Synthetic RNA-based devices --Programming cellular networks using synthetic riboregulators --Synthetic Microbiology Heinrich-Heine-Universität Düsseldorf 24.06.2014 Synthetic RNA-based devices --Programming cellular networks using synthetic riboregulators --Synthetic Microbiology Heinrich-Heine-Universität Düsseldorf 24.06.2014 Synthetic RNA-based biocomputing devices Synthetic RNA signaling networks TBI Vienna Düsseldorf/ Berlin www.ribonets.eu 1/ 19 CNRS Paris Synthetic RNA-based biocomputing devices Outline Overview: base-pairing sRNAs in bacteria One example from the cyanobacterium Synechocystis sp. PCC 6803 PsrR1 – a negative regulator of photosynthesis-related mRNAs In silico design of RNA devices Example: Repression through switching Strategies for in vivo analysis Application: metabolic engineering 2/ 19 RNA - a versatile regulator trans-encoded base-pairing sRNAs ´negative regulation´ • Blocking ribosome binding site (RBS) • Targeting coding sequence (CDS) degradation of sRNA-mRNA duplex by RNases ´positive regulation´ • Preventing the formation of an inhibitory structure Waters & Storz, 2009 3/ 19 release of RBS RNA - a versatile regulator base-pairing with target mRNAs limited complementarity multiple target sequences 6-7 nt for ‘‘seed-pairing’’ decrease/increase ribosome binding and/or mRNA stability E. coli 80-100 sRNAs Gottesman & Storz, 2011 Y. pseudotubercolosis 165 sRNAs Koo et al., 2011 Synechocystis 6803 Mitschke et al., 2011 4/ 19 314 trans-sRNAs 1011 asRNAs for 866 genes Natural RNA regulators in cyanobacteria (Synechocystis sp. PCC 6803) Example 1: trans-encoded sRNA PsrR1 PsrR1 accumulates under High Light & Ci limitation PsrR1 psaL PsrR1 Northern Blot Anti-SD Mitschke, Georg, Scholz, Sharma, Dienst, Bantscheff, Voß, Steglich, Wilde, Vogel, Hess. PNAS 2011 Georg, Dienst, Schürgers, Kuchmina, Wallner, Klähn, Knoop, Lokstein, Hess, Wilde. Submitted 5/ 19 Natural RNA regulators in cyanobacteria (Synechocystis sp. PCC 6803) Example 1: trans-encoded sRNA PsrR1 PsrR1 targets psaL mRNA EMSA Mitschke, Georg, Scholz, Sharma, Dienst, Bantscheff, Voß, Steglich, Wilde, Vogel, Hess. PNAS 2011 Georg, Dienst, Schürgers, Kuchmina, Wallner, Klähn, Knoop, Lokstein, Hess, Wilde. Submitted 6/ 19 Natural RNA regulators in cyanobacteria (Synechocystis sp. PCC 6803) Example 1: trans-encoded sRNA PsrR1 Constitutive and pulsed overexpression provides insight into PsrR1 function PC Chl a control psrR1+ psrR1+ Car Western Blot BN PAGE Mitschke, Georg, Scholz, Sharma, Dienst, Bantscheff, Voß, Steglich, Wilde, Vogel, Hess. PNAS 2011 Georg, Dienst, Schürgers, Kuchmina, Wallner, Klähn, Knoop, Lokstein, Hess, Wilde. Submitted Annegret Wilde 7/ 19 Wolfgang R. Hess RNA-based biocomputing devices Synthetic RNA signaling networks TBI Vienna Düsseldorf/ Berlin www.ribonets.eu 11/ 19 CNRS Paris RNA-based biocomputing devices In silico design of RNA devices Christoph Sven Stefan analysis and selection of the best solutions by machine learning approach Höner zu Siederdissen, C., Hammer, S., Abfalter, I., Hofacker, I. L., Flamm, C., & Stadler, P. F. (2013). Biopolymers, 99(12), 1124-1136. 12/ 19 RNA-based biocomputing devices Status: In total 6 RNAdev candidates: Direct OFF Switch C99 and F34 Direct ON Switch A94 and E63 Indirect OFF Switch D50 and H60 13/ 19 RNA-based biocomputing devices Example: Repression through switching - indirect Solution H60 sRNA 5‘UTR SD SD Sequence constraints: • Leading A • Terminator loop sequence (GCGAAAGC) Sequence constraints: • • • • pos 1-14 (ACCCGTTTTTTTGG) SD sequence (AAGGAG) Length between RBS and start codon (7N) Start Codon (ATG) RNA-RNA duplex 14/ 19 RNA-based biocomputing devices Example: Repression through switching in vivo analysis RNAdev 1 IPTG ATc RNAdev 1 YFP YFP + yfp E. coli E. coli RNAdev 2 ATc IPTG CFP cfp E. coli 15/ 19 yfp RNAdev 2 CFP + cfp E. coli RNA-based biocomputing devices Example: comparator device in vivo analysis YFP yfp Input signals CFP cfp RNAdev 2 E. coli YFP RNAdev 1 IPTG yfp + + cfp cfp E. coli 16/ 19 CFP Output signals + + ATc RNAdev comparator yfp RNA-based biocomputing devices Example: comparator device in vivo analysis … in Cyanobacteria yfp RNAdev comparator Input signals ATc RNAdev 2 RNAdev 1 IPTG + + cfp yfp zFP cfp Synechocystis xFP yfp cfp Synechocystis 17/ 19 Output signals + + xFP ? zFP ? Application: metabolic engineering production of antimalarial drug artemisinin (Keasling, 2012; Martin et al., 2003) problem: intermediate toxic compounds accumulate solution: RNA-based comparator J. Keasling 18/ 19 Application: metabolic engineering our strategy: extended comparator → self-adjustment & balancing of two branches 19/ 19 Our Team Düsseldorf Ilka Axmann Berlin Jennifer Andres Janos Jablonski Katharina Wiebe Dennis Dienst Tim Kolmsee Stefanie Hertel Christian Beck Adrian Kölsch Beate Heilmann Rainer Machné Julian Balzer Anika Wiegard Jan-Philipp Kunz … and collaborators: IMET Jülich: Karl-Erich Jaeger Thomas Drepper Anita Loeschcke Dennis Binder 15/ 19 TBI Vienna: Christoph Flamm Sven Findeiß Stefan Hammer CNRS Paris: André Estévez-Torres Jonathan Lee Tin Wah Thank you for your attention !!!
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