Korean Chemical Engineering Research

The Korean Institute of Chemical Engineers (한국화학공학회)
권호 표지
DOI QR코드

DOI QR Code

Quantification of DNA Delivery Efficiency Labeled with Fluorescent Dye in Digital Electroporation System

디지털 전기천공시스템에서 형광 염료로 표지 된 DNA 전달 효율의 정량화

  • Bae, Seo Jun (Department of Chemical Engineering, Pukyong National University) ;
  • Im, Do Jin (Department of Chemical Engineering, Pukyong National University)
  • Received : 2020.03.25
  • Accepted : 2020.04.20
  • Published : 2020.08.01
Download PDF
⟨ Previous Next ⟩

Abstract

In the previous study, there was a big difference between the tendency of the delivery efficiency of Yo-Pro-1 and the expression efficiency of the CFP gene, but there was a problem that could not provide a clue to this problem. Therefore, this study aimed to present a clue to this problem by quantifying and comparing the delivery efficiency after labeling DNA using a fluorescent dye, which was one of the methods for quantifying biomolecules. As a fluorescent dye for labeling, Yo-Pro-1 was used, and the delivery efficiency of the fluorescent dye Yo-Pro-1 and the labeled DNA was compared. The delivery efficiency of Yo-Pro-1 and labeled DNA according to the voltage condition of the digital electroporation system was maximized at 96 V, and the delivery efficiency tended to decrease as the voltage increased further. In the comparison of the delivery efficiency of Yo-Pro-1 and labeled DNA according to the number of voltage application conditions, the delivery efficiency was maximized at the number of 8 voltage application times for both delivery materials, and the delivery efficiency tended to decrease as the number of voltage application increases further. Through the two results, it was confirmed that the delivery efficiency using Yo-Pro-1 in the digital electroporation system represents the delivery efficiency of the system well. In addition, through the results of this study, the difference between the tendency of the delivery efficiency of Yo-Pro-1 and the expression efficiency of the CFP gene shown in the preceding study was not the result of the difference in the delivery efficiency of the delivery material, but it can be predicted to be due to a problem with the expression process of the genetic material that had been delivered.

선행된 연구에서 Yo-Pro-1의 전달 효율의 경향과 CFP 유전자의 발현 효율의 경향이 큰 차이를 보였지만 이 문제에 대한 원인을 제시할 수 없었다. 따라서 본 연구에서는 형광 염료를 이용하여 DNA에 표지 후 전달 효율을 정량화함으로써 이 문제에 대한 원인을 찾고자 한다. 표지를 위한 형광 염료로 Yo-Pro-1을 사용하였으며, Yo-Pro-1과 표지 된 DNA의 전달 효율을 비교하였다. 전압 조건에 따른 전달효율 비교에서는 Yo-Pro-1과 Yo-Pro-1으로 표지 된 DNA의 전달 효율 모두 96 V에서 전달 효율이 최대가 되었으며 전압이 더 증가하면 전달 효율이 오히려 감소하는 경향을 보였다. 전압 인가 횟수 조건에 따른 전달 효율 비교에서는 Yo-Pro-1과 Yo-Pro-1으로 표지 된 DNA의 두 전달 물질 모두 8회의 전압 인가 횟수에서 전달 효율이 최대가 되었으며 전압 인가 횟수가 더 증가하면 전달 효율이 감소하는 경향을 보였다. 두 결과를 통해 디지털 전기천공시스템에서 Yo-Pro-1을 사용한 전달 효율 측정이 DNA의 전달 효율을 잘 대변하는 것을 확인하였다. 또한, 본 연구의 결과를 통해 선행된 연구에서 보인 Yo-Pro-1의 전달 효율의 경향과 CFP 유전자의 발현 효율의 경향 차이는 전달 물질의 전달 효율 차이에서 기인한 결과가 아닌 전달 된 유전 물질의 발현 과정에서의 문제로 인한 결과임을 추론해 볼 수 있었다.

Keywords

References

  1. Ma, F., Li, Y., Tang, B., and Zhang, C. Y., "Fluorescent Biosensors Based on Single-Molecule Counting," Acc. Chem. Res. 49, 1722-1730(2016). https://doi.org/10.1021/acs.accounts.6b00237
  2. Ueno, T., Nagano, T. "Fluorescent Probes for Sensing and Imaging," Nat. Methods 8(8), 642-645(2011). https://doi.org/10.1038/nmeth.1663
  3. Proudnikov, D. and Mirzabekov, A., "Chemical Mehotds of DNA and RNA Fluorescent Lableing," Nucleic Acids Res., 24(22), 4535-4542(1996). https://doi.org/10.1093/nar/24.22.4535
  4. Pieter, E. O., Mohaddeseh, A. A., Ibrahim, K., Nhu, T. N. P., and Andrew, G. E. "Chemical Analysis of Single Cells," Anal. Chem. 91, 588-621(2019). https://doi.org/10.1021/acs.analchem.8b04732
  5. Peng, T. and Hang, H. C., "Site-specific Bioorthognal Labelign for Fluorescence Imaging of Intracellular Proteins in Living Cells," J. Am. Chem. Soc. 138, 14423-14433(2016). https://doi.org/10.1021/jacs.6b08733
  6. Schvartz, T., Aloush, N., Goliand, I., Segal, I., Nachmias, D., Arbely, E., and Elia, N., "Direct Fluorescent-dye Labeling of ${\alpha}$-tubulin in Mammalian Cells for Live Cell and Superresolution Imaging," Biol. Cell 28, 2747-2756(2017).
  7. Weiss, L. E., Naor, T., and Shechtman, Y., "Observing DNA in Live Cells," Biochem. Soc. Trans., 46(3), 729-740(2018). https://doi.org/10.1042/BST20170301
  8. Marie, D., Vaulot, D., and Partensky, F. "Application of the Novel Nucleic Acid Dyes YOYO-1, YO-PRO-1, and PicoGreen for Flow Cytometric Analysis of Marin Prokaryotes," Appl. Environ. Microbiol. 62, 1649-1655(1996). https://doi.org/10.1128/AEM.62.5.1649-1655.1996
  9. Lee, S., Kopp, F., Chang, T. C., Sataluri, A., Chen, B., Sivakumar, S., Yu, H., Xie, Y. and Mendell, J. T., "Noncoding RNA NORAD Regulates Genomic Stability by Sequestering PUMILIO Proteins," Cell 164, 69-80(2016). https://doi.org/10.1016/j.cell.2015.12.017
  10. Fujisawa, S., Romin, Y., Barlas, A., Petrovic, L. M., Turkekul, M., Fan, N.,Xu, K., Garcia, A. R., Monette, S., Klimstra, D. S., Erinjeri, J. P., Solomon, S. B., Manova-Todorova, K. and Sofocleous, C. T., "Evaluation of YO-PRO-1 as an Early Marker of Apoptosis Following Radiofrequency Ablation of Colon Cancer Liver Metastases," Cytotechnology, 66, 259-273(2014). https://doi.org/10.1007/s10616-013-9565-3
  11. Larsson, A., Carlsson, C., Jonsson, M., and Albinosson, B. "Characterization of the Binding of the Fluorsecent Dyes YO and YOYO to DNA by Polarized Light Spectroscopy," J. Am. Chem. Soc. 166, 8459-8465(1994).
  12. Kim, Y. H., Kwon, S. G., Bae, S. J., Park, S. J. and Im, D. J., "Optimization of the Droplet Electroporation Method for Wild Type Chlamydomonas Reinhardtii Transformation," Bioelectrochemistry 126, 29-37(2019). https://doi.org/10.1016/j.bioelechem.2018.11.010
  13. Im, D. J., "Next Generation Digital Microfluidic Technology: Electrophoresis of Charged Droplets," Korean J. Chem. Eng., 32, 1001-1008(2015). https://doi.org/10.1007/s11814-015-0092-0
  14. Im, D. J., "Charging of an Ionic Liquid Droplet in a Dielectric Medium," Clean Technology 20, 354-358(2014). https://doi.org/10.7464/ksct.2014.20.4.354
  15. Im, D. J., Noh, J., Moon, D. and Kang, I. S., "Electrophoresis of a Charged Droplet in a Dielectric Liquid for Droplet Actuation," Anal. Chem. 83, 5168-5174(2011). https://doi.org/10.1021/ac200248x
  16. Im, D. J., Ahn, M. M., Yoo, B. S., Moon, D., Lee, D. W. and Kang, I. S., "Discrete Electrostatic Charge Transfer by the Electrophoresis of a Charged Droplet in a Dielectric Liquid," Langmuir, 28, 11656-11661(2012). https://doi.org/10.1021/la3014392
  17. Im, D. J., Yoo, B. S., Ahn, M. M., Moon, D. and Kang, I. S., "Digital Electrophoresis of Charged Droplets," Anal. Chem. 85, 4038-4044(2013). https://doi.org/10.1021/ac303778j
  18. Ahn, M. M., Im, D. J. and Kang, I. S., "Geometric Characterization of Optimal Electrode Designs for Improved Droplet Charging and Actuation," Analyst 138, 7362-7368(2013). https://doi.org/10.1039/c3an01623d
  19. Lee, D. W., Im, D. J. and Kang, I. S., "Measurement of the Interfacial Tension in an Ionic Liquid-Dielectric Liquid System Using an Electrically Deformed Droplet," J. Phys. Chem. C., 117, 3426-3430(2013). https://doi.org/10.1021/jp312212e
  20. Ahn, M. M., Im, D. J., Kim, J. G., Lee, D. W. and Kang, I. S., "Extraction of Cations from an Ionic Liquid Droplet in a Dielectric Liquid under Electric Field," J. Phys. Chem. Lett., 5, 3021-3025(2014). https://doi.org/10.1021/jz501511z
  21. Ahn, M. M., Im, D. J., Yoo, B. S. and Kang, I. S., "Characterization of Electrode Alignment for Optimal Droplet Charging and Actuation in Droplet-based Microfluidic System," Electrophoresis 36, 2086-2093(2015). https://doi.org/10.1002/elps.201500141
  22. Choi, C. Y. and Im, D. J., "Contact Charging and Electrophoresis of a Glassy Carbon Microsphere,Choi, C. Y. and Im, D. J., "Contact Charging and Electrophore," Korean Chem. Eng. Res., 54(4), 568-573(2016). https://doi.org/10.9713/kcer.2016.54.4.568
  23. Im, D. J., Jeong, S.-N., Yoo, B. S., Kim, B., Kim, D.-P., Jeong, W.-J. and Kang, I. S., "Digital Microfluidic Approach for Efficient Electroporation with High Productivity: Transgene Expression of Microalgae without Cell Wall Removal," Anal. Chem. 87, 6592-6599(2015). https://doi.org/10.1021/acs.analchem.5b00725
  24. Kurita, H., Takahashi, S., Asada, A., Matsuo, M., Kishikawa, K., Mizuno, A. and Numano, R., "Novel Parallelized Electroporation by Electrostatic Manipulation of a Water-in-Oil Droplet as a Microreactor," PLOS ONE 10, e0144254(2015). https://doi.org/10.1371/journal.pone.0144254
  25. Jung, J. H. and Lee, C. S., "Droplet Based Microfluidic System," Korean Chem. Eng. Res., 48(5), 545-555(2010).
  26. Im, D. J. and Jeong, S.-N., "Transfection of Jurkat T Cells by Droplet Electroporation," Biochem. Eng. J., 122, 133-140(2017). https://doi.org/10.1016/j.bej.2017.03.010
  27. Bae, S. J. and Im, D. J., "Evaluation of Cell Viability and Delivery Efficiency in Electroporation System According to the Concentration of Propidium Iodide and Yo-Pro-1," Korean Chem. Eng. Res., 57(6), 898-906(2019). https://doi.org/10.9713/kcer.2019.57.6.898
  28. Flors, C., "DNA and Chromatin Imaging with Super-resolution Fluorescence Microscopy Based on Single-molecule Localization," Biopolymers 95, 290-297(2011). https://doi.org/10.1002/bip.21574