Syntheses of Nucleosides Designed for Combinatorial DNA Sequencing
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Nucleoside triphosphates I with 3′-O-blocking groups that are both photolabile and fluorescent were required to investigate the viability of a strategy for sequencing DNA in a combinatorial fashion (see Figure 1). Four compounds were prepared to realize this goal. Two of them, 14a and 14t, had dansyl-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 enzymes, but the analogues were not incorporated. These results were surprising in view of the fact that previous studies had shown that 3′-O-(2″-nitrobenzyl)adenosine triphosphate II was incorporated by Bst DNA polymerase I. However, molecular simulations with the coordinates of a T7 polymerase crystal 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 conclude that both the nucleoside triphosphates and the DNA polymerase enzyme must be modified if the proposed DNA sequencing scheme is to be viable.
author list (cited authors)
Welch, M. B., Martinez, C. I., Zhang, A. J., Jin, S., Gibbs, R., & Burgess, K.