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

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?

Halfsite Dilutions

Concentration

Plus

Minus

MFI

Minus

Plus

Concentration (nM) Concentration (nM)

Concentration (nM) Concentration (nM)

MFI

MFI

MFI

MFI

WT Y2

E148D L156R

Plus site Kd = 12,383 nM

Minus site Kd = 503 nM

Plus site Kd = 4,591 nM

Minus site Kd = 128 nM

Plus site Kd = 4,071 nM

Minus site Kd = 116 nM

Plus site Kd = 9,539 nM

Minus site Kd = 373 nM

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?

WT Y2 E148D L156R

Overlay of Mg Ca

Flow cleavage assay optimization: (Hoku)

Min

us

Teth

ered

to

c-M

yc

Min

us

Unt

ethe

red

PE-SA (on minus half site)

Ale

xa-6

47 (o

n pl

us h

alf s

ite e

nd)

- 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

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)

Ale

xa-6

47 (o

n pl

us h

alf s

ite e

nd)

Min

us

Teth

ered

to

c-M

yc

Min

us

Unt

ethe

red

30 min Ca tethered

0

20

40

60

80

100

120

0 20 40 60 80 100

WTE148DNon-cleaving

30 min Mg tethered

0

20

40

60

80

100

120

0 20 40 60 80 100

WTE148DNon-cleaving

30 min Ca untethered

0

20

40

60

80

100

120

0 20 40 60 80 100

WTE148DNon-cleaving

30 min Mg untethered

0

20

40

60

80

100

120

0 20 40 60 80 100

WTE148DNon-cleaving

WT / E148D mixing assays (post-incubation)

0 overhangs

Kd = 21,280 nM

1 overhang

Kd = 5,688 nM

2 overhangs

Kd = 2,301 nM

3 overhangs

Kd = 5,299 nM

4 overhangs

Kd = 1,297 nM

5 overhangs

Kd = 703 nM

Overhang WT binding (full WT Ani target plus variable length overhangs)

Gate

d 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 …

dsAn

i SCI

D-64

7

dsAni WT-PE

2.0.B1 2.1.C0 2.1.C2 2.1.C3 2.1.C4 2.1.C5

SCID Target

C0 C1 C2 C3 C4 C5

WT Target

C0 C1 C2 C3 C4 C5B1 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

TGAGGAGGTTTCTCTGTAAAAGGAAGGATTCTCTGTAA

WT AniSCID

Comparison of selection methods: direct vs. counterselection

Blue: SCID Oligo bindingRed: WT Oligo bindingBlack: ratio SCID/WTGreen: ratio WT/SCID

“You get what you select for”

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”

0

100

200

300

400

500

600

700

Start Round 2 Round 3

Median Fluorescence Intensity (normalized)

SCIDWT

Start Round 2 Round 3Mutagenized (no selection)

- moderately aggressive gating to avoid bottlenecking our evolutionary pool- mutagenesis every round- Result: moderate population level improvement in MFI

Improvement by evolution?

Future Directions

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

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