A chemically synthesized RNA minihelix, 32 nucleotides in length with a fair amount of GC-rich content needs to undergo some high temperature-based experiments. Before I could do that I want an estimate of the melting temperature of this RNA minihelix so as to not disrupt its secondary structure which is crucial for the experiments. How do I determine the melting temperature of such RNA minihelix or what temperature can be attained without disrupting its secondary structure?
I don't know what you mean by "minihelix", is this a single strand folded onto itself like a hairpin? If so, one way to get a rough estimate is to separate the stem from the loop in a text editor, that is made only of CG and AT base pairs and submit the stem part to a on-line Tm calculator . You may need to provide the buffer conditions that your experiment will be running, especially sodium. The resulting Tm will be a lower estimate of the actual melting temperature since the loop further stabilises your structure.
A few locations of on-line calculators: https://bioinf.fisica.ufmg.br/app/comparetm.pl (this is from our group), https://www.atdbio.com/tools/oligo-calculator, http://www.unafold.org/twostate.php, https://www.biosyn.com/tew/melting-temperature-(tm)-calculation-for-bna-oligonucleotides.aspx
If you need further help please contact me [email protected]
Do a melting experiment in the same solvent in which you are going to do the high-temperature manipulations in the future. Once you have the melting curve ready, you will know in which temperature range what is the amount of secondary structure.
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