Discovery Research via in vivo Evolution
Huang Lei, Tian He, Wen Ya, and Zhang Yi
Peking University, and
National Institute of Biological Sciences, Beijing
2008 03 02
Discovery Research in Biology
• To answer the question: ‘what’/‘whether’
• Example 1: what activates receptor X? – Whether drug alpha activates X?
• Example 2: what suppresses gene Y?– Whether gene beta suppresses Y?
• Example 3: what maintains stem cell state?– Whether kinase gamma maintains stem cell
state?
Strategies for Discovery Research
• Two strategies towards the goal:
• Guess: answering whether, intelligent but very few novel insight
• Screen: answering what, laboring but can give anti-intuition insight
• Hereinafter we concentrate on screening
Complexity Theory for Screen
• You always have it in your first 100 lines or you never have it -- Seymour Benzer on flies
• Complexity theory: when dimension grows, for serial screening, complexity grows in geometrical metrics
• Monte Carlo method complexity
• Simulated annealing: decelerating Monte Carlo method
Example of Simulated Annealing in Biological System
• Adaptive Immunity
Molecular components of adaptive immunity
• Somatic hypermutation
Molecular components of adaptive immunity
• DNA break and repair
AID at the center of adaptive immunity
•AID converts C to U, causing U:G mispairs.
•The mispairs are repaired through the base excision repair (BER) or the mismatch repair (MMR) pathways
•Mutations are introduced through the intervention of translesion DNA polymerases.
How does AID works?C U
UNG regulates transition/transversion ratio
Limiting AID function
• Transcription rate of the target gene: AID only targets ssDNA
• AID promoters and enhancers
• Epigenetic insulators
• Specific sequence bias
-Hotspots: DGYW/WRCH
(R = A/G, Y = T/C, W = A/T, D = A/G/T).
A Problem:
How to restrict AID function within the targeted sequence? The genomic damage must be avoided!
Possible solution:
Mimic the Immunoglobin structure?
in vivo evolution application based on adaptive immunity
Problems (and solutions?)
• Mammalian cells grow slow– Bacteria/yeast grow fast
• Mammalian cells are expensive– Bacteria/yeast are cheap
• Eukaryote protein has to be correctly folded and glycosylated – Yeast better than bacteria?
AID can work in yeast
An Example
Class of drug target SpeciesNumber of
molecular targets
Targets of approved drugs Pathogen and human 324
Human genome targets of approved drugs
Human 266
Targets of approved small-molecule drugs
Pathogen and human 248
Targets of approved small-molecule drugs
Human 207
Targets of approved oral small-molecule drugs
Pathogen and human 227
Targets of approved oral small-molecule drugs
Human 186
Targets of approved therapeutic antibodies
Human 15
GPCR, deorphanization and drug discovery
• GPCR: G protein coupled receptors
• A huge gene family
• Important pharmacological target
Sexual Reproduction in yeast-- a GPCR signaling pathway
How to get it done in yeast?
GPCR signaling mating pathway expression of heterologous GPCRs
Four modification for heterogolous GPCRs
Introducing heterologous GPCRs
add a cleavable leader sequence to aid transport to the plasma membrane
remove regions not required for interacting with the ligand or G protein.
Modifying the G protein develop chimeric G alpha subunits to incorporate receptor binding pr
operties of mammalian subunits into a Gpa1 subunit that retains efficient interaction with the yeast G beta gamma
Four modification for heterogolous GPCRs
Knockout some native genes and incorporating reporter genes
knock out Ste2, Sst2, Far1
combine reporter genes behind PRE
Autocrine system establish an autocrine system
combine the ligand to a factor or alpha factor facilitating its secreting
but restrict on the membrane
What can we do with it ??
Our Plan …
hAID
lacZ lacI
lacO
IRES
PRE
Peptide-alpha factor
One example using GPCR protocol for artificial evolution
Ade2
Ade2
His3
IRES
Protocol…
The whole system
Signalling
No binding between peptide and GPCR
x
Initially………………..
hAIDlacO
Peptide-alpha factorAde2
Ade2
xlacZ lacI
IRES
PRE His3
IRES
No GPCR signalling: hAID is expressed to mutate peptide ligand
Signalling
Binding between peptide and GPCR
Until the peptide become an agonist of GPCR….
hAIDlacO
PRE
Peptide-alpha factor
GPCR is activated, AID is silenced…
Ade2
Ade2
x
Fus1
lacZ lacI
IRES
PRE His3
IRES
His3 lacZ
lacZ readout with fluorescence……… or visual detection directly
Positive and negative selections
• Positive selection:– his3 mediated histidine- survival– High lacZ activity
• Negative selection:– Raise in complete medium (let it grow!)– Low or no lacZ activity
Applications for drug discovery
• Peptidergic ligand for specific GPCR
• Optimizing peptidergic ligand hits
• Finding conserved motif for agonist/antagonist
Assay procedure: it is easy!
• Transform GPCR to ready-knockout lines• Assay for constitutive activity• Transform the peptide-encoding vector libr
ary into a nice coupled GPCR line• Grow the transformant in large vial with ev
olution medium (His-, 3AT+)• After sometime, collect the solution and pl
ate for colonies• Sequence individual colony for hits
Further development on compound structure
GPCR other than ligand
Taking the complexity of the GPCR pathway into account
We can first use the simple yeast two- or three- hybrid systems for a test.
Yeast two-hybrid system
hAID
lacZ lacI
lacO
IRES
UAS
Peptide-Gal4-AD
For example,the core circuit could be adopted into Y2H
Ade2
Ade2
His3
IRES
The methods in two-hybrid systemsGenerally, the cDNA encoding the DBD-X fusion prot
ein and the cDNA encoding the AD-Y fusion protein are inserted into two plasmids, respectively, and then both transformed into the yeast cells.
Sexual Reproduction in yeast
Interaction mating methods can also be used in two-hybrid systems.
The AD and DBD fusion proteins begin in two different haploid yeast strains with opposite mating types, a and α, respectively.
To test for interaction,the hybrid proteins are brought together by mating, a process in which two haploid cells fuse to form a single diploid cell.
Further, Yeast
three-hybridsystem
• RNA aptamer screen
• … or: RNA-interacting protein?
Application of Y3H
Applications other than GPCR
• Nuclear receptor ligand screen
• Protein interaction screen
• Novel bacterial transcriptional biosensors?
• Whatever you can think about! :)
Summary
• We present a simple core genetic circuit which can evolve any desired target in vivo
• We present a unified, inexpensive solution for both academical and industrial needs
• In vivo evolution brings greater capacity and flexibility to screening
• Further assay development based on mammalian systems such as immune cell lines
Acknowledgements
• Wang Yiping
• Youri Pavlov
• Rao lab members
• iGEM 2007 members :)
• PKU iGEM 2008 society :)