Isolation, characterization and molecular cloning of Duplex-Specific Nuclease from the hepatopancreas of the Kamchatka crab

Abstract

BackgroundNucleases, which are key components of biologically diverse processes such as DNA replication, repair and recombination, antiviral defense, apoptosis and digestion, have revolutionized the field of molecular biology. Indeed many standard molecular strategies, including molecular cloning, studies of DNA-protein interactions, and analysis of nucleic acid structures, would be virtually impossible without these versatile enzymes. The discovery of nucleases with unique properties has often served as the basis for the development of modern molecular biology methods. Thus, the search for novel nucleases with potentially exploitable functions remains an important scientific undertaking.ResultsUsing degenerative primers and the rapid amplification of cDNA ends (RACE) procedure, we cloned the Duplex-Specific Nuclease (DSN) gene from the hepatopancreas of the Kamchatka crab and determined its full primary structure. We also developed an effective method for purifying functional DSN from the crab hepatopancreas. The isolated enzyme was highly thermostable, exhibited a broad pH optimum (5.5 – 7.5) and required divalent cations for activity, with manganese and cobalt being especially effective. The enzyme was highly specific, cleaving double-stranded DNA or DNA in DNA-RNA hybrids, but not single-stranded DNA or single- or double-stranded RNA. Moreover, only DNA duplexes containing at least 9 base pairs were effectively cleaved by DSN; shorter DNA duplexes were left intact.ConclusionWe describe a new DSN from Kamchatka crab hepatopancreas, determining its primary structure and developing a preparative method for its purification. We found that DSN had unique substrate specificity, cleaving only DNA duplexes longer than 8 base pairs, or DNA in DNA-RNA hybrids. Interestingly, the DSN primary structure is homologous to well-known Serratia-like non-specific nucleases structures, but the properties of DSN are distinct. The unique substrate specificity of DSN should prove valuable in certain molecular biology applications.

Rational Design of Duplex Specific Nuclease for One-Step Isothermal Viral RNA Detection

“…The impact of varying Mg 2+ concentration on DSN activity has been well documented [15]. Maximum DSN digestion activity is reached at ~20mM Mg 2+ and begins to reduce beyond this concentration [15]. Notably, increasing the amount of Mg 2+ in the reaction mix aided in improving the efficiency of detection in the presence of ATA, and a total Mg 2+ concentration of 10mM provided superior sensitivity for our norovirus assay ( Figure 5C).…”

“…The impact of varying Mg 2+ concentration on DSN activity has been well documented [15]. Maximum DSN digestion activity is reached at ~20mM Mg 2+ and begins to reduce beyond this concentration [15].…”

“…To minimize these limitations, isothermal RNA amplification techniques have been developed [7][8][9][10][11][12][13][14], but these still remain dependent on nucleic acid replication and are therefore hindered by polymerase speed and fidelity. Recent RNA detection methods that have employed duplex specific nuclease (DSN) isolated from the Paralithodes camtschaticus crab [15,16], remain limited to microRNA [17] and are thus unsuitable for longer RNA templates like virus genomic or mRNA targets. DSN is an attractive candidate for the latter, since it preferentially cleaves DNA in a DNA-RNAduplex, leaving ssRNA and dsRNA virtually unchanged [15].…”

“…Recent RNA detection methods that have employed duplex specific nuclease (DSN) isolated from the Paralithodes camtschaticus crab [15,16], remain limited to microRNA [17] and are thus unsuitable for longer RNA templates like virus genomic or mRNA targets. DSN is an attractive candidate for the latter, since it preferentially cleaves DNA in a DNA-RNAduplex, leaving ssRNA and dsRNA virtually unchanged [15]. DSN acts as an endonuclease on DNA, cleaving within the phosphate backbone [16], although the direction of digestion is unknown.…”

Construction and analysis of a subtractive cDNA library of early embryonic development in duck

“…DSN shows maximal activity at 65°C and specifically cleaves DNA into DNA:DNA or DNA:RNA forms (Zhulidov et al, 2004;Anisimova et al, 2008). However, some cDNA fragments broke as a result of the experimental procedure.…”