I'm planning to synthesize biotin-labelled hairpin RNA by in vitro transcription (use biotin-UTP) and wondering if the biotin modification affects hairpin annealing (i.e. base-pairing).
Thank you for your help!!
Most body-labelling by biotinylated nucleotides is done by a mix of native and modified UTP (e.g. 10:1). Check the details of your protocol. If so, only 1/10 of the U residues in each transcript are modified and the net effect on melting and hybridisation should be minimal.
End-labelling is better, but will give a much weaker signal - only one modification per molecule.
From what I remember, yes it does, but the labelled bases you buy are labelled in a way to give minimal interference. You may have noticed when you searched to buy biotin-UTP some different numbers that refer to where the biotin has been conjugated. So it should be OK but to be sure you just have to try and test it. you may be able to alter your labelled to unlabelled UTP ratio if you have problems.
End-labelling might be a safer bet?
Hello Leonard,
Yes, it might interfere with the annealing. It will be better, as suggested by Nathan, to go for end labeling.
Most body-labelling by biotinylated nucleotides is done by a mix of native and modified UTP (e.g. 10:1). Check the details of your protocol. If so, only 1/10 of the U residues in each transcript are modified and the net effect on melting and hybridisation should be minimal.
End-labelling is better, but will give a much weaker signal - only one modification per molecule.
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