3/29/06 LaBean COMPSCI 296.5RNA Self-assembly(2004) Science 306, 2068-20723/29/06 LaBean COMPSCI 296.5RNA Self-assembly(2004) Science 306, 2068-20723/29/06 LaBean COMPSCI 296.5RNA Self-assembly(2004) Science 306, 2068-20723/29/06 LaBean COMPSCI 296.5stemmer2003Rational designDirectedevolution3/29/06 LaBean COMPSCI 296.5RNA Physical Catalyst(2004) Science 304, 850-852 *UTP is 5-(4-pyridylmethyl)-uridine 5’ triphosphatePd2(DBA)3 is dibenzylide-acetone palladium (0)3/29/06 LaBean COMPSCI 296.5*UTP is 5-(4-pyridylmethyl)-uridine 5’ triphosphatePd2(DBA)3 is dibenzylide-acetone palladium (0)RNA Physical Catalyst3/29/06 LaBean COMPSCI 296.5Ellington & SzostakNature. 1990 Aug 30;346(6287):818-22.In vitro selection of RNA molecules that bind specificligands.Ellington AD, Szostak JW.Department of Molecular Biology, Massachusetts General Hospital, Boston 02114.Subpopulations of RNA molecules that bind specifically to a variety oforganic dyes have been isolated from a population of random sequenceRNA molecules. Roughly one in 1010 random sequence RNA moleculesfolds in such a way as to create a specific binding site for small ligands.3/29/06 LaBean COMPSCI 296.5Ellington & SzostakNature. 1990 Aug 30;346(6287):818-22.Random DNARandom RNATranscriptionColumn fractionationUnbound (discard)[flow-through high salt]Bound RNA[high salt wash, water elute]T7 PromotercDNA PrimingReverseTranscriptionDye column3/29/06 LaBean COMPSCI 296.5Ellington & SzostakNature. 1990 Aug 30;346(6287):818-22. 100 random nucleotides (+ constant regions) Complexity of initial pool ~1015 (1060 possible) Poor transcription; PCR amp; ~1013 diversity ~4x1014 RNA molecules applied to column <0.1% of initial RNA library bound then after 4 to 5cycles of bind, RT, PCR, Transcribe, rebind ~50% ofRNA molecules bound. Binding is specific for dye selected. For 1 column 20 clones were sequenced ~16-20nucleotides were invariant over the set of clones. APTAMER from Latin ‘aptus’, to fit.3/29/06 LaBean COMPSCI 296.5Ellington & SzostakNature. 1990 Aug 30;346(6287):818-22. AC A C G C A C U C A GAAAA GUAC A C C UCACA A-C CC A UCC AUCC CCAAC CAG GC CGG GCC A A ||| |||| ||||| ||| || ||| ||| U G AGG UAGG GGUUG GUC-CG GCC CGG U C A-UAA ACCC C-GAC C-A-A AAU CA G-C G-C C | G-C C-G C-G C-G U-A U-A A \ A G G-C A-U G-U G GB4 Reactive blue binder3/29/06 LaBean COMPSCI 296.5Tuerk & Gold SELEX- systematic evolution of ligands byexponential enrichment. Selected RNA for binding to T4 DNA polymerase. 65,536 possible sequences. Wild-type T4 bacteriophage mRNA sequence foundand a variant with 50% identity to wild-type.3/29/06 LaBean COMPSCI 296.5Tuerk & Gold3/29/06 LaBean COMPSCI 296.5Tuerk & Gold3/29/06 LaBean COMPSCI 296.5Bartel & Szostak 220 random positions 1.6x1015 diversity3/29/06 LaBean COMPSCI 296.5Bartel & Szostak3/29/06 LaBean COMPSCI 296.5In vitro evolution of nucleic acids.Random poolSelect for desired phenotypeSelected poolMultiplecyclesFinal selected ensembleAmplify and/or mutate geneotype3/29/06 LaBean COMPSCI 296.5Review In vitro selection requires linking of genotype andphenotype. Binding targets: simple ions, small molecules,peptides, proteins, organelles, and cells.3/29/06 LaBean COMPSCI 296.5ReviewAMP binding aptamer Selectivity: theophyllineaptamer shows 10,000-folddecrease in binding tocaffeine which differs byone additional methyl group.3/29/06 LaBean COMPSCI 296.5Review3/29/06 LaBean COMPSCI 296.5In vitro selection and directedevolution of peptides and proteins Goal: find protein/peptide with desired function(binding, catalyst, stability, etc.) In vivo vs In vitro selection. Rational design vs. Library methods. Combinatorial peptide libraries. Geysen. Discontinuous epitope mapping. Solid phasepeptide synthesis. Birth of combinatorial chemistry. Phage display. mRNA fusion. Ribosome stalling. Antibody engineering and evolution. Primerless PCR. DNA shuffling. Sexual PCR.3/29/06 LaBean COMPSCI 296.5Scott & SmithFilamentous bacteriophage (M13, fd)Cloning into p3 (pIII) coat proteinNNK codons: N=ACTG, K=GT.Encodes 32 triples, all 20 aminoacids and only one stop codon.antigenantibodyepitopeantigen combining region3/29/06 LaBean COMPSCI 296.5Scott & Smith 64 million possible hexapeptides Library contained 1.3 x 1014 phage from2 x 108 clones3/29/06 LaBean COMPSCI 296.5Phage display Enzyme selection. Note: hidden in vivoselection too...3/29/06 LaBean COMPSCI 296.5 mRNA-proteinfusion Ribosomedisplay Other. Cro?3/29/06 LaBean COMPSCI 296.53/29/06 LaBean COMPSCI 296.5Phage display of Ab libraries3/29/06 LaBean COMPSCI 296.5Artificial immune maturation Gene library from human,animal, or synthetic source. Maturation: Saturation mutagenesis ofCDRs. Chain shuffling. Growth in mutagenic E. colistrains.3/29/06 LaBean COMPSCI 296.5Diabodies and triabodies In vitro selection/evolution of bifunctional antibodyfragments. Universal protein-based adapter/address system.3/29/06 LaBean COMPSCI 296.5Stemmerhttp://www.maxygen.com/science-pub.php Gene reassembly Whole plasmid reassembly3/29/06 LaBean COMPSCI 296.5Stemmer Can natural diversity accelerate the evolution process? Goal: obtain moxalactamase activity from fourcephalosporinase genes. DNA shuffling within genesor between genes...3/29/06 LaBean COMPSCI 296.5Stemmer3/29/06 LaBean COMPSCI 296.5Stemmer Can natural diversity accelerate the evolution process? Goal: obtain moxalactamase activity from four cephalosporinasegenes. DNA shuffling within genes or between genes... One cycle: ~8-fold improvements from separately evolved genes. One cycle: 270- to 540-fold improvement from the four genesshuffled together. Best clone contained 8 segments from 3 of the 4 genes & 33amino-acid point mutations.3/29/06 LaBean COMPSCI 296.5stemmer20033/29/06 LaBean COMPSCI 296.5stemmer20033/29/06 LaBean COMPSCI 296.5Belcher(2000) Nature 405, 665-6683/29/06 LaBean COMPSCI 296.5Belcher GaAs
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