Download Asymmetry in Binding and Cleavage Specificities Found for Homing

Asymmetry in
Binding and
Cleavage
Specificities Found
for Homing
Endonuclease I-AniI
KM
kcat
Asymmetry in kcat
and KM
Specificity Shifts for Designed Enzymes
Modulating kcat or KM
-8G:C
+8C:G
Current Projects
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Design and selection toward target site
Built-in negative Selection System
Homologues and their target sites
Second shell effects (learning from
homologues)
WT site:
TGAGGAGGTTTCTCTGTAAA
FANCA_32:
TTAGCAGCTCCCTCTGTCTC
Arshiya Quadri
Benchmarking Selection System
M5 Pendo Plasmid with competent cellls
containing pccdb Ts
90
% Colony Survival
80
70
60
50
40
30
20
10
0
Wt
"-8g"
"-6c"
"-3c"
"+3a"
"+8c"
Target Site Positions
M5 (with and without I55V) survives with a single-target site, so used in all selections
Built-in negative selection
bla
p15A origin
HE ORF
pENDO-HE
Unwanted HE
target sites
pBAD promoter
(arabinose
inducible)
araC
Arshiya Quadri
Results of Negative Selection
45
40
% Colony Survival
35
30
25
20
15
10
5
0
Wt
"-8g"
"-5c"
"+3a"
"+8c"
Target Site Position
Benchmark: Recovery of -8G and +8C highly specific designs in this selection -> putting
design directly in (works) and using a randomized library to try and recover them
(going to sequencing). Using this selection in combination with other selection for -9T
and for improving single-base pair designs.
Second shell effects and homologues
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Current endonuclease design is generally
limited to target DNAs close to the sequence
observed in crystal structures
Redesign of the overall curvature of
LAGLIDADG endonucleases to expand the
range of targets to DNA sequences assuming
different conformations
Influence of second shell effects (core
especially)
Vdi and Mso - Fabio
Onu and Ani and other uncharacterized
homologues - Summer
Computational: analyzing homologues
sequences and incorporating information
into designs (Justin and Fabio)
Experimental: transplanting residues and
selection (Summer and Fabio)
• Identifying homologue target sites is not trivial
• New method: using the selection system to identify the target sites
Flexible-backbone design and second-shell mutations
(justin)
i.e. how to make Rosetta stabilize new backbones in a realistic and conservative fashion
wildtype I-MsoI,
wildtype -11 CYT, -10 ADE
The protocol that
generated this result was:
designed I-MsoI,
-11 THY, -10 THY
superposition
“ccd” backbone design w/ 2nd shell mutations,
with BLOSUM62 constraints because Rosetta over-mutates
iterative
multistate design of DNA contacts only, for specificity