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

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

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. 3D2D

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 cutpaste

15. Cut & Paste for designing a Scaffold

16. P1 P2 P3 P1 P2&P3 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) Scaffold RNA

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

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

19. 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 ~ 5 nm FRET Lambda

25. in silico

26. Cut and Paste

27. in vitro

28. ~ 5 nm FRET Lambda Prototype for multifunctional RNP

30. 475 nm 527 nm Prototype for multifunctional RNP

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

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

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. Science, 2005, 310, 1132 Multifunctional Nanoparticle

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

36. 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. L7Ae-Box C/D S15-rRNA ThrRS-mRNA

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

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 ・・・・ Death Signal RNA binding protein Marker Protein 1st step 2nd step

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) Nanobio RNP architecture Design and construction of 3D objects (10~100 nm) e.g. Nano cupsule, Nano switch….

50. What is your greatest ambition? I would like to understand in chemical detail how RNA or some simpler polymer capable of evolution through natural selection established itself on the primitive Earth. Leslie E. Orgel 1927. 01. 12 - 2007. 10. 27 "Evolution is cleverer than you are"Evolution

51. Appendix

52. RNP design RNARNA-protein interactions in naturally occuring RNPs RNP

53. Design of new signal-transduction circuit Target: Cancer cell Method ： Signal Detector RNA that induces apoptosis RNA binding protein Step 1 Marker protein RNP switch 1 RNA mRNA

54. Design of new signal-transduction circuit Target: Cancer cell Method ： Signal Detector RNA that induces apoptosis RNA binding protein Step 1 Marker protein RNP switch 1 RNA mRNA Parts

55. Design of new signal-transduction circuit Target: Cancer cell Method ： Signal Detector RNA that induces apoptosis RNA binding protein Step 1 Marker protein RNP switch 1 RNA mRNA Device

56. Design of new signal-transduction circuit Target: Cancer cell Method ： Signal Detector RNA that induces apoptosis RNA binding protein Step 1 Marker protein RNP switch 1 RNA mRNA System

57. Design of new signal-transduction circuit Target: Cancer cell Method ： Signal Detector RNA that induces apoptosis Bim Apoptosis RNA binding protein Step 1 Step 2 Marker protein RNP switch 1 RNP switch 2 RNA mRNA

58. in silico RNA biology

59. RNP double mutant Type E RNP Rational design of RNP

60. RNP switch ☞ RNA の構造変換をデザインする Appendix 1

61. RNP switch Protein RNA RNP switch ☞ RNA の構造変換をデザインする 拡張性： mRNA labeling 、 pre-siRNA ⇨ RNAi 制御 2

62. RNA aptamer マーカータンパク質に特異的に結合する RNA を 分子デザインされた combinatrial library から取得する。 Protein RNA 3

63. RNA aptamer マーカータンパク質に特異的に結合する RNA を 分子デザインされた combinatorial library から取得する。 Protein RNA 3

64. RNA aptamer マーカータンパク質に特異的に結合する RNA を 分子デザインされた combinatorial library から取得する。 Protein RNA ☞ Protein RNA 4

65. 目的 : RNP を利用した、 miRNA(siRNA) のプロセッシング制御・モニタリング 従来法 : 目的のタイミングでの miRNA プロセッシング技術はない。 本技術 : miRNA 機能に影響しない「ループ部分」に RRE(Rev 結合 ) モチーフ を導入し、 RNP を形成させる。内在性 miRNA の制御には、 ループ部分に対するアンチセンス +RRE モチーフの融合 RNA を同様に利用できる。 RNase(Dicer) プロセッシング 成熟 miRNA mRNA の切断 / 翻訳抑制 A. 通常のマイクロ RNA 生成機構 B. RNP によるマイクロ RNA 機能制御 RNase(Dicer) Rev ペプチド （ anti-sense) RRE モチーフ 活性調節 競合ペプチドにより ON/OFF 制御 ・ RNase 制御ドメイン ・蛍光、発光蛋白質 成熟 miRNA miRNA 前駆体 miRNA プロセッシング制御・ 動態モニター 5