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A. R. N. RNA/RNP synthetic biology. Tan Inoue Kyoto University. 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. - PowerPoint PPT Presentation
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RNA/RNP synthetic biology
Tan InoueKyoto University
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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.
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http://syntheticbiology.org/
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.
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RNA protein
RNP
Circuit
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Background Ribozyme → modular units
RNA design designed RNA→ Ribozyme
RNP design prototype RNP → multifunctional RNP
New parts Circuit NanoRNP
Background
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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)
3D 2D
Background
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Ribozyme can be designed and constructedby connecting the modular units.
Background Ribozyme → modular units
RNA design designed RNA→ Ribozyme
RNP design prototype RNP → multifunctional RNP
New parts Circuit NanoRNP
RNA design w/ Graphic Software
cut paste
G
GGGG
GGGU
UUU
AAAUUU
UUU
UUUAAAUUUAAA
CCCGGG
UUUUUUU
GGG CCC
GGGCCCG
GGGCCCC
GGGCCC
AAAAAAAAAAAA
AAAUUU
UUUGGGCCC
CCC
GGGCCCC
GGGG
GGGCCCCCCGGG
UUU
UUU AAAAAAAAAA
GGG
UUU
U
UUUU UUUGGGGAAAAAAA
GGGCCCGGGCCC
AAAUUU
GGGGGG CCCUUU
CCCGGGUUU
AAAUUUU
CCC
GGGCCCC
GGGUUU
CCCGGGUUUAAAA
AAA
CCCAAAA
CCC GGGGGG CCC
AAAA
AAAUUUG
GGGU
UUUCCCCA
AAAUUUUUUUUAAAAA
AAAC
CCC
AAA
5'-55''---3'
-3'--3''
GGGAAAAAAAA
AAACCCUUUUUUUUUUUGGGGAAAAGGGGAAAAUUUUG
GGGCCCCCCCCU UUUUUUUGGGGCCCCA
AAAAAAAAAAAGGGGGGGGGGGGUUUUAAAAUUUUG
GGGGGGGUUUUAAAAAAAAUUUUA
AAAAAAAGGGGCCCCUUUUGGGGAAAAC
CCCGGGGGGGGAAAACCCCAAAAG
GGGGGGGGGGGC
CCCUUUUCCCCUUUUGGGGAAAAA
AAACCCCCCCCAAAAU
UUUGGGGAAAACCCCUUUUU
UUUGGGGCCCCCCCCGGGGAAAACCCCAAAAU
UUUUUUUCCCCUUUUA
AAAGGGGAAAACCCCA
AAAAAAAC
CCCUUUUGGGGA
AAAAAAAUUUUCCCCCCCCUUUUGGGGA
AAAAAAAC
CCCCCCCGGGGAAAACCCCGGGGCCCCAAAAC
CCCCCCCAAAAAAAAUUUUC
CCCCCCCUUUUGGGGGGGGUUUUAAAAC
CCCUUUUC
CCCUUUUGGGGUUUUUUUUGGGGAAAAUUUUA
AAAUUUUG
GGGGGGGAAAAUUUUGGGGCCCCA
AAAGGGGUUUUU
UUUGGGGCCCCGGGGG
GGGGGGGA
AAAA
AAAGGGGA
AAAG
GGGG
GGGGGGGG
GGGA
AAAAAAAU
UUUU
UUUCCCC
A
AAA
5'-
5'-55''---3'
-3'--33''
GAAA/11-nt motif
triple helical scaffold
P4-P6 domain RNA
(160 nts)
Designed type B RNA
(96 nts)
P6b
P6a
P6
P4
P5P5a
P5b helix I
helix II
helix III
Cut & Paste for designing a Scaffold
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
→
scaffold Ribozyme in 3D
reaction site
catalytic module
Ribozyme in 2D
catalytic module=
Scaffold to Ribozyme
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 design designed RNA→ Ribozyme
RNP design prototype RNP → multifunctional RNP
New parts Circuit NanoRNP
Why RNP?Combination of designed RNA & protein molecule whose function and structure are known
is highly versatile.
Background Ribozyme → modular units
RNA design designed RNA→ Ribozyme
RNP design prototype RNP → multifunctional RNP
New parts Circuit NanoRNP
Prototype for multifunctional RNP
~ 5 nm
FRET
Lambda
in silico
Cut and Paste
in vitro
~ 5 nm
FRET
Lambda
Prototype for multifunctional RNP
FRET
0
0.5
1
1.5
2
2.5
3
450 500 550
îgí∑Å@ÅinmÅj
åuåıã≠ìx
RNA 0 nMRNA 250 nM
Prototype for multifunctional RNP
FRET
0
0.5
1
1.5
2
2.5
3
450 500 550
îgí∑Å@ÅinmÅj
åuåıã≠ìx
RNA 0 nMRNA 250 nM
475 nm
527 nm
Prototype for multifunctional RNP
Background Ribozyme → modular units
RNA design designed RNA→ Ribozyme
RNP design prototype RNP → multifunctional RNP
New parts Circuit NanoRNP
ProteinProtein
Protein
Protein
RNA
ProteinRNA Protein
Multifunctional RNP
e.g. Recognition + Labeling + Killing activity
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
Science, 2005, 310, 1132
Multifunctional Nanoparticle
Science, 2005, 310, 1132
Multifunctional Nanoparticle Multifunctional RNP⇔
Type B Design
CA
AA
A
A
AA
C
C
C
CC
UU
UU
C
UGG
G
G
G
GC
G
C
CC
CC
A
AA
A
GG
G
G
U
U
U
U
U
U
G
A
GG
G
U
A
A
AG
G C
CU
GG G
G
GGC
CC
U A
C GGC
U AA
AAG
G
CAG
GCU
GA U
CG CG C
CA U
GA U
A UU AA UU AA UU AA UU AA UU AA UU AU AA UU AA UU AA UU AA UU AA UU AA U
A A
boxB
RRE
FRET :TypeB
0
0.2
0.4
0.6
0.8
1
1.2
1.4
440 460 480 500 520 540 560 580 600
no RNAtypeE RNAtypeB RNA
0.9
0.95
1
1.05
1.1
1.15
1.2
1.25
1.3
no RNA typeE RNA 100nM typeB RNA 100nM
FRET value(I527/I475)
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 design designed RNA→ Ribozyme
RNP design prototype RNP → multifunctional RNP
New parts Circuit NanoRNP
To design NEW Parts, we are
looking for usable RNP motifs.
Collection of RNP motifs
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Collection of RNP motifs
L7Ae-Box C/D S15-rRNA ThrRS-mRNA
…NNNNNAUG mRNA
Ligand binding motif
Start codon
Input ligands :
・ RNA binding proteinRibosome
ligand
Output : GFP
For constructing Riboswitch-based synthetic circuits
Background Ribozyme → modular units
RNA design designed RNA→ Ribozyme
RNP design prototype RNP → multifunctional RNP
New parts Circuit NanoRNP
Circuit : AND OR Circuit with riboswitch w/protein
・・・・ ・・・・ ・・・・ ・・・・
Death Signal
RNA binding protein
Marker Protein
1st step
2nd step
Background Ribozyme → modular units
RNA design designed RNA→ Ribozyme
RNP design prototype RNP → multifunctional RNP
New parts Circuit NanoRNP
Bionano RNP
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DNA RNP
Frank Gehry’s Architecture Frederick R. Weisman Museum of Art
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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….
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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"
Appendix
RNP design
RNA RNA-protein interactions in naturally occuring RNPs
RNP
Design of new signal-transduction circuit
Target: Cancer cellMethod: Signal Detector RNA that induces apoptosis
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RNA binding protein
Step 1 Marker protein
RNP switch 1
RNA
mRNA
Design of new signal-transduction circuit
Target: Cancer cellMethod: Signal Detector RNA that induces apoptosis
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RNA binding protein
Step 1 Marker protein
RNP switch 1
RNA
mRNAParts
Parts
Parts
Design of new signal-transduction circuit
Target: Cancer cellMethod: Signal Detector RNA that induces apoptosis
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RNA binding protein
Step 1 Marker protein
RNP switch 1
RNA
mRNA
Device
Design of new signal-transduction circuit
Target: Cancer cellMethod: Signal Detector RNA that induces apoptosis
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RNA binding protein
Step 1 Marker protein
RNP switch 1
RNA
mRNA
System
Design of new signal-transduction circuit
Target: Cancer cellMethod: Signal Detector RNA that induces apoptosis
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BimApoptosis
RNA binding protein
Step 1 Step 2Marker protein
RNP switch 1 RNP switch 2
RNA
mRNA
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in silico RNA biology
0.99
1.04
1.09
1.14
1.19
1.24
1.29
1.34
1.39
0 100 200 300RNA濃度 (nM)
FRET V
alu
e (
I5
27
nm/I4
75
nm)
Type E RNP
double mutantType E RNP
Rational design of RNP
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RNP switch
RNP switch☞
RNA の構造変換をデザインする
Appendix 1
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RNP switch
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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
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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
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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
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3
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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。
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Protein
RNA
☞
Protein
RNA
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4
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