RNA/RNP synthetic biology

Transcription

1 RN/RNP synthetic biology R N Ç Ç±ÇÃÉsÉNÉ`ÉÉǾå ÇÈÇžÇ½Ç ÇÕïKóvÇ Ç Tan Inoue Kyoto niversity

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.

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 RN protein

4 Background RN design Ribozyme? modular units designed RN? 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, (1987) Reconstitution of a group I intron self- van der Horst, G., Christian,. & Inoue, T. splicing reaction with an activator RN. Proc. Natl. cad. Sci.. S.., 88, (1991) Ikawa, Y., Shiraishi, H. & Inoue, T. Minimal catalytic domain of a group I self-splicing intron RN. Nature Struct. Biol., 7, (2000)

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

13 Background RN design Ribozyme? modular units designed RN? Ribozyme RNP design prototype RNP? multifunctional RNP New parts Circuit NanoRNP

14 RN design w/ Graphic Software cut paste

15 Cut & Paste for designing a Scaffold P6b G /11-nt motif P5b P6a P6 helix I G helix III G P4 triple helical scaffold P5a P5 helix II Designed type B RN P4-P6 domain RN (96 nts) (160 nts)

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

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

18 Design of trans-rn ligase cis RN ligase trans-rn ligase Ikawa et al., PNS, 101:

19 Modularity of ribozyme

20 Modularity of ribozyme

21 Background RN design Ribozyme? modular units designed RN? Ribozyme RNP design prototype RNP? multifunctional RNP New parts Circuit NanoRNP

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

23 Background RN design Ribozyme? modular units designed RN? Ribozyme RNP design prototype RNP? multifunctional RNP New parts Circuit NanoRNP

24 Prototype for multifunctional RNP Lambda ~ 5 nm FRET

25 in silico

26 Cut and Paste

27 in vitro

28 Prototype for multifunctional RNP ~ 5 nm Lambda FRET

29 Prototype for multifunctional RNP FRET RN 0 nm RN 250 nm???? ?? (nm)

30 Prototype for multifunctional RNP 475 nm FRET???? nm RN 0 nm RN 250 nm ?? (nm)

31 Background RN design Ribozyme? modular units designed RN? Ribozyme RNP design prototype RNP? multifunctional RNP New parts Circuit NanoRNP

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

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

34 Multifunctional Nanoparticle Science, 2005, 310, 1132

35 Multifunctional Nanoparticle? Multifunctional RNP Science, 2005, 310, 1132

36 Type B Design C C C C C C C G G G G G G C G C C C C C G G G G G G G G G G C C G G G G G G C C C C G G C G G C G G C G C G C G C C G boxb RRE

37 FRET :TypeB no RN typee RN typeb RN FRET value(i527/i475) no RN typee RN 100nM typeb RN 100nM

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

39 Background Ribozyme? modular units RN design designed RN? 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 TIFFÅiàèkǻǵÅj êlí ÉvÉçÉOÉâÉÄ

42 Collection of RNP motifs L7e-Box C/D S15-rRN ThrRS-mRN

43 For constructing Riboswitch-based synthetic circuits Input ligands: RN binding protein Ribosome ligand Ligand binding motif NNNNNG Start codon Output: GFP mrn

44 Background RN design Ribozyme? modular units designed RN? Ribozyme RNP design prototype RNP? multifunctional RNP New parts Circuit NanoRNP

45 Circuit : ND OR Circuit with riboswitch w/protein 1st step Marker Protein RN binding protein 2nd step Death Signal

46 Background RN design Ribozyme? modular units designed RN? Ribozyme RNP design prototype RNP? multifunctional RNP New parts Circuit NanoRNP

47 Bionano RNP Ç Ç±ÇÃÉsÉNÉ`ÉÉǾå ÇÈÇžÇ½Ç ÇÕïKóvÇ Ç DN RNP

48 TIFFÅià èkç»çµåj êlí ÉvÉçÉOÉâÉÄ Frank Gehry s rchitecture Frederick R. Weisman Museum of rt

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 Leslie E. Orgel TIFF (LZW) êlí ÉvÉçÉOÉâÉÄ "Evolution is cleverer than you are"

51 ppendix

52 RNP design RNP RN RN-protein interactions in naturally occuring RNPs

53 Design of new signal-transduction circuit Target: Cancer cell Method: Signal Detector RN that induces apoptosis Ç Ç±ÇÃÉsÉNÉ`ÉÉǾå ÇÈÇžÇ½Ç ÇÕïKóvÇ Ç RN mrn Step 1 Marker protein RNP switch 1 RN binding protein

54 Design of new signal-transduction circuit Target: Cancer cell Method: Signal Detector RN that induces apoptosis Ç Ç±ÇÃÉsÉNÉ`ÉÉǾå ÇÈÇžÇ½Ç ÇÕïKóvÇ Ç RN mrn Parts Step 1 Marker protein Parts RNP switch 1 Parts RN binding protein

55 Design of new signal-transduction circuit Target: Cancer cell Method: Signal Detector RN that induces apoptosis Ç Ç±ÇÃÉsÉNÉ`ÉÉǾå ÇÈÇžÇ½Ç ÇÕïKóvÇ Ç RN mrn Step 1 Marker protein RNP switch 1 Device RN binding protein

56 Design of new signal-transduction circuit Target: Cancer cell Method: Signal Detector RN that induces apoptosis Ç Ç±ÇÃÉsÉNÉ`ÉÉǾå ÇÈÇžÇ½Ç ÇÕïKóvÇ Ç RN mrn Step 1 System Marker protein RNP switch 1 RN binding protein

57 Design of new signal-transduction circuit Target: Cancer cell Method: Signal Detector RN that induces apoptosis Ç Ç±ÇÃÉsÉNÉ`ÉÉǾå ÇÈÇžÇ½Ç ÇÕïKóvÇ Ç RN mrn Marker protein Step 1 Step 2 Ç Ç±ÇÃÉsÉNÉ`ÉÉǾå ÇÈÇžÇ½Ç ÇÕïKóvÇ Ç RNP switch 1 RNP switch 2 RN binding protein Bim poptosis

58 in silico RN biology

59 Rational design of RNP FRET Value (I527nm/I475nm) Type E RNP double mutant Type E RNP RN?? (nm)

60 RNP switch Ç Ç±ÇÃÉsÉNÉ`ÉÉǾå ÇÈÇžÇ½Ç ÇÕïKóvÇ Ç RN????????????? RNP switch ppendix 1

61 RNP switch Ç Ç±ÇÃÉsÉNÉ`ÉÉǾå ÇÈÇžÇ½Ç ÇÕïKóvÇ Ç RN???????????? RN Protein? RNP switch???: mrn labeling? pre-sirn? RNi?? 2

62 RN aptamer??????????????????rn??????????combinatrial library??????? Protein RN 3

63 RN aptamer Ç Ç±ÇÃÉsÉNÉ`ÉÉǾå ÇÈÇžÇ½Ç ÇÕïKóvÇ Ç??????????????????RN??????????combinatorial library??????? Protein RN 3

64 RN aptamer??????????????????rn??????????combinatorial library??????? Protein? Protein RN RN 4

65 .???????RN???? B. RNP???????RN???? mirn??? ( anti-sense) RRE???? RNase(Dicer)??????? RNase(Dicer)???? Rev???? RNase?????????????? mirn????????????????? mirn?????????on/off?? mrn??? /?????? mirn?? : RNP?????? mirn(sirn)??????????????????? :??????????mirn???????????????? : mirn???????????????? RRE(Rev?? )????????? RNP?????????? mirn?????????????????????+rre??????? RN?????????? 5