Duke CPS 296.5 - RNA Self-assembly

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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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Duke CPS 296.5 - RNA Self-assembly

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