5-methylcytosine regardless of the nature of the base opposite 5-methylcytosine. In addition, the 5′-neighboring thymine also reacted in spite of the formation of a Watson-Crick base pair with the complementary adenine base. This may be because the accessibility of oxidants to the C5-C6 double bond of the thymine base adjacent to the mismatched base pair is higher compared with a thymine base in a fully matched duplex. ODN(TCG) also showed oxidation of the mismatch-neighboring thymine base, but the efficiency was lower. Oxidation at cytosine was negligible, as well as of other unmethylated cytosine sequences. To learn more about the reactivity of mismatchneighboring thymine, we prepared an artificial methylation sequence GMT, where a thymine base was located at the 3′ side of 5-methylcytosine, and the oxidation site was examined (Supporting Information Figure S2). In the GMT sequence, 5-methylcytosine was predominantly oxidized. Oxidation at the 3′-neighboring thymine was much lower. The site selectivity of oxidation for the GMT sequence is quite different from oxidation at the 5′-neighboring thymine, but this is an expected result because the thymine forms a stable base pair with the complementary adenine. Compared with the oxidation of the
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Figure 5. Osmium complexation of single-stranded ODN(TNG). Reaction time was 15 s. Lane 1, G + A sequencing lane; lane 2, N ) M; lane 3, N ) C.
Figure 3. The reactivity at each base of the duplex 32P-ODN(XNG)/ ODN′(CN′X′). The heights of bars were determined from the ratio of the band intensity at the corresponding base among the total band intensity of each lane.
Figure 4. Relationship between the value of Tm and osmium complexation at 5-methylcytosine. Circles, ODN(GMG) hybridized with ODN′(CN′C); squares, ODN(AMG) hybridized with ODN′(CN′T); triangles, ODN(CMG) hybridized with ODN′(CN′G); crosses, ODN(TMG) hybridized with ODN′(CN′A).
thymine located at the 3′ side of the mismatched 5-methylcytosine, the reactivity of the 5′-neighboring thymine is higher and unique. We have reported that osmium complexation proceeds rapidly at thymines and 5-methylcytosines in a single-stranded DNA, and also that methylated and unmethylated cytosines are clearly distinguished by the oxidation rate (12). In addition, we have shown that enhancement of osmium oxidation is observed for
5′ thymine of TT and TM sequences, whereas it is low at the thymine of the TC sequence (22). In brief, the reactivity at thymine greatly depended on the pyrimidine bases located at the 3′ side. This phenomenon seems to indicate that a methyl group at C5 of the 3′-neighboring pyrimidine base enhanced the reactivity of thymine. The result of osmium oxidation of single-stranded ODN(TNG) is shown in Figure 5. This figure exhibits not only the high reactivity of thymine bases, but also the enhancement of reaction at thymine by a methylated cytosine located at the 3′ side of thymine. The unique reactivity of mismatched ODN(TNG) described above can be explained by these phenomena. Mismatched ODN(TMG) reacted at 5-methylcytosine, but the 5′-neighboring thymine base was also oxidized in spite of the Watson-Crick base pair formation of thymine. The high accessibility of oxidants to thymine is supported by the predominant formation of a (5R,6S)-thymine glycol complex (23) showing that the oxidant approached the target thymine from the 3′ mismatched base pair side. In addition, the fact that a methyl group of 5-methylcytosine enhanced the reactivity of 5′-neighboring thymine would also support the reaction at thymine of mismatched ODN(TMG). On the other hand, the reaction efficiency of ODN(TCG) was lower at thymine and negligible at cytosine. The thymine can be oxidized because of high accessibility from the 3′ mismatched base pair side, but the oxidation efficiency decreased because of lack of enhancement of oxidation ability by the “methyl group effect”. The reason the modification of cytosine with a methyl group enhanced the reactivity of 5′-neighboring thymine is unclear, but the methyl group of a 3′-neighboring 5-methylcytosine base may play an important role in electronic or hydrophobic promotion of oxidant approach or might have worked as a bridgehead for the approach of oxidants to 5′-neighboring thymine (22). The low reactivity of thymine of the GMT sequence can also be explained by the difficulty of approach of oxidants from the 3′ side and loss of the “methyl group effect” by the 3′-neighboring base. ICON Probe. The ICON probe is a conceptually new DNA for sequence-specific short-term methylation analysis supported by a chemical basis (21). The formation of a methylated/ unmethylated cytosine-adenine mismatched base pair in hybridization of the probe and the target DNA is applied for sequence-selective methylation detection because this mismatched base pair is well-known to adopt a wobble-type structure with the cytosine displaced laterally into the major groove and the adenine into the minor groove by hydrogen bonding between the 6-amino group of adenine and the N3 of cytosine (24, 25). Osmium complexation using the probe may also result in oxidation of the thymine base in the TNG sequence. We examined the oxidation site of TNG sequences using ICON probes under the same reaction conditions as described above except for the absence of bipyridine, which
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prior estimation of the reaction abilities of probes and helps in the design of highly methylation sensitive probes. Supporting Information Available: PAGE analysis of osmium oxidation. This material is available free of charge via the Internet at http://pubs.acs.org.
LITERATURE CITED
Figure 6. Osmium oxidation using the ICON probe. (a) The sequence of the duplexes formed by hybridization of 32P-labeled target DNA and ICON probes. (b) The reactivity at each base. Lane 1, G + A sequencing lane for ICON(TMG) and ICON(TCG); lane 2, ICON(TMG); lane 3, ICON(TCG); lane 4, ICON(GMG); lane 5, G + A sequencing lane for ICON(GMG).
was replaced by the ICON probe (Figure 6). For a TMG sequence, oxidation was observed at both thymine and 5-methylcytosine. In contrast, for a TCG sequence, oxidation was negligible at both thymine and cytosine. Because the bipyridine ligand of the ICON probe is fixed near the target 5-methylcytosine base because of the linker length, the reaction efficiency at the thymine neighboring the target is significantly decreased. The reaction at thymine was suppressed in the TCG sequence. However, even though the bipyridine was fixed, oxidation of thymine was still observed for the TMG sequence. Oxidation suppression at the neighboring thymine is due to the combination of three effects: Watson-Crick base pair formation at thymine, small enhancement of thymine reactivity by 3′-neighboring cytosine, and limitation of the reaction site by the bipyridineconnecting linker. As a result, the function of the ICON probes, which discriminate methylated cytosine from unmethylated cytosine using osmium complexation efficiency, is completely maintained, although complexation at the thymine of TMG occurs. There is no problem in quantification of 5-methylcytosine through interstrand cross-linking between the target methylated DNA and the ICON probe. In conclusion, the efficiency of osmium complex formation at 5-methylcytosine in mismatched DNA duplexes is a key point for the design of sequence-specific detection of DNA methylation. We have described the osmium oxidation efficiency at 5-methylcytosines in a series of duplexes with a single base mismatch. The complexation site and efficiency in mismatched duplexes changed depending on the type of 5′-neighboring base of the 5-methylcytosine forming a mismatched base pair. In particular, when the base neighboring the 5′ side of the mismatched base pair was thymine, oxidation at thymine was also observed. The nature of the mismatched base pairs and their neighboring bases influences the reaction site and efficiency. However, the variation of the reaction site does not degrade the characteristic function of ICON probes, in that it captures only methylated DNA although 5′-neighboring thymine may react. The osmium complexation efficiency and selectivity depending on the nature of mismatched base pairs and neighboring sequences is demonstrated. This knowledge facilitates
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