We can make lots of modification on 3' or 5' of the DNA oligonucleoties. Say, I add a modification group to 3' of oligonucleotide (such as thiol, amino, biotin, phosphate, or a fluorophore) . Does this modification inhibit/promote the degradation of DNA by exonuclease ExoI that degrades single-stranded DNA in a 3'→5' direction? In another case, If I attached the DNA to nanoparticles at 3', does nanoparticles protect DNA from degradation by exonuclease?
In many cases, this will inhibit exonuclease activity. For example, ExoI is blocked by 3' phosphoryl or acetyl groups (http://www.jbc.org/content/239/8/2628.long).
For ExoI, I would assume that this holds true for most other modifications.
Regarding nanoparticles, it is reported that binding an RNA oligo to gold nanoparticles enhances their stability in 10% serum up to 6 times. Here is the link (http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2843496/ )
I agree with Christan, many modifications can block exonuclease activity especially if they form any phosphotriesters because no exonucleases seem to be able to get around those very well. I think you'll just have to check for each particular modification/exonuclease combination.
I agree that many of the modifications would block exo I- as would attachment of the 3' OH to a nanoparticle. You may also be able to increase stability by modification of the penultimate phosphate linkage as that is the site of action for the enzyme.
Very interesting questions. Exol is affected by the modifications you have specified in the 3' end of the oligonucleotide. From my experience, I can say that the activity is maximally affected by attaching thiol or fluorophore groups. Secondly, if you have succeeded in attaching DNA to nano particles (please specify the bonding and which type of particles), exonuclease might be affected if the 3' end is not free.
ExoI can be blocked by modifications or nanoparticle conjugation as discussed. Another possibility is engineering in a reverse 3'-3' linkage at the terminus. There is some discussion of these and other strategies at the IDT web site: http://www.idtdna.com/
This is also a very interesting issue from the biosafety viewpoint. It is generally assume that oligonucleotides are present only transiently in the cell, a very important argument for having organisms developed by oligo-directed mutagenesis considered as non-GMO. I would be interested to hear views about the possible consequences in term of safety an increased stability of the oligonucleotide could have.
In our experience, modification near the 3' end of the oligo with a dye-modified thymidine does not seem to significantly increase the stability of the DNA with respect to Exo III digestion. Obviously, this will change depending on the chemistry of the modification. Alternatively, incorporating DNA into polymeric nanoparticles via attachment of a polymer at the 5' end of the oligo seems to increase the stability with respect to Exo III digestion significantly (~4x more stable see: Nuclease-Resistant DNA via High-Density Packing in Polymeric Micellar Nanoparticle Coronas ). Similiar effects have been realized on gold nanoparticles for both DNA and RNA (see work of Mirkin et. al.: Polyvalent DNA nanoparticle conjugates stabilize nucleic acids, note DNase I is an endonuclease). This type of stabilization is distinct from stabilization achieved via chemical modification of the nucleobase sequence itself in that the stabilization seems to be a result of high local DNA density resulting in restricted access/binding/activity of nucleases.
Daniel, as already mentioned by many others, there are lots of DNA modifications affecting exonuclease activities. Affections can occur with enzymes from different companies to different amounts (of course if it is meant to be the same enzyme!). In my experience you can check for information in the appendices of the catalogue of New England Biolabs, which provides lots of precious and meaningful information. You can also contact the support, which were always very helpful.
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