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Functional RNA - Introduction Part 2

Functional RNA - Introduction Part 2. Biochemistry 4000 Dr. Ute Kothe. in vitro selection of RNAs. SELEX = Systematic evolution of ligands by exponential enrichment. Generates Aptamers

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Functional RNA - Introduction Part 2

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  1. Functional RNA- Introduction Part 2 Biochemistry 4000 Dr. Ute Kothe

  2. in vitro selection of RNAs SELEX = Systematic evolution of ligands by exponential enrichment • Generates Aptamers • = oligonucleotides (RNA or ssDNA) which bind to their target with high selectivity and sensitivity because of their 3-dimensional shape • Targets: • single molecules to whole organisms • Chiral molecules • Recognition of distinct epitopes • Applications: • pharmaceutical research • drug development • proteomics • molecular biology

  3. SELEX • Library: 1013 – 1015 sequences • In vitro selection • Binding to target • Partitioning from unbound oligos • Elution of selected oligos • Amplification • PCR for DNA or RT-PCR for RNA • Conditioning: transformation of dsDNA into new pool of ssDNA or RNA for seletion • Iterative process

  4. Random oligonucleotide library Chemically synthesized DNA oligonucleotides: Randomized sequence flanked by 2 fixed sequences used as primer binding sites

  5. Selection of catalytic RNA • more complex RNA • – often random pool is further enlarged by mutagenic PCR • reaction must result in self-modification • such that active molecules can be selected • Example: Selection of an RNA ligase ???

  6. In vitro evolution of proteins • Principle: • selection based on protein properties, genes must be selected simultaneously • Physical linkage between genotype & phenotype Methods: • Cell-surface display • Phage display • mRNA display • Ribosome display • In vitro compartmentalization

  7. Selection of proteins: mRNA Display • random mRNA is translated in vitro • mRNA is linked to DNA oligo • with puromycin • puromycin covalently attaches • mRNA to produced protein Puromycin: analog of Tyr-tRNA can not be hydrolyzed

  8. Selection of proteins: mRNA Display • By binding to • target of interest • specific for • Each problem

  9. In vitro evolution of proteins Ribosome Display In vitro compartmentalization • mRNA linked tomicrobeads emulsified with substrate-biotin conjugate • product-biotin binds to beads via streptavidin • detection of product by fluorescent-labeled anti-product antibody, sorting by FACS • In vitro translation of mRNA without stop codon • mRNA is linked to protein in ternary complex with ribosome

  10. k1 k2 k3 k4 E + S ES ES* EP E + P k-1 k-2 k-3 k-4 Enzyme/ribozyme kinetics Kinetics = study of chemical reaction rates Why Kinetics? • Understanding of enzyme function: affinity, maximum catalytic rate • Identification of intermediates • Insight into catalytic mechanism • Investigation of inhibitors, activators

  11. k1 k2 E + S ES EP E + P k-1 Michaelis-Menten Kinetics Assumed Mechanism: Assumption of steady-state, i.e. [ES] = constant, then: k-1 + k2 KM = k1 kcat [E0] [S] v = KM + [S] vmax = kcat [E0] • Follow reaction under multiple-turnover conditions to obtain kcat & KM • Problem: KM╪ KD and kcat╪ k2 (kchem) if not Michaelis-Menten mechanism • no information on intermediate steps and their rate constants

  12. k1 k2 k3 k4 E + S ES ES* EP E + P k-1 k-2 k-3 k-4 Pre-steady state Kinetics Solution: Follow reaction • in real-time, i.e. pre-steady state by rapidly mixing substrates and enzymes and detection in ms to s range • under single-turnover conditions ([E] >> [S]) • Quench-Flow: observation of chemcial reactions (S P) • Stopped-Flow: observation of conformational changes by absorbance or fluorescence

  13. Rate constants First order reaction: v = d[P] / dt = - d[S] / dt = k [S] S P • ln[S] = ln [S0] –kt • [S] = [S0] exp (-kt) Second order reaction: v = d[P] / dt = - d[S1] / dt = - d[S2] / dt = k [S1] [S2] S1 + S2 P • [S1] = ??? • measure at pseudo-first order conditions: [S1] >> [S2] • [S1] = constant • v = - d[S2] / dt = k’ [S2] with k’ = k [S1] • [S2] = [S20] exp (-k’t) • measure apparent rate constant k’ at various [S1] to determine rate constant k

  14. Quench-Flow • rapidly mix samples • stop reaction after desired time (ms) with quencher (strong acid, base etc.) • analyze (radioactive) reaction product by HPLC, thin-layer chromatography etc. • One time point at a time, several mixing events required to obtain time curve

  15. Quench-Flow data EPSP synthase: PEP + S3P I EPSP + Pi shikimate 3-phosphate (S3P), 5-enolpyruvoylshikimate 3-phosphate (EPSP)

  16. Stopped-flow • Rapidly mix samples, • stop the flow of mixed solutions such that it stays in cuvette • Detect change in fluorescence/absorbance in real time • One mixing event generates data of whole time curve

  17. Stopped-Flow data • Analyze data by exponential fitting: • F = Amp * exp (-kapp*t) • Generates apparent rate constant kapp (e.g. for particular concentrations) • Titrate different substrate concentrations to determine real rate constant k from kapp

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