I am going to be designing DNA oligos for zinc finger sites and I need to avoid edge effects. Are there general principles for the composition of nonspecific flanking sites? I've seen some groups use a restriction site overhang 5' to the binding site (i.e. four single-stranded nucleotides) all the way to nine double stranded base pairs.
As far as I understand you want to design a double stranded DNA fragment containing a recognition sequence of a TF. In general, the design (length, flanking sequences etc.) depends on the experiments you want to do. Please, provide more information. What you mean by "edge effects"? “DNA ends” and “non-specific flanking sequences” are different things.
You are correct in your understanding. I will be doing isothermal titration calorimetry (ITC) experiments.
I am probably using the wrong term with "edge effects". I don't imagine that an oligo that contains the recognition sequence alone, flanked by 5' phosphates and 3' hydroxyls, would simulate physiological conditions. However, neither do I want to design segments of DNA that are impractically long (and expensive!).
Dear Michael,
The design of your DNA oligos must depend upon the zinc finger domain/protein which you are using for ITC. You must have some predicted number of bases which your zinc finger (there are different types of zinc fingers) might bind (for example, one C2H2 zinc finger can recognise 3 bases). I hope you are dealing with more than one zinc finger. Also most of the zinc fingers recognise bases in grooves of DNA, so the formation of DNA structure is important for zinc finger to bind. I guess you have got the hint now. The extra bases can be anything, without forming the secondary structure or short enough, not to make steric hindrance. 4-6 non-specific bases would not give any "edge effects" as you said.. :-)
I hope this might help you! I guess more information would be helpful to have more productive discussion.
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