Synthetic RNA-based devices --- Programming cellular networks using synthetic riboregulators ---

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1 Synthetic RNA-based devices --- Programming cellular networks using synthetic riboregulators --- Synthetic Microbiology Heinrich-Heine-Universität Düsseldorf

2 Synthetic RNA-based devices --- Programming cellular networks using synthetic riboregulators --- Synthetic Microbiology Heinrich-Heine-Universität Düsseldorf

3 Synthetic RNA-based biocomputing devices Synthetic RNA signaling networks TBI Vienna CNRS Paris Düsseldorf/ Berlin 1/ 19

4 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

5 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 release of RBS 3/ 19

6 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 srnas Gottesman & Storz, 2011 Y. pseudotubercolosis 165 srnas Koo et al., 2011 Synechocystis 6803 Mitschke et al., trans-srnas 1011 asrnas for 866 genes 4/ 19

7 Natural RNA regulators in cyanobacteria (Synechocystis sp. PCC 6803) Example 1: trans-encoded srna PsrR1 PsrR1 accumulates under High Light & C i 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

8 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

9 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 Wolfgang R. Hess 7/ 19

10 RNA-based biocomputing devices Synthetic RNA signaling networks TBI Vienna CNRS Paris Düsseldorf/ Berlin 11/ 19

11 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), / 19

12 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

13 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

14 RNA-based biocomputing devices Example: Repression through switching in vivo analysis ATc RNAdev 1 yfp YFP IPTG RNAdev 1 + yfp YFP E. coli E. coli IPTG RNAdev 2 cfp CFP ATc + RNAdev 2 cfp CFP E. coli E. coli 15/ 19

15 Input signals RNA-based biocomputing devices Example: comparator device in vivo analysis RNAdev comparator ATc IPTG yfp + + RNAdev 2 RNAdev cfp E. coli yfp cfp yfp cfp YFP CFP YFP CFP Output signals E. coli 16/ 19

16 Input signals RNA-based biocomputing devices Example: comparator device in vivo analysis in Cyanobacteria RNAdev comparator ATc IPTG yfp + + RNAdev 2 RNAdev cfp yfp cfp Synechocystis yfp cfp Synechocystis xfp zfp xfp zfp?? Output signals 17/ 19

17 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

18 Application: metabolic engineering our strategy: extended comparator self-adjustment & balancing of two branches 19/ 19

19 Our Team Düsseldorf and collaborators: IMET Jülich: Karl-Erich Jaeger Thomas Drepper Anita Loeschcke Dennis Binder Ilka Axmann Jennifer Andres Janos Jablonski Katharina Wiebe Dennis Dienst Julian Balzer Anika Wiegard Jan-Philipp Kunz TBI Vienna: Christoph Flamm Sven Findeiß Stefan Hammer Berlin Tim Kolmsee Stefanie Hertel Christian Beck Adrian Kölsch Beate Heilmann Rainer Machné CNRS Paris: André Estévez-Torres Jonathan Lee Tin Wah 15/ 19

20 Thank you for your attention!!!

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