In-situ hydrophobic environment triggering reactive fluorescence probe to real-time monitor mitochondrial DNA damage

Abstract

Mitochondrial DNA has a special structure that is prone to damage resulting in many serious diseases, such as genetic diseases and cancers. Therefore, the rapid and specific monitoring of mitochondrial DNA damage is urgently needed for biological recognition. Herein, we constructed an in situ hydrophobic environment-triggering reactive fluorescence probe named MBI-CN. The fluorophore was 2-styrene-1H-benzo[d]imidazole, and malononitrile was introduced as a core into a molecule to initiate the hydrolysis reaction in the specific environment containing damaged mitochondrial DNA. In this design, MBI-CN conjugates to mitochondrial DNA without causing additional damages. Thus, MBI-CN can be hydrolyzed to generate MBI-CHO in an in situ hydrophobic environment with mitochondrial DNA damage. Meanwhile, MBI-CHO immediately emitted a significative fluorescence signal changes at 437 and 553 nm within 25 s for the damaged mitochondria DNA. Give that the specific and rapid response of MBI-CN does not cause additional damages to mitochondrial DNA, it is a potentially effective detection tool for the real-time monitoring of mitochondrial DNA damage during cell apoptosis and initial assessment of cell apoptosis.

Graphical abstract

Keywords

hydrolysis reaction / mitochondrial DNA damage / in situ hydrophobic environment trigger / fluorescence probe / apoptosis

References

[1] Kuchlyan J, Martinez-Fernandez L, Mori M, Gavvala K, Ciaco S, Boudier C, Richert C, Didier P, Tor Y, Improta R, Mély Y. What makes thienoguanosine an outstanding fluorescent DNA probe. Journal of the American Chemical Society, 2020, 142(40): 16999–17014

[2] Gao F L, Li L J, Fan J L, Cao J F, Li Y Q, Chen L Y, Peng X J. An off-on two-photon carbazole-based fluorescent probe: highly targeting and super-resolution imaging of mtDNA. Analytical Chemistry, 2019, 91(5): 3336–3341

[3] Yakes F M, Van Houten B. Mitochondrial DNA damage is more extensive and persists longer than nuclear DNA damage in human cells following oxidative stress. Proceedings of the National Academy of Sciences of the United States of America, 1997, 94(2): 514–519

[4] Vladimir B S, Evgeny E, Natalia A, Maria A A, Sergey I N, Georgii A B, Igor A, Shamil R S. Error-prone bypass of DNA lesions during lagging-strand replication is a common source of germline and cancer mutations. Nature Genetics, 2019, 51(1): 36–41

[5] Anderson S, Bankier A T, Barrell B G, De Bruijn M H, Coulson A R, Drouin J, Eperon I C, Nierlich D P, Roe B A, Sanger F, Schreier P H, Smith A J H, Staden R, Young I G. Sequence and organization of the human mitochondrial genome. Nature, 1981, 290(5806): 457–465

[6] Bibb M J, Van Etten R A, Wright C T, Walberg M W, Clayton D A. Sequence and gene organization of mouse mitochondrial DNA. Cell, 1981, 26(2): 167–180

[7] Assaf C B, Maayan R, Yifat S O, Michael M I, Dan S, Malka C, Aaron B, Gideon Z, Donna S S, Batsheva K. Nucleotide deficiency promotes genomic instability in early stages of cancer development. Cell, 2011, 145(3): 435–446

[8] Makoto R H, Jeffrey J K, Erin J W, Sudarshan R, Ryan T S, Wayne G, Aaron J T, Barbara W, Christopher M L, Kunhong X, . A stress response pathway regulates DNA damage through β2-adrenoreceptors and β-arrestin-1. Nature, 2011, 477(7364): 349–353

[9] Furda A M, Marrangoni A M, Lokshin A, Van Houten B. Oxidants and not alkylating agents induce rapid mtDNA loss and mitochondrial dysfunction. DNA Repair, 2012, 11(8): 684–692

[10] Feng B D, Wang K, Liu J W, Mao G J, Cui J Q, Xuan X P, Jiang K, Zhang H. Ultrasensitive apurinic/apyrimidinic site-specific ratio fluorescent rotor for real-time highly selective evaluation of mtDNA oxidative damage in living cells. Analytical Chemistry, 2019, 91(21): 13962–13969

[11] Neiman M, Taylor D R. The causes of mutation accumulation in mitochondrial genomes. Proceedings. Biological Sciences, 2009, 276(1660): 1201–1209

[12] Furda A M, Marrangoni A M, Lokshin A, Van Houten B. Oxidants and not alkylating agents induce rapid mtDNA loss and mitochondrial dysfunction. DNA Repair, 2012, 11(8): 684–692

[13] Schon E A, DiMauro S, Hirano M. Human mitochondrial DNA: roles of inherited and somatic mutations. Nature Reviews. Genetics, 2012, 13(12): 878–890

[14] Schuermann D, Scheidegger S P, Weber A R, Bjoras M, Leumann C J, Schar P. 3CAPS—a structural AP-site analogue as a tool to investigate DNA base excision repair. Nucleic Acids Research, 2016, 44(5): 2187–2198

[15] Bender A, Krishnan K J, Morris C M, Taylor G A, Reeve A K, Perry R H, Jaros E, Hersheson J S, Betts J, Klopstock T, Taylor R W, Turnbull D M. High levels of mitochondrial DNA deletions in substantia nigra neurons in aging and parkinson disease. Nature Genetics, 2006, 38(5): 515–517

[16] Rebbeck C A, Leroi A M, Burt A. Mitochondrial capture by a transmissible cancer. Science, 2011, 331(6015): 303

[17] Ascaso F J, Lopez-Gallardo E, DelPrado E, Ruiz-Pesini E, Montoya J. Macular lesion resembling adult-onset vitelliform macular dystrophy in Kearns-Sayre syndrome with multiple mtDNA deletions. Clinical & Experimental Ophthalmology, 2010, 38(8): 812–816

[18] Badura-Stronka M, Wawrocka A, Zawieja K, Silska S, Krawczynski M R. Severe manifestation of Leber’s hereditary optic neuropathy due to 11778G>A mtDNA mutation in a female with hypoestrogenism due to perrault syndrome. Mitochondrion, 2013, 13(6): 831–834

[19] Sun W, Li M, Fan J L, Peng X J. Activity-based sensing and theranostic probes based on photoinduced electron transfer. Accounts of Chemical Research, 2019, 52(10): 2818–2831

[20] Sun W, Guo S G, Hu C, Fan J L, Peng X J. Recent development of chemosensors based on cyanine platforms. Chemical Reviews, 2016, 116(14): 7768–7817

[21] Lin F, Zhou Y F, Li Q S, Zhou X S, Shao Y, Haber-meyer B, Wang H, Shi X H, Xu Z A. Prototropically allosteric probe for superbly selective DNA analysis. Analytical Chemistry, 2017, 89(17): 9299–9306

[22] Zou X X, Shi Y L, Zhu R, Han J H, Han S F. Organelle-redirected chameleon sensor-enabled live cell imaging of mitochondrial DNA. Analytical Chemistry, 2019, 91(24): 15899–15907

[23] Abeywickrama C S, Bertman K A, Plescia C B, Stahelin R V, Pang Y. Structural effect on the cellular selectivity of an NIR-emitting cyanine probe: from lysosome to simultaneous nucleus and mitochondria selectivity with potential for monitoring mitochondria dysfunction in cells. ACS Applied Bio Materials, 2019, 2(11): 5174–5181

[24] Briggs C, Jones M. SYBR Green I-induced fluorescence in cultured immune cells: a comparison with acridine orange. Acta Histochemica, 2005, 107(4): 301–312

[25] Davis S K, Bardeen C J. Cross-linking of histone proteins to DNA by UV illumination of chromatin stained with hoechst 33342. Photochemistry and Photobiology, 2003, 77(6): 675–679

[26] Pfeifer G P, You Y H, Besaratinia A. Mutations induced by ultraviolet light. Mutation Research, 2005, 571(1-2): 19–31

[27] Smith P J, Blunt N, Wiltshire M, Hoy T, Teesdale-Spittle P, Craven M R, Watson J V, Amos W B, Errington R J, Patterson L H. Characteristics of a novel deep red/infrared fluorescent cell-permeant DNA probe, DRAQ5, in intact human cells analyzed by flow cytometry, confocal and multiphoton microscopy. Cytometry, 2000, 40(4): 280–291

[28] Burke C S, Byrne A, Keyes T E. Highly selective mitochondrial targeting by a ruthenium(II) peptide conjugate: imaging and photoinduced damage of mitochondrial DNA. Angewandte Chemie International Edition, 2018, 57(38): 12420–12424

[29] Cao J J, Zheng Y, Wu X W, Tan C P, Chen M H, Wu N, Ji L N, Mao Z W. Anticancer cyclometalated iridium(III) complexes with planar ligands: mitochondrial DNA damage and metabolism disturbance. Journal of Medicinal Chemistry, 2019, 62(7): 3311–3322

[30] Zheng Y, Zhang D Y, Zhang H, Cao J J, Tan C P, Ji L N, Mao Z W. Photodamaging of mitochondrial DNA to overcome cisplatin resistance by a Ru II–Pt II bimetallic complex. Chemistry (Weinheim an der Bergstrasse, Germany), 2018, 24(71): 18971–18980

[31] Feng B D, Wang K, Yang Y G, Wang G, Zhang H, Liu Y F, Jiang K. Ultrasensitive recognition of AP sites in DNA at the single-cell level: one molecular rotor sequentially self-regulated to form multiple different stable conformations. Chemical Science (Cambridge), 2019, 10(44): 10373–10380

[32] Peng X J, Wu T, Fan J L, Wang J Y, Zhang S, Song F L, Sun S G. An effective minor groove binder as a red fluorescent marker for live-cell DNA imaging and quantification. Angewandte Chemie International Edition, 2011, 50(18): 4180–4183

[33] Friedberg E C. DNA damage and repair. Nature, 2003, 421(6921): 436–440

[34] Song Y L, Tian T, Shi Y Z, Liu W L, Zou Y, Khajvand T, Wang S L, Zhu Z, Yang C Y. Enrichment and single-cell analysis of circulating tumor cells. Chemical Science (Cambridge), 2017, 8(3): 1736–1751

[35] Leung C H, Zhong H J, He H Z, Lu L, Chan D S H, Ma D L. Luminescent oligonucleotide-based detection of enzymes involved with DNA repair. Chemical Science (Cambridge), 2013, 4(10): 3781–3795

[36] Liu C X, Wang Y F, Zhang X, Wu F, Yang W, Zou G G, Yao Q, Wang J Q, Chen Y Q, Wang S R, Zhou X. Enrichment and fluorogenic labelling of 5-formyluracil in DNA. Chemical Science (Cambridge), 2017, 8(6): 4505–4510

[37] Visvardis E E, Tassiou A M, Piperakis S M. Study of DNA damage induction and repair capacity of fresh and cryopreserved lymphocytes exposed to H₂O₂ and γ-irradiation with the alkaline comet assay. Mutation Research, 1997, 383(1): 71–80

[38] Berova N, Nakanishi K, Woody R W. Circular Dichroism: Principles and Applications. New York: Wiley-VCH, 2000, 703–718

[39] Monnot M, Mauffret O, Lescot E, Fermandjian S. Probing intercalation and conformational effects of the anticancer drug 2-methyl-9-hydroxyellipticinium acetate in DNA fragments with circular dichroism. European Journal of Biochemistry, 1992, 204(3): 1035–1039

[40] Karami K, Shirani-Sarmazeh Z, Hosseini-Kharat M, Lipkowski J, Saeidifar M. Synthesis, spectral characterization, crystal structure and in vitro DNA/protein binding studies of phosphorous ylide palladacyclic complexes containing azide group. Journal of Photochemistry and Photobiology. B, Biology, 2015, 144: 11–19

[41] Baase W A, Johnson W C Jr. Circular dichroism and DNA secondary structure. Nucleic Acids Research, 1979, 6(2): 797–814

[42] Sarkar D, Das P, Basak S, Chattopadhyay N. Binding interaction of cationic phenazinium dyes with calf thymus DNA: a comparative study. Journal of Physical Chemistry B, 2008, 112(30): 9243–9249

[43] Poulsen B C, Estalayo-Adrián S, Blasco S, Bright S A, Kelly J M, Williams D C, Gunnlaugsson T. Luminescent ruthenium polypyridyl complexes with extended ‘dppz’ like ligands as DNA targeting binders and cellular agents. Dalton Transactions (Cambridge, England), 2016, 45(45): 18208–18220

[44] Fishel M L, Seo Y R, Smith M L, Kelley M R. Imbalancing the DNA base excision repair pathway in the mitochondria; targeting and overexpressing N-methylpurine DNA glyeosylase in mitochondria leads to enhanced cell killing. Cancer Research, 2003, 63(3): 608–615

[45] Scaduto R C Jr, Grotyohann L W. Measurement of mitochondrial membrane potential using fluorescent rhodamine derivatives. Biophysical Journal, 1999, 76(1): 469–477

RIGHTS & PERMISSIONS

Higher Education Press