A novel theophylline triggered RNaseP riboswitch. Stefan Hammer Bled 2018

Transcription

1 A novel theophylline triggered RNaseP riboswitch Stefan Hammer Bled 2018

2 Transcription/Translation 3' RNA polymerase DNA helix 3' factor trnas Ribosomes Tyr Phe Met RNA strand Translation Proteins + NH3 + NH3 Transcription

3 Riboswitches UUUUU + ligand aptamer anti-terminator RBS + ligand terminator RBS UUUUU expression platform Transcriptional expression platform Translational

4 Artificial Riboswitch Theophylline triggered Transcriptional Reporter Gene spacer transcription termination 3'part terminator UUUUUU + theophylline ~4x AUG UUUUUU RBS Wachsmuth, Manja, Sven Findeiß, Nadine Weissheimer, Peter F. Stadler, and Mario Mörl De Novo Design of a Synthetic Riboswitch That Regulates Transcription Termination. Nucleic Acids Research 41 (4):

5 RNaseP riboswitch idea leader- st em aptamer x trna RNaseP processing + theophylline leader trna

6 trna processing mechanism

7 Copy&Paste Design

8 Timing is important! spacer transcription termination leader- st em 3'part terminator aptamer UUUUUU + theophylline x trna UUUUUU AUG RBS RNaseP processing + theophylline leader trna

9 Design Pipeline Biological Analysis Sequence Sampling Optimization Method Objective Function In silico Analysis Biological Testing Findeiß, Sven, Stefan Hammer, Michael T. Wolfinger, Felix Kühnl, Christoph Flamm, and Ivo L. Hofacker In Silico Design of Ligand Triggered RNA Switches. biorxiv, January,

10 Constrained Sampling leader- st em aptamer spacer (0-8nt) leader trna trna Sequence + Structure constraints RNAblueprint Sequences Hammer, Stefan, Birgit Tschiatschek, Christoph Flamm, Ivo L. Hofacker, and Sven Findeiß RNAblueprint: Flexible Multiple Target Nucleic Acid Sequence Design. Bioinformatics 33 (18):

11 Objective Functions Accessibility score [0,1]: leader- st em aptamer x trna State score [0,1]: RNaseP processing + theophylline leader trna

12 Design Candidates 10 original/27-66 maximize_states_score/27-66 maxmimize_accessbility_score/27-66 remove_1nt_terminator/27-65 remove_1nt_terminator maximize_states_score/27-65 remove_1nt_terminator maxmimize_accessbility_score_1/27-65 C C U U G G C remove_1nt_terminator maxmimize_accessbility_score_2/27-65 C C U U G G C remove_1nt_terminator maxmimize_accessbility_score_3/27-65 C C U U G G C remove_1nt_terminator maxmimize_accessbility_score_4/27-65 C C U U G G C remove_2nt_terminator/27-64 remove_2nt_terminator maxmimize_accessbility_score/27-64 C C U U G G C remove_2nt_terminator maximize_states_score/27-64 remove_3nt_terminator/27-63 remove_3nt_terminator maximize_states_score/27-63 remove_3nt_terminator maxmimize_accessbility_score/27-63 C C U U G G C remove_4nt_terminator/27-62 remove_4nt_terminator maximize_states_score/27-62 remove_4nt_terminator maxmimize_accessbility_score/27-62 C C U U G G C remove_5nt_terminator/27-61 remove_5nt_terminator maximize_states_score/27-61 remove_5nt_terminator maxmimize_accessbility_score/27-61 C C U U G G C remove_6nt_terminator/27-60 remove_6nt_terminator maximize_states_score/27-60 remove_6nt_terminator maxmimize_accessbility_score/27-60 C C U U G G C remove_7nt_terminator/27-59 remove_7nt_terminator maximize_states_score/27-59 remove_7nt_terminator maxmimize_accessbility_score/27-59 C C U U G G C remove_8nt_terminator/27-58 remove_8nt_terminator maxmimize_accessbility_score/27-58 C C U U G G C AG A A AU C U C U G A AGU GC U GC C A AGG AG A A AU C U U U GGGGU GU U GU U G AGG AG A A AU C U C U GGGGU GU U GU U G AGG AG A A AU C U C U G A AGU GC U GC C A - GG AG A A AU C U U U GGGGU GU U GU U G - GG AG A A AU C U U U GGGGU GU U GU U A - GG AG A A AU C U C U GGGGU GU U GU U A - GG AG A A AU C U U U G AGGU GU U GU U A - GG AG A A AU C U C U G A AGU GU U GU U G - GG AG A A AU C U C U G A AGU GC U GC C - - GG AG A A AU C U C U GGGGU GU U GU C - - GG AG A A AU C U U U GGGGU GU U GU C - - GG AG A A AU C U C U G A AGU GC U GC GG AG A A AU C U U U GGGGU GU U GC GG AG A A AU C U U U GGGGU GU U GC GG AG A A AU C U C U G A AGU GC U G GG AG A A AU C U U U GGGGU GU U G GG AG A A AU C U U U G A AGU GU U G GG AG A A AU C U C U G A AGU GC U GG AG A A AU C U U U GGGGU GC U GG AG A A AU C U U U GG AGU GC U GG AG A A AU C U C U G A AGU GC GG AG A A AU C U U U GGGGU GC GG AG A A AU C U U U GG AGU GC GG AG A A AU C U C U G A AGU G GG AG A A AU C U U U GG AGU G GG AG A A AU C U U U GG AGU G GG AG A A AU C U C U G A AGU GG AG A A AU C U U U GG AGU GG Consensus AG A A AU C U U U GG AGU GU U GU U A AGG

13 Analysis and Filtering trna structure must be maintained Verify strong switching behavior Avoid undesired structures in ensemble Test variation of theophylline binding energies Co-transcriptional analysis without ligand

14 Laboratory Analysis

15 Thanks to Sven Findeiß Felix Kühnl Anna Ender Maja Etzel Mario Mörl Peter Stadler

16 Cotranscriptional View sensing window downhill folding window functional window anti-terminator seed + ligand anti-terminator aptamer aptamer UUUUU anti-terminator terminator aptamer transcription elongation terminator UUUUU expression platform