COVID-19 mRNA Aşı Geliştirilmesinde Kullanılacak Olan Yüksek Konsantrasyondaki Plazmid DNA’sının Optimizasyonu: Alkalen Lizis Metodu ve Ticari Kit Sonuçlarının Kıyaslanması

Abstract

Kararlı IVT mRNA molekülü oluşturulurken başlangıçta plazmid DNA’sından kopyalanan ORF antijen sekansının UTR bölgeleri ile ligaze edilebilmesi için yüksek konsantrasyon ve saflıkta plazmid DNA'nın elde edilmesi gerekmektedir. Bu çalışmada, COVID-19 mRNA aşısının ilk basamağı olan mRNA molekülünün oluşturulması adımında, ORF hedef antijen sekansını içeren pDNA’nın alkalen lizis method ve ticari kit ile izolasyonu sonucundaki kıyaslama ve optimizasyonu yapılmıştır. Hedef antijen ORF sekansını içeren plazmid DNA bakterisi uygun üreme koşullarında çoğaltılmıştır. Farklı OD600 nm değerlerinde (0.02-0.05, 0.05-0.1, 0.1-0.2, 0.2-0.3, 0.3-0.4, 0.4-0.5) durdurulan bakteri kültürlerinden ticari kit ve alkalen lizis yöntemi ile plazmid DNA izolasyonu yapılmıştır. Elde edilen pDNA’lar bir agaroz jelde görüntülendikten sonra, spektroskopik ölçüme tabi tutularak saflığı ve konsantrasyonu ölçülmüştür. Stab kültür SOC medium içerisinde canlandırıldıktan sonra, kit ile izolasyon sonrasında tek formda, alkalen lizis metodu ile pDNA izolasyonu sonrasında birden çok formda (lineer, süper kıvrımlı, sirküler) bant oluşur. Her iki yöntem için de ideal OD600 nm 0.3-0.4 olarak bulunmuştur. Kit ile izolasyon sonucunda daha yüksek saflıkta ancak düşük konsantrasyonda pDNA elde edilmiştir. İdeal OD600 nm değeri, pDNA'nın konsantrasyonunu ve saflığını etkileyen önemli bir parametredir. Alkalen lizis yöntemi, kit izolasyonuna kıyasla mRNA aşı geliştirmeye alternatif olarak kullanılabilecek ucuz ve güçlü bir tekniktir.

Keywords

• Plazmid DNA
• izolasyon
• alkalen lizis
• kit
• mRNA aşısı

Project Number

2021/2663

Optimization of High Concentration Plasmid DNA for Use in COVID-19 mRNA Vaccine Development: Comparison of Between Alkaline Lysis Method and Commercial Kit Results

Abstract

While forming the stable IVT mRNA molecule, high concentration and purity plasmid DNA must be obtained to ligase the ORF antigen sequence initially copied from the plasmid DNA with the UTR regions. In this study, in the stage of creating the mRNA molecule, which is the first step of the COVID-19 mRNA vaccine, comparison and optimization of the pDNA containing the ORF target antigen sequence were performed as a result of isolation with alkaline lysis method and commercial kit. Plasmid DNA bacteria containing the target antigen ORF sequence were grown under appropriate conditions. Plasmid DNA was isolated by commercial kit and alkaline lysis method from bacterial cultures stopped at different OD600 nm values (0.02-0.05, 0.05-0.1, 0.1-0.2, 0.2-0.3, 0.3-0.4, 0.4-0.5). After the obtained pDNAs were visualized on an agarose gel, their purity and concentration were measured by spectroscopic measurement. After the stab culture is resuscitated in SOC medium, bands are formed in a single form after isolation with the kit, and in multiple forms (linear, supercoiled, circular) after pDNA isolation by alkaline lysis method. The ideal OD600 nm for both methods was 0.3-0.4. As a result of isolation with the kit, higher purity on the contrary low concentration pDNA was obtained. The ideal OD600 nm value is a critical parameter that affects the concentration and purity of pDNA. The alkaline lysis method is a cheap and powerful technique that can be used as an alternative for mRNA vaccine development compared to kit isolation.

Keywords

• Plasmid DNA
• isolation
• alkalen lysis
• kit
• mRNA vaccine

Supporting Institution

Kocaeli University Scientific Research Projects Coordination Office

Project Number

2021/2663

References

1. Adami RC, Collard WT, Gupta SA, KwoK KY, Bonadio J, Rice KG, 1998: Stability of peptide-condensed plasmid DNA formulations. Journal of pharmaceutical sciences, 87(6), 678-683.
2. Aleksić JM, Stojanović D, Banović B, Jančić R, 2012: A simple and efficient DNA isolation method for Salvia officinalis. Biochem Genet, 50(11-12):881-892.
3. Anand P, Stahel VP, 2021: The safety of Covid-19 mRNA vaccines: A review. Patient safety in surgery, 15(1), 1-9.
4. Chancham P, Hughes JA, 2001: Relationship between plasmid DNA topological forms and in vitro transfection. Journal of liposome research, 11(2-3), 139-152.
5. Duran T, Aydın Terzioğlu M, Çine N (2021). Lipid nanoparçacık tabanlı mRNA aşı teknolojisi ve SARS-CoV-2 (COVID-19) mRNA aşıları: Güncel COVID-19 Bilgileri, Canatan D, Alkan ŞŞ, (Ed), 47-56. Türkiye Klinikleri, Ankara.
6. Glasel JA, 1995: Validity of nucleic acid purities monitored by 260nm/280nm absorbance ratios. Biotechniques,18(1), 62-63.
7. Green MR, Sambrook J, 2016: Preparation of plasmid DNA by alkaline lysis with sodium dodecyl sulfate: minipreps. Cold Spring Harbor Protocols,(10), pdb-prot093344.
8. Hassan R, Husin A, Sulong S, Yusoff S, Johan MF, Yahaya BH, Ang CY, Ghazalı S, Cheong SK, 2015: Guidelines for nucleic acid detection and analysis in hematological disorders. The Malaysian journal of pathology, 37(2), 165–173.

9. Hirose S, Tsuda M, Suzuki Y, 1985: Enhanced transcription of fibroin gene in vitro on covalently closed circular templates. Journal of Biological Chemistry, 260(19), 10557-10562.
10. Kim J, Eygeris Y, Gupta M, Sahay G, 2011: Self-assembled mRNA vaccines. Advanced drug delivery reviews, 170, 83-112. Konarska MM, Padgett RA, Sharp PA, 1984: Recognition of cap structure in splicing in vitro of mRNA precursors. Cell, 38(3), 731-736.
11. Krieg PA, Melton DA, 1984: Functional messenger RNAs are produced by SP6 in vitro transcription of cloned cDNAs. Nucleic Acids Research, 12(18), 7057-7070.
12. Liu MA, 2011: DNA vaccines: an historical perspective and view to the future. Immunological reviews, 239(1), 62-84.
13. Liu MA, 2019: A comparison of plasmid DNA and mRNA as vaccine technologies. Vaccines, 7(2), 37.
14. Lucena-Aguilar G, Sánchez-López AM, Barberán-Aceituno C, Carrillo-Ávila JA, López-Guerrero JA, Aguilar-Quesada R, 2016: DNA source selection for downstream applications based on DNA quality indicators analysis. Biopreservation and Biobanking,14(4), 264-270.
15. Nouvel A, Laget J, Duranton F, 2021: Optimization of RNA extraction methods from human metabolic tissue samples of the COMET biobank. Scientific reports,11(1), 1-12.
16. Pardi N, Hogan MJ, Porter FW, Weissman D, 2018: mRNA vaccines—a new era in vaccinology. Nature reviews Drug discovery, 17(4), 261-279.
17. Pardi N, Weissman D (2017). Nucleoside modified mRNA vaccines for infectious diseases: In RNA Vaccines, 109-121, Humana Press, New York, NY.
18. Pascolo S, 2006: Vaccination with messenger RNA. DNA Vaccines, 23-40.
19. Schlake T, Thess A, Fotin-Mleczek M, Kallen KJ, 2012: Developing mRNA-vaccine technologies. RNA biology, 9(11), 1319-1330.
20. Schlake T, Thess A, Thran M, Jordan I, 2019: mRNA as novel technology for passive immunotherapy. Cellular and Molecular Life Sciences, 76(2), 301-328.
21. Stulnig TM, Amberger A, 1994: Exposing contaminating phenol in nucleic acid preparations. Biotechniques,16(3):402-404.
22. Usman T, Yu Y, Liu C, Fan Z, Wang Y, 2014: Comparison of methods for high quantity and quality genomic DNA extraction from raw cow milk. Genetics and molecular research, 13(2), 3319–3328.
23. Verbeke R, Lentacker I, De Smedt SC, Dewitte H, 2019: Three decades of messenger RNA vaccine development. Nano Today, 28, 100766.
24. Villarreal DO, Talbott KT, Choo DK, Shedlock DJ, Weiner DB, 2013: Synthetic DNA vaccine strategies against persistent viral infections. Expert review of vaccines, 12(5), 537-554.
25. Yang B, Jeang J, Yang A, Wu TC, Hung CF, 2014: DNA vaccine for cancer immunotherapy. Human vaccines & immunotherapeutics, 10(11), 3153-3164.
26. Zou SL, Zhang K, You L, Zhao XM, Jing X, Zhang MH, 2012: Enhanced electrotransformation of the ethanologen Zymomonas mobilis ZM4 with plasmids. Engineering in Life Sciences, 12(2), 152-161.