Duplex-specific nuclease

cat.# EA001; EA002; EA003

- Specific to double-stranded DNA
- Thermostable
- Inhibited by EDTA

Duplex-specific nuclease (DSN) is an enzyme purified from hepatopancreas of Kamchatka crab (Shagin et al., 2002). DSN shows a strong preference for cleaving double-stranded (ds) DNA and DNA in DNA-RNA hybrid duplexes, compared with single-stranded (ss) DNA and RNA. Moreover, the cleavage rate of short, perfectly matched DNA duplexes by this enzyme is considerably higher than that for nonperfectly matched duplexes of the same length. DSN finds use in various applications to isolate single-stranded DNA from complex nucleic acids, for example in cDNA normalization method (Zhulidov et al., 2004, Zhulidov et al., 2005; Bogdanova et al., 2008), for quantitative telomeric overhang determination (Zhao et al., 2008), and for SNP detection (Shagin et al., 2002).

Main properties of DSN

Substrate specificity: DSN exhibited strong cleavage preference for ds DNA substrates. No significant cleavage activity on RNA substrates and ss DNA is observed with working DSN concentrations (Zhao et al., 2008). dsDNA:ssDNA cleavage ratio is about 1000. Moreover, the nuclease effectively cleaves DNA molecules in DNA-RNA hybrid duplexes.

	Action of DSN on ss DNA of phage M13 and ds DNA of phage λ. Lanes 1, 2 - negative controls, incubation without nuclease. Lane 1: phage M13 DNA alone, Lane 2 - mixture containing phage M13 and λ DNA. Lanes 3; 4 - digestion of phage M13 and λ DNA mixture by DSN at 70°C for 1.5 min (lane 3) and 5 min (lane 4).

Analysis of DSN action on synthetic oligonucleotide substrates revealed that the enzyme discriminates between perfectly matched short DNA-DNA duplexes (10-12 bp) and duplexes of the same length with at least one mismatch. It requires at least 10 bp DNA or 15 bp DNA-RNA perfect duplex for cleavage.

DSN action on one mismatch-containing and perfectly matched DNA duplexes. Duplexes formed by 5-carboxyfluorescein (Fl)-5'-gccctatagt-3'-TAMRA signal probe and complementary targets ( 5’-actcactataCggcgaat-3’ and 5’-actcactatagggcgaat-3’) were incubated with DSN at 35°C for 15 min. Emission spectra were obtained on the spectrofluorimeter, with excitation at 480 nm. Dotted line - substrate fluorescence in the absence of enzyme; firm line - substrate fluorescence after incubation with DSN.

Biochemical properties

DSN acquires its enzymatic activity in the presence of divalent cations (Mn²⁺, Co²⁺, or Mg²⁺). Mg²⁺ ion concentration for most applications should be at least 5 mM. DSN is inhibited by EDTA.

The temperature optimum for activity is 60°C. Despite a high optimal temperature, DSN retained only 10% activity as early as at 80°C. This sharp decrease in activity may be attributable, at least in part, to dsDNA substrate denaturation.

The optimal pH for DSN activity was estimated as 6.6. At pH values between 3 and 5, DSN displayed only 10% of its maximal activity. The nuclease is stable at a wide range of pH (from 4 to 12) and temperatures below 65°C. About 60% of DSN activity remains after 30-min incubation at 70°C, and 40% - after incubation at 80°C.

Effects of various conditions on DSN activity. Activity of DNAse on ds DNA substrate was measured using modified Kunitz assay (Kunitz, 1950)

Incubation of DSN with aggressive chemicals like 1% SDS, 10 mM β-mercaptoethanol, and 0.3% hydrogen peroxide at 37°C resulted in only a moderate drop in activity, and ~90% activity was maintained after 30 min incubation. However, a sharp decrease in activity was observed upon chemical treatment at 60°C. SDS completely inhibited DSN activity, while β-mercaptoethanol and hydrogen peroxide induced approximately 70% and 80% loss in activity, respectively.

DSN is highly sensitive to ionic force (e.g., a 10 times decrease in catalytic activity is observed in the presence of 0.2 M NaCl). The addition of chaotropic agents or polyamines to the reaction mixture also resulted in suppression of enzyme activity.

DSN is tolerant to proteinase K treatment (incubation at 37°C for 30 min).