Fig. 4. Representative in-synthesis modifications of DNA (denoted as X). (A) Scheme showing the general strategy for 5′-modification of DNA using a modified phosphoramidite without hydroxyl-handle for further chain elongation. (B) 3′-modification of DNA using a modified solid support. (C) Internal modification of DNA using a non-nucleobase phosphoramidite containing the modification. (D) Internal modification of DNA using a modified nucleobase in order to maintain Watson–Crick–Franklin base pairing in the oligonucleotide product. In this case modified dT is shown, but all four bases can in principle be employed, although dT and dA remain the most popular sites for modification. Note that the oligonucleotide products on the right-hand side are rotated 180° to display the sequences in the 5′ to 3′ direction. (E) Branching modifications can also be employed in DNA synthesis. Reproduced with permission from ref. 51 Copyright 2019 American Chemical Society.