What are Nicking Enzymes and How are They Used?

Learn more about nicking endonucleases (aka nicking enzymes), how they work and their role in different applications including virus detection and SDA.


Most restriction enzymes cleave both strands of double-stranded DNA. However, nicking endonucleases, or nicking enzymes, cleave only one strand, thereby “nicking” the double-stranded DNA. NEB has engineered nicking enzymes from other restriction enzymes, and also has cloned, expressed and purified naturally-occurring DNA nicking enzymes.

Nicking enzymes are used in a variety of applications. For example, BbvCI- and BspQI-derived nicking variants have been used in DNA optical mapping to study chromosome DNA rearrangement in cancer cells. Nt.CviPII has been used in the study of open chromatin in normal human and cancer cells. Nt.BstNBI, which is also available in a WarmStart version, has been useful in the detection of flu and coronaviruses.

Enzymes prefixed by “Nt.”, such as “Nt.BstNBI”, will nick only the top strand, while enzymes prefixed with “Nb.”, such as “Nb.BbvCI”, will nick only the bottom strand. Similar to other restriction enzymes, a nicking enzyme recognizes a specific DNA sequence and cleaves.Nickases recognize strictly non-palindromic sequences, hence their ability to distinguish the top from bottom strand. Currently, only 2 nicking enzyme structures are known.

Structural work on Nt.BspD6I indicates that Nt.BstNBI recognizes the double-stranded DNA sequence GAGTC and nicks downstream at position N4. The target recognition domain, or TRD, binds to the specific DNA sequence and activates its catalytic center, resulting in the nicking of only the top strand. Because Nt.BstNBI lacks a second, smaller subunit that could nick the bottom strand, the enzyme only nicks the top strand.

Recently, NEB has innovated with the launch of a WarmStart version of Nt.BstNBI. WarmStart technology inhibits enzyme activity below 40°C and allows room-temperature setup without the reaction starting prematurely. This greatly facilitates its use in applications such as strand displacement amplification, or SDA, and exponential amplification reaction, also known as EXPAR chain reaction. NEB continues to engineer and discover more nicking enzymes, particularly in response to specific customer needs.

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