I have purchased an FMR1 oligonucleotide with 105 bases. I want to immobilize it on the gold electrode for DNA probe use.
after studying some procedures, we have found that introducing the thiol group to the DNA, causes a chemical bond with gold, thus we can somehow stick the DNA to the gold electrode.
However, most of the studies, introduce the thiol group during the synthesis process of oligonucleotide itself. but in our case, the oligonucleotide is already purchased without having any thiol group. thus, we look to find a way to connect the thiol group to oligonucleotide. or just any other simple way to immobilize the DNA on gold.
does anybody have an idea?
thank you for your time.
Unfortunately, attachment of a linker to a pre-synthesized oligonucleotide via chemical means is not feasible. My suggestion is to order a new oligonucleotide, this time with an SH modification. You may also try to find a source of a nucleoside triphosphate bearing an SH modification and use an enzymatic reaction catalyzed by kinase to attach that at the 3'-terminus. I am not sure though such NTP is commercially available.
I disagree that this is not possible to use the unmodified oligonucleotide, but Andrei is correct in saying it is more convenient to synthesize the oligo with one non-deoxynucleoside synthesis unit. The general approach, including several examples where SH-conjugated oligos bind to gold surfaces, is described in a 2020 review (TC Roberts, et al, Nature Reviews Drug Delivery, 2022, 19, 673-694 - DOI: https://www.nature.com/articles/s41573-020-0075-7).
Several methods for introducing thiols are described in the website of a commercial producer of oligos: (https://www.biosyn.com/faq/What-is-a-thiol-modifier-in-oligonucleotide-synthesis.aspx)
Other possibility is to enzymatically phosphorylate either the 5' or 3' terminal and then activate the resulting monophosphate to attach a sulfur-containing linker. These two steps can be combined by activation of a protected thioalkane phosphate ester with BrCN. A good starting place is Gottikh, et al, Nucleosides Nucleotides, 1996, 15(6) 1137-1147; paper avail for download on this site (https://www.researchgate.net/publication/247532332_Cyanogen_Bromide-Induced_Chemical_Ligation_Mechanism_and_Optimization_of_the_Reaction_Conditions) This will require a linker group such as [CH3PhO]2(PhO)C-S-(CH2)nOP(=O)
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