Choosing the Best Exonuclease for Your Workflow
Posted on Tuesday, October 29, 2024
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Topic: Tips for the lab
Selecting the exonuclease that meets your application needs is easier with NEB’s technical insight and user-friendly selection tools. Drawing from our long history in enzymology and solutions for basic research and applied scientific development, we’re sharing answers to some common questions about exonucleases and providing the resources you can use for successful exonuclease selection.
Exonucleases are essential enzymes in many in vivo biological processes, including DNA damage repair, error correction, and recombination. Exonucleases hydrolyze phosphodiester bonds in nucleic acids, ultimately digesting the polymer into mononucleotides or short oligos, depending on the enzyme. Exonucleases require a free nucleic acid end to initiate activity, which differentiates them from endonucleases, which can initiate in the middle of a nucleic acid polymer and can even digest closed circular molecules. Exonucleases can exhibit directionality (also referred to as polarity), digesting in either the 5´ to 3´ direction, the 3´ to 5´ direction, or bifunctionally. Exonucleases can prefer single-stranded (ss) or double-stranded (ds) DNA, or can digest both with near equal activity.
Exonucleases can digest in multiple directions and have several important applications.
In addition to their critical roles in many organisms, exonucleases are also important enzymatic tools for selectively degrading nucleic acids in many in vitro molecular biology applications, such as next-generation sequencing (NGS), plasmid cleanup, site-directed mutagenesis, and gene synthesis. Check out some frequently asked questions about working with exonucleases below.
How do I choose the right exonuclease for my workflow?
NEB has several resources to help you choose the correct exonuclease.
Selection Chart
The Properties of Exonucleases and Nonspecific Endonucleases chart provides an overview of the activity for each enzyme, including the polarity, activity on different forms of ssDNA and dsDNA, the expected products of digestion, and any relevant notes for each specific enzyme. This chart is a great resource to start your selection process.
Interactive Tool
The Exo Selector webtool allows you to specify the different nucleic acid forms present in your reaction mixture and indicate which you want to digest and which you want to retain. The tool will then suggest the appropriate enzyme(s) to achieve your desired outcome. For example, if you are trying to remove excess ssDNA primers from a polymerase chain reaction (PCR) but retain the dsDNA PCR products, the Exo Selector tool will suggest exonucleases which meet this requirement. Exonuclease I is the enzyme typically used for this application, but the Exo Selector tool presents all options that satisfy the specified requirements, allowing you to choose the enzyme that will be most compatible with your workflow.
Webinar
To see specific examples of exonuclease use in various workflows, refer to our Exonucleases and Endonucleases as Molecular Tools Webinar. Here, you can learn more about a workflow using molecular inversion or padlock probes; after the circularization of a single stranded probe, you can enrich for circular ssDNA using Exonuclease VII which degrades any remaining unligated linear probe DNA.
Are exonucleases blocked by modifications? Which modifications block exonuclease progress?
Some, but not all, exonucleases are sensitive to modification of the nucleic acid substrate. In some applications, such as ssDNA production or second strand synthesis in NGS library preparation, you may wish to retain one molecule of nucleic acid while digesting another, despite both being potential substrates for your exonuclease of choice. In these cases, selective modification of a nucleic acid can prevent degradation by exonucleases. NEB scientists have evaluated the activity of exonucleases on nucleic acid substrates containing a variety of chemical modifications, including modifications to the phosphodiester backbone, the nucleobase, and the sugar moiety.
The most common modification for blocking or halting exonuclease activity is the phosphorothioate (pt) bond. The pt linkages must be installed at the end(s) where the exonuclease to be blocked initiates. A single pt bond is insufficient to block cleavage by most exonucleases. Tests confirm five consecutive pt bonds are required to effectively block degradation. Importantly, even multiple pt bonds are not sufficient to block certain exonucleases that can scan past blocking linkages to begin digestion deeper in the polymer, such as Exo VII, and some also have endonuclease activity, such as T5 Exonuclease. We also found that bulky substituents at the 2´ position of the sugar, such as a 2´-O-methoxyethyl (MOE) modification, can provide even more robust protection against the activity of many exonucleases. We recommend incorporating at least three MOEs in a row in the appropriate position to block/halt the exonuclease of interest. Both pt bonds and MOEs are standard modifications offered by many oligonucleotide synthesis companies. Modification of the nucleobase is generally not an effective strategy for blocking exonucleases.
For a comprehensive summary of our results, please see the feature article, The effect of nucleic acid modifications on digestion by DNA exonucleases.
Modified bonds, such as 5 pt bonds, and modified sugars, such as 3 MOEs, can both block exonuclease digestion.
Can exonucleases digest DNA/RNA hybrids?
The ability to digest DNA/RNA hybrids varies between different exonucleases. In most cases, exonucleases do not change specificity on DNA/RNA hybrid helices. ssDNA-specific exonucleases are generally blocked by hybridization to an RNA strand, while dsDNA specific exonucleases will digest the DNA strand of DNA/RNA hybrid helices and exhibit some ability to degrade the RNA strand. Several nucleases, including T7 Exonuclease, Exonuclease III, and T5 Exonuclease, have significant activity on the RNA strand of a DNA/RNA helix. If it is desirable to digest the DNA strand of a DNA/RNA helix while leaving the RNA portion completely intact, we recommend Duplex DNase.
What should I do if I see unwanted digestion?
If you are seeing digestion of nucleic acids you want to retain, we recommend titering the enzyme (gradually decreasing the amount of enzyme added) to empirically determine the appropriate amount required for your particular application. In some cases, you can also modulate the amount of activity by increasing or decreasing reaction time or temperature.
It is important to note that many exonucleases have multiple activities, which can lead to unwanted digestion products. For example, in addition to exonuclease activity, Exonuclease III is reported to have RNase H, 3´ phosphatase, and AP endonuclease activities (Rogers and Weiss, 1980). Additionally, there is a wide variety of processivity among exonucleases, with some acting in an extremely processive manner and others that catalyze only a few rounds of digestion before dissociating. This processivity can be impacted by reaction conditions, such as temperature and buffer, as well as sequence specificities that are not fully characterized.
Why are exonucleases important?
Exonucleases are critical enzymes used for selective degradation of nucleic acids in a variety of applications, such as PCR, NGS, and gene synthesis. It is important to note the specific preferences of each exonuclease, as awareness of these activities and nuances will inform your choice of enzyme. Additionally, it is important to recognize that it may be necessary to empirically determine the best conditions for your experiment.
For a full list of NEB exonucleases, please refer to our product listing.
Further resources:
- Activities of Exonucleases and Non-specific Endonucleases
- Common Applications for Exonucleases and Endonucleases
- NEB technical support, available at 1-800-632-7799
References:
- Rogers, G.S. and Weiss, B. (1980). L. Grossman and K. Moldave(Ed.), Methods Enzymol.. 65, 201-211. New York: Academic Press.
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