In a single-photon counting instrument, where we perform single molecule fluorescence measurements, we are systematically detecting fluorescence excitation -> emission at ~ 490 nm -> 520 nm coming from unlabeled DNA and RNA oligonucleotides, from several batches of the same and different lengths and sequences. The contribution of the oligonucleotides to the background fluorescence is detected already at nM concentrations and become cumbersome in the 0.5 mM - 1 mM range. We are using mostly IDT oligos, HLPC purified.
We tried removing the fluorescent contaminant/s through concentration/dilution in a proper MW filter without any success.
Does anybody know if some steps/reagents employed in the synthesis (from phosphoramidites) could contribute some fluorescence in these wavelenghts (490 nm -> 520 nm)?
According to what they told us, the company that provided the oligos is trying to figure out which could be the source of it.
Any suggestion is appreciated.
Are you using any colored plastics in your procedures? They can have fluorescent contaminants leaching out of them.
Hi Jeremías,
It seems that the signal is proportional with sample concentration, from what you describe. Have you tried to take excitation/emission spectra of your oligo solutions on a fluorescence-spectrometer and see where the exc./em. max peaks are? This might help in trying to troubleshoot where the contamination is coming from. Spectra would give a hint whether the compound is (possibly) always the same in every sample or it changes batch to batch.
What is the lifetime you observe when you do TCSPC measurements?
You mention that you purchase HPLC purified oligos, but you could try to purify them further:
- a relatively easy way is to re-precipitate the DNA with EtOH, and wash it with 70% EtOH to remove impurities; the protocol from Cold Spring Harbor is quite straightforward for this: http://cshprotocols.cshlp.org/content/2016/12/pdb.prot093377.full?sid=f08cad84-47f0-4387-bc10-272ec72efab6 (most likely your stock solutions are very high in concentration, ~mM from your description, therefore you can skip the "carrier" as that might also introduce contaminations)
- you could purify your DNA using denaturing polyacrilamide gel electrophoresis, but that is a bit more laborious to setup if you the lab is not equipped for it
I am not too familiar with handling and working with RNA, but I think that there are similar protocols for that too.
One last thing that you might consider is that DNA itself might show fluorescence when excited in the 300-500nm range, please see this publication for further details:
https://www.pnas.org/content/113/35/9716
How does the intensity of the signal you observe compares with the background? And with the actual signal that you are trying to observe?
Hope this helps,
Alessio
Hi Alessio Andreoni , thanks for the tips and for that PNAS reference. I will take a look to it carefully, it would be great if fluorescence is coming from the actual oligonucleotide bases. My concern with the work -so far- is that they didn't mention any special purification step to remove possible contaminants form oligonucleotides, and in my hands I found very different fluorescence contributions in different batches. Maybe I can ask the author.
Regarding your first comments, yes, it is pretty much a linear dependence of intensity as a function of oligo concentration (with some downward deviations from linearity at the higher concentrations I could reach).
Obtaining excitation/emission spectra is something that we really want to do. With the amounts of oligos we get (which are crazy expensive for RNAs) we were not able to have ~ mM concentration in the cuvettes we have. IDT people couldn't get it yet -according to what they told me.
I hope to go back to it and try some smaller cuvette or microplate reader, after this horrible covid-19 pandemia.
I didn't make yet the proper analysis to get the characteristic lifetimes but they look in the order of 3-5 ns, similar to what we get for the fluorophores we usually use.
Thanks again for the input.
Best,
Jere
Do HPLC (better with fluorescene detection), maybe there is/are fluorescent impurity in your oligo. Sometimes they may accidently use a column used to purify labeled olig before, then trace amount of fluorencent impurity may come off the column, get into your unlabeld oligo.
Good luck!
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