I am having problems with R-loops dissociating and would like to ask if anyone has successfully made stable ones. These R-loops are composed of a double stranded DNA "bubble" with an RNA strand annealed to one of the DNA stands. I've made them successfully and gel-purified them, but they dissociate during storage. I've tried to store them in RNAse-free water at -20°C, and also TES buffer in the fridge (thinking that freeze-thaw and lack of salt could be issues). I still have dissociated strands. Any help/advice would be appreciated!
Hi Agnes,
For sure salt will help a bit at stabilizing the structure.
Do you know what is dissociating? the RNA from DNA bubble or DNA itself?
How do you see that? Have you got RNA or DNA labelled or both?
Is your heteroduplex RNA/DNA GC rich? maybe that would also help.
Cheers
Christophe
Hello Agnes, in my lab we make model R-loops quite frequently, using various sequences of RNA annealed to DNA, and don't require any chemical modifications of the DNA or RNA. After annealing the strands (typically these are 25 to 65 nt in length, sometimes with a 5' handle and or a 3' hairpin) overnight to room temperature in sodium citrate buffer we purify the annealed substrate through a 10% acrlymaide TBE gel and then elute the R-loop by diffusion at 4 degrees into buffer that is Tris.Hcl pH7.5 + 100 mM sodium chloride. These remain stable for 2-3 weeks usually. Maybe this gives you some help that you can apply to your method
best,
ed
Thanks all for your helpful suggestions!
Matteo: we haven't considered modified backbones because we're investigating cleavage reactions, but will think about this a bit more.
Christophe: I have all three strands labeled (one per R-loop), and can see that some of the R-loop is intact, some appear to be the DNA-RNA hybrid, and some are ssDNA or ssRNA (but no dsDNA bubble without RNA). I checked the DNA sequence and it's GC rich in the homologous arms (which are 25bp), but the mismatch in the bubble is all T's on one strand. Could this be the source of the problem?
Edward: My method is very similar to yours: annealing in citrate buffer (SSC), purification on 10% PAGE, elution into Tris with 50mM NaCl overnight in fridge, EtOH ppt, and storage in fridge in Tris with 50mM NaCl. Do you skip the ethanol precipitation?
Hi Agnes, I do miss out the ethanol precipitation, although I cannot be sure that this is the reason for your problem. I just asked a PhD student in the lab who made some last week, and he said he tends to make them fresh and use them for just 4-5 days and then dipsoses of them before making more, because they tend to fall apart slowly.
ed
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Molecular Biological Techniques