Part:BBa_K4778006

DSN

Duplex-specific nuclease (DSN) is an enzyme purified from hepatopancreas of Red King (Kamchatka) crab [1]. DSN exhibits strong cleavage preference for ds DNA substrates. It can show minor activity against ss DNA when both DSN enzyme and substrate are present at high concentrations, however this activity is not detectable in presence of competitive ds DNA and RNA [2]. DSN does not cleave RNA, but effectively cleaves DNA in DNA-RNA hybrid duplexes [1].

Biology

Anisimova et al. [3] and Qiu et al. [4] summarized the relevant properties of DSN enzymes. DSN enzyme protein is a monomer with a molecular weight of 41.5kD and an isoelectric point of 4.2. DSN enzyme activation requires the addition of magnesium ions (at least 5mmol/L, the optimal concentration is 7mmol/L). Ca2+ cannot directly activate DSN enzyme, but it can significantly enhance the activation ability of Mn2+, Co2+ and Mg2+ on the enzyme, resulting in a synergistic effect. DSN enzyme activity decreases as the ion concentration increases. The effective pH value range is 3-9, and the optimal pH value is 6.6. EDTA can completely inhibit enzyme activity, and DSN enzyme is sensitive to polyamines and chaotropic agents. DSN enzyme can distinguish double-stranded hybrids between completely complementary pairs and incomplete complementary pairs, and can achieve single-base discrimination. Substrate length requirement: Minimum 9bp [3] or 10bp [4] DNA in the DNA double strands. The shorter DNA double strands will not be cut and remain intact. The effective cutting length of the DNA/RNA hybrid double strands is 15bp [4] .

Usage

Duplex-specific nuclease (DSN) is a type of nuclease, that is isolated from the hepatopancreas of the Kamchatka crab. DSN displays a strong preference for cleaving double-stranded DNA or DNA in DNA-RNA heteroduplexes, and is practically inactive toward single-stranded DNA or RNA. Moreover, this enzyme shows excellent discrimination capability between perfectly and imperfectly matched (up to one mismatch) short duplexes. Owing to its unique feature of cleaving DNA, DSN enzyme is widely applied in the fields of biomedicine and molecular biology, including full-length cDNA library normalization,genomic single-nucleotide polymorphism (SNP) detection and high throughput sequencing. The recent research on DSN are mainly focused on the applications in microRNAs (miRNAs) detection using a DSN-mediated signal amplification strategy. miRNAs are a group of short, endogenous, noncoding RNAs that play vital regulatory roles in physiologic and pathologic processes, including hematopoietic differentiation, cell cycle, regulation, and metabolism. So miRNA is one of the most important biomarkers in individualized treatment, which has great value in terms of improving the diagnosis and treatment of diseases. However, detection of miRNAs is challenging owing to their unique characteristics, including a small size, sequence homology among family members, low abundance in total RNA samples, and susceptibility to degradation in solution. In recent years, isothermal signal amplification and detection of trace miRNA in fluids are reported by many researchers using DSN-mediated biosensors.

Results

In order to verify whether DSN has good activity in our system, we tested the cleavage activity of DSN enzyme to linear probe-miR complex at 37 ℃. The results showed that (Fig1) the experimental group produced obvious sgRNA bands (the bands indicated by the arrow), which indicated that DSN could function normally and efficiently in the first generation system we designed.

Fig1. Denaturing PAGE. Arrow denotes sgRNA. We tested the cleavage activity of DSN enzyme on the circular probe-miRNA complex at 37℃(Fig2).The results showed that the experimental group produced distinct sgRNA bands (arrows indicated bands), indicating that DSN can function normally and efficiently in our modified system. We also compared the linear probe with the circular probe. Under the same reaction conditions, after the miRNA-linear probe complex was cut by DSN, the amount of remaining linear probes was reduced, but no obvious sgRNA bands were observed. After the miRNA-circular probe complex cleaved by DSN, obvious sgRNA bands were generated. This shows that the circular probe is more stable and more efficient.

Fig2. Denaturing PAGE. Arrow denotes sgRNA.

References

[1]D.A. Shagin et al. (2002). Genome Res, 12 (12): 1935–1942 / pmid: 12466298

[2]Y. Zhao et al. (2008). Nucleic Acids Res, 36 (3): e14 / pmid: 18073199

[3]Anisimova VE, Rebrikov DV, Shagin DA, et al. Isolation, characterization and molecular cloning of Duplex-Specific Nuclease from the hepatopancreas of the Kamchatka crab[J]. BMC Biochemistry, 2008, 9(1): 14. DOI:10.1186/1471-2091-9-14

[4]Qiu X, Zhang H, Yu H, et al. Duplex-specific nuclease-mediated bioanalysis[J]. Trends in Biotechnology, 2015, 33(3): 180-188. DOI:10.1016/j.tibtech.2014.12.008