Effects of Lyse-It on endonuclease fragmentation, function and activity

Autoři: Tonya M. Santaus aff001;  Fan Zhang aff001;  Shan Li aff003;  O. Colin Stine aff003;  Chris D. Geddes aff001
Působiště autorů: Chemistry and Biochemistry Department, University of Maryland, Baltimore County, Baltimore, Maryland, United States of America aff001;  Institute of Fluorescence, University of Maryland, Baltimore County, Baltimore, Maryland, United States of America aff002;  Epidemiology and Public Health Department, University of Maryland School of Medicine, Baltimore, Maryland, United States of America aff003
Vyšlo v časopise: PLoS ONE 14(9)
Kategorie: Research Article
doi: 10.1371/journal.pone.0223008


Nucleases are enzymes that can degrade genomic DNA and RNA that decrease the accuracy of quantitative measures of those nucleic acids. Here, we study conventional heating, standard microwave irradiation, and Lyse-It, a microwave-based lysing technology, for the potential to fragment and inactivate DNA and RNA endonucleases. Lyse-It employs the use of highly focused microwave irradiation to the sample ultimately fragmenting and inactivating RNase A, RNase B, and DNase I. Nuclease size and fragmentation were determined visually and quantitatively by SDS polyacrylamide gel electrophoresis and the mini-gel Agilent 2100 Bioanalyzer system, with a weighted size calculated to depict the wide range of nuclease fragmentation. Enzyme activity assays were conducted, and the rates were calculated to determine the effect of various lysing conditions on each of the nucleases. The results shown in this paper clearly demonstrate that Lyse-It is a rapid and highly efficient way to degrade and inactivate nucleases so that nucleic acids can be retained for down-stream detection.

Klíčová slova:

Oxygen – Reactive oxygen species – Vibrio cholerae – Nucleases – Ribonucleases – Microwave radiation – Deoxyribonucleases – SDS polyacrylamide gel electrophoresis


1. Ressler VT, Raines RT. Consequences of the Endogenous N-Glycosylation of Human Ribonuclease 1. 2019:987.

2. Raines RT. Ribonuclease A. 1998:1045.

3. On the Structure of Bovine Pancreatic Ribonuclease B. Isolation of a Glycopeptide. 2018. edsstc.

4. Arnold U, Ulbrich-Hofmann R. Kinetic and Thermodynamic Thermal Stabilities of Ribonuclease A and Ribonuclease B. 1997:2166.

5. Del Vecchio P, Catanzano F, de Paola B, Barone G. Thermodynamic stability of ribonuclease B. 2000:363.

6. Kreuder V, Dieckhoff J, Sittig M, Mannherz HG. Isolation, characterisation and crystallization of deoxyribonuclease I from bovine and rat parotid gland and its interaction with rabbit skeletal muscle actin. European Journal of Biochemistry. 1984;139(2):389–400. doi: 10.1111/j.1432-1033.1984.tb08018.x 6230231.

7. Yang W. Nucleases: diversity of structure, function and mechanism. Quarterly Reviews of Biophysics. 2010;44(1):1. doi: 10.1017/S0033583510000181 20854710

8. Parsiegla G, Noguere C, Santell L, Lazarus RA, Bourne Y. The Structure of Human DNase I Bound to Magnesium and Phosphate Ions Points to a Catalytic Mechanism Common to Members of the DNase I-like Superfamily. 2012:10250.

9. Scientific T. Product Information: DNase I, RNase-free Thermo Scientific: Thermo Scientific; 2016 [cited 2019 April 22nd]. https://www.thermofisher.com/document-connect/document-connect.html?url=https%3A%2F%2Fassets.thermofisher.com%2FTFS-Assets%2FLSG%2Fmanuals%2FMAN0012000_DNase_I_RNasefree_1UuL_UG.pdf&title=VXNlciBHdWlkZTogRE5hc2UgSSwgUk5hc2UtZnJlZSwgMVUvdUw=.

10. Zhang Y, Agreda P, Kelley S, Gaydos C, Geddes CD. Development of a Microwave—Accelerated Metal-Enhanced Fluorescence 40 Second, <100 cfu/mL Point of Care Assay for the Detection of Chlamydia Trachomatis. IEEE Transactions on Biomedical Engineering. 2011;58(3):781. doi: 10.1109/TBME.2010.2066275 20709634

11. Wang Y, Li Y, Santaus TM, Newcomb CE, Liu J, Geddes CD, et al. In situ enzymatic conversion of Nannochloropsis oceanica IMET1 biomass into fatty acid methylesters Microwave-accelerated method for ultra-rapid extraction of Neisseria gonorrhoeae DNA for downstream detection. Bioenergy research. 2016;510:33–40. Epub 2016/06/22.

12. Santaus TM, Melendez JH, Negesse MY, Harvery A, Cyr M, Ladd P, et al. Lyse-It™: A Rapid Platform for Cellular Lysing and Tunable DNA/Protein Fragmentation. In: Geddes CD, editor. Microwave Effects on DNA and Proteins: Springer; 2017. p. 275–96.

13. Melendez JH, Santaus TM, Brinsley G, Kiang D, Mali B, Hardick J, et al. Microwave-accelerated method for ultra-rapid extraction of Neisseria gonorrhoeae DNA for downstream detection. Analytical Biochemistry. 2016;510:33–40. doi: 10.1016/j.ab.2016.06.017 27325503

14. Santaus TM, Li S, Ladd P, Harvey A, Cole S, Stine OC, et al. Rapid sample preparation with Lyse-It(R) for Listeria monocytogenes and Vibrio cholerae. PLoS One. 2018;13(7):e0201070. Epub 2018/07/26. doi: 10.1371/journal.pone.0201070 30044836 a patent related to microwave-based lysing and DNA fragmentation (“Assays for pathogen detection using microwaves for lysing and accelerating metal-enhanced fluorescence”, US9500590B2). Those patents are licensed to Lyse-It LLC, a Maryland-based biotechnology company, which Professor Geddes currently holds stock in. This does not alter our adherence to PLOS ONE policies on sharing data and materials. 30044836

15. Corporation WB. Deoxyribonuclease I Worthington Biochemical Corporation2019 [cited 2019 April 22nd]. http://www.worthington-biochem.com/DNASE/default.html.

16. Waris G, Alam K. Attenuated antigenicity of ribonucleoproteins modified by reactive oxygen species. 1998:33.

17. Knight JA. Diseases related to oxygen-derived free radicals. Annals Of Clinical And Laboratory Science. 1995;25(2):111–21. 7785961.

18. Probes M. Indicators for Highly Reactive Oxygen Species. Journal of Biological Chemistry; 2003. p. 3170.

19. Krumova K, Cosa G. Overview of Reactive Oxygen Species. 2016:3.

20. Halliwell B. Oxygen and nitrogen are pro-carcinogens. Damage to DNA by reactive oxygen, chlorine and nitrogen species: measurement, mechanism and the effects of nutrition. 1999:37.

21. Tripathy S, Mohanty PK. Reactive Oxygen Species (ROS) are Boon or Bane. International Journal of Pharmaceutical Sciences and Research. 2017;8(1):1–16.

22. Ray RS, Mujtaba SF, Dwivedi A, Yadav N, Verma A, Kushwaha HN, et al. Singlet oxygen mediated DNA damage induced phototoxicity by ketoprofen resulting in mitochondrial depolarization and lysosomal destabilization. Toxicology. 2013;314:229–37. doi: 10.1016/j.tox.2013.10.002 24128752

23. Santaus T. Focused microwaves for cellular lysing, DNA/RNA and protein/enzyme fragmentation. In preparation2019.

24. Santaus TM, Suri P, Bhagat S, Geddes CD. Elucidation of the non-thermal mechanism for DNA/RNA fragmentation and protein degradation using Lyse-It ®. In Preparation2019.

25. Agnez-Lima LF, Melo JTA, Silva AE, Oliveira AHS, Timoteo ARS, Lima-Bessa KM, et al. Review: DNA damage by singlet oxygen and cellular protective mechanisms. Mutation Research-Reviews in Mutation Research. 2012;751:15–28.

26. Breen AP, Murphy JA. Reactions of oxyl radicals with DNA. Free Radical Biology & Medicine. 1995;18(6):1033–77. doi: 10.1016/0891-5849(94)00209-3 7628729.

27. Chang S, Hu L, Xu Y, Li X, Ma L, Feng X, et al. Inorganic nitrate alleviates total body irradiation-induced systemic damage by decreasing reactive oxygen species levels. International Journal of Radiation Oncology, Biology, Physics. 2018. doi: 10.1016/j.ijrobp.2018.11.021 30458235

28. Schuch AP, Moreno NC, Schuch NJ, Menck CFM, Garcia CCM. Review Article: Sunlight damage to cellular DNA: Focus on oxidatively generated lesions. Free Radical Biology and Medicine. 2017;107:110–24.

29. Scientific F. Promega Wizard Genomic DNA Purification Kits Thermo Fisher Scientific2015 [cited 2015].

30. Santaus TM, Li S, Saha L, Chen WH, Bhagat S, Stine OC, et al. A Comparison of Lyse-It to Other Cellular Sample Preparation, Bacterial Lysing, and DNA Fragmentation Technologies. In Submission PLoS One. 2019.

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2019 Číslo 9

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