yupeng. yeast surface aga1p s s s s aga2p i-anii myc 3’-actcctccaaagagacatt...

Yupeng

Yeast surface

Aga1ps

ss

s

Aga2p

I-AniI

Myc

3’-ACTCCTCCAAAGAGACATT5’-TGAGGAGGTTTCTCTGTAABiotin

Ani-wt: T G A G G A G G T T T C T C T G T A Am-XID: A G T G C C T G T T T C T C T T G A C

m-wt: C

-10 -9 -8 –7 –6 –5 -4 –3 –2 –1 +1 +2 +3 +4 +5 +6 +7 +8 +9

Engineering I-AniI HEs for XID site

I-Anil Yeast surface display

HA

Ani XID C terminal Design (Jim Havranek from Baker Lab)

1 12710 20 30 40 50 60 70 80 90 100 110(1)IESIINTSYFSAWLVGFIEAEGCFSVYKLNKDDDYLIASFDIAQRDGDILISAIRKYLSFTTKVYLDKTNCSKLKVTSVRSVENIIKFLQNAPVKLLGNKKLQYKLWLKQLRKISRYSEKIKIPSNYwt (1)IESIINTSYFSAWLVGFIEAEGCFSVTKKEKKREEKSAVYSIAQRDGDILISAIRKYLSFTSKVYLDKTNCSKLQVTSIRSVENIIKFLQNAPVKLLGNKKLQYLLWLKQLRKISRYSEKIKIPSNY1 (1)IESIINTSYFSAWLVGFIEAEGCFSVDKNRKKTDKRRAKFEIAQRDGDILISAIRKYLSFTSKVYLDKTNCSKLQVTNVRSVENIIKFLQNAPVKLLGNKKLQYLLWLKQLRKISRYSEKIKIPSNY2 (1)IESIINTSYFSAWLVGFIEAEGCFAVTKEDKKRRPYRASYEIAQRDGDILISAIRKYLSFTSKVYLDKTNCSKLQVQNIRSVENIIKFLQNAPVKLLGNKKLQYLLWLKQLRKISRYSEKIKIPSNY3 (1)IESIINTSYFSAWLVGFIEAEGCFSVTKNNKYRKKQDAKFSIAQRDGDILISAIRKYLSFTTKVYLDKTNCSKLQATKIRSVENIIKFLQNAPVKLLGNKKLQYLLWLKQLRKISRYSEKIKIPSNY4 (1)IESIINTSYFSAWLVGFIEAEGCFSVTKKNKKRKDQDAKFSIAQRDGDILISAIRKYLSFTSKVYLDKTNCSKLEVTRIRSVENIIKFLQNAPVKLLGNKKLQYLLWLKQLRKISKYSEKIKIPSNY5 (1)IESIINTSYFSAWLVGFIEAEGCFSVDKNRKKTDKRRAKFQIAQRDGDILISAIRKYLSFTSKVYLDKTNCSKLQVTNVRSVENIIKFLQNAPVKLLGNKKLQYLLWLKQLRKISRYSEKIKIPSNY6 (1)

STS 3 STS 4

Ani XID C variants yeast surface expression.

Myc-Ani

Myc-AniMyc-Ani

Myc-Ani Myc-Ani Myc-Ani

Myc-AniMyc-Ani

1 2

3 4 5 6

Vector digestion

Yeast transformtion to establish Ani XID-C library (9×106)

Six combo error prone PCR

First round sorting based on Myc-Ani expression.

Second round sorting based on binding affinity toward Ani +6T+7G+9C.

Ani wt oligo

Ani XID oligo(+6T +7G +9C)

Ani wt Library w/o sorting Ani-Myc sorting Ani XID +6T+7G+9C sorting

Binding affinity of Ani variants toward Ani wt and Ani +6T+7G+9C oligos.

1 12710 20 30 40 50 60 70 80 90 100 110(1)IESIINTSYFSAWLVGFIEAEGCFSVYKLNKDDDYLIASFDIAQRDGDILISAIRKYLSFTTKVYLDKTNCSKLKVTSVRSVENIIKFLQNAPVKLLGNKKLQYKLWLKQLRKISRYSEKIKIPSNYwt (1)IESIINTSYFSAWLVGFIEAEGCFSVTKKEKKREEKSAVYSIAQRDGDILISAIRKYLSFTSKVYLDKTNCSKLQVTSIRSVENIIKFLQNAPVKLLGNKKLQYLLWLKQLRKISRYSEKIKIPSNY1 (1)IESIINTSYFSAWLVGFIEAEGCFSVDKNRKKTDKRRAKFEIAQRDGDILISAIRKYLSFTSKVYLDKTNCSKLQVTNVRSVENIIKFLQNAPVKLLGNKKLQYLLWLKQLRKISRYSEKIKIPSNY2 (1)IESIINTSYFSAWLVGFIEAEGCFAVTKEDKKRRPYRASYEIAQRDGDILISAIRKYLSFTSKVYLDKTNCSKLQVQNIRSVENIIKFLQNAPVKLLGNKKLQYLLWLKQLRKISRYSEKIKIPSNY3 (1)IESIINTSYFSAWLVGFIEAEGCFSVTKNNKYRKKQDAKFSIAQRDGDILISAIRKYLSFTTKVYLDKTNCSKLQATKIRSVENIIKFLQNAPVKLLGNKKLQYLLWLKQLRKISRYSEKIKIPSNY4 (1)IESIINTSYFSAWLVGFIEAEGCFSVTKKNKKRKDQDAKFSIAQRDGDILISAIRKYLSFTSKVYLDKTNCSKLEVTRIRSVENIIKFLQNAPVKLLGNKKLQYLLWLKQLRKISKYSEKIKIPSNY5 (1)IESIINTSYFSAWLVGFIEAEGCFSVDKNRKKTDKRRAKFQIAQRDGDILISAIRKYLSFTSKVYLDKTNCSKLQVTNVRSVENIIKFLQNAPVKLLGNKKLQYLLWLKQLRKISRYSEKIKIPSNY6 (1)

STS 3 STS 4D

Future experiments:Characterizing binding affinity and cleavage activity of individual variant.Engineering Ani XID N variants.

0

5

10

15

20

25

30

1 2 3 4 5 6

STS3

STS4

JH Design 1 2 3 4 5 6

Co

py

nu

mb

er

Ani XID variants sequence analysis (45 variants)

P

0

1

2

3

4

5

1 2 3 4 5 6

STS3

STS4

JH Design 1 2 3 4 5 6

Fo

ld e

nri

chm

ent

Jordan

backbonegroove

mutated backbonemutated groovetargeted

0

2000

4000

6000

8000

10000

12000

14000

16000

0.01 0.1 1 10 100 1000

[dsOligo] nM

ReoAni WT

ReoAni NS

E148D WT

E148D NS

L156R WT

L156R NS

C150R WT

C150R NS

90nM25nM15nM

0

2000

4000

6000

8000

10000

0.01 0.1 1 10 100 1000

[dsOligo] nM

ReoAni WT

E148D

L156R

E148D L156R

E148D C150R L156R

E148D K155N L156R

E148D L156R C198R

C150R L156R

L156R I164V

L156R Q171H

L156R D173N

C150S L156R C198R

4nM

Ani WT E148D L156R L156R L156RL156RL156R L156R L156R L156R L156R

L156RE148D

E148DC150R K155N

E148D E148D

C198RC150R

I164V Q171H D173N

C150S

C198R

CTD mutants of I-Ani1: Selection by YSD for higher affinity to target

10nM

0

1000

2000

3000

4000

5000

6000

7000

8000

9000

1 10 100 1000[dsOligo] nM

Nor

mal

ized

MFI

F13Y

S111Y

F13Y S111Y

WT

YSD Kd titration for Reo’s “Y2” mutant

90nM50nM10nM

-10 -9 -8 -7 -6 -5 -4 -3 -2 -1 +1 +2 +3 +4 +5 +6 +7 +8 +9 ACGT

0.00000

0.00500

0.01000

0.01500

0.02000

0.02500

0.03000

1/K d

Position

YSD Bindability of I-Ani1 One-off Site Variants

A

C

G

T

Melissa

Current Projects

Expression of I-SceI (wt coding sequence) in S. cerevisiae

Design of REO-I-SceI (Restriction Enzyme-saturated ORF) for target of canine X-SCID site

Future projects: REO-I-CreI monomer and REO-I-MsoI monomer

Design of REO-I-SceI

• Optimized codon usage for S. cerevisiae expression

• Eliminated splice donors and acceptors

• Inserted silent restriction sites around desired motifs.

I-SceI