I have a linear ssDNA oligo that is designed such that the ends are complementary, and conducive to hairpin formation (see attached figure). My question is, which has a higher likelihood of formation, a hairpin, or a self-dimer? Would the likelihood of hairpin formation increase if I decrease the concentration of the ssDNA oligo during the annealing process? If I enter the sequence in IDT oligo analyzer, the gibbs free energy for formation of self-dimer is significantly lower compared to hairpin.
The melting temperature of the complementary part of my oligo is 50 deg. The annealing will be done in nuclease free water, without any Mg+.
To some degree the likelihood of self dimer vs hairpin formation will be DNA dependent, high DNA concentrations are likely to drive dimer formation. However hairpin formation also needs to account for the energetic cost of folding the DNA strand back upon itself. What is the energetic cost of the bend and the cost of the repulsion of the non paired bases on the oligo (although maybe the IDT oligo analyzer is taking that into account, I'm not sure).
Michael J. Benedik , Yes, I shall play around with the concentration and see if that helps. Thank you.
Hi Ranjit Gulvady
The hairpin formation will significantly dominate.
I know exactly what you mean. In my work "Electronic pH switching of DNA triplex reactions" I have employed a 53 nt oligo that was folding into 24 bp hairpin with 5 nt loop. I have run subsequently electrophoresis to confirm the mobility of 24 bp DNA duplex.
Antonio S Minero , thank you for your response, it's very useful for me!
Did you also find that you need to play around with the concentration of the 53 nt oligo to eliminate/reduce the possibility of self-dimerization, or was the hairpin formation preferred irrespective of the concentration?
I used 1 µM concentration of that oligo (H53). So I think it will be same for the lower concentrations but might be different for higher concentrations...
So an oligonucleotide with a self-complementary sequence X-Y is forming hairpin X/Y. However, a potential competing reaction would be formation XY/YX. Remember that between X and Y, you have a spacer (loop). That spacer keeps the self-complementary parts in the closest possible proximity, so formation of hairpin dominates.
I have also observed that the 24bp hairpin had melting temperature by about 12 degrees higher than the non-looped duplex with the same sequence.
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