We are examining structures of non-B-DNA such as DNA or DNA-RNA G quadruplex and would like to test if these occur in cells using antibodies. I know some G guadruplex antobides exist but we are interested in novel structures that would likely require new antibodies. Are these robust ways to successfully make structure-specific anti-DNA antibodies that we could try?
Hello John...
Phage display would be rank 1.
Will be difficult with immunization methods
Good luck
Here are two papers on the selection of quadruplex-specific antibody-scFv by phage-display
Schaffitzel, C., Berger, I., Postberg, J., Hanes, J., Lipps, H. J., and Plückthun, A. (2001). In vitro generated antibodies specific for telomeric guanine-quadruplex DNA react with Stylonychia lemnae macronuclei. Proc. Natl. Acad. Sci. U.S.A. 98, 8572-8577. https://www.bioc.uzh.ch/plueckthun/pdf/APpub0202.pdf
and selection of quadruplex-specific DARPins by ribosome display:
Scholz, O., Hansen, S., and Plückthun, A. (2014). G-quadruplexes are specifically recognized and distinguished by selected designed ankyrin repeat proteins. Nucleic Acids Res 42, 9182-9194. https://www.bioc.uzh.ch/plueckthun/pdf/APpub0364.pdf
out of our lab.
Dear John,
For this specific task, I would approach a lab that offers a synthetic library of some sorts (be it scFv, nanobodies, Fabs etc) and a stringent selection process such as phage display. While there are some anti-DNA antibodies out of immunisations, DNA is probably still not the most immunogenic molecule, so a classical mAb campaign may well fail. Also, using in vitro selection, you are able to control buffers etc, which may be helpful for maintaining the specific DNA structure. And you can counter-select against other DNA structures.
Good luck with this project!
Hi John
As some of the replies mention, DNA (and RNA) are poor immunogens, because they are self-antigens which would generate an autoimmune response. In fact, the few initially described come from selection of clones of autoimmune mice (as Falk rightly suggest as an alternative), but not elicited to recognize a particular antigen. Of course selection from phage display is a great choice, it depends on the expertise you have, the time you are prepared to invest and what is your goal with those antibodies. I am sure there are a number of labs with that expertise that you could collaborate with, as Christian suggests.
If you want to generate the antibodies naturally, say in mice (that is elicited against a particular epitope), it is obviously non-trivial, but we have succeeded in doing so by immunizing a particular DNA that binds to a DNA binding domain strongly . In fact, it appears that the protein has a lot to do (I noticed that Falk also suggest a polycharged cationic carrier). These days I would try assembling a larger and stable particle, either by an engineered carrier or simply chemical crosslinking. This would ensure long lasting circulation in the system for immunogenic epitopes to te selected. The work is mostly summarized in one of the references I attach (the first, and the most recent).
We obtain what it appears a couple of antibodies with some sequence specificity for double stranded DNA, but they ended up having a strong affinity and specificity against the 5" trinucleotide. The interaction is fundamentally based on stacking between base aromatics and a few trps and tyrs in the right place in the antibody. Given the size of the required contact area for a double stranded B DNA, I suspect sequence specificity with high affininty for such a ligand is not likely to happen. But this is just speculation.
I would not be surprised if some of the existint a-DNAbs work for your particular goal, or, more laborious, using these as frames for selection of particular sequences.
Cheers
Gonzalo
Article Specific Recognition of a DNA Immunogen by its Elicited Antibody
Article Generation of Sequence-specific, High Affinity Anti-DNA Antibodies
As my first post indicated, in-vitro selection is crucial, whether on a naive library or an immune library generated from mice immunized with the selected target. However, in addition to the poor immunogenicity of nucleic acids, it may not be possible to maintain the desired DNA/RNA form in vivo. In vitro selection allows both positive selection on the desired target and negative selection on the unwanted target, as long as it is possible to maintain the target in the desired conformation throughout the selection.
The Schaffitzel paper cited above presents two antibody scFvs selected in vitro from a synthetic naive antibody library, the HuCAL library (Morphosys), one recognizing both parallel and antiparallel d(T4G4) quadruplexes, the other is specific to parallel quadruplexes.
The DARPin paper describes the selection of bindersin vitro against two different quadruplex targets, (TTAGGG)4 TT which can form one quadruplex unit with different topologies and (TTAGGG)12 that may form up to three quadruplex units, including compact forms with two or three contiguous quadruplexes.
It shows the detailed characterization of the interactions of the selected binders with human sequences thought to form quadruplexes, e.g. human promoter regions of RET, HIF-1α, VEGF, c-KIT1, c-KIT2,ILPR and c-MYC sequences.
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Biomolecules
- Nucleic Acids
- DNA