systematic evolution of ligands by exponential.PPTVIP

Systematic Evolution of Ligands by ExponentialEnrichment: RNA Ligands to BacteriophageT4 DNA Polymerase CRAIG TUERK AND LARRY GOLD So what was being studied? The interaction of bacteriophage T4 DNA polymerase (gp43) and the ribosome binding site of the mRNA that it encodes But a bit of background first… Binding gp43 overlapping the SD sequences prevents translation and promotes autogenous regulation The minimum target size of the RNA is 36nt which includes a 5bp helix and 8nt loop To investigate what is responsible for this loop bias, the researches created the SELEX method to select preferred binding sequences from a random sequence pool But what the heck is SELEX? Sytematic Evolution of Ligands by EXponential enrichment. The method relies on mechanisms that are often ascribed to evolution to accomplish its goal. Variation Selction Replication Creation of Variant Sequences Created a 110nt ssDNA transcript by ligating three oligonucleotides together and two bridging oligos Oligo 4 was randomized for the 8nt of the loop sequence (AAUAACUC), creating 65,536 sequences WT sequence was also engineered and used for comparison with the results. Comparison of dissociation constants Kd for WT:gp43 = 5x10^-9 Kd for variant sequences = 3.2x10^-7 WT binding is about 60x stronger than variant sequences, but only 100x stronger than nonspecific binding So this leads us to a goal To enrich for the highest affinity RNA ligands to gp43 Experiment Three experiments were carried out at different concentrations ratios of RNA to gp43. A=10 B=1000 C=100 RNA was added to gp43 in excess of binding sites so that competitive binding occurred. Amount of RNA retrieved was roughly equal to the amount of gp43 in the reaction What happened? After four rounds of selection the labeled products exhibited binding that was indistinguishable from WT binding Though it is not explained HOW they know this Three rounds of B were gel purified and sequenced The final round of all the experiments were puri