Biotin- and Chloroalkane-Modified Nucleotides for Dual Display of Proteins on DNA Through Streptavidin and Halotag Binding.

We designed and synthesized 2'-deoxyribonucleoside triphosphates (dNTPs) bearing chlorohexyl and biotin modifications attached to position 7 of 7-deazaadenine or to position 5 of cytosine. The modified dNTPs served as substrates for KOD XL DNA polymerase in the enzymatic construction of DNA bearing either one or a combination of the two modifications. The chlorohexyl-linked DNA underwent covalent reactions with halotag protein, while the biotin-linked DNA formed stable non-covalent complexes with monomeric streptavidin. The dually modified DNA bearing both modifications at different distances apart from each other were used for sequence-specific, spatially defined display of both halotag and streptavidin proteins. The study provides the foundation for display of proteins on DNA rulers.