Syntheses of nucleosides designed for combinatorial DNA sequencing

Nucleoside triphosphates I with 3'-O-blocking groups that are both photolab ile and fluorescent were required to investigate the viability of a strateg y for sequencing DNA in a combinatorial fashion (see Figure 1). Four compou nds were prepared to realize this goal. Two of them, 14a and 14t, had dansy l-functionalized, 3'-O-(2"-nitrobenzyl) ether groups, while the other two, 18a and 18t, had similar pendant carbonate groups. Tests for incorporation of these analogues were performed by using five different DNA replicating e nzymes, but the analogues were not incorporated. These results were surpris ing in view of the fact that previous studies had shown that 3'-O-(2"-nitro benzyl)adenosine triphosphate II was incorporated by Bst DNA polymerase I. However, molecular simulations with the coordinates of a T7 polymerase crys tal structure as a model demonstrates that analogues 14a, 14t, 18a and 18t are too large to fit into the enzyme active site, whereas accommodation of the unsubstituted 2-nitrobenzyl compound II is much less demanding. We conc lude that both the nucleoside triphosphates and the DNA polymerase enzyme m ust be modified if the proposed DNA sequencing scheme is to be viable.