Advertisement

Construction of DNA-NanoLuc luciferase conjugates for DNA aptamer-based sandwich assay using Rep protein

  • Masayasu MieEmail author
  • Takahiro Niimi
  • Yasumasa Mashimo
  • Eiry Kobatake
Original Research Paper
  • 105 Downloads

Abstract

Objective

We developed a DNA-NanoLuc luciferase (NnaoLuc) conjugates for DNA aptamer-based sandwich assay using the catalytic domain of the replication initiator protein derived from porcine circovirus type 2 (pRep).

Results

For construction of DNA aptamer and NanoLuc conjugate using the catalytic domain of Rep from PCV2. pRep fused to NanoLuc was genetically constructed and expressed in E. coli. After purification, the activities of fused pRep and NanoLuc were evaluated, and DNA-NanoLuc conjugates were constructed via the fused pRep. Finally, constructed DNA-NanoLuc conjugates were applied for use in a DNA aptamer-based sandwich assay. Here, pRep was used not only for conjugation of the NanoLuc to the detection aptamer, but also for immobilization of the capture aptamer on the plate surface.

Conclusion

We have demonstrated that DNA-NanoLuc conjugates via the catalytic domain of PCV2 Rep could be applied for DNA aptamer-based sandwich assay system.

Keywords

DNA aptamer DNA–protein conjugate Rep NanoLuc luciferase DNA aptamer-based sandwich assay 

Notes

Acknowledgements

This work was supported in part by JSPS KAKENHI Grant Numbers 16K01388 (M.M.), 15K13781 and 6289310J (E.K.).

Supplementary Information

Supplementary Figure 1—The gene of the catalytic domain of PCV2 Rep comprising residues 1–116 (pRep). The amino acid sequence of the pRep was shown as red. The sequence of the pRep optimized for E.coli expression was shown as black (Opt).

Supplementary Figure 2—Immobilization of DNA-protein conjugates on the hydrophobic plate surface.

Supplementary Figure 3—Evaluation of specific binding abilities of Thrombin DNA aptamer conjugated to protein.

Supplementary material

10529_2018_2641_MOESM1_ESM.docx (213 kb)
Supplementary material 1 (DOCX 212 kb)

References

  1. Bock LC, Griffin LC, Latham JA, Vermaas EH, Toole JJ (1992) Selection of single-stranded DNA molecules that bind and inhibit human thrombin. Nature 355:564–566CrossRefGoogle Scholar
  2. Hanai R, Wang JC (1993) The mechanism of sequence-specific DNA cleavage and strand transfer by phi X174 gene A* protein. J Biol Chem 268:23830–23836Google Scholar
  3. Jungmann R, Avendano MS, Woehrstein JB, Dai M, Shih WM, Yin P (2014) Multiplexed 3D cellular super-resolution imaging with DNA-PAINT and Exchange-PAINT. Nat Methods 11:313–318CrossRefGoogle Scholar
  4. Koussa MA, Sotomayor M, Wong WP (2014) Protocol for sortase-mediated construction of DNA-protein hybrids and functional nanostructures. Methods 67:134–141CrossRefGoogle Scholar
  5. Lee KA, Ahn JY, Lee SH, Singh Sekhon S, Kim DG, Min J, Kim YH (2015) Aptamer-based Sandwich assay and its clinical outlooks for detecting lipocalin-2 in hepatocellular carcinoma (HCC). Sci Rep 5:10897CrossRefGoogle Scholar
  6. Lovendahl KN, Hayward AN, Gordon WR (2017) Sequence-directed covalent protein-DNA linkages in a single step using HUH-Tags. J Am Chem Soc 139:7030–7035CrossRefGoogle Scholar
  7. Mashimo Y, Maeda H, Mie M, Kobatake E (2012) Construction of semisynthetic DNA–protein conjugates with Phi X174 Gene-A* protein. Bioconjug Chem 23:1349–1355CrossRefGoogle Scholar
  8. Nakata E et al (2012) Zinc-finger proteins for site-specific protein positioning on DNA-origami structures. Angew Chem Int Ed 51:2421–2424CrossRefGoogle Scholar
  9. Sagredo S, Pirzer T, Aghebat Rafat A, Goetzfried MA, Moncalian G, Simmel FC, de la Cruz F (2016) Orthogonal protein assembly on DNA nanostructures using relaxases. Angew Chem Int Ed 55:4348–4352CrossRefGoogle Scholar
  10. Sanhueza S, Eisenberg S (1984) Cleavage of single-stranded DNA by the varphiX174 A protein: the A-single-stranded DNA covalent linkage. Proc Natl Acad Sci USA 81:4285–4289CrossRefGoogle Scholar
  11. Sano T, Smith CL, Cantor CR (1992) Immuno-PCR: very sensitive antigen detection by means of specific antibody-DNA conjugates. Science 258:120–122CrossRefGoogle Scholar
  12. Tasset DM, Kubik MF, Steiner W (1997) Oligonucleotide inhibitors of human thrombin that bind distinct epitopes. J Mol Biol 272:688–698CrossRefGoogle Scholar
  13. Tuleuova N, Jones CN, Yan J, Ramanculov E, Yokobayashi Y, Revzin A (2010) Development of an aptamer beacon for detection of interferon-gamma. Anal Chem 82:1851–1857CrossRefGoogle Scholar
  14. van Buggenum JA et al (2016) A covalent and cleavable antibody-DNA conjugation strategy for sensitive protein detection via immuno-PCR. Sci Rep 6:22675CrossRefGoogle Scholar
  15. Vega-Rocha S, Byeon IJ, Gronenborn B, Gronenborn AM, Campos-Olivas R (2007) Solution structure, divalent metal and DNA binding of the endonuclease domain from the replication initiation protein from porcine circovirus 2. J Mol Biol 367:473–487CrossRefGoogle Scholar
  16. Vivekananda J, Kiel JL (2006) Anti-Francisella tularensis DNA aptamers detect tularemia antigen from different subspecies by Aptamer-Linked Immobilized Sorbent Assay. Lab Invest 86:610–618CrossRefGoogle Scholar

Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  1. 1.Department of Life Science and Technology, School of Life Science and TechnologyTokyo Institute of TechnologyMidori-Ku, YokohamaJapan

Personalised recommendations