FIGURE 4.

(A) Representative NAIM gels for quantification of phosphorothioate and 2′-deoxy effects in the improved ligase. Secondary-structure cartoons, colored as in Figure 1, provide landmarks. 6% gels were used to resolve the 3′ half of the ligase (left), and 15% gels were used to resolve the 5′ half of the ligase (right). White arrowheads mark positions of particularly strong phosphorothioate interference; red arrowheads mark positions of particularly strong 2′-deoxy interference. (B) [α-Phosphorothioate]-2′-deoxyadenosine triphosphate (dATPαS), one of the eight nucleotide analogs used for NAIM. Use of the α-phosphorothioate-bearing ribonucleotides ATPαS, CTPαS, GTPαS, and UTPαS permits quantification of phosphorothioate interference effects and establishes a baseline for comparison with the α-phosphorothioate-bearing deoxyribonucleotides dATPαS, dCTPαS, dGTPαS, and dUTPαS to determine 2′-deoxy interference effects (Conrad et al. 1995; Strobel and Shetty 1997; Ryder and Strobel 1999). The stereoisomer shown bears a pro-S_p sulfur substitution; this isomer is the only isomer recognized by T7 RNA polymerase, but because polymerization proceeds with inversion of stereochemistry, all sulfur substitutions in the resulting RNA are in the pro-R_p position (Verma and Eckstein 1998).