thiol-modified oligonucleotide: thiol deprotection - (May/02/2012 )
I'm trying to link chemically a protein with a DNA oligonucleotide. I think one of the simplest way would be to use Michael addition, where thiol group reacts with C=C electron deficient bond. Befor trying to work with proteins, which could be hard, I'm trying to ligate two DNA oligonucleotides, one modified with thiol group, another is modified with acrydite group:
DNA1--SH + CH2=CH--C(O)--DNA2 ---> DNA1--S--CH(CH3)--C(O)--DNA2
Thiol-modified DNA comes in protected form, like this: DNA1--S--S--ProtectionGroup. Before performing the reaction of interest, one should deprotect thiol group, which is done by reaction with DTT and further desalting.
The reaction I'm trying to do fails, and I don't know whether the reason for faiure is Michaelis addition itself, or failed deprotection, or failed desalting.
So here my question comes: is there any simple way to test, that deprotection was succesfully done and DTT was completely removed from the system?
Thank you very much for help!
I have had issues removing DTT with desalting. If your DTT isn't completely removed, the thiols from DTT can also react with your acrydite group instead of just your deprotected DNA. Instead of using DTT you could try TCEP agarose (I get mine from Pierce), where TCEP is the reducing agent and is bound to agarose, so after reduction you can simply filter out the agarose beads.