Directed Evolution Try to direct evolution (on shorter time scales) to produce new biologically based materials. Take advantage of the power of mutagenesis and screen/selection to engineer new biomolecules. Rational design of new proteins, etc are limited by our incomplete knowledge of relations between sequence, structure and function. Directed Evolution is an alternative and also a complementary approach. Diverse applications: Industrial Biomedical Basic Research Examples of goals: Improve existing enzymes to function under different environmental conditions (pH, temperature, etc.) Create new enzymes to catalyze specific reactions Industrial enzymes used in over 500 products from detergents to beer making Advantages of enzymes over conventional catalysts: Renewable resource Biodegradable High selectivity Work under mild conditions Examples include replacement of phosphates in laundry detergents with proteases and cellulases, replacement of emulsifiers with lipases in bread making, replace sodium hydroxide with amylases and pectinases. Can also create proteins that specifically bind target ligands (diagnostic applications, biosensors, templates for self-assembly, …) To implement directed evolution, must have a means to link genotype and phenotype. Some methods for linking genotype and phenotype: SELEX with DNA or RNA Phage Display Ribosome Display Direct screening or selecting of cells When some or all of the steps are carried out in vitro then often called in vitro evolution Earliest example (that I am aware of) was from 1967, PNAS 58:217-224 Sol Spiegelman and colaborators: “An extracellular darwinian experiment with a self-duplicating nucleic acid molecule” Evolved Q replicase – an RNA dependent RNA polymerase “What will happen to the RNA molecules if the only demand made on them is the Biliblical injuction, multiply, with the biological proviso that they do so as rapidly as possible?” Start with the RNA genome of phage Q. Note, random RNA fragments cannot replicate by Q. Add replicase, incubate 20min Remove small amount of RNA product and add to new tube, add replicase and incubate 20min repeat. After 74th serial transfer 83% of original genome was eliminated. Replicates 15 times faster than original RNA. SELEX (Systematic Evolution of Ligands by Exponential Enrichment) – a particularly simple means to link phenotype and genotype e.g. select for functional DNA molecules genotype and phenotype are directly linked to the DNA start with random library of DNA sequences and select for those that bind substrate (“aptamers” - “apt” - fit) can be used to find the best DNA sequence that binds a DNA binding protein can also be used to create diagnostic tools. Similar procedures can be performed with RNA - in fact for applications RNA is often more popular. The extra hydroxyl on RNA can make it easier to bind, also provides greater chemical reactivity. Note - with RNA or DNA libraries can start with an enormous number of distinct molecules (32-mer 432 1019 - in fact it would be difficult to really have quite this level of diversity in your library but even something several orders of magnitude smaller is still enormous. Can also screen or select for enzymatic activity: ribozymes Now need to be a little more clever in designing the selection or screen If want a riboszyme that cleaves a particular chemical bond in response to a stimulus (e.g. pH) then can construct a library that is immobilized via this bond, apply stimulus and collect whatever comes out of column. Example of Selex application: find DNA sequences that will bind to spores of B. anthracis (anthrax) www.arches.uga.edu/~mfield/selex.htm Conjugate one set of aptamers to microscopic magnetic beads and another set to a reporter enzyme. If spores are present then magnet will pull out reporter enzyme (e.g. chemiluminescent signal) Phage Display Display peptide or protein on surface of bacterial virus (in principle can use other viruses but phage viruses easiest to prepare etc.) Some proteins on viral coats can accommodate peptides or proteins and will present them on the surface. The phage genome (or alternatively phagemid) contains the sequence for the protein or peptide so isolation of the phage with desired phenotype will also provide the genotype. Most popular is filamentous phage f1 or M13. pIII on the end or pVIII along the length of the rod-like virion for pVIII ~10% can be loaded with alternate peptide Advantage of phage display: easy to screen over 109 sequences Can either clone library directly into phage genome or use a phagemid (plasmid that contains f1 ori) with replication deficient helper phage 5 copies of pIII and pVI 2800 copies pVIII - all can accommodate peptides http://www.biochem.unizh.ch/plueckthun/teaching/Teaching_slide_shows/filamentous_phages/index.htm www.neb.com/nebecomm/products/productE8100.asp Phage library selected for binding anti -endorphin antibody with NEB phage display library www.neb.com/nebecomm/products/productE8100.asp http://www.unizh.ch/~pluckth/slide_shows/Slides/ribo/ Can also express libraries directly in bacteria or yeast and screen or select for desired phenotype - all depends on having a good screen or selection. e.g. yeast surface display. Boder and Wittrup used expressed antibody library against fluorescein on the surface of yeast. Screened for fluorescein binding by flow cytometry. Achieved (femptomolar binding affinity!) Can screen for improved green fluorescent proteins by simply monitoring fluorescence of cells expressing libraries of mutants and selecting the brightest cells. http://cheme.che.caltech.edu/groups/fha/ Yeast surface display (available from Invitrogen) fusion to a-aglutinin http://www.invitrogen.com/content.cfm?pageid=3458 How to generate libraries? can use any mutagenesis technique - e.g. chemical mutagens, mutator strains, UV light, site directed mutagenesis with randomized oligonucleotides, mutagenic pcr, DNA shuffling, … Error Prone PCR Type of mutation Number times observed e.g. substitute Mn for Mg in reaction buffer, 5-fold excess of dTTP and dCTP. AT and TA 34 GA and CT 26 AG and TC 24 AC and TG 6 GC and CG 5 GT and CA 2 Template length EP-PCR doublings Mutations per nucleotide position 5 100 bp 200 bp 400 bp 800 bp 1600 bp 0.0033 0.33 0.66 1.3 2.6 5.3 10 0.0066 0.66 1.3 2.6 5.3 11 20 0.013 1.3 2.6 5.3 11 21 30 0.020 2.0 4.0 7.9 16 32 50 0.033 3.3 6.6 13 26 53 Current Protocols in Molecular Biology, 2004 DNA Shuffling of family of genes to evolve class C cephalosporinase to moxalactam resistance Crameri, et al. 1998 With directed evolution you get what you select for! Very often the system will find a way to solve the challenge you pose in a way that you have not anticipated. Example: Saggio and Laufer, 1993 Used phage displaying a library of peptides to look for binding to a rat mono-clonal antibody against nicotinic acetylcholine receptor. Antibody was conjugated to biotin which was then immobilized on a surface bound with avidin. They ended up selecting for peptides that bind to biotin instead of to the antibody. Note, with all of the techniques we have discussed there is an amplification step between rounds of selection (Selex – pcr; phage display – growth of phage; direct selection of cells – cell growth) so you may end up selecting for what is most easily amplified, not what best satisfies your selection. Selections are never white or black!