1. Scharenberg Lab Technical update on yeast display (Hoku/Jordan) –Overhang length can influence binding (see meeting notes) –Half sites can be interrogated independently using yeast display (Hoku) –Cleavage assay - optimized (Hoku) SCID target progress (Jordan) –specificity analysis of best designed variant –Results from 4 rounds of iterative mutagenesis/sorting of best designed variant

2. Flow cytometric analysis of half site interactions - base pair alterations in the I-Ani minus half site produce large affinity changes; whereas base pair alterations in the plus half site typically do not - Obvious explanation: the above is due to differences in the individual half site affinities - From an engineering standpoint, this facet of I-Ani makes masks our ability to do to detailed evaluation of plus half site designs and evolution Strategy: - interrogate surface displayed I-Ani affinity with oligos which extend from central four across one half site --> Can half sites be separately/individually interrogated?

3. Halfsite Dilutions Concentration Plus Minus MFI Minus Plus

4. Concentration (nM) MFI WT Y2 E148D L156R Plus site K d = 12,383 nM Minus site K d = 503 nM Plus site K d = 4,591 nM Minus site K d = 128 nM Plus site K d = 4,071 nM Minus site K d = 116 nM Plus site K d = 9,539 nM Minus site K d = 373 nM

5. Thoughts and speculation: I-Ani sits down on minus half site DNA, and the plus half site flops in the breeze - if a compatible plus half site sequence of DNA is present, it cleaves? –Could this be why Ani structures are blurry? –Are we really separately interrogating the plus half site (e.g. could minus half site-induced conformational changes be important?) What does Rosetta say about the plus half site? –Can we design a tighter plus half site binder which would still cleave and give better structures?

6. WT Y2 E148D L156R Overlay of Mg Ca Flow cleavage assay optimization: (Hoku) Minus Tethered to c-Myc Minus Untethered PE-SA (on minus half site) Alexa-647 (on plus half site end) - Many failures with yeast for unclear reasons: (Jordan and Andy) - Half site data: Hoku noted we have been labeling the minus half which doesn’t come off easily, perhaps we would do better if we labeled the plus half site? T=0

7. 30 minutes post-incubation (at 37°C) WT Y2 E148D (inactive) L156R Overlay of Mg Ca Conclusion: Hoku got it right, the minus half site sticks very well even after cleavage, while the plus dissociates rapidly after cleavage PE-SA (on minus half site) Alexa-647 (on plus half site end) Minus Tethered to c-Myc Minus Untethered

8. WT / E148D mixing assays (post-incubation)

9. 0 overhangs K d = 21,280 nM 1 overhang K d = 5,688 nM 2 overhangs K d = 2,301 nM 3 overhangs K d = 5,299 nM 4 overhangs K d = 1,297 nM 5 overhangs K d = 703 nM Overhang WT binding (full WT Ani target plus variable length overhangs)

10. Gated on Myc+ Original Library on Y2 L156R background: sorted for Myc+ only After 3 rounds sorting for dsAni SCID binding, epPCR/reshuffling library, unsorted Counterselection … dsAni SCID-647 dsAni WT-PE 2.0.B12.1.C02.1.C22.1.C32.1.C42.1.C5 SCID Target C0C1C2C3C4C5 WT Target C0C1C2C3C4C5 B1 ds ss cut SCID target design project Start with 160 designs (JH), shuffle in yeast with additional diversity from ep-PCR Output: - no primary design came through selection - final population was dominated by a few designed STS motifs - no cleavage activity TGAGGAGGTTTCTCTGTAA AAGGAAGGATTCTCTGTAA WT Ani SCID

11. Comparison of selection methods: direct vs. counterselection Blue: SCID Oligo binding Red: WT Oligo binding Black: ratio SCID/WT Green: ratio WT/SCID “You get what you select for”

12. SCID target: specificity analysis We have binding selectivity for what we selected for and against what we selected against (dsSCID vs. dsAni –what does that mean for selectivity against “one offs”? SCID oligo: Blue: others are “one offs” -10-9-8-7 -6-5-4-3 Conclusion: selectivity of best binder is dominated by -10 position “You get what you select for”

13. StartRound 2Round 3 Mutagenized (no selection) - moderately aggressive gating to avoid bottlenecking our evolutionary pool - mutagenesis every round - Result: moderate population level improvement in MFI Improvement by evolution?

14. Future Directions - refined selectivity via counterselection against one-offs (in progress) - redesign native plus half site?

15. D16E/A/G/V/N (38%) L36R (19%) S37G/N (19%) K39E/R/N (12%) I64T (enriched from 33% to 98%) K60T/M (10%) D73N (5%) I53V/T (19%) I44V (depleted from 33% to 5%) mutually exclusive Round 4 output Red areas of DNA - altered base pairs