RNA/RNP synthetic biology
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- Elijah Long
- 5 years ago
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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)
6 3D 2D
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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