EngineeringRNA

EngineeringRNA - RNA Engineering BioE131 Microarrays Beads...

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RNA Engineering BioE131
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PCR Microarrays siRNA Beads Widely-used engineered RNAs
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Possibilities ... Hammerhead ribozyme Ribosomal RNA Hepatitis Delta Virus ribozyme
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Repressilator Elowitz & Leibler, Nature, 2000
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Post-transcriptional regulation Keene, 2007 “RNA regulons”
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RNA localization in Drosophila bicoid osk (Micklem et al, 2000) orb (Navarro et al, 2004) K10 ftz (Lall et al, 1999) nanos (Forrest & Gavis, 2003)
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Zipcodes K10 nanos (Crucs et al, 2000) ~40Myr
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Regulatory RNA genes From www.ionchannels.org Lai et al, 2000 Also (in flies) rox , hsr -omega, bithoraxoid (?). .. miRNAs. ..
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RNA vs DNA engineering RNA DNA Localization Degradation Regulation of translation Etc. cis -acting transcription factors
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Protein engineering
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RNA vs protein engineering RNA Proteins Easy to design specific binding partners Can predict structure Ideal for some processes (e.g. post- transcriptional regulation) Much broader functional repertoire Essential for many processes
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RNA structure jargon (a.k.a. “stem loop”) “Wobble” base pair “Watson-Crick” base pairs Pseudoknot
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Subtleties of RNA folding energetics Favored loops, e.g. tetraloop: Favored bulges: Base pair “stacking”: Van der Waals, steric interactions
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Aptamers Structured RNA or DNA Forms binding pocket for specific ligand Generated by in vitro selection c.f. antibodies Saccharide aptamer Bacteriophage MS2 coat protein aptamer
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In vitro selection Increasingly automated process
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Riboswitches Naturally occurring aptamers Coupled to genetic switches Regulators of gene expression Widespread in prokaryotes 2% of genes in Bacillus subtilis Known in eukaryotes ( Arabidopsis, Neurospora. .. )
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Adenine riboswitch In the presence of sufficient adenine, the terminator stem- loop does not form and the mRNA is transcribed When adenine is not present, the stem-loop forms and transcription is prematurely terminated (B.subtilis )
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Riboswitch- ligand complex is 3-dimensional Ligand is hypoxanthine, a metabolite of guanine
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Theophylline (dimethylxanthine) Anti-asthmatic drug; naturally found in tea Riboswitch: presence of theophylline prevents transcription Works by blocking the Ribosome Binding Site ( RBS ), a.k.a. the
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EngineeringRNA - RNA Engineering BioE131 Microarrays Beads...

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