Stoddard Lab Activities

1. Structural biology of design work: I-MsoI cluster designs vs. individual designs (Justin Ashworth and Greg Taylor)

2. Binding specificity selections translated into cleavage selections with I-AniI (Amanda Mak: FAH / Audrey McConnell-Smith: CFTR)

3. Direct selection of cleavage activity via protein-DNA interface mutations only with I-AniI (Ryo Takeuchi: CTLMA2)

4. Structural biology and selection experiments with novel naturally occuring LD monomer, I-OnuI (Ryo Takeuchi: MAOB target)

Multiple rounds of B-cell display and binding selection shift I-AniI specificity towards CFTR target

WT I-AniI Target Site

CFTR Target Site

-5 consistent point mutations in

final selected population-

Final selected I-AniI population

UnselectedWT I-AniI population

CFTR TargetWT Target

Consensus I-AniI variant sequence from binding selection specifically nicks and cleaves CFTR target

WT I-AniI Target Site

CFTR Target Site

Initial clone from binding selection displays site-specific

DNA cutting

Wild-type enzyme does not cleave

CFTR site

Redesigned enzyme

nicks and cleaves

CFTR site

Redesign for the left half site of CTLMA2

WT: TGAGGAGGTTTCTCTGTAAA || | ||| ||||| ||CTLMA2: AGAAGCCGTTCATCTGTCAA

-10A/-7A -5C/-4CT22Y/K23G/T29CV

in addition to F13Y/S111YG33R/I55TR/V56I/S57EQN/R59S/A68R

in addition to F13Y/S111Y

Isolated I-AniI variant Isolated I-AniI variant

T22Y/K23G/T29CV/G33R/I55TR/V56I/S57EQN/R59S/A68Rin addition to F13Y/S111Y

-10A/-7A/-5C/-4C (CTLMA2L)

Shuffling the amino-acid substitutions for -10A/-7A and for -5C/-4C could not accomplish cleavage of the target site with the combined 4 base mismatches.

How to recover the cleavage activity?

Spatial clash between the side chains of the substituted amino-acid residues in the 2 independent selections may inhibit the binding?

K94G15A147

E148

D16

K227

The change in the DNA bending pattern may make some residues improperly positioned in the active site?

Mutated positions in the selection for -10A/-7A

Mutated positions in the selection for -5C/-4C

Crystallography of a monomeric LAGLIDADG endonuclease, I-OnuI

The crystals of SeMet I-OnuI/DNA complex are being grown for phasing.

GST/I-OnuI

I-OnuI

C F E

Native crystal (2.35 A, P212121)

GST/I-OnuI

I-OnuI

C F E

Growing!

SeMet-labeled crystal

Human monoamine oxidase B (MAOB) gene includes similar sequence to I-OnuI target.

MAOB is the drug target for Parkinson’s disease and some neurodegenarative diseases.

X chromosome:

MAOB:

MAOB target: CAAAAGGTTAAATATGTGGACC |||||||| |||| |||||I-OnuI target: TAAAAGGTTGAATAAGTGGAAA

Exon 3