I am trying to study the binding of a transcription factor to a synthetic DNA oligo. I want to modify my oligo in such a way, that some part of the oligo is blocked/or made inaccessible to the transcription factor for binding, while a part of it still remains available for binding. Any help would be much appreciated.
Thanks
You can either introduce some mutations that will alter the binding of your TF of interest or introduce a modified nucleotide. There are a whole lot of them (see for example http://www.trilinkbiotech.com/nucleotides/, first result of a simple google search with "modified nucleotides" as input), so yopu may find what you're looking for. Good luck!
well if you make a synthetic oligo you can hang there everything chemically you can imagine and which blocks the binding - so ask what they can put to your synthetic oligo which blocks binding efficiently and yes http://www.trilinkbiotech.com/nucleotides is one website, but there are others as well, you will find even by a simple google search...
Is it safe same to assume then that the TF is binding in the major groove of DNA since you mentioned methylation?
i would suggest checking out IDT (Integrated DNA Technologies). They offer a large selection of modifications. You could try using a fluorescent tag which could both block the TF from binding.
well, what is safe about a binding of any protein if you want to test with modifications whether it binds or not?
so, answer: it might be an educated guess, it might be correct to assume so...
consequently, be open to the unexpected and find out...
sorry seems to be an odd answer, but always pull the carpet below your feet and start thinking from scratch. so many people start project with such guesses, but what if they are wrong and you do not realise? so better to be always alert. if the educated guess and what people believe is right, the better. if not, then you proof an assumption wrong and that might really be major breakthrough then - so always also look for internal controls or consistency in your experimental setups. so have a broad view...
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sorry strange answer perhaps, well, the more reviews of paper or research proposals I do, the more I find things where I shake my head because people either do not know or assume things, or did not check or whatever - but perhaps I am also just getting old...
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The reason I mentioned except from methylation is because there are reports suggesting that methylation does not impair binding. The TF of my interest is known to bind to both major and minor grooves.
This is not my field of expertise, but I think that the synthetic nucleotides, isocytosine and isoguanine, may be of use, as Tobias mentioned.
Or, is there perhaps a way to design your oligo is such a way that promotes self folding at one end and still allow the binding to the DNA with the other? I am not sure if this is even feasible, but it sounds kind of cool.
jessy, whether methylation or any other modification, e.g. as mitchell proposes, impairs or even fosters binding is a matter of the concrete DNA sequence and protein. so what might be true for one, might not be for another and vize versa. of course there are general rules of experience, but do they hold in your case ??? so I guess you have to try it out...
what Noa says is definately a possibility, the point is, of course, that a change in the 3D structure could also lead to an enhancement of binding...
and also think of the fact that it is not only the structure which is changing, but also the dynamics, so how atomes, bases, or the entire chain of bases internally move - and this also influences the binding parameters...
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