rna/rnp synthetic biology

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.



RNA protein

RNP

Circuit



Background Ribozyme → modular units

RNA design designed RNA→ Ribozyme

RNP design prototype RNP → multifunctional RNP

New parts 　　 Circuit 　　　　　　　 NanoRNP

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)

http://www.nature.com/cgi-taf/DynaPage.taf?file=/nsb/journal/v7/n11/abs/nsb1100_1032.html&dynoptions=doi1072232781



Background　



　 Ribozyme can be designed and constructedby connecting the modular units.

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

Why RNP?Combination of designed RNA & protein molecule whose function and structure are known

is highly versatile.

Prototype for multifunctional RNP

~ 5 nm

FRET

Lambda

in silico

Cut and Paste

in vitro

~ 5 nm

FRET

Lambda


FRET

0

0.5

1

1.5

2

2.5

3

450 500 550

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åuåıã≠ìx

RNA 0 nMRNA 250 nM


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


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

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

Circuit : AND OR Circuit with riboswitch w/protein

・・・・・・・・・・・・・・・・

Death Signal

RNA binding protein

Marker Protein

1st step

2nd step

Bionano RNP



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"

http://en.wikipedia.org/wiki/Evolution

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









RNA binding protein

Step 1 Marker protein

RNP switch 1

RNA

mRNA











RNA binding protein


RNP switch 1

RNA

mRNAParts

Parts

Parts











RNA binding protein


RNP switch 1

RNA

mRNA

Device











RNA binding protein


RNP switch 1

RNA

mRNA

System













BimApoptosis

RNA binding protein

Step 1 Step 2Marker protein

RNP switch 1 RNP switch 2

RNA

mRNA



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







RNP switch

RNP switch☞

RNA の構造変換をデザインする

Appendix 1







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





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



RNA aptamer


分子デザインされた combinatorial library から取得する。

Protein

RNA





3



RNA aptamer


分子デザインされた combinatorial library から取得する。



Protein

RNA

☞

Protein

RNA



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

rna/rnp synthetic biology

Documents

rna ligasecis rna ligase

activator rna

rna ligase ikawa

redesign rewiring of

selfsplicing intron

multifunctional rnpprototype

multifunctional rnpgraph1000

natural biological systems