Citation: Qian Wang, Jiang-Hui Ding, Jun Xiong, Yang Feng, Bi-Feng Yuan, Yu-Qi Feng. Site-specific quantification of 5-carboxylcytosine in DNA by chemical conversion coupled with ligation-based PCR[J]. Chinese Chemical Letters, ;2021, 32(11): 3426-3430. doi: 10.1016/j.cclet.2021.05.020 shu

Site-specific quantification of 5-carboxylcytosine in DNA by chemical conversion coupled with ligation-based PCR

Qian Wang ^{a, 1} ,
Jiang-Hui Ding ^{a, 1} ,
Jun Xiong ^a ,
Yang Feng ^a ,
Bi-Feng Yuan ^{a, b, *,} ,
Yu-Qi Feng ^{a, b}

a.
Sauvage Center for Molecular Sciences, Department of Chemistry, Wuhan University, Wuhan 430072, China
b.
School of Health Sciences, Wuhan University, Wuhan 430071, China

bfyuan@whu.edu.cn

Received Date: 21 March 2021
Revised Date: 1 April 2021
Accepted Date: 12 May 2021
Available Online: 23 May 2021

Figures(4)

5-Methylcytosine (5mC) is the most important epigenetic modification in mammals. The active DNA demethylation could be achieved through the ten-eleven translocation (TET) protein-mediated oxidization of 5mC with the generation of 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). It has been known that 5mC, 5hmC and 5fC play critical roles in modulating gene expression. However, unlike the 5mC, 5hmC, and 5fC, the functions of 5caC are still underexplored. Investigation of the functions of 5caC relies on the accurate quantification and localization analysis of 5caC in DNA. In the current study, we developed a method by chemical conversion in conjugation with ligation-based real-time quantitative PCR (qPCR) for the site-specific quantification of 5caC in DNA. This method depends on the selective conversion of 5caC to form dihydrouracil (DHU) by pyridine borane treatment. DHU behaves like thymine and pairs with adenine (DHU-A). Thus, the chemical conversion by pyridine borane leads to the transformation of base paring from 5caC-G to DHU-A, which is utilized to achieve the site-specific detection and quantification of 5caC in DNA. As a proof-of-concept, the developed method was successfully applied in the site-specific quantification of 5caC in synthesized DNA spiked in complex biological samples. The method is rapid, straightforward and cost-effective, and shows promising in promoting the investigation of the functional roles of 5caC in future study.
- 5-Methylcytosine,
- 5-Carboxylcytidine,
- Dihydrouracil,
- Mass spectrometry,
- PCR,
- Chemical conversion
1. [1]
  C. Luo, P. Hajkova, J.R. Ecker, Science 361(2018) 1336-1340. doi: 10.1126/science.aat6806
2. [2]
  T. Liu, C.J. Ma, B.F. Yuan, Y.Q. Feng, Sci. China Chem. 61(2018) 381-392. doi: 10.1007/s11426-017-9186-y
3. [3]
  F. Spada, S. Schiffers, A. Kirchner, et al., Nat. Chem. Biol. 16(2020) 1411-1419. doi: 10.1038/s41589-020-0621-y
4. [4]
  B.F. Yuan, Chem. Res. Toxicol. 33(2020) 695-708. doi: 10.1021/acs.chemrestox.9b00372
5. [5]
  X. Wu, Y. Zhang, Nat. Rev. Genet. 18(2017) 517-534.
6. [6]
  Y. Feng, N.B. Xie, W.B. Tao, et al., CCS Chem 2(2020) 994-1008.
7. [7]
  T. Carell, M.Q. Kurz, M. Muller, M. Rossa, F. Spada, Angew. Chem. Int. Ed. 57(2018) 4296-4312. doi: 10.1002/anie.201708228
8. [8]
  Q.Y. Li, N.B. Xie, J. Xiong, B.F. Yuan, Y.Q. Feng, Anal. Chem. 90(2018) 14622-14628. doi: 10.1021/acs.analchem.8b04833
9. [9]
  C.G. Spruijt, F. Gnerlich, A.H. Smits, et al., Cell 152(2013) 1146-1159. doi: 10.1016/j.cell.2013.02.004
10. [10]
  J. Xiong, B.F. Yuan, Y.Q. Feng, Chem. Res. Toxicol. 32(2019) 808-819. doi: 10.1021/acs.chemrestox.9b00042
11. [11]
  X. Lu, D. Han, B.S. Zhao, et al., Cell Res 25(2015) 386-389. doi: 10.1038/cr.2015.5
12. [12]
  D. Zhou, B.M. Alver, S. Li, et al., Genome Biol 19(2018) 43. doi: 10.1186/s13059-018-1420-6
13. [13]
  T.M. Storebjerg, S.H. Strand, S. Hoyer, et al., Clin. Epigenetics 10(2018) 105. doi: 10.1186/s13148-018-0540-x
14. [14]
  Q.Y. Cheng, J. Xiong, C.J. Ma, et al., Chem. Sci. 11(2020) 1878-1891. doi: 10.1039/C9SC05094A
15. [15]
  M.D. Lan, B.F. Yuan, Y.Q. Feng, Chin. Chem. Lett. 30(2019) 1-6. doi: 10.1016/j.cclet.2018.04.021
16. [16]
  C.B. Qi, J.H. Ding, B.F. Yuan, Y.Q. Feng, Chin. Chem. Lett. 30(2019) 1618-1626. doi: 10.1016/j.cclet.2019.02.005
17. [17]
  C.B. Qi, H.P. Jiang, J. Xiong, B.F. Yuan, Y.Q. Feng, Chin. Chem. Lett. 30(2019) 553-557. doi: 10.1016/j.cclet.2018.11.029
18. [18]
  X.J. You, T. Liu, C.J. Ma, et al., Anal. Chem. 91(2019) 10477-10483. doi: 10.1021/acs.analchem.9b01318
19. [19]
  J. Xiong, T.T. Ye, C.J. Ma, et al., Nucleic Acids Res 47(2019) 1268-1277. doi: 10.1093/nar/gky1218
20. [20]
  B. Chen, J. Xiong, J.H. Ding, B.F. Yuan, Y.Q. Feng, Chem. Res. Toxicol. 32(2019) 2078-2085. doi: 10.1021/acs.chemrestox.9b00249
21. [21]
  Q.Y. Cheng, J. Xiong, F. Wang, B.F. Yuan, Y.Q. Feng, Chin. Chem. Lett. 29(2018) 115-118. doi: 10.1016/j.cclet.2017.06.009
22. [22]
  Y. Tang, S.J. Zheng, C.B. Qi, Y.Q. Feng, B.F. Yuan, Anal. Chem. 87(2015) 3445-3452. doi: 10.1021/ac504786r
23. [23]
  Y. Tang, J. Xiong, H.P. Jiang, et al., Ana. l Chem 86(2014) 7764-7772. doi: 10.1021/ac5016886
24. [24]
  X. Lu, C.X. Song, K. Szulwach, et al., J. Am. Chem. Soc. 135(2013) 9315-9317. doi: 10.1021/ja4044856
25. [25]
  K. Tanaka, A. Okamoto, Bioorg. Med. Chem. Lett. 17(2007) 1912-1915. doi: 10.1016/j.bmcl.2007.01.040
26. [26]
  M. Saikia, Q. Dai, W.A. Decatur, et al., RNA 12(2006) 2025-2033. doi: 10.1261/rna.208906
27. [27]
  F. Tang, S. Liu, Q.Y. Li, et al., Chem. Sci. 10(2019) 4272-4281. doi: 10.1039/C8SC04946G
28. [28]
  B. Chen, B.F. Yuan, Y.Q. Feng, Anal. Chem. 91(2019) 743-756. doi: 10.1021/acs.analchem.8b04078
29. [29]
  Y. Xiao, Y. Wang, Q. Tang, et al., Angew. Chem. Int. Ed. 57(2018) 15995-16000. doi: 10.1002/anie.201807942
30. [30]
  W. Liu, J. Yan, Z. Zhang, et al., Chem. Sci. 9(2018) 3354-3359. doi: 10.1039/C7SC05233B
31. [31]
  J.H. Ding, C.J. Ma, M.Y. Chen, et al., Anal. Chem. 92(2020) 2612-2619. doi: 10.1021/acs.analchem.9b04454
32. [32]
  Y. Liu, P. Siejka-Zielinska, G. Velikova, et al., Nat. Biotechnol. 37(2019) 424-429. doi: 10.1038/s41587-019-0041-2
33. [33]
  C.X. Song, K.E. Szulwach, Q. Dai, et al., Cell 153(2013) 678-691. doi: 10.1016/j.cell.2013.04.001
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9. [9]
  Chenghe Yang , Yi Lü , Rui Liu . The Rise to Fame of Digital PCR. University Chemistry, 2025, 40(4): 340-345. doi: 10.12461/PKU.DXHX202406111
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16. [16]
  . . University Chemistry, 2024, 39(5): 0-0.
17. [17]
  . 第41卷第5期封面和目次. Acta Physico-Chimica Sinica, 2025, 41(5): -.
18. [18]
  Youlin SI , Shuquan SUN , Junsong YANG , Zijun BIE , Yan CHEN , Li LUO . Synthesis and adsorption properties of Zn(Ⅱ) metal-organic framework based on 3, 3', 5, 5'-tetraimidazolyl biphenyl ligands. Chinese Journal of Inorganic Chemistry, 2024, 40(9): 1755-1762. doi: 10.11862/CJIC.20240061
19. [19]
  Zhen Liu , Zhi-Yuan Ren , Chen Yang , Xiangyi Shao , Li Chen , Xin Li . Asymmetric alkenylation reaction of benzoxazinones with diarylethylenes catalyzed by B(C₆F₅)₃/chiral phosphoric acid. Chinese Chemical Letters, 2024, 35(5): 108939-. doi: 10.1016/j.cclet.2023.108939
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