N6-methylation assessment in E. coli 23s rRNA utilizing a bulge loop

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N6-methylation assessment in E. coli 23s rRNA utilizing a bulge loop in an RNA-DNA hybrid Kyoko Yoshioka, and Ryoji Kurita Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b01223 • Publication Date (Web): 30 May 2018 Downloaded from http://pubs.acs.org on May 30, 2018

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Analytical Chemistry

TITLE N6-methylation assessment in E. coli 23S rRNA utilizing a bulge loop in an RNA-DNA hybrid

AUTHORS Kyoko Yoshioka and Ryoji Kurita*

National Institute of Advanced Industrial Science and Technology (AIST) and DAILAB, Tsukuba Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki, 305-8566, Japan

*Corresponding Author Phone: +81-29-861-6158 E-mail: [email protected]

1 ACS Paragon Plus Environment

Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ABSTRACT

We propose the sequence selective assay of N6-methyl-adenosine (m6A) in RNA without a PCR or reverse transcription, by employing a hybridization assay with a DNA probe designed to form a bulge loop at the position of a target modified nucleotide. The m6A in the bulge in the RNA-DNA hybrid was assumed to be sufficiently mobile to be selectively recognized by anti-m6A antibody with a high affinity. By employing a surface plasmon resonance measurement or using a microtiter plate immunoassay method, a specific m6A in E. coli 23S rRNA sequence could be detected at a nano-molar level, when synthesized and purified oligo RNA fragments were used for measurement. We have successfully achieved the first selective detection of a specific m6A2030 in 23S rRNA from real samples of E. coli total RNA by using our immunochemical approach.


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INTRODUCTION

Over the last 50 years, more than a hundred varieties of RNA nucleobase modifications have been identified and their biological functions studied.1,2 Modifications in ribosomal RNA (rRNA) and transfer RNA play a homeostatic role in quality control in ribosome assembly and accuracy of gene translation, respectively.3,4 The most common and abundant modifications, especially in messenger RNA and non-coding RNA are made of N6-methyl adenosine (m6A). However, their biological significance had long remained unelucidated. In recent years, a combination of techniques, namely immunoprecipitation with m6A antibody and high-throughput sequencing, has provided a transcriptome profile of m6A.5,6 The dynamic metabolism of mRNA modifications is known to be related to the epigenetic regulation of gene expression. In addition, enzymes in the m6A metabolism, that is, m6A demethylases (FTO and ALKBH5) and a m6A methyltransferase (METTL3), were found to play crucial roles in several diseases including obesity, diabetes, and leukemia.7—15 Therefore, the N6-methylation of adenosine in RNA now constitutes an epigenetic biomarker. The m6A analytical methods mentioned above exhibit a high-throughput performance, but only give general information about the amount and distribution of m6A in an RNA sample. When a specific nucleotide modification in an RNA sequence is revealed to be an epigenetic marker, a sequence-selective analysis of the target at a single-nucleotide resolution becomes necessary. However, there are several difficulties as regards the analysis of an RNA modification. For example, the N6-methylation of adenosine does not affect base-pairing ability. Furthermore, with a reverse transcription of RNA into a complementary DNA, information about the modified nucleotide is lost. Only a few analytical methods are known.16—18 For example, Liu et al.17 developed a method that directly determines the methylation site of adenosine and its methylation ratio in a specific RNA sequence at a single nucleotide resolution. This approach involves site-specific cleavage and radioactive-labeling followed by ligation-assisted



extraction and thin-layer chromatography (the SCARLET method). However, this method has an experimental procedure that includes many complicated steps and requires radioactive (32P) labelling. Golovina et al.18 determined the presence of m6A in a specific RNA sequence with the high-resolution melting (HRM) method using a difference between the melting temperatures of hybrids of a probe and sample RNA. However, this technique appears to need a high concentration (0.4 µM) RNA sample and is time consuming. Therefore, a simpler method for detecting a specific m6A must be developed. In our previous reports,19—21 we proposed a sequence-selective DNA methylation assay technique with an anti-methylcytosine antibody using bulge specific immuno-recognition. A target methylcytosine in a bulge in a duplex was specifically recognized by an antibody. Sequence selectivity was allowed by hybridization with a complementary sequence consisting in a bulge-inducing DNA probe. In this study, we have adopted this concept to the immuno-detection of RNA modification. The target modified nucleotide is a specific m6A related to antibiotics in the E. coli 23S rRNA sequence. We confirmed the availability of this immuno-detection method for RNA modification by performing a surface plasmon resonance measurement or using a microtiter plate.


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EXPERIMENTAL SECTION

Analyte RNA and probe DNA designs. The analyte RNA and biotinylated DNA probe oligonucleotide were synthesized and purified with high-performance liquid chromatography (Gene Design Inc.). The sequences of the analyte RNA oligonucleotide (27-mer) and DNA probes are shown in Table 1. The target N6-methylated adenosine is m6A2058 in E. coli 23S ribosomal RNA (rRNA). For comparison, unmethylated RNA oligonucleotide was also synthesized. The DNA probes were 5’-biotinylated and had a five-thymidine linker. The one bulge inducing DNA probe lacked a thymidine nucleotide, which would make a base-pair with the target m6A. A full-match DNA probe was also synthesized.

Affinity analysis using surface plasmon resonance. An RNA oligonucleotide analyte and a biotinylated DNA probe were mixed in Dulbecco’s phosphate buffered saline (PBS, Sigma-Aldrich). The mixed solution was heated at 95 °C for 5 min, and then gradually cooled to room temperature for hybridization. Surface plasmon resonance (SPR) measurements were performed with a Biacore system (BIACORE T100 and T200, GE Healthcare). A streptavidin-coated sensor chip (SA chip) was immobilized with biotinylated RNA-DNA duplexes (10 nM in PBS) for 30 min at a flow rate of 10 µL/min. The running buffer was PBST (containing 0.05 (v/v) % Tween 20). Anti-m6A antibody in PBST was injected for 5 min, and then the flow was changed in the running buffer for 3 min. The sensor surface was regenerated by the injection of 50 mM Gly-NaOH solution (pH 10.6) for 30 s at a flow rate of 60 µL/min. Bound antibodies were confirmed to be dissociated from the duplexes by a regeneration step while maintaining the hybridization of the DNA-RNA duplex.

Immunoassay of m6A on a microtiter plate



Fifty µL of the hybridized RNA-DNA duplex solution was added to a streptavidin-coated microtiter plate (Sumitomo Bakelite) and incubated for 30 min to immobilize the duplex via an avidin-biotin interaction. Each well was rinsed three times with 300 µL of washing buffer (PBST) with a plate washer (Biorad, model 1575), and then 50 µL of 500 ng/mL anti-m6A antibody (abcam, ab190886) in PBST was added and incubated for 30 min at 37 °C. Each well was rinsed with PBST, and then 50 µL of 500 ng/mL horseradish peroxidase (HRP)-labeled secondary antibody (donkey anti-rabbit IgG, ab6802 by abcam) was added and incubated for 30 min at 37 °C. Each well was rinsed and incubated with 50 µL of 3, 3’, 5, and 5’-tetramethyl benzidine (TMB, BETHYL Laboratories Inc.) for 10 min at room temperature. Finally, the absorbance of each well was read at 450 nm with a microplate reader (Biorad, model 680) once the HRP reaction had been stopped with 50 µL of 1 N HCl.

RNA preparation for m6A methylation assessment A real total RNA sample from E. coli cells (DH5) were used for m6A methylation assessment in this study. E. coli cells (DH5) were cultured in Luria-Bertani broth (DAIGO, Wako Pure Chemical Industries, Ltd.) at 37 °C with reciprocal shaking at 180 rpm. Total RNA from E. coli cells (DH5) was purified with a commercial purification kit (RNeasy Mini Kit, QIAGEN). A purified E. coli total RNA was analyzed for its purity by electrophoresis with an Agilent 2100 Bioanalyzer in accordance with the supplier’s protocols. The RNA concentration was determined by measuring the absorbance at 260 nm with NanoDrop 1000 (Thermo Scientific). Commercially available total RNA (Ambion, AM7940) from E. coli cells (DH5α) was also purchased from Applied Biosystems for comparison. The RNA had already been purified by the supplier and was ready for any application. Before the assessment of m6A methylation, two total RNAs were fragmented with the RNA restriction enzyme (mRNA Interferase MazF, Takara Bio Inc.). It exhibits endoribonuclease activity, which specifically cleaves single-stranded RNA at the 5’end of the ACA sequence. A denatured (for 2 min


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at 70 °C) total RNA (100 nM) was incubated with 10 units of MazF for 15 min at 37 °C. After the reaction, salt and enzyme were removed from the reaction mixture with a purification kit (RNeasy MinElute Clean up Kit, QIAGEN). RNA fragmentation was analyzed with a bioanalyzer.



RESULTS AND DISCUSSION

Immuno-recognition of m6A in a bulge loop of RNA-DNA duplex. Our first target nucleobase in this experiment was m6A2058 in an E. coli 23S ribosomal RNA (rRNA) sequence. The N6-methylation of this adenosine has been well studied, and it was revealed that it is related to the antibiotics resistance of E. coli and an escape from natural immunity system of their host.22—27 The oligo RNAs consisting of A2042—U2068 in E. coli 23S rRNA sequence were synthesized. We designed a DNA probe sequence that lacked a pairing nucleotide with the target m6A2058, resulting in a one-bulge loop formation at the position and had a biotin group and 5-T linker in its 5’-terminus (Table 1). The hybridized biotinylated DNA-RNA duplexes were immobilized onto the surface of a streptavidin modified sensor chip (SA chip for Biacore) or a streptavidin coated microtiter plate. Next, anti-m6A antibody recognition was monitored with SPR responses or HRP-IgG secondary antibody binding. Figure 1 shows the amounts of antibody binding to the DNA-RNA hybrids. We found that only m6A in the bulge loop was recognized by an antibody and that a full match pairing disturbed antibody binding. We confirmed that there was no cross reactivity to unmethylated adenosine of the anti-m6A antibody. The same results were obtained from the SPR measurements (fig. S1).

It is well known that a complementary

hybrid of RNA and DNA adopts an almost A-form structure.28, 29 We have previously reported specific recognition by an antibody for a methylcytosine in a bulge loop of a DNA-DNA duplex, which has a B-form structure.19—21 In this study, we have confirmed for the first time the applicability of our detection principle consisting of specific recognition by an antibody to a nucleotide in a bulge loop in an A-form RNA-DNA duplex. We mixed solutions of the m6A RNA-DNA probe duplex and the unmethylated A RNA-DNA probe duplex to obtain various methylation ratios at a target adenosine, and then immobilized them on streptavidin coated sensor surfaces


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with various m6A concentrations. Figure 2 shows SPR sensorgrams of m6A antibody binding on a 50% methylated m6A surface. After 300 sec of antibody binding, the flow solution changed to running buffer, and then the antibody had dissociated. By adopting a Langmuir binding model (1:1 binding) of curve fitting to the adsorption-dissociation curves, we were able to calculate the kinetic parameters (kinetic rate constants (ka and kd) and dissociation constant (KD)) of antibody binding. The sensorgrams were well fitted to a 1:1 binding model and the affinity between m6A in a bulge loop and an antibody was found to be sufficiently high (dissociation constant value; KD= ~1 x 10-8 (M)) as shown in Table 2 (the SPR sensorgrams for 5% m6A and 20% m6A surfaces are presented in fig. S2.). In our previous studies19—21 regarding the immuno-detection of methylated cytosine in DNA, we achieved the sequence-selective detection of a specific m5C using bulge inducing DNA probes in a DNA-DNA duplex. We showed that a nucleobase in a bulge loop had sufficient mobility to be recognized by its antibody. On the other hand, a nucleobase pairing with its partner made a stacking structure with neighboring nucleobases and lost its mobility. We adopted the same explanation for the specific detection of m6A in RNA-DNA hybrids in this stud. Namely, an m6A in a bulge loop could rotate freely in the RNA-DNA hybrids, and binding by an antibody was made easy. We also found that RNA-DNA hybrids (27 base pairs) with one mismatch (one bulge) maintained a rather stable structure during the experimental steps (immobilization, washing, and immuno-recognition).

Immuno-detection of m6A on a microtiter plate Figure 3 shows an m6A calibration curve obtained with our immunoassay. From the calibration curve, the detection range was 0.5—5 nM and the calculated detection limit was 0.5 nM of m6A oligo RNA (at noise/sensitivity = 3). Generally, one eukaryote cell is known to contain 4 × 106—5 × 106 rRNA molecules. For example, to prepare a 1 mL sample of 0.5 nM rRNA, ca. 7.5 x 104 cells are required. Therefore, our



immunoassay method makes it possible to confirm whether or not a specific nucleotide in rRNA is modified without any further RNA amplification, when 105 cells order of sample are available for RNA purification. The number of eukaryote cells obtained with semi-confluent growth in a cell culture dish (diameter = 100 mm) is ~ 106 or more. Therefore, our immunoassay method has sufficient sensitivity to detect one specific modified nucleotide in an RNA sample purified from a cell culture in one dish.

Methylation ratio determination of a specific adenosine in RNA sequence. Figure 4 shows the amounts of anti-m6A antibody bound in relation to m6A concentrations modified on sensor surfaces assessed with microtiter plate immuno-detection. Mixed solutions of biotinylated RNA-DNA probe hybrids (total RNA concentration = 10 nM) in various m6A ratios were applied to the sensor surfaces. Linear relationships were obtained between the m6A ratios and antibody binding. In the microtiter plate immunoassay, the detection range was 5 – 50% m6A and the color formation of TMB by an HRP reaction was almost saturated at a 100% m6A surface with our assay protocols in this study. These results indicated that 5% m6A in mixed oligo RNAs (10 nM) could be detected by both immunoassays (with SPR and a microtiter plate) using a one-bulge inducing DNA probe.

Immuno-detection of a specific m6A from E. coli real RNA samples. Next, we attempted the immuno-detection of m6A in real E. coli 23S rRNA. The probe DNA in Table 1 is for m6A2058 determination in 23S rRNA, which is related to bacterial resistance to macrolide antibiotics. However, A2058 is not methylated in wild E. coli. Therefore, in this paper, we demonstrated the detection of A2030 in 23S rRNA since this adenosine is confirmed to be completely methylated in the wild-type E. coli strain.30 Probe DNA for the selective detection of m6A2030 was simply designed by eliminating thymidine nucleotide, which would make a base-pair with the target


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m6A2030. The sequences of the newly synthesized biotinylated DNA probes were as follows; the full-match probe: biotin-5’-tttttgtacactgcatcttcacagcgagttca-3’, and the one-bulge inducing probe: biotin-5’-tttttgtacactgcatct_cacagcgagttca-3’. Schematic images of hybrids between the new DNA probes and E. coli rRNA samples with/without MazF treatment (MazF(+) or MazF(–)), and the recognition of m6A by the antibody are presented in 5 (a). We obtained purified RNA samples from a culture of E. coli DH5 strain, which is known to be a wild-type strain and not resistant to antibiotics. From 0.5 mL of overnight culture, ca. 10 µg of RNA was obtained and the purity of the sample was comparable with that of commercially available RNA (Figure S3). We tried to clarify the methylation state of target A2030 in 23S rRNA of a real sample of E. coli RNA with our immuno-detection method. Figure 5 (b) shows the results for m6A2030 detection using the new DNA probes. By hybridization with the one-bulge inducing DNA probe (10 nM), m6A2030 in an MazF fragment and an intact RNA (5 ng µL-1) was recognized by an anti-m6A antibody. In contrast, with the full-match DNA probe, the amount of detected m6A was almost the same as the background level. From the absorbance of MazF treated fragments at 450 nm, ca. 2 nM m6A was detected with the one-bulge inducing DNA probe. The size of 23S rRNA is 2904nt, and so the molecular weight of 23S rRNA is approximately 930891. From an electropherogram of total RNA, 40 % of the total RNA is calculated to be 23S rRNA. From a rough estimation of the molecular weight of rRNA, 5 ng µL-1 RNA equals 2.2 nM 23S rRNA. From these estimations, we could conclude that single m6A2030 was selectively detected by our method. With the full-match DNA probe and an intact RNA (without MazF treatment), a few m6As were positively detected. This was because other m6A, for example, m6A1618 in 23S rRNA, should be recognized by an antibody shown in fig. 5(a). The signals for intact rRNA (MazF(-) samples) were smaller than that for the MazF(+)-bulge although a long rRNA was captured. This is because parts of the ssRNA would form a 3D structure, and the antibody binding for m6A was hindered. To elucidate the detection specificity of our method, we also performed a control



experiment for a non-methylated adenosine target (A2058 in 23S rRNA). The result is shown in fig. S4. The difference between the signals of the one-bulge inducing probe and the full-match probe is clearly small compared with that in fig. 5(b). This suggests that our assay selectively detect the methylation status of the target adenosine. By MazF fragmentation and probe DNA hybridization, we have successfully detected a specific m6A in a real E. coli RNA sample.


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CONCLUSIONS

In this report, we described the sequence selective immuno-detection of an m6A in RNA by using a DNA probe designed to induce a one-bulge loop in RNA-DNA duplexes. We assumed that an m6A in a bulge loop could be easily recognized with a high affinity by an antibody because of its free mobility in a duplex. We confirmed that an m6A that made a full-match base pairing with T was not immuno-detected. With this immuno-detection method, 5% of m6A in a mixed sample with unmethylated A (10 nM oligo RNA) could be detected by SPR measurement or by color formation with HRP activity. The detection limit of an m6A in a specific RNA sequence was 0.5 nM. With our immunoassay method, we have succeeded in the selective detection of a specific m6A in 23S rRNA from E. coli total RNA real samples.



ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI:

Additional information; the SPR sensorgrams of an anti-m6A antibody binding to RNA-DNA probe hybrids (m6A-1 bulge, A-1 bulge, and m6A-full match) (fig. S1) and to the sensor surfaces of various m6A concentrations (5% and 20% m6A) (fig. S2), the electropherograms of RNA fragments after digestion by a MazF restriction enzyme (fig. S3), and the results of m6A detection from a purified E. coli RNA sequence with the DNA probe targeting A2058 in 23S rRNA (fig. S4).


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ACKNOWLEDGMENTS This study was financially supported by JSPS KAKENHI, Grant No. 26410168. We thank Mr. D. Meacock for revising the language of the manuscript.



FIGURE CAPTIONS

Figure 1. Bulge-selective recognition of m6A in RNA-DNA duplex by an anti-m6A antibody.

Figure 2. SPR sensorgrams of antibody (1—10 nM) binding to an m6A mixed (50% m6A) surface and kinetics parameters of antibody binding affinity.

Figure 3. Calibration curve for m6A in a bulge on a microtiter plate.

Figure 4. Variations in amount of anti m6A antibody estimated on a micro-tilter plate when varying the methylation ratio of adenosine in target RNA. Concentration of oligo RNA mixture was 10 nM.

Figure 5. (a) Schematic images of hybrids of E. coli RNAs (MazF(+) or MazF(–)) and the DNA probes (full match or bulge inducing).

(b) M6A detection in E. coli real

RNA sample (MazF(–)) or digested RNA fragments (MazF(+)) with the bulge inducing DNA probe targeting m6A2030 and the full-match DNA probe. The RNA concentration was 5 ng mL-1. The DNA probe concentration was 10 nM. Absorbance data at 450 nm is presented after the subtraction of a result without RNA.


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Page 20 of 28

Table 1. Target m6A sequence and DNA probe design.

RNA-m6A

5’-ACCCGCGGCAAGACGGAAAGACCCCGU-3’

RNA-A

5’-ACCCGCGGCAAGACGGAAAGACCCCGU-3’

probe DNA-full match

biotin_5’-tttttacggggtctttccgtcttgccgcgggt-3’

probe DNA-1 bulge

biotin_5’-tttttacggggtctt_ccgtcttgccgcgggt-3’


A=m6A

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Figure 1



Figure 2


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Table 2. Kinetics parameters of antibody binding affinity estimated from Figure 2.

m6A (%)

ka (1/Ms)

kd (1/s)

KD (M)

5

5.81 x 105

0.92 x 10-2

1.59 x 10-8

20

1.21 x 106

1.63 x 10-2

1.35 x 10-8

50

2.45 x 106

2.00 x 10-2

8.17 x 10-9



Figure 3


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Figure 4.



Figure 5 (a)


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Figure 5 (b)



for TOC only


Page 28 of 28

N6-methylation assessment in E. coli 23s rRNA utilizing a bulge loop

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