Addition of 2', 3' cis-dialdehydes, 2', 3' cis-diols and phosphoryl groups to the 3' end of oligonucleotides using periodate-oxidized nucleoside triphosphates and terminal deoxynucleotidyl transferase

Researchers have developed a straightforward method to introduce cis dialdehydes, phosphoryl groups, and cis diols to the 3' end of oligonucleotides. This process utilizes periodate-oxidized nucleoside triphosphates (oNTPs) and terminal deoxynucleotidyl transferase (TdT). To generate oligonucleotides with cis dialdehyde modifications, TdT is incubated with an oligonucleotide for several minutes, followed by the addition of an oNTP and incubating at 30°C for 30 minutes to an hour.

Heating the reaction mixture at 90-95°C for 10 minutes after introducing the cis dialdehydes results in oligonucleotides with phosphoryl groups at the 3' end.

To synthesize 3' cis diol-modified oligonucleotides, researchers start with 3' cis diol nucleotides (HO-NTPs). The cis dialdehyde-modified oligonucleotides and cis diols can then be employed for various studies, such as examining the interaction of proteins with the 3' end of DNA or RNA. The researchers demonstrate the effectiveness of using an oligonucleotide modified with oGMP at the 3' end as an affinity label for TdT, identifying a peptide fragment that contains two of the three aspartate residues present in the TdT active site.