RNA/RNP synthetic biology

1. A R N RNA/RNP synthetic biology Tan Inoue Kyoto University

2. What is Synthetic Biology? the design and construction of new biological parts, devices, and systems. B) the re-design (rewiring) of existing, natural biological systems for useful purposes. http://syntheticbiology.org/

3. What is Synthetic Biology? the design and construction of new biological parts, devices, and systems. B) the re-design (rewiring) of existing, natural biological systems for useful purposes. Circuit RNP RNA protein

4. Background Ribozyme → modular units RNA design designed RNA→ Ribozyme RNP design prototype RNP → multifunctional RNP New parts Circuit NanoRNP

5. Background Ribozyme is physically separable : modular units. Kay, P. S. & Inoue, T.Catalysis of splicing-related reactions between dinucleotides by a ribozyme. Nature, 327, 343-346 (1987) van der Horst, G., Christian, A. & Inoue, T.Reconstitution of a group I intron self-splicing reaction with an activator RNA. Proc. Natl. Acad. Sci. U. S. A., 88, 184-188 (1991) Ikawa, Y., Shiraishi, H. & Inoue, T. Minimal catalytic domain of a group I self-splicing intron RNA. Nature Struct. Biol., 7, 1032-1035 (2000)

6. 3D 2D

12. Background Ribozyme can be designed and constructed by connecting the modular units.

13. Background Ribozyme → modular units RNA design designed RNA→ Ribozyme RNP design prototype RNP → multifunctional RNP New parts Circuit NanoRNP

14. RNA design w/ Graphic Software cut paste

15. Cut & Paste for designing a Scaffold

16. Scaffold RNA P1 P3 P2&P3 P1 Consisting of 3 helices (P1-P3) P2 and P3 are stacked coaxially by triple helical scaffold motif (blue) P1 and P3 are assembled by GAAA-11nt interaction (red) P2

17. Scaffoldto Ribozyme Ribozyme in 3D Ribozyme in 2D scaffold reaction site = → catalytic module catalytic module

18. Design of trans-RNA ligase cis RNA ligase trans-RNA ligase Ikawa et al., PNAS, 101:13750-5. 2004

19. Modularity of ribozyme

20. Modularity of ribozyme

21. Background Ribozyme → modular units RNA design designed RNA→ Ribozyme RNP design prototype RNP → multifunctional RNP New parts Circuit NanoRNP

22. Why RNP? Combination of designed RNA & protein molecule whose function and structure are known is highly versatile.

23. Background Ribozyme → modular units RNA design designed RNA→ Ribozyme RNP design prototype RNP → multifunctional RNP New parts Circuit NanoRNP

24. Prototype for multifunctional RNP FRET ~ 5 nm Lambda

25. in silico

26. Cut and Paste

27. in vitro

28. Prototype for multifunctional RNP FRET ~ 5 nm Lambda

29. Prototype for multifunctional RNP

30. Prototype for multifunctional RNP 475 nm 527 nm

31. Background Ribozyme → modular units RNA design designed RNA→ Ribozyme RNP design prototype RNP → multifunctional RNP New parts Circuit NanoRNP

32. Multifunctional RNP e.g. Recognition + Labeling + Killing activity Protein Protein Protein RNA Protein RNA Protein Protein

33. Design Target: e.g. Cancer Cell Method：Designed RNA +Functional Protein Recognition: e.g. antibody Killing:Apoptosis inducer e.g. Bim Imaging: e.g. GFP

34. Multifunctional Nanoparticle Science, 2005, 310, 1132

35. Multifunctional RNP ⇔ Multifunctional Nanoparticle Science, 2005, 310, 1132

36. A A G G A U A C G boxB G C C G G C G U A A A A A G U A C G U A U C G C G U A U A A U G C U A A U A U C G U A C G A U A U U A C G A U A G RRE U A U A U G G U A G C U A C G A U A U A G C A U A U U A A U A U G C U A G C A U G C U A A U A U C G U A A U A U C G A C G A A C G U G U A U U C G G C C U A U A G C G U A C Type B Design

37. FRET :TypeB

38. Design Target: e.g. Cancer Cell Method：Designed RNA +Functional Protein Recognition: e.g. antibody Killing:Apoptosis inducer e.g. Bim Imaging: e.g. GFP

39. Background Ribozyme → modular units RNA design designed RNA→ Ribozyme RNP design prototype RNP → multifunctional RNP New parts Circuit NanoRNP

40. To design NEW Parts, we are looking for usable RNP motifs.

41. Collection of RNP motifs

42. Collection of RNP motifs ThrRS-mRNA S15-rRNA L7Ae-Box C/D

43. For constructing Riboswitch-based synthetic circuits Input ligands： ・RNA binding protein Ribosome ligand Ligand binding motif …NNNNNAUG mRNA Start codon Output： GFP

44. Background Ribozyme → modular units RNA design designed RNA→ Ribozyme RNP design prototype RNP → multifunctional RNP New parts Circuit NanoRNP

45. Circuit : AND OR Circuit with riboswitch w/protein 1st step Marker Protein RNA binding protein ・・・・ ・・・・ ・・・・ ・・・・ 2nd step Death Signal

46. Background Ribozyme → modular units RNA design designed RNA→ Ribozyme RNP design prototype RNP → multifunctional RNP New parts Circuit NanoRNP

47. Bionano RNP DNA RNP

48. Frank Gehry’s Architecture Frederick R. Weisman Museum of Art

49. Future directions: 1) Synthetic biology with RNP Design and synthesis of RNP for regulating gene expression e.g. riboswitch, synthetic circuit… 2) NanobioRNP architecture Design and construction of 3D objects (10~100 nm) e.g. Nano cupsule, Nano switch….

50. Leslie E. Orgel 1927. 01. 12 - 2007. 10. 27 "Evolution is cleverer than you are" What is your greatestambition? I would like tounderstand in chemical detailhow RNA or some simplerpolymer capable of evolutionthrough natural selectionestablished itself on the primitiveEarth.