Table 4.
Stability analysis
| High–low (stability) |
|||
|---|---|---|---|
| AA | G4 design | NNB | G4 |
| Positions with wild-type bias | |||
| D23 | 10% D | 0% | +3% |
| A24 | 8% A | −4% | +1% |
| P25 | 42% P | 0% | +4% |
| V29 | A|L|S|V | +10|−19|+18|+5 | + 29|−11|−27|+12 |
| Y31 | S|Y | — | 0% |
| G52 | 49% G | +7% | +10% |
| S53 | 51% S | +5% | +20% |
| S55 | 51% S | +18% | +44% |
| T56 | 49% T | +10% | +8% |
| G77 | 12% G | +1% | +5% |
| G79 | 12% G | +17% | +17% |
| S85 | 100% S | +18% | — |
| Positions without wild-type bias | |||
| Y | 19% | −1% | −4% |
| S | 10% | +5% | +2% |
| G | 6% | 0% | +1% |
| D | 10% | +2% | +5% |
| R | 4% | −1% | +2% |
| C | 10% | 1% | −2% |
| H | 6% | 0% | 0% |
The NNB and G4 libraries were independently sorted for clones of low stability and high stability. Sequences of about 50 clones from each sorted population were analyzed. “AA” indicates the wild-type amino acid at positions with wild-type bias or amino acids of elevated frequency at positions without wild-type bias. “G4” design indicates the designed frequency of the indicated amino acid. “NNB” and “G4” indicate the difference in amino acid frequency between the high- and low-stability populations from the indicated library, respectively.