Figure 1. Schematic diagram of DNase I cleaving dsDNA in the presence of Mg²⁺ and Mn²⁺

Common DNase I is primarily purified from bovine pancreas or is a recombinant enzyme. Compared to DNase I purified from bovine pancreas, recombinant DNase I has relatively lower endogenous RNase levels or can be formulated as RNase-free products, making it more suitable for RNase-sensitive applications, such as the removal of DNA from RNA samples.

Applications

Regarding applications, DNase I is well-known for its use in experiments related to maintaining RNA integrity, such as extracting RNA free of DNA, preparing RNA templates without gDNA before reverse transcription, and degrading DNA templates after in vitro transcription. Additionally, it can be used to digest DNA probes in rRNA removal, for DNA labeling through nick translation, analyzing DNA-protein interactions using the footprinting method, generating random DNA libraries, reducing the viscosity of cell lysates or protein extracts, digesting cell adhesions as an additive in cell culture, and partially cleaving genomic DNA as a positive control in TUNEL assays for apoptosis detection. In summary, DNase I can be used in almost any application requiring enzymatic digestion of DNA. Below, a brief introduction to several common applications will be provided.

• Removal of gDNA before RNA extraction or reverse transcription

RNA, as a commonly studied sample in laboratories, greatly influences the quality of experimental data due to its own quality. It is generally impossible to completely avoid the residual of gDNA during the RNA extraction process. Therefore, before conducting challenging downstream applications (such as mRNA expression analysis, transcriptome analysis, etc.), it is usually recommended to treat RNA samples with DNase I to digest residual gDNA. The digestion of gDNA can be carried out during RNA extraction, after RNA extraction, or before RNA reverse transcription.

Figure 2. DNase I-based gDNA removal process

• Removal of template DNA in in vitro transcription

In vitro transcription (IVT) primarily uses DNA as a template to produce RNA under the influence of appropriate substrates and buffers. Commonly used RNA polymerases in this process include T7, T3, and SP6, which are responsible for catalyzing RNA synthesis. However, the synthesized RNA product may contain DNA residues, and eliminating these residues is beneficial for downstream experiments.

Especially in the development process of mRNA vaccines, removing these DNA residues is a critical step that directly affects the difficulty of subsequent purification processes and the purity of the final product. To efficiently eliminate template DNA, DNase I is typically used for treatment to ensure that the RNA sample is free of DNA residues, a step that helps improve the overall accuracy and efficiency of the experiment.

Figure 3: In vitro transcription process using linearized plasmid as a template^[4]

• rRNA removal in RNA library construction and sequencing 

In organisms, rRNA is highly abundant and highly conserved, which is of little significance for obtaining biological information research. However, 95% of the total RNA extracted during experiments is human rRNA, and the presence of these rRNAs may interfere with the detection of target RNAs. Therefore, in RNA library preparation and sequencing, rRNA is usually removed first. Currently, the main method for rRNA removal is RNAase digestion, and its main operational steps and principles are as follows:

1. Extract Total RNA;
2. Hybridize single-stranded DNA probes with rRNA molecules (Note: Design and synthesize rRNA-specific single-stranded DNA probes);
3. RNase H degrades the hybridized rRNA;
4. DNase I degrades the DNA probes;
5. rRNA is successfully removed, leaving behind non-rRNA RNA templates.

Figure 4: Schematic diagram of the rRNA removal principle based on enzymatic method^[5]

• Nick translation for DNA labeling

Nick translation is the most commonly used method for labeling deoxyribonucleic acid probes in the laboratory. This method is achieved through the combined action of DNase I and E.coli DNA Polymerase I.

The main principle is as follows:

1. First, use an appropriate concentration of DNase I to create several single-strand nicks in each strand of the double-stranded DNA to be labeled, forming 3' hydroxyl termini at the nick sites.
2. Then, use the 5'→3' exonuclease activity of coliDNA Polymerase I to remove a nucleotide from the 5' end of the nick, while the 5'→3' polymerase activity of E. coli DNA Polymerase I introduces a labeled nucleotide to the 3' end of the nick to repair it. As the nick moves along the DNA strand, the labeled nucleotides are incorporated into the newly synthesized DNA strand.

Figure 5: Schematic diagram of the principle of DNA labeling by nick translation^[6]

• DNase I footprinting assay experiment

The DNase I footprinting assay is a precise method for identifying the binding sites of DNA-binding proteins on DNA molecules. The principle is that proteins bound to a DNA fragment protect the bound region from being degraded by DNase I. After enzymatic digestion, the remaining fragment (the "footprint") can be used to determine its sequence. In the gel image, there are no bands corresponding to the regions where the protein is bound.

The main principle is as follows:

1. Label the single-stranded ends of the double-stranded DNA molecule to be tested.
2. Mix the protein with DNA and add an appropriate amount of DNase I for enzymatic digestion, forming DNA fragments of varying lengths. The amount of enzyme should be controlled to ensure that adjacent DNA fragments differ by only one nucleotide, and a control without added protein should be included in parallel.
3. Remove the protein from the DNA, separate the denatured DNA by PAGE electrophoresis, and autoradiograph to interpret the nucleotide sequence of the footprint region by comparison with the control group.

Figure 6: Schematic diagram of the principle of DNase I footprinting assay^[7]

The DNase I Selection Guide of Yeasen

To meet various application needs, Yeasen offers recombinant DNase I in E. coli, and can provide GMP-grade DNase I to meet the requirements of mRNA in vitro transcription and other applications.

Product positioning	Application	Product name	catalog number
RNase Free	removing DNA from RNA and protein preparations	Recombinant DNase I (RNase-free)	10325ES
GMP-grade, RNase Free	In vitro transcription of mRNA	UCF.ME® Deoxyribonuclease I (DNase I) GMP-grade	10611ES

 

References

[1] Ndiaye C, Mena M, Alemany L, et al. HPV DNA, E6/E7 mRNA, and p16INK4a detection in head and neck cancers: a systematic review and meta-analysis[J]. The Lancet Oncology, 2014, 15(12): 1319-1331.

[2] Broccolo F, Fusetti L, Rosini S, et al. Comparison of oncogenic HPV type‐specific viral DNA load and E6/E7 mRNA detection in cervical samples: results from a multicenter study[J]. Journal of medical virology, 2013, 85(3): 472-482.

[3] Huang Z, Fasco M J, Kaminsky L S. Optimization of Dnase I removal of contaminating DNA from RNA for use in quantitative RNA-PCR[J]. Biotechniques, 1996, 20(6): 1012-1020.

[4] Kang D D, Li H, Dong Y. Advancements of in vitro transcribed mRNA (IVT mRNA) to enable translation into the clinics[J]. Advanced Drug Delivery Reviews, 2023, 199: 114961.

[5] Wiame I, Remy S, Swennen R, et al. Irreversible heat inactivation of DNase I without RNA degradation[J]. Biotechniques, 2000, 29(2): 252-256.

[6] Adolph S, Hameister H. In situ nick translation of metaphase chromosomes with biotin-labeled d-UTP[J]. Human genetics, 1985, 69: 117-121.

[7] Song C, Zhang S, Huang H. Choosing a suitable method for the identification of replication origins in microbial genomes. Front Microbiol, 2015, 6: 1049.