TABLE 5.
Effects of modifying Exo−Kl-1 sequence on affinity for Exo−Kl polymerase
| Exo−Kl-1 modification | Sequencea | Fold decrease in affinityb |
|---|---|---|
| Exo−Kl | 5′-gcctgcaggtcgactctagaCAACCATCGAAGACTA | NA |
| 3′-cggacgtccagctgagatctGTTGGTAGCTTCTGATATCGTCCGT | ||
| Random substrate | 5′-gcctgcaggtcgactctagaNNNNNNNNNNNNNNNN | >4 |
| 3′-cggacgtccagctgagatctNNNNNNNNNNNNNNNNNNNNNNNNN | ||
| 36-nt primer-42-nt template | 5′-gcctgcaggtcgactctagaCAACCATCGAAGACTA | 2.5 |
| 3′-cggacgtccagctgagatctGTTGGTAGCTTCTGATATCGTG___ | ||
| 36-nt primer-44-nt template | 5′-gcctgcaggtcgactctagaCAACCATCGAAGACTA | 1 |
| 3′-cggacgtccagctgagatctGTTGGTAGCTTCTGATATCGTGCC_ | ||
| +1 shift | 5′-ggcctgcaggtcgactctagaCAACCATCGAAGACT | 4 |
| 3′-ccggacgtccagctgagatctGTTGGTAGCTTCTGATATCGTCCG | ||
| −1 shift | 5′-cctgcaggtcgactctagaCAACCATCGAAGACTAT | 2 |
| 3′-ggacgtccagctgagatctGTTGGTAGCTTCTGATATCGTCCGTC | ||
| −4 shift | 5′-gcaggtcgactctagaCAACCATCGAAGACTATAGC | 3 |
| 3′-cgtccagctgagatctGTTGGTAGCTTCTGATATCGTCCGTCCGT | ||
| −8 shift | 5′-gtcgactctagaCAACCATCGAAGACTATAGCAGGC | >4 |
| 3′-cagctgagatctGTTGGTAGCTTCTGATATCGTCCGTCCGTCCGT | ||
| Modified T7Pcore | 5′-gcctgcaggtcgactctagaCAACCATCGAAGCAGC | >4 |
| 3′-cggacgtccagctgagatctGTTGGTAGCTTCGTCGCGAGTCCGT | ||
| Modified template overhang | 5′-gcctgcaggtcgactctagaCAACCATCGAAGACTA | 2 |
| 3′-cggacgtccagctgagatctGTTGGTAGCTTCTGATCGATGTAAG | ||
| 5′ modified non-T7 sequence | 5′-gcctgcaggtcgactctagaACCAACGATCCTACTA | 2 |
| 4′-cggacgtccagctgagatctTGGTTGCTAGGATGATATCGTCCGT |
Lowercase letters are bases derived from the fixed region of the starting material, while bases derived from the random region are capitalized. “N” signifies that the base was random in the starting material (either A, T, C, or G), while “N” is the complement of that base in the template strand. Underlined regions highlight changes from the Exo−Kl-1 sequence.
Affinities measured in gel shift assays were measured with 2 nM primer-template and under the conditions described in Materials and Methods. The derived Kd values were useful for comparisons between these sequences but were not determined under conditions that would necessarily yield accurate Kd values. Therefore, they are referred to as apparent Kd values that are specific for the gel shift experiment (Kd,app,gelshift). Values listed are approximate changes relative to the Exo−Kl-1 sequence (measured Kd,app,gelshift, 125 ± 25 nM; averages from at least 3 experiments ± SD), with higher numbers indicating fold lower affinity. NA, not applicable (reference sequence).