I'm measuring the CD spectra of a series of RNA hairpins. These RNAs have the same sequence in all residues except in one, as each of them carries a single point mutation. My idea was that these point mutations would tend to destructure the hairpin compared to the WT. I expected to see a decrease in the intensity of the peaks (I have a positive peak at 263-265nm and a negative peak at 210nm when I scan from 200 to 320nm). However, for two of them, I see a shift of the 263-265nm peak to 269nm coupled with an increase in the signal intensity. What is this shift of the peak and why is the signal so intense? I'm a beginner in this, so if you have any ideas to share I'd be very grateful. I have been reading some papers, but can't find a good description of what this peak shift might mean in terms of structural rearrangement of an RNA hairpin. Only that the CD spectra of these two RNAs resemble the shape of A-DNA CD.
Yes, they all have the same absorbance spectra :-) Also the CD peak at 210 only changes in intensity between the different mutants, but no shifts there. The shift only ocurrs from 263 to 269 nm.
Hi, Amparo.
May be you need a complementary technique. Recently I`m working with Raman and SERS as a complementary techniques for CD, try to measure a Raman spectra. If you need i can help you, contact me on [email protected]
The CD technique is very sensitive to impurities/interferents (in these I include salts and other molecles with a CD spectra in that region). Are you sure you have the same RNA concentration in all samples? Is your buffer the same in all samples? That might influence significantly your results. Also, make sure you have a clean cell, by measuring a cell with milliQ water (blank sample). Check the spectra obtained. Below 220 nm, you absorve everything, so interferences are more frequent. Depending on te concentration you can have shifts that do not mean anything, because CD generates data with a lot of noise. Make several spectras of the same sample. What are your conditions (bandwidth, step size, time per point)?
Hope that helped!
Hi, thanks all for your replies. I was using CD as a confirmatory technique for results I obtained measuring the fluorescence of 2-AP in those RNAs, in a fluorimeter. The truth is that I'm not really getting what I expected; ie, less CD signal where I had more 2-AP signal. The buffer is exactly the same for all samples, I made a fresh batch and measured all my RNAs on the same day with buffer from this batch. This shift occurs at 269 nm and is identical for two of my RNAs (i measured 8 in total). I did not measure these two consecutively, but others were measured in between where the shift didn't occur. I will measure everything again using a lower concentration (atm, I'm using 30 uM for a 21-mer; giving me between 3 mdeg and 8 mdeg depending on the mutant). To make sure I'm not detecting dust on the cells :-) But meantime, for the CD experts, can an RNA peak be shifted without changes in buffer/addition of molecules, but just by changing its sequence? And if it does, any clue as to what a shift to the right might mean for RNA? Thanks again.
well if its real it means the chirality of the RNA is changing, because that's essentially what CD measures right? its only a 21 mer, so that means your stem is what, 8-9 bp? so small changes can have big effects on a short hairpin. I'm guessing your mutation is in the middle of the stem?
Yes, the stem is 9-bp long. One of the peak-shifting mutations is in nt 3 starting from the 5' end, and the other one is in nt 3 starting from the last pair before the loop. So they are relatively symetrical (bottom-up speaking). I have another mutation exactly in the middle of the stem, and with this one the CD signal at 265nm decreased a lot (probably because the hairpin lost a lot of structure), but there was no peak shift here. Do you know any CD papers that I could consult about this change in the chirality of RNA? I've spent the day googling and googling, but I haven't got anything inspiring yet (maybe because of my lack of CD jargon).
Ana, sorry I forgot to write my CD conditions. Here they are: band width: 1nm, response: 8 sec, speed: 20 ns/min, data pitch: 0.2 nm, accumulation: 10. I scan from 320 to 200, Tª is 10ºC, volume is 200 uL, [ ] is 30 uM.
I found a review on the subject (http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2665218/), Kypr et al., Nucl. Acids Res., 2009, 37:1713-1725. It seems to cover transitions in nucleic acid duplexes pretty well, such as A-B and B-Z transitions, and some info on RNA hairpins. It also mentions isodichroic points (Figure 2), where your spectra is really a combination of two spectra, that's why there may be a shift. I would look up the definition of isodichroic (see question 4, http://www.brynmawr.edu/chemistry/NSFpchem/solutions/SolutionsHelixCoil.pdf). So your mutations may be causing formation of two RNA structural states and you are seeing the ensemble. This could perhaps be due to strand slipping (check possibility of alternative base-pairing) or non-canonical base-pairing of your mutations (mismatched bases still hydrogen bond in odd ways). Hope this is a start. I can email you the review if you don't have access.
Thanks! I found the review. Reading it for the 2nd time now (the 1st time left me a bit overwhelmed :-) ). It's great, right what I needed. Thanks very much again!
I used to do CD of small RNAs. I know the frustration of trying to interpret results. Best of luck to you!
Honestly CD is not the best way to go. With a mutation you may affect all kinds of things. Yes structure is one of them you may get different stacking more B like more A like etc. but you also affect stability. Especially if your stem is short, you may have more single strands (i.e stability). Also you may have multimers... I have done a fair amount of CD on DNA and the technique is good if you know what you are looking for....
The best way to check such a small oligo is NMR. Before you say no consider this.
You only look for iminoprotons (base pairing). If you have access to a 400 or higher MHz machine you can do this at 300uM or less (depending on probe heads). On a 600 you can do this at 50-100uM RNA concentration. You have to be in water, not D2O. This can show you A) what base pairs you have and B) where they are perturbed. Also it can tell you if you actually have a hairpin under these conditions.
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