1. YSD: Engineering Molecular Interactions Target protein is on the surface – biophysical characterization of binding by flow cytometry High-throughput & versatile molecular biology applications Target protein is not exposed to host genome; toxic intermediate states allowed

2. YSD of I-Ani1: Analysis of Binding Affinity & Cleavage dsOligo ssOligo Cleaved dsOligo TGAGGAGGTTTCTCTGTAA TGAGAAGGTTTCTCTGTAA K D WT = 80nM [dsAni WT] in nM Epitope-Normalized MFI K D -6A = 2  M

3. 1.Jim Havranek: Computational redesign of STS1 & 2 2.Strategy to generate a YSD library of Jim’s designs 3.Screen for variants that bind the  c (“SCID”) sequence 4.Specificity profiling and cleavage analysis 5.Optimization (epPCR) &/or iteration Strategy for Generating a  c -specific variant of I-Ani1 WT = TGAGGAGGTTTCTCTGTAA  c = AAGGAAGGCTTCTCTGTAA -10-9-8-7-6-5-4-3-2-1 1 2 3 4 5 6 7 8 9

4. 160 designs

5. GGATGGAGCCTTTRHTATCAGGAAGCAGGGCAAGARATTGCAGTATGATTTATACATTGAGCTGAGCA = STS1: 80 oligos = 350 variants TATTGGCATCGTAGAATTCAGGAAGAGAAACGAGATTGAAATGGTTGMATTGARSATCAVSGATAAGAATCAT = STS2: 75 oligos = 250 variants Lib Size: 80,000 (STS1 x STS2) Strategy for Generating a Large YSD Library of Designed HEs WT = TGAGGAGGTTTCTCTGTAA  c = AAGGAAGGCTTCTCTGTAA

6. STS1 PCRSTS2 PCR Myc-FITC HA-APC Sorted for JH160 STS1+STS2 lib 1.0.B1 Sorted: Lib 1.0.B1A2 + Galactose Sorting STS1/2 Library: 1) Expression; 2) Binding; 3) Specificity Sorted: Lib 1.0.B1A1

7. JH160 Lib1.0.B1A2 (WT background)JH160 Lib2.0.B1A2 (Y2 background) F13Y

8. Analysis of Specificity of Individual Clones WT cc dsOligo ssOligo Cleaved

9. Employing Counter-selective Sorting to Isolate Specific Binders Pool of high-affinity (non-selective) variants WT Oligo-AF647 SCID Oligo-PE Anti-Myc-FITCepPCR + combo library

10. Round 1Round 2Round 3 SCID oligo MycWT oligo SCID oligo MycWT oligo SCID oligo MycWT oligo SCID oligo MycWT oligo SCID oligo MycWT oligo SCID oligo MycWT oligo Because I always remake my libraries on the WT enzyme background, there’s always a bit of contamination with yeast that have recombined back in the WT sequence that was cut out…this actually serves as a good internal control for what a specific enzyme should look like WT background Y2+L156R background

12. Does Direct Readout Mediate all of the Binding Specificity of NTD? What’s different between the NTD and CTD of I-Ani1?

13. Does Direct Readout Mediate all of the Binding Specificity of NTD?

14. What Control’s the Specificity at the NTD of I-Ani1? D73

15. D73G: Reduced Specificity of NTD WT D73G -10-9-8-7-6-5-4-3 +4+3+6+5+7+9+8

16. Protein-Protein vs. Protein-DNA Interactions N 20 N 20 N 20 N 4 +/- hydrophobic polar - only CH 3 or CH polar Rotamers  G = 0 Rigid  G > 0 DNA surfaces exhibit much less structural and electrochemical diversity / A 2  G (specific vs. non-specific) is lower in DNA-protein interactions For protein-DNA interactions: specificity is not a simple function of affinity…

18. Barcoding Yeast for Parallel Analysis of Variant HEs Alexa-647 Alexa-350 [dsOligo] Epitope-Normalized MFI Myc-FITC dsOligo-PE

19. Overview 1.DNA-protein interactions 2.Yeast surface display of I-Ani1 3.Hypothesis-driven studies on specificity 4.Engineering novel DNA-protein interactions