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A star-shaped DNA probe based on strand displacement for universal and multiplexed fluorometric detection of genetic variations

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Abstract

A star-shaped fluorescent DNA probe (S-probe) is described that can recognize target DNA and discriminate it from interfering DNA via strand displacement including branch migration and toehold exchange. The artificially designed S-probe does not harm the strand displacement while it allows the fluorescently labelled strand and the quencher-labelled strand to be shared among different S-probes targeting different genetic variations. Generally, multiplexed detection of different MT/WT pairs requires different fluorophore-labelled and quencher-labelled strands. The two labelled oligonucleotides of S-probe have sequences decoupled from the target/interfering DNA sequence, so the same fluorescent and quencher strands can be used for different S-probes that target different sequences. The sensitivity, specificity, and general applicability of the method toward BRCA 41293497 mutation, KRAS G13D mutation and two types of EGFR mutations (T790 M and L858R) were experimentally demonstrated. The limit of quantification of the MT concentration is 2 nM, and the detection limit of the low abundance of the target sequence is 5% (40 nM of MT strand in the background of 760 nM of WT strand). The fluorometric assay with excitation/emission wavelengths of 485/582 nm was successfully applied to clinical samples spiked with mutant-type and wild-type DNA. The unique structure of the S-probe provides a useful tool for the regulation of the strand displacement reaction. Conceivably, the star-shaped DNA probe can be widely adopted to multiplexed detection of genetic variations and provide novel insights into the regulation of strand displacement processes as utilized in DNA based nanomachines.

A star-shaped fluorescent DNA probe (S-probe) with a detection limit of 2 nM was adopted to multiplexed detection of genetic variations via strand displacement including branch migration and toehold exchange.

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References

  1. Cohen JD, Li L, Wang Y, Thoburn C, Afsari B, Danilova L, Douville C, Javed AA, Wong F, Mattox A, Hruban RH, Wolfgang CL, Goggins MG, Dal Molin M, Wang T-L, Roden R, Klein AP, Ptak J, Dobbyn L, Schaefer J, Silliman N, Popoli M, Vogelstein JT, Browne JD, Schoen RE, Brand RE, Tie J, Gibbs P, Wong H-L, Mansfield AS, Jen J, Hanash SM, Falconi M, Allen PJ, Zhou S, Bettegowda C, Diaz LA Jr, Tomasetti C, Kinzler KW, Vogelstein B, Lennon AM, Papadopoulos N (2018) Detection and localization of surgically resectable cancers with a multi-analyte blood test. Science 359(6378):926–930. https://doi.org/10.1126/science.aar3247

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Tibshirani R, Hastie T, Narasimhan B, Chu G (2002) Diagnosis of multiple cancer types by shrunken centroids of gene expression. Proc Natl Acad Sci U S A 99(10):6567–6572. https://doi.org/10.1073/pnas.082099299

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Bettegowda C, Sausen M, Leary RJ, Kinde I, Wang Y, Agrawal N, Bartlett BR, Wang H, Luber B, Alani RM, Antonarakis ES, Azad NS, Bardelli A, Brem H, Cameron JL, Lee CC, Fecher LA, Gallia GL, Gibbs P, Le D, Giuntoli RL, Goggins M, Hogarty MD, Holdhoff M, Hong S-M, Jiao Y, Juhl HH, Kim JJ, Siravegna G, Laheru DA, Lauricella C, Lim M, Lipson EJ, Marie SKN, Netto GJ, Oliner KS, Olivi A, Olsson L, Riggins GJ, Sartore-Bianchi A, Schmidt K, Shih I-M, Oba-Shinjo SM, Siena S, Theodorescu D, Tie J, Harkins TT, Veronese S, Wang T-L, Weingart JD, Wolfgang CL, Wood LD, Xing D, Hruban RH, Wu J, Allen PJ, Schmidt CM, Choti MA, Velculescu VE, Kinzler KW, Vogelstein B, Papadopoulos N, Diaz LA Jr (2014) Detection of circulating tumor DNA in early- and late-stage human malignancies. Sci Transl Med 6(224):224ra24. https://doi.org/10.1126/scitranslmed.3007094

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Diaz LA, Bardelli A (2014) Liquid biopsies: genotyping circulating tumor DNA. Journal of Clinical Oncology 32(6):579–586. https://doi.org/10.1200/jco.2012.45.2011

    Article  PubMed  PubMed Central  Google Scholar 

  5. Diehl F, Schmidt K, Choti MA, Romans K, Goodman S, Li M, Thornton K, Agrawal N, Sokoll L, Szabo SA, Kinzler KW, Vogelstein B, Diaz LA Jr (2008) Circulating mutant DNA to assess tumor dynamics. Nat Med 14(9):985–990. https://doi.org/10.1038/nm.1789

    Article  CAS  PubMed  Google Scholar 

  6. Schwarzenbach H, Hoon DSB, Pantel K (2011) Cell-free nucleic acids as biomarkers in cancer patients. Nat Rev Cancer 11(6):426–437. https://doi.org/10.1038/nrc3066

    Article  CAS  PubMed  Google Scholar 

  7. Wang H, Zhou G, Gai H, Chen X (2012) A fluorescein-based probe with high selectivity to cysteine over homocysteine and glutathione. Chem Commun 48(67):8341–8343. https://doi.org/10.1039/c2cc33932c

    Article  CAS  Google Scholar 

  8. Vogelstein B, Papadopoulos N, Velculescu VE, Zhou S, Diaz LA Jr, Kinzler KW (2013) Cancer genome landscapes. Science 339(6127):1546–1558. https://doi.org/10.1126/science.1235122

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Chung HS, Eaton WA (2013) Single-molecule fluorescence probes dynamics of barrier crossing. Nature 502(7473):685–688. https://doi.org/10.1038/nature12649

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Okamoto A, Kanatani K, Saito I (2004) Pyrene-labeled base-discriminating fluorescent DNA probes for homogeneous SNP typing. J Am Chem Soc 126(15):48204827. https://doi.org/10.1021/ja039625y

    Article  CAS  Google Scholar 

  11. Zhang DY, Chen SX, Yin P (2012) Optimizing the specificity of nucleic acid hybridization. Nat Chem 4(3):208–214. https://doi.org/10.1038/nchem.1246

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Tyagi S, Kramer FR (1996) Molecular beacons: probes that fluoresce upon hybridization. Nat Biotechnol 14(3):303–308. https://doi.org/10.1038/nbt0396-303

    Article  CAS  PubMed  Google Scholar 

  13. Tyagi S, Bratu DP, Kramer FR (1998) Multicolor molecular beacons for allele discrimination. Nat Biotechnol 16(1):49–53. https://doi.org/10.1038/nbt0198-49

    Article  CAS  PubMed  Google Scholar 

  14. Zhou H, Liu J, Xu J-J, Chen H-Y (2011) Highly sensitive Electrochemiluminescence detection of single-nucleotide polymorphisms based on isothermal cycle-assisted triple-stem probe with dual-nanoparticle label. Anal Chem 83(21):8320–8328. https://doi.org/10.1021/ac2022629

    Article  CAS  PubMed  Google Scholar 

  15. Xiao X, Wu T, Xu L, Chen W, Zhao M (2017) A branch-migration based fluorescent probe for straightforward, sensitive and specific discrimination of DNA mutations. Nucleic Acids Res 45(10):e90. https://doi.org/10.1093/nar/gkx117

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Wang JS, Zhang DY (2015) Simulation-guided DNA probe design for consistently ultraspecific hybridization. Nat Chem 7(7):545–553. https://doi.org/10.1038/nchem.2266

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Seeman NC (2010) Nanomaterials based on DNA. In: Kornberg RD, Raetz CRH, Rothman JE, Thorner JW (eds) Annual review of biochemistry, vol 79, pp 65-87. doi:https://doi.org/10.1146/annurev-biochem-060308-102244

  18. Bath J, Turberfield AJ (2007) DNA nanomachines. Nature Nanotechnol 2(5):275–284. https://doi.org/10.1038/nnano.2007.104

    Article  CAS  Google Scholar 

  19. Ge J, Bai DM, Geng X, Hu YL, Cai QY, Xing K, Zhang L, Li ZH (2018) Fluorometric determination of nucleic acids based on the use of polydopamine nanotubes and target-induced strand displacement amplification. Mikrochim Acta 185(2):105. https://doi.org/10.1007/s00604-017-2632-2

    Article  CAS  PubMed  Google Scholar 

  20. Xiaoting Ji, Haoyuan Lv, Minghui Ma, Binglin Lv, Caifeng Ding (2017) An optical DNA logic gate based on strand displacement and magnetic separation, with response to multiple microRNAs in cancer cell lysates. Microchim Acta 184:2505–2513. https://doi.org/10.1007/s00604-017-2248-6

    Article  CAS  Google Scholar 

  21. Wang X, Liu W, Yin B, Sang Y, Liu Z, Dai Y, Duan X, Zhang G et al (2017) An isothermal strand displacement amplification strategy for nucleic acids using junction forming probes and colorimetric detection. Microchim Acta 184:1603–1610. https://doi.org/10.1007/s00604-017-2158-7

    Article  CAS  Google Scholar 

  22. Xu H, Jiang Y, Liu D, Liu K, Zhang Y, Yu S, Shen Z, Wu ZS (2018) Twin target self-amplification-based DNA machine for highly sensitive detection of cancer-related gene. Anal Chim Acta 1011:86–93. https://doi.org/10.1016/j.aca.2018.01.022

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

The work is financially supported by the National Science Foundation of China (No. 21705053), Natural Science Foundation of Hubei Province (No. 2017CFB117), Hubei Province health and family planning scientific research project (No. J2017Q017), Wuhan Youth Science and Technology Plan (2017050304010293) and the Beijing National Laboratory for Molecular Sciences (BNLMS).

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Correspondence to Xianjin Xiao.

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Liu, N., Xu, K., Liu, L. et al. A star-shaped DNA probe based on strand displacement for universal and multiplexed fluorometric detection of genetic variations. Microchim Acta 185, 413 (2018). https://doi.org/10.1007/s00604-018-2941-0

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