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Scharenberg Lab

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)

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Scharenberg Lab

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  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 --> Can half sites be separately/individually interrogated? Strategy: - interrogate surface displayed I-Ani affinity with oligos which extend from central four across one half site

  3. MFI Halfsite Dilutions Plus Minus Concentration Minus Plus

  4. WT Y2 MFI MFI Concentration (nM) Concentration (nM) Plus site Kd = 12,383 nM Minus site Kd = 503 nM Plus site Kd = 4,591 nM Minus site Kd = 128 nM E148D L156R MFI MFI Concentration (nM) Concentration (nM) Plus site Kd = 9,539 nM Minus site Kd = 373 nM Plus site Kd = 4,071 nM Minus site Kd = 116 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. Overlay of Mg Ca Flow cleavage assay optimization: (Hoku) • - 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 WT Y2 E148D L156R Minus Untethered Alexa-647 (on plus half site end) Minus Tethered to c-Myc PE-SA (on minus half site)

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

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

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

  10. SCID target design project WT Ani TGAGGAGGTTTCTCTGTAA AAGGAAGGATTCTCTGTAA SCID • Start with 160 designs (JH), shuffle in yeast with additional diversity from ep-PCR After 3 rounds sorting for dsAni SCID binding, epPCR/reshuffling library, unsorted Original Library on Y2 L156R background: sorted for Myc+ only Counterselection … 2.0.B1 2.1.C0 2.1.C2 2.1.C3 2.1.C4 2.1.C5 Gated on Myc+ dsAni SCID-647 dsAni WT-PE • Output: • no primary design came through selection • final population was dominated by a few designed STS motifs • no cleavage activity SCID Target WT Target B1 C0 C1 C2 C3 C4 C5 B1 C0 C1 C2 C3 C4 C5 ds ss cut

  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”? -10 -9 -8 -7 -6 -5 -4 -3 SCID oligo: Blue: others are “one offs” Conclusion: selectivity of best binder is dominated by -10 position “You get what you select for”

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

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

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

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