1 reply to this topic

[renada.ba]

First of all, I would like to congratulate for this so helpful forum. I am a Ph.D student at the department of organic chemistry and biochemistry. I am starting now my studies and a part of my project is to find a method to conjugate a 14 mer oligonucleotide with a 42 residues polypeptide helix-loop-helix conformation. I am thinking of conjugating them by a hydrazone bound, where a maleimide-hydrazine is incorporated in the cysteine residue at the loop region and a formylbenzoate group modifies the oligonucleotide. The reaction is SN2 so I was thinking of using DMSO as a solvent for the conjugation. This molecule will be used as a Proximity ligation assay Prob for PCR experiments. I was wondering how safe
it is to use this polar aprotic solvent for the reaction of the biomolecules. I know that the DMSO denatures the biomolecules. But  by literature I have found that for PCR experiment they use to store the DNA in 20% DMSO and also my group use this solvent for the modification of the polypeptide with organic molecules. I would not prefer to use buffer solution because I am trying to find a way to increase the yield of the reaction.  Do you think that the method that I am thinking will work? I am looking forward to hearing from you. Thank you in advance!

[sergii]

As I understand, you're afraid that polypeptide you're going to use will be denatured by DMSO. I don't know exact answer for your question, and I'm not familiar with proximity ligation assay. I'd try to study the reaction using only correspondingly modified DNA strands, and after figuring out the best conditions, I'd move to polypeptide.

I'm working with the similar problem of connecting protein to DNA. I'm trying MIchael addition, having cysteine from protein side and maleimide modification from DNA side (you actually can do oposite). This reaction is reported to go in water buffer with pH 7.5-8. Another option I'm considering is to use one of the reaction of click chemistry (Staudinger ligation or Huisgen cycloaddition), which goes with very high yield.

There's cool review on methods of DNA-protein link: Functionalization of DNA nanostructures with proteins; by Barbara Sacca and Christof M. Niemeyer; Chem Soc Rev 2011, 40, 5910-5921; DOI: 10.1039/c1cs15212b