After heating the oligo, do not immediately transfer it to ice, that only stabilizes the single strand. You should let it slow cool from 70 to RT for about 2 hours and then put it in ice to stabilize the dsDNA formation.

Hi, Thanks for the suggestion. I am actually doing that. I perform dsDNA annealing by heating oligo s to 95 degree for 10 mins and allowing to cool RT for 4 hours. The RNA oligo is heated to 70degree and then transferred to ice to resolve any secondary structure and maintain it that way.

Do you run also the labeled ss oligo alone?

Do you run the EMSA in cold room with cold buffer?

If ss, ds and ds+RNA were run in the same condition, but i) the high mobility band appeared only in the ds+RNA lanes, ii) the high mobility bands run exactly as your labeled ss-oligo, you can reasonably argue that the RNA oligo fully displace the dsDNA.

Maybe performing a time course you should observe the formation of trimeric complex at earlier time points.

Hello Emmanuele,

Yes, I run the EMSA in cold room with cold buffer.

I did run the ssDNA under same conditions and can tell with a fair amount of certainty that the high mobility band that I observe only in the ds+RNA runs exactly as my labelled ss-oligo.

I was considering performing a time course but for longer since I came across an earlier article reporting that Triplex stabilised at 6 hours. However, most protocols have incubation time of around 2-3 hours. I will include an earlier time point as well as see if I observe the complex.

Thanks!

Hi Bhavana,

I want to first congratulate you on describing your problem in such details; rarely here I see questions that are articulated so clearly. I hope you have solved your problem, as I see it is a bit old now.

From your last response to Emmanuele, it appears that your labeled species in all reactions containing the TFO RNA is single-stranded. That means your RNA prep has something that is (1) disrupting the annealed DNA duplex, and (2) not allowing the TFO RNA to anneal to the DNA. I am not sure what it could be. You might contact the facility that provided you the TFO RNA to clear any doubt that it had any contaminants. It won't be bad to re-purify your RNA (by columns; not from gel).

You might know from the ancient Southern blotting (nobody talks about it anymore) that low salt solutions are actually high-stringency washes. That means, the duplex stability requires a favorable salt concentration. So, increasing NaCl from your current 25mM to 50 or 100mM might help stabilize the duplex. Further, why do you have MgCl2 in your annealing reactions? You know that Mg2+ ions can bridge adjacent phosphates and compact the DNA. Such deformation might interfere with duplex/triplex formation. I am not sure, though, that the persistence length would allow deformation of your short oligos. Still, I would suggest you replace MgCl2 with, say, 1mM EDTA. I am also sure that your TFO RNA prep has no trace of MgCl2, but has some EDTA. So, once the duplex/triplex are formed in presence of EDTA (and no Mg2+), you might want to add MgCl2 to stabilize the structure.

You might want to test whether your TFO RNA is capable of duplexing with ssDNA. If it is, then your scheme of reactions should not fail. Now, is your labed ssDNA oligo complementary to the TFO RNA? In that case, you can easily test annealing capability of your TFO RNA. To do that, add TFO RNA (just as you do; heated to 70C, cooled, added to labeled ssDNA; but make doubly sure that it has no trace of Mg2+). After the annealing reaction, treat half of the reactions with RNaseH; at this step, you will require some MgCl2. Run on the gel (i) labeled ssDNA, (ii) annealed labeled ssDNA-unlabeled ssDNA duplex (as annealing control), (iii) annealed labeled ssDNA-RNA duplex, and (iv) ssDNA-RNA duplex treated with RNaseH. If you see a slower migrating species in (ii) and (iii) only but both (i) and (iv) migrate similarly faster, then your TFO RNA is perfectly annealing to the DNA.

Having ascertained that your TFO RNA is good, you can now redo your actual experiment.

Also, sometimes contaminants (excess salt, buffer, chelators) interfere with gel migration. So, if your bands are perfect bands, and not like splotch/blob/smudge, then you need not worry; but if your bands do not look like perfect bands, you might want to reprecipitate your reactions (with certified RNA-grade glycogen), wash well with EtOH and resolve on your gel. If you repurify your TFO RNA as suggested above, you might already have resolved any electrophoretic anomaly.

Good luck.

Hi Anil,

Thank you for taking the time to go into the depths of the problem. I actually managed to get good triplex shift in my reaction maintaining the same buffer conditions and increasing the incubation time for triplex formation to 6 hrs at room temperature.

But I did end up running all the the controls you have mentioned on a gel trying to get to the above mentioned solution.

Thanks once again!