RNARNP synthetic biology R N A Tan Inoue
RNA/RNP synthetic biology R N A Tan Inoue Kyoto University
What is Synthetic Biology? A) the design and construction of new biological parts, B) devices, and systems. B) the re-design (rewiring) of existing, natural biological systems for useful purposes. http: //syntheticbiology. org/
What is Synthetic Biology? A) the design and construction of new biological parts, B) devices, and systems. B) the re-design (rewiring) of existing, natural biological systems for useful purposes. Circuit RNP RNA protein
Background Ribozyme → modular units RNA designed RNA→ Ribozyme RNP design prototype RNP → multifunctional RNP New parts Circuit Nano. RNP
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 selfsplicing 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)
3 D 2 D
Background Ribozyme can be designed and constructed by connecting the modular units.
Background Ribozyme → modular units RNA designed RNA→ Ribozyme RNP design prototype RNP → multifunctional RNP New parts Circuit Nano. RNP
RNA design w/ Graphic Software cut paste
Cut & Paste for designing a Scaffold
Scaffold RNA P 1 P 3 P 1 P 2&P 3 Consisting of 3 helices (P 1 -P 3) P 2 and P 3 are stacked coaxially by triple helical scaffold motif (blue) P 2 P 1 and P 3 are assembled by GAAA-11 nt interaction (red)
Scaffold to Ribozyme in 3 D scaffold Ribozyme in 2 D reaction site → catalytic module = catalytic module
Design of trans-RNA ligase cis RNA ligase trans-RNA ligase Ikawa et al. , PNAS, 101: 13750 -5. 2004
Modularity of ribozyme
Modularity of ribozyme
Background Ribozyme → modular units RNA designed RNA→ Ribozyme RNP design prototype RNP → multifunctional RNP New parts Circuit Nano. RNP
Why RNP? Combination of designed RNA & protein molecule whose function and structure are known is highly versatile.
Background Ribozyme → modular units RNA designed RNA→ Ribozyme RNP design prototype RNP → multifunctional RNP New parts Circuit Nano. RNP
Prototype for multifunctional RNP ~ 5 nm Lambda FRET
in silico
Cut and Paste
in vitro
Prototype for multifunctional RNP ~ 5 nm Lambda FRET
Prototype for multifunctional RNP
Prototype for multifunctional RNP 475 nm 527 nm
Background Ribozyme → modular units RNA designed RNA→ Ribozyme RNP design prototype RNP → multifunctional RNP New parts Circuit Nano. RNP
Multifunctional RNP e. g. Recognition + Labeling + Killing activity Protein RNA Protein
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
Multifunctional Nanoparticle Science, 2005, 310, 1132
Multifunctional Nanoparticle ⇔ Science, 2005, 310, 1132 Multifunctional RNP
Type B Design G A G G A U A U A U A A A G box. B G G C U U C C C G AG A A AU A C U U C G A U G C A A U U C G A G C U A C G U A G RRE A U U G G A G C A C G U A A G C U U A A A U U G C A G C U G C A A U U C G A A C G U G U U A G C U C G C A U C G G U A C
FRET : Type. B
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
Background Ribozyme → modular units RNA designed RNA→ Ribozyme RNP design prototype RNP → multifunctional RNP New parts Circuit Nano. RNP
To design NEW Parts, we are looking for usable RNP motifs.
Collection of RNP motifs
Collection of RNP motifs L 7 Ae-Box C/D S 15 -r. RNA Thr. RS-m. RNA
For constructing Riboswitch-based synthetic circuits Input ligands: Ribosome ・RNA binding protein ligand Ligand binding motif …NNNNNAUG m. RNA Start codon Output: GFP
Background Ribozyme → modular units RNA designed RNA→ Ribozyme RNP design prototype RNP → multifunctional RNP New parts Circuit Nano. RNP
Circuit : AND OR Circuit with riboswitch w/protein 1 st step Marker Protein RNA binding protein ・・・・ 2 nd step Death Signal ・・・・
Background Ribozyme → modular units RNA designed RNA→ Ribozyme RNP design prototype RNP → multifunctional RNP New parts Circuit Nano. RNP
Bionano RNP DNA RNP
Frank Gehry’s Architecture Frederick R. Weisman Museum of Art
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 3 D objects (10~100 nm) e. g. Nano cupsule, Nano switch….
Leslie E. Orgel 1927. 01. 12 - 2007. 10. 27 "Evolution is cleverer than you are"
Appendix
RNP design RNP RNA-protein interactions in naturally occuring RNPs
Design of new signal-transduction circuit Target: Cancer cell Method:Signal Detector RNA that induces apoptosis RNA m. RNA Step 1 Marker protein RNP switch 1 RNA binding protein
Design of new signal-transduction circuit Target: Cancer cell Method:Signal Detector RNA that induces apoptosis RNA m. RNA Step 1 Parts Marker protein Parts RNP switch 1 Parts RNA binding protein
Design of new signal-transduction circuit Target: Cancer cell Method:Signal Detector RNA that induces apoptosis RNA m. RNA Step 1 Marker protein RNP switch 1 Device RNA binding protein
Design of new signal-transduction circuit Target: Cancer cell Method:Signal Detector RNA that induces apoptosis RNA m. RNA Step 1 System Marker protein RNP switch 1 RNA binding protein
Design of new signal-transduction circuit Target: Cancer cell Method:Signal Detector RNA that induces apoptosis RNA m. RNA Step 1 Marker protein Step 2 RNP switch 1 RNA binding protein RNP switch 2 Bim Apoptosis
in silico RNA biology
Rational design of RNP FRET Value (I 527 nm /I 475 nm ) 1. 39 Type E RNP 1. 34 double mutant Type E RNP 1. 29 1. 24 1. 19 1. 14 1. 09 1. 04 0. 99 0 100 200 RNA濃度 (n. M) 300
RNP switch RNAの構造変換をデザインする ☞ RNP switch Appendix 1
RNP switch RNAの構造変換をデザインする Protein ☞ 拡張性:m. RNA labeling、pre-si. RNA⇨RNAi制御 RNP switch 2
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