Yupeng. Yeast surface Aga1p s s s s Aga2p I-AniI Myc 3’-ACTCCTCCAAAGAGACATT...
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Transcript of 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