Selective labelling aldehydes in DNA - Analytical Chemistry (ACS

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Selective labelling aldehydes in DNA Chaoxing Liu, Xiaomeng Luo, Yuqi Chen, Fan Wu, Wei Yang, Yafen Wang, Xiong Zhang, Guangrong Zou, and Xiang Zhou Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b04822 • Publication Date (Web): 16 Nov 2018 Downloaded from http://pubs.acs.org on November 16, 2018

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Analytical Chemistry

Selective labelling aldehydes in DNA Chaoxing Liu1, ‡, Xiaomeng Luo1, ‡, Yuqi Chen1, Fan Wu1, Wei Yang1, Yafen Wang1, Xiong Zhang1, Guangrong Zou1, and Xiang Zhou1, * 1

College of Chemistry and Molecular Sciences, Key Laboratory of Biomedical Polymers of Ministry of Education, The Institute for Advanced Studies, Hubei Province Key Laboratory of Allergy and Immunology, Wuhan University, Wuhan, Hubei, 430072, P. R. China. *Corresponding author: Xiang Zhou ([email protected]) ABSTRACT: A naphthalimide hydroxylamine probe has been designed and synthesized to first selective labelling the whole natural aldehydes present in DNAs including 5-formylcytosine, 5-formyluracil and abasic sites. The fluorescence characteristics of the generated nucleosides have been detailedly examined and the reaction activities of hydroxylamine, amine groups towards aldehydes in DNA have been discussed with others which will be a vital reference for designing chemicals for selective labelling DNAs.

Aldehydes are highly important groups for the natural nucleobase modifications in DNA for researches on epigenetics.1 Aldehydes present in the natural nucleobase modifications, 5-formylcysine (5fC),2 5-formyluracil (5fU)3 and abasic sites (AP)4 are of extreme importance. The 5formylpyrimidine including 5fC and 5fU exists in the genome of normal healthy cells, such as mouse embryonic stem cells (mESCs),5 and diseased cells, such as HeLa cells,6 HEK293T cells,7 liver cancer tissues and colorectal cancer tissues.8 5fC is an important epigenetic marker which plays a crucial role in gene regulation,9 cell differentiation,10 and alteration of DNA secondary structure.11 5fC can be generated from the oxidation of 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) via TET enzyme12 or gamma rays.13 In addition, vitamin C (Vc) can directly enhance the catalytic activity of Tet dioxygenases for the oxidation of 5-methylcytosine (5mC), thus elevating 5-formylcytosine.14 And 5fC might produce 5fU via AID/APOBEC enzymes by removing its amino group.16 What’s more, it can also be removed by the action of apurinic/apyrimidinic endonuclease to produce AP. 5fU was once considered an important genetic damage marker bearing strong genotoxicity.20 5fU can be generated from thymidine by ultraviolet radiation,21 gamma-ray damage,6 reactive oxygen attack, Fenton reagent treatment,22 and thymidine-7 hydroxylase oxidation.23 5fU can introduce gene mismatches,24 conjugate with amino acids,25 and alter DNA structure.26 AP can be generated from hydrolysis of glycosidic bonds of canonical bases spontaneously, internal natural nucleobase modifications during base excision repair via Tdg and modified bases caused by external alkylating agents and radiation through base excision repair.27 AP is recognized as a vital damaged base and an intermediate of active demethylation processes in Tdg-mediated pyrimidine modifications excision resulting in genetic mutations, diseases, epigenetic reprogramming and potentially harmful strand break.28,29

discovery of natural nucleobase modification and a better understanding of their functions in epileptics.3 Detection and quantification of aldehydes in DNA play a significant part in studies on causal relationships among natural nucleobase modifications, DNA lesions and epileptics.3 Chemical probes specific for aldehydes in DNA are powerful tools for studying them since probe-mediating detection is convenient, effective and easy-operated. Several ways have emerged as strategies to improve the detection of aldehydes in DNA, including: (1) chemical labeling or adding an ion additive (bicarbonate, etc.) in combination with liquid chromatography coupled to electrospray ionization tandem mass spectrometry analysis,6,8,30-34 (2) fusion or conjugation to chemicals generating fluorescence switch-on phenomenon detection target aldehydes in DNA,13,35-38 (3) conjugating with reagents for enrichment or single-base analysis of them.13,39-44 However, there are no chemicals which have been designed and reported to selectively label all these aldehydes present in DNA. As mentioned above, 5fC and 5fU both serve as substrates for Tdg enzymes and DNA glycosylases in the formation of abasic sites (APs). 5fC, 5fU and AP are all intermediates, involved in the active demethylation of DNA.15,17,26 Though differentiating 5fC, 5fU or AP is important and they are different forms of DNA damage, the effects caused by aldehydes present in DNAs have many similar specialties. The aldehyde groups lead to revisable DNA-protein cross-link (DPC) and contribute to transcriptional regulation and chromatin remodeling. In addition, these same lysine residues in AP-protein cross-link intermediates would cause DNA strand scission at abasic sites (APs).18,19 It is reported that aldehyde groups in DNA would alter the structure and stability of DNA,11,26 lead to DNA strand break at the modification sites and cause DNA damage.28,29 To design and synthesize a compound which can estimate the whole contents of aldehydes in DNA is significant and useful.

The chemicals as selective labelling 5fC, 5fU or AP reagents have expanded in recent years due to the urgent need of

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130.73, 129.73, 127.75, 124.13, 121.96, 120.31, 108.29, 103.49, 74.05, 53.87, 53.39, 43.07, 37.97, 37.47, 22.82 ppm. HRMS C22H28N5O4+ [M+H]+ calculated 426.21358, found 426.21440. ODN reaction protocols. ODNs (100 µM, 1 µL), compound (10 mM in DMSO, 1 µL), NaOAc buffer (1 M, pH=5.0, 5 µL) and 43 µL ddH2O were added together into 1.5 mL microcentrifuge tube at 37°C for 5h in a thermomixer (Eppendorf, 1200 r.p.m.), respectively. Scheme 1. Structure of aldehydes present in the natural nucleobase modifications, from left to right: 5-formylcysine (5fC), 5-formyluracil (5fU) and abasic sites (AP). Herein, we have created a highly reactive aldehydesdetecting probe by introducing hydroxylamine group to a naphthalimide structure (Figure 1). Synthesized probe contains a naphthalimide fluorophore for effective sensing, a pyrrolidine residue for enhancing water-solubility and high chargeability and a hydroxylamine group for high selectivity and great reaction activity with aldehydes in DNA under warm conditions.

Figure 1. Reaction of naphthalimide hydroxylamine probe with 5fC, 5fU and AP.

EXPERIMENTAL SECTION Synthesis of naphthalimide hydroxylamine probe. (NTritylaminooxy) acetic acid was synthesized according to previous report.45 Compound 2 (500 mg, 1.4 mmol), (NTritylaminooxy) acetic acid (566 mg, 1.7 mmol) and HATU (1.07 g, 2.8 mmol) was dissolved in 18 ml DMF at room temperature. Subsequently, a few drops of trimethylamine were added into the mixture. After stirring for 6 h, the solvent was removed in oil vacuo pump and the residue didn’t be separated directly. Then, to a solution of the residue in CH2Cl2 (48 mL), 0.5 M HCl in MeOH (48 mL) was added, and the whole was stirred at room temperature for another 16 h. The mixture was concentrated in vacuo, and the residue was purified by silica gel column chromatography (DCM/MeOH/Et3N =50:1:0.1%) to give 3 (390 mg, 65%) as an orange solid. 1H NMR (400 MHz, CD3OD) δ 8.28 (dd, J = 21.8, 7.8 Hz, 2H), 8.15 (d, J = 8.5 Hz, 1H), 7.47 (t, J = 7.9 Hz, 1H), 6.68 (d, J= 8.6 Hz, 1H), 4.18 (t, J = 7.1 Hz, 2H), 4.05 (s, 2H), 3.55 (t, J = 5.6 Hz, 2H), 3.48 (t, J = 5.7 Hz, 2H), 2.72 (m, J = 25.6, 18.6 Hz, 6H), 1.78 (s, J = 28.7 Hz, 4H). 13C NMR (101 MHz, CD3OD) δ 172.83, 164.73, 164.15, 150.92, 134.40,

Denaturing PAGE analysis. ODNs were in 20 µL 50% deionized formamide. A 20% denaturing PAGE was prepared by using 1xTBE buffer (89 mM Boric acid, 89 mM Tris base, 2 mM EDTA) containing 7.0 M urea. The denaturing PAGE was carried out in 1xTBE buffer at a constant voltage of 350 V for about 2 h at room temperature. We scanned the final PAGE products with Pharos FX Molecular imager operated in the fluorescence mode (λex=488 nm). Then the gel was stained with nucleic acid stains to visualize other DNA bands (λex=532 nm). HPLC analysis of ODNs. ODNs were reacted with hydroxylamine compound respectively using the ODN reaction protocol and then detected by reverse-phase HPLC chromatography (Shimadzu LC-6AD) which equipped with an Inertsil ODS-SP column (5 μm, 250×4.6 mm) (GL Science lnc. Japan) with mobile phase A (100 mM TEAA buffer, pH=7.0) and B (acetonitrile) with a flow rate of 1 mL/min at 35°C (B conc.: 5-5-30%/0-5-30 min for ODN-AP and ODN5fC or 10-10-50-70-100%/0-5-30-35-40 min for ODN-5fU). Quantitative analysis by polyacrylamide gel electrophoresis analysis. A series of gradient concentrations of ODNs after incubation with hydroxylamine compound were purified by Micro Bio-Spin columns (Bio-Rad, USA) and then quantified by 20% denaturing PAGE. We scanned the final PAGE products with Pharos FX Molecular imager operated in the fluorescence mode (λex=488 nm) and calculated the relationship between percentage of gray value and DNA concentration. Detection of calf thymus DNA samples. Calf thymus DNA (1 ug) was made up to 24 uL with NaOH (0.05 M final concentration) on ice, then 1 uL of KRuO4 solution (15 mM in 0.05 M NaOH) was added and the reaction was held on ice for 1 hour, with vortexing every 10 minutes. The reaction was purified by Micro Bio-Spin columns (Bio-Rad, USA). Then the aforementioned purified DNA or the same amount of unoxidized calf thymus DNA were mixed with NaOAc buffer (100 mM, pH 5.0) and the compound (200 uM) respectively and made up to 50 uL aqueous solution. The reaction was performed at 37°C for 5 h in a thermomixer (Eppendorf, 1200 r.p.m.). After the reaction was completed, the mixture was purified with Micro Bio-Spin columns (Bio-Rad, USA) to remove the excess compound. Enzymatic digest of ODNs protocol. DNAs in the presence of Degradase Plus (1 μL) and 10×Degradase Plus reaction buffer (2.5 μL) (Zymo Research) in a final volume of 25 μL was incubated at 37°C for 2 h and digested to its corresponding nucleosides. The digested mixture was filtered by an ultrafiltration tube (3 kDa cutoff, Amicon, Millipore) to remove the enzymes to yield the corresponding nucleosides for further LC-MS detection. Agarose gel electrophoresis. ODNs were in 10 µL 20% 6x


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Analytical Chemistry loading buffer. A 2% agarose gel was prepared by dissolving 1.2 g agarose into 60 ml 1 x TBE buffer containing 5 uL nucleic acid stains. The 2% agarose gel was carried out in 1xTBE buffer at a constant voltage of 120 V for about 1 h at room temperature. We scanned the final agarose gel with Pharos FX Molecular imager operated in the 488 nm and 532 nm excitation model.

RESULTS AND DISCUSSION Synthesis of naphthalimide hydroxylamine and naphthalimide amino probe and their nucleoside adducts. In order to solve this problem, a naphthalimide hydroxylamine probe 3 and naphthalimide amino probe 2 have been designed and synthesized (Figure 2). 5-Formyl-2'-deoxycytidine, 5formyl-2'-deoxyuridine and 2-deoxy-d-ribose can react with naphthalimide hydroxylamine probe to generate fluorescent nucleotides in methanol solution with high yield (Figure S2). However, 2-deoxy-d-ribose only need room temperature stirring, while 5-formylpyrimidine needs p-anisidine and acetic acid as catalysts to speed up chemical reactions. Compared to 5-formyl-2'-deoxyuridine (50°C), the higher temperature (60°C) is helpful to naphthalimide hydroxylamine probe reaction with 5-formyl-2'-deoxycytidine. For naphthalimide amino probe (Figure S1), the satisfactory yield and reaction conditions can also be found during the conjugating with 5formyl-2'-deoxycytidine. However, the reducing agent sodium borohydride is needed to ensure the stability of Schiff base between the formyl group in 5-formyl-2'-deoxyuridine or 2deoxy-d-ribose and amino residue of the probe. Though amino group is also effective to attach with aldehydes, hydroxylamine group is more suitable for using in designing reagents to detect aldehydes present in DNA. And the reactivity difference between naphthalimide hydroxylamine probe to the aldehydes is slight. The warm reaction conditions and high yields are acceptable. N

O O O

Br

N

N

H 2N

O N O

2-Methoxyethanol,60oC

H 2N

NH2

O N O

fluorescence intensity increased slightly in the pH 5.0 acetate buffer. And the fluorescence of the probe in deionized water is linearly related to its concentration in the range of 0-5 uM (Figure S4). In another word, naphthalimide hydroxylamine probe can conjugate with 5fC, 5fU or AP to generate fluorescent nucleosides with large Stokes shift which showed stable UV absorbance spectra and fluorescence spectra even in the various buffers. Furthermore, to estimate the fluorescence characteristic of the fluorescent nucleosides in the different complex environment, we also studied the fluorescence spectra in aqueous solution containing various common metal ions (Ru3+, Pd2+, Ag+, Cd3+, Ba2+, Pt3+, Hg2+, Pb2+, Mg2+, Al3+, Ca2+, Sc3+, V3+, Cr3+, Mn2+, Fe2+, Fe3+, Co2+, Ni2+, Cu2+, Zn2+, Sr3+). To our surprise, the fluorescent signals mostly showed stability. For naphthalimide hydroxylamine and 5fC adduct, Pd2+ quenched its fluorescence, and the response to Mn2+ showed highest fluorescence. The adduct in Other metal ions showed equal fluorescence to the adduct in deionized water (Figure 3a). For naphthalimide hydroxylamine and 5fU adduct, fluorescence intensity decreased during the addition of metal ions, while climbed highest in the deionized water. Most metal ions caused the same influence to it except the Pd2+ completely quenched the whole fluorescence (Figure 3b). On the contrary, for naphthalimide hydroxylamine and AP adduct, fluorescence intensity increased slightly after addition of metal ions. And Pd2+ still quenched its fluorescence (Figure 3c). Moreover, the Pd2+ mediated quenching fluorescence with each generated fluorescent nucleoside might be reversible. Fluorescence recovery phenomenon of naphthalimide hydroxylamine and AP adduct can be obtained with the addition of 10 equiv. of EDTA (Figure 3d), nevertheless, fluorescence of naphthalimide hydroxylamine and 5fC or 5fU adduct failed to completely recovered (Figure S5). This might be used in further modulating biochemistry reactions. Figure 3. Fluorescence emission spectra (λex: 450 nm, λem: 540 nm) of naphthalimide hydroxylamine and 5fC (a), 5fU (b) or AP (c) adduct upon addition of various common metal ions.

2-Methoxyethanol,90oC HN

Br

2

1

NH2

N

N

O N O

O N O +

Tr N O H

O OH

HATU

HCl, CH3OH

DMF,rt

CH2Cl2 ,rt

HN

HN

NH NH2

2

O

O NH

2

3

Figure 2. Synthesis of probe 3. Studies of UV absorbance spectra and fluorescence spectra of generated nucleotides. With the highly reactive probe in hand, we assessed UV absorbance spectra and fluorescence spectra of generated nucleotides in different buffers. The changes of the UV absorbance spectra among naphthalimide hydroxylamine probe and 5fC, 5fU or AP adducts were negligible from pH 5.0 to 8.0 in the whether phosphate buffer, TEAA buffer, MES buffer or acetate buffer. And the maximum UV absorption wavelength was detected as 450 nm (Figure S3). For fluorescence spectra, the peak of fluorescence emission was not shifted which was detected as 540 nm, but

The blank line is fluorescence emission spectrum of the solvent only and the control line is fluorescence emission spectrum of fluorescent adduct without addition of metal ions. (d) The fluorescence quenching signal of the naphthalimide hydroxylamine and AP was reversible upon addition of 10 equiv. of EDTA.



Studies of naphthalimide hydroxylamine probe selective labelling aldehydes in DNAs. First, we tested the reactivity of the hydroxylamine probe against ssDNA (ODN-AP) under different reaction conditions to such as buffer, temperature, reaction time.49 In three groups of experiments, the probe all showed excellent reactivity and almost completely reacted with ODNs (Figure S9). In consideration of the better stability of 5fC and 5fU than AP, a common and more modest reaction condition was determined in order to detect these aldehydemodified nucleotides in DNA at the same time. The best reaction yields (up to nearly 100%) were achieved with experiments performed in NaOAc buffer pH 5.0, at 37°C for 5 h. Then, a 9-mer oligonucleotide (ODN-5fC, bearing one 5fC site) was used as a model test. After incubation with naphthalimide hydroxylamine probe in sodium acetate buffer (pH 5.0) for 5 h, a complete conversion to new product (the 5fC site was reacted with naphthalimide hydroxylamine probe to generate the corresponding fluorescent nucleobase) was recorded by RP-HPLC (monitored at 260 nm) in comparison to the original signal of ODN-5fC (Figure 4a). The other cytosine counterparts, such as ODN-C, ODN-5mC, ODN5hmC (the 5fC site was replaced by C, 5mC or 5hmC) which bear no aldehyde group can’t be labelled by this probe through the denaturing polyacrylamide gel electrophoresis (PAGE) analysis (Figure 4d). Naphthalimide hydroxylamine probe selectively labelled ODN-5fC and generated a band in the 488 nm excitation model (Figure 4d). We can get all bands through 532 nm excitation model after nucleoside staining to ensure the control DNAs exactly exist. And the probe labelled ODNs migrated slowly since its larger molecular weight. What’s more, the MALDI-TOF analysis (Figure S6) confirmed the exact chemical labeling with ODNs bearing 5fC moieties. And enzymatically digested mononucleosides were also analyzed by reverse phase-HPLC and LC-MS to confirm the reaction of 5fC, 5fU and AP respectively (Figure S12, S13, S14). In the same way, the reaction activities towards ODN-5fU and ODN-AP were also studied. The RP-HPLC monitored at 260 nm demonstrated the high yields of naphthalimide hydroxylamine probe labelling ODN-5fU (Figure 4b) and ODN-AP (Figure 4c). The selectivity tests have been done to reveal naphthalimide hydroxylamine probe only reacts with aldehydes present in DNAs. The control DNAs containing methyl, hydroxymethyl groups can’t be labelled by the probe in the same reaction conditions through PAGE analysis (Figure 4e, S10). And the MALDI-TOF analysis is also provided to test and verify the exact chemical labeling with ODNs bearing 5fU moieties (Figure S7) and AP (Figure S8). It revealed naphthalimide hydroxylamine probe had higher selectivity in aldehyde than hydroxymethyl, methyl present in DNAs.

Figure 4. RP-HPLC trace at λ=260 nm of ODN-5fC (a), ODN-5fC (b) and ODN-AP (c) before and after reaction with naphthalimide hydroxylamine probe under optimized conditions. Illustration of strategy for NOT logic gate. (d) Denaturing PAGE analysis of ODN-5fC (lane 4), ODN-C (lane 1), ODN-5mC (lane 2) and ODN-5hmC (lane 3) after incubation with naphthalimide hydroxylamine probe. (e) Denaturing PAGE analysis of ODN-5fU (lane 4), ODN-U (lane 1), ODN-T (lane 2) and ODN-5hmU (lane 3) after incubation with naphthalimide hydroxylamine probe. Qualitative and quantitative analysis of aldehydes in DNAs. To investigate whether the reagent is able to quantity aldehydes directly in DNA, we did a series of quantitative analysis by denaturing PAGE analysis through gradient concentrations of ODNs bearing 5fC, 5fU or AP after incubation with hydroxylamine compound. After being purified by ice ethanol, we then quantified them by 20% denaturing PAGE. The final PAGE products were scanned with Pharos FX Molecular imager operated in the fluorescence mode (λex=488 nm). The relationship between percentage of gray value and DNA concentrations can be calculated. A linear correlation between the concentration of ODNs and fluorescence intensity was confirmed, ranging from 10 pmol to 50 pmol (Figure 5, S11). The quantitative and qualitative assays and the high signal-to-noise ratio (detection limit 10 pM) in detecting aldehydes in DNA demonstrated that this naphthalimide hydroxylamine probe was a useful chemical in this application. To further explore the probe to complex biological samples, we prepared KRuO4-oxidized calf thymus DNA for detecting aldehydes formation. KRuO4 was reported to cause many DNA damages such as oxidizing 5hmC to 5fC, 5hmU to 5fU.44 KRuO4-oxidized calf thymus DNA was treated with the probe together with the control unoxidized DNAs, respectively. It showed that the amount of aldehydes presented in KRuO4-oxidized DNAs had doubled compared to unoxidized samples (Figure S15). Moreover, our synthesized probe was compared to CBAN13 that can selectivity detect 5fC in the previous research (Figure S15). The fluorescence intensity of the hydroxylamine probe labelled DNA showed greater changes than CBAN labelled DNA before and after KRuO4-oxidization, demonstrating the probe acting on three aldehyde-modified nucleobases at the same time.


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Analytical Chemistry

ACKNOWLEDGMENT We thank the National Natural Science Foundation of China (21432008, 91753201 and 21721005).

REFERENCES

Figure 5. Correlation of the Gray Value (fluorescence mode, λex: 488 nm) of various DNA concentration ODN-5fC (a), ODN-5fU (b) or ODN-AP (c) after incubation with naphthalimide hydroxylamine probe.

CONCLUSION The reaction activities of aldehydes presented in DNA were studied and the hydroxylamine group of the naphthalimide hydroxylamine probe has been compared with the amino group. We found the naphthalimide hydroxylamine probe can selectively label aldehydes presented in DNA in contrast with hydroxymethyl, methyl groups. The precious reports of the hydroxylamine probes selectively tagging only 5fC,43,46 5fU47,48 or AP4 might because of the specific severe reaction conditions or didn’t concern about the whole aldehydes presented in DNA. We found hydroxylamine group can react with 5fC, 5fU and AP without much difference which is in reasonable agreement with the researches by Balasubramanian S.44 The hydrazine35,36 and o-phenylenediamine39,44 groups are more suitable in the application of being designed for selective labelling certain aldehydes presented in DNAs. The hydroxylamine groups bear the great reaction activities to label all aldehydes presented in DNAs and other hydroxymethyl, methyl groups can’t react with them. Our researches revealed hydroxylamine groups can effectively conjugate with all aldehydes presented in DNAs and compare the reaction activities of hydroxylamine, amine with 5fC, 5fU and AP which can be a basic reference for designing chemicals for selective labelling 5fC, 5fU and AP. What’s more the fluorescence characteristics of the generated nucleosides have been detailedly examined which bear the potential applications in biochemistry, analytical chemistry and cell imaging.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Synthesis and characterization data, DNA MALDI-TOF mass spectra, UV absorption spectra and fluorescence emission spectra, PAGE analysis (file type, i.e., PDF).

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]

Author Contributions ‡These authors contributed equally.

Notes The authors declare no competing financial interest

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