Asymmetry in Binding and

Cleavage Specificities Found

for Homing Endonuclease I-AniI

KMkcat

Asymmetry in kcat and KM

Specificity Shifts for Designed Enzymes Modulating kcat or KM

-8G:C

+8C:G

Current Projects

• Design and selection toward target site• Built-in negative Selection System• Homologues and their target sites• Second shell effects (learning from

homologues)

FANCA_32: TTAGCAGCTCCCTCTGTCTC

WT site: TGAGGAGGTTTCTCTGTAAA

Benchmarking Selection System

M5 (with and without I55V) survives with a single-target site, so used in all selections

Arshiya Quadri

Wt "-8g" "-6c" "-3c" "+3a" "+8c"0

10

20

30

40

50

60

70

80

90

M5 Pendo Plasmid with competent cellls containing pccdb Ts

Target Site Positions

% C

olon

y Su

rviv

al

Built-in negative selection

pENDO-HE

p15A origin

HE ORF

pBAD promoter (arabinose inducible)

bla

araC

Unwanted HE target sites

Results of Negative Selection

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.

Arshiya Quadri

Wt "-8g" "-5c" "+3a" "+8c"0

5

10

15

20

25

30

35

40

45

50

Target Site Position

% C

olon

y Su

rviv

al

Second shell effects and homologues• 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

designed I-MsoI,-11 THY, -10 THY

superposition

The protocol that generated this result was:

“ccd” backbone design w/ 2nd shell mutations, with BLOSUM62 constraints because Rosetta over-mutates

multistate design of DNA contacts only, for specificityiterative