I have a 1kb RNA strand which I have already tried heating at 65C for 10 minutes. I generally use 10mM Ni or Mg to bind on mica surface. However, I recently also tried heating the RNA and binding it on APTES treated mica. Unfortunately, I have not been able to obtain single linear RNA strands.
Not surprising at all. Put your sequence into the MFOLD server http://mfold.rna.albany.edu/ and you will see why. You are fighting the delta-G of conformation when your strand cools.
Long or short, you won't be able to generate a linear ssRNA strand very easily. In cells, mRNA has chaperone proteins on it. And still, there are introns that form loops and modify the translated sequence. Here's an example I did yesterday. It's rhinovirus 14's sequence.
Here's an idea though. Try binding the 5' end of your ssRNA to a surface, and biotinylate the 3' end. http://www.piercenet.com/method/methods-labeling-nucleic-acids Get a Streptavidin-quantum dot conjugate. You can probably do this with any of them, but the longer the wavelength the larger (hence more massive) the quantum dot is.http://www.lifetechnologies.com/order/catalog/product/Q10173MP It will be easier to see the flourescence of a sparse single qdots in the green.
Attach your surface to a plate on something that can spin at pretty high RPM.
Heat your surface to 60C in or so to melt the RNA. (In water.) Then spin up your plate and cool it below 40C while spinning, and raise it out of the water. Keep it spinning while it dries. I don't know if the bases will try to hydrogen bond to the mica or not, but probably. And you'll have van der Waals on your side.
Whether hydrogen bonding and van der Waals will overcome the attraction of compatible base pairs for each other is an open question. I think it should, mostly. But you may well find that the strand wants to kink up and is able to drag the streptavidin+qdot across the surface of your mica. You can see if that happens by taking multiple time-lapse fluorescent microscopy photos, at intervals of, say, 5 to 60 minutes. If they move, that's your answer.
If they do drag the dots around, you could try flash-freezing in liquid nitrogen after drying the spinning disc. Then you would need to work with your AFM at low temp too. Or, you could try something like using a polystyrene surface that has carboxyl groups on it, and during the spin, in the water, do an amine reaction to bind the streptavidin to the surface. That's not the best AFM base though.
Another thought is to use streptavidin + magnetic beads. http://www.lifetechnologies.com/order/catalog/product/11205D
If that doesn't work,then try it with much shorter strands, on the order of 10-20 bases. Those should linearize better.
If that doesn't work, all may not be lost. You could do a study of the most common conformations and compare those to what MFOLD calculates.
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