Nucleases are a broad and diverse class of enzymes that hydrolyze the phosphodiester bonds of DNA and RNA.
In nature, they play crucial roles in genetic quality control, such as in DNA proofreading during replication, base, nucleotide, mismatch, and double-strand repairs, homologous recombination, and turnover.
Nucleases have also found widespread use in molecular and cell biology applications which require precise manipulation of nucleic acids, such as restriction digestion, degradation of selected nucleic acids, and trimming.
Nucleases are diverse in their functions and selectivity for certain types of nucleic acid substrates, and thus can be further classified into distinct categories.
Nuclease Types:
-
Endonucleases:
Endonucleases cleave DNA and RNA from within the middle of the chain, with varying levels of site recognition.
Restriction endonucleases, or
restriction enzymes
, are sequence specific, and are widely used in cloning and gene analysis. Others, like DNase I and Benzonase are indiscriminate and are used to fully digest DNA or RNA samples.
-
Exonucleases:
In contrast to endonucleases, exonucleases cleave off nucleotides one at a time from the 3’ or 5’ ends of DNA and RNA chains. Applications of exonucleases include selective degradation of single-stranded DNA and removal of overhangs.
-
DNases:
DNases, or deoxyribonucleases, selectively digest DNA over RNA and different types can cut either internally or from the ends. In research, DNase I is often used in removing contaminating DNA from RNA and protein samples, cleaning cell cultures, and in assays involving DNA fragmentation.
-
RNases:
Ribonucleases, in turn, are selective to digesting RNA over DNA. RNases (commonly RNase A and RNase H) are often used for removing contaminating RNA from samples and certain RNA assays. RNases (with the exception of RNase H) typically are more selective for single-stranded than hybridized RNA.
- Strand-specific Nucleases:
A number of nucleases have found use for their selectivity for either single- or double-stranded nucleic acids.
Micrococcal nuclease,
S1 nuclease, and
Mung Bean nuclease
are selective for single-stranded DNA and RNA (including DNA-RNA hybridizations), and are used for selective digestion and removal of overhangs.
Duplex-specific nucleases
, in turn, prefer double-stranded DNA or RNA.
- Other nucleases:
Cas9
, or “CRISPR associated protein 9,” is a Streptococcus pyogenes-derived nuclease that is used in the CRISPR/Cas9 genome editing technique.
Cas9 nuclease, together with a sequence-specific guide RNA, can be used to mutate, insert or delete a specific region with the genome.
Considerations in Nuclease Selection:
A number of commercial sources for nucleases are now available, so it is wise to pay attention to certain key details before making a purchase.
First of all, is the enzyme active?
Check for reported units of activity to ensure that the nuclease has been tested and will work reliably in your desired application.
What are the optimal reaction conditions?
Will it require key metal ion cofactors like calcium or zinc?
Will the chemical environment and pH range of your samples support the nuclease activity?
Some enzymes may also feature thermostable properties or guaranteed
RNase-free formulations.
When in doubt, don't hesitate to contact the manufacturers for any further clarifications.