Triplex-forming properties and enzymatic incorporation of a base-modified nucleotide capable of duplex DNA recognition at neutral pH

The sequence-specific recognition of duplex DNA by unmodified parallel triplex-forming oligonucleotides is restricted to low pH conditions due to a necessity for cytosine protonation in the third strand. This has severely restricted their use as gene-targeting agents, as well as for the detection and/or functionalisation of synthetic or genomic DNA. Here I report that the nucleobase 6-amino-5-nitropyridin-2-one (Z) finally overcomes this constraint by acting as an uncharged mimic of protonated cytosine. Synthetic TFOs containing the nucleobase enabled stable and selective triplex formation at oligopurine-oligopyrimidine sequences containing multiple isolated or contiguous GC base pairs at neutral pH and above. Moreover, I demonstrate a universal strategy for the enzymatic assembly of Z-containing TFOs using its commercially available deoxyribonucleotide triphosphate. These findings seek to improve not only the recognition properties of TFOs but also the cost and/or expertise associated with their chemical syntheses.

Automated summary

The introduction of the nucleobase 6-amino-5-nitropyridin-2-one (Z) enables stable and selective triplex formation of synthetic TFOs at neutral pH and above, overcoming the limitation of cytosine protonation at low pH. Furthermore, a universal strategy for enzymatic assembly of Z-containing TFOs using commercially available deoxyribonucleotide triphosphate has been demonstrated, aiming to improve the recognition properties of TFOs and reduce the cost and expertise associated with their chemical syntheses.