dna computing and robotics based on deoxyribozymes

DNA Computing and Robotics

Based on Deoxyribozymes

DAO-E Sierpinski triangle experiments

Paul Rothemund, Nick Papadakis, Erik Winfree, PLoS Biology 2: e424 (2004)

340nm

• Part 1: How do we program mixtures of molecules in solution?

• Part 2: How do we program behavior of an individual molecule?

Introduction to deoxyribozymes:

AT

A T

G

C

C

G

A

T

A

T

C

G

T

A

T

A

C

GA TCGTTGAAG

Complementary Single strandedDNA (or RNA)

CT

CTTC

TAGTGA

C

A

CG

G

GC

A GC

TC

GAA

T

Deoxyribozyme

TA

CACT

F

TrAG

AG

AGAG

R

+

Substrate recognition region

Catalytic core

S


TA

CACTF

TrA

OF

CC

A

CG

G

GC

A GC

TC

GAA

T

TAT

ATGCTAGCAT

GA TA TG CA TG C

rAG

F

R

Cleavage

Joyce 1995, 1997

GAAGAG

G

R

Introduction to deoxyribozymes II:

CT

CTTC

TAGTGA

C

A

CG

G

GC

A GC

TC

GAA

T

Deoxyribozyme

TA

CACT

F

TrAG

AG

AGAG

R

+


Catalytic core

S


TA

CACTF

TrA

OF

CC

A

CG

G

GC

A GC

TC

GAA

T

TAT

ATGCTAGCAT

GA TA TG CA TG C

rAG

F

R

Cleavage

Joyce 1995, 1997

+

E

S


E*SSubstrate recognition region

kon

Catalytic core

P1

P2

kcat

+P1

P2

koff's

…and we can combine them with stem loops:

P1

P2

S

P1

P2

kcat

P1

P2

koff's

E

E

ES

P1

P2

…into a two-state switch:

0 1

Detector gateorsensororYES gateor…

Complete set of switches:

i1 i2 o0

1

1111

000 0

00

AND Gate:NOT Gate:

i1 o0

01

1

ANDANDNOT:

i1 i20

1

1100

000 0

00

i1 o0

1 11 1

1 1111

00

0

000

000

1

i3

Before we give examples of gates, a reminder:

CT

CTTC

TAGTGA

C

A

CG

G

GC

A GC

TC

GAA

T

Deoxyribozyme

TA

CACT

F

TrAG

AG

AGAG

R

+


Catalytic core

S


TA

CACTF

TrA

OF

CC

A

CG

G

GC

A GC

TC

GAA

T

TAT

ATGCTAGCAT

GA TA TG CA TG C

rAG

F

R

Cleavage GAAGAG

G

R

1 is an increase in fluorescence, 0 no such increase!

A bit more about FRET:

d1 < d2

GC

TA

C G

A T

C G

CG

CG

GC

TA

G C

AT

A

Fluorescein

TAMRA

480 nm520 nm

580 nm

d1C

C G

G

AT

CG

TA

GC

TA

C G

A T

C G

CG

CG

GC

TA

G C

AT

A

Fluorescein

TAMRA

480 nm520 nm

580 nm

d2

, FRET is stronger

Cleavage

ACAA

TGGA

CACA

TTGA

TA C A C T FTrA

CTCTTC

TAGTGA

C

A

CG

G

GC

A GC

TC

GAA

T

AGAAG

G G

GA

CC

A

CG

G

GC

A GC

TC

GAA

T

AGAAG

CA TT AT AG CG CT

AGC

TA

TA

AT

AT

CG

TA

TA

TA

CACT

F

TrAG

AG

AGAG

R A*S*IA

TAT

ATGCTAGCAT

GA TA TG CA TG C

rAG

F

R

+

Cleavage

Inactive Form

Active Form

A GG

AGG

IA

A

OF

S

IA

0 1

I1

Stojanovic et al., ChemBioChem 2001Stojanovic et al., J. Am. Chem. Soc. 2002

0

50000

100000

150000

200000

250000

0 100 200 300 400t (min)

FU

Catalytic Molecular Beacons as Sensor Gates

Joyce 1995, Breaker 1999, Tyagi, Kramer 1996

01

I2

C

ATGT

C CC

A

G

CGA

G ATC C

GGC

C

ATA

TTC

GCTAGCA 3'T

F

TC

TrAG

AG

AGAG

R3'

A

CAATG

CGA

CT

GT

TTGA

AT

C

ATGT

CCCA

G

CGA

GATC C

G

G

C

C

ATA

TTC

GCTAGCA 3'T

F

TC

TrAG

AG

AGAG

R3'

C

GA

T

GTAACCTGTCTTGAA

CATTGGACAGAACTT

B*IB

TA C A C T FTrA

B

IB

OF

NOT IB

IB

Inactive Form

Active Form

Cleavage

NOT Gates – Inverters:

Stojanovic et al., J. Am. Chem. Soc. 2002

0

50000

100000

150000

200000

0 100 200 300t(min)

FU

0 10

I2

I1I1

I2

0

TA

CACT

F

ACAA

TGGA

CACA

TTGA

TrAG

AG

AGAG

R

TA

CACTF

TrA

ACAAT

G A

CT G T

TTG

A

CTCTTC

TAGTG

C

A

CG

G

GC

A GC

TC

GAA

T

AGAAG

CACT

+IA AND IB

A B

TA

TT

G

IA

IB

OFS

Dual Input Molecular Computation Elements: AND Gate

-20000

0

20000

40000

60000

80000

100000

120000

140000

160000

180000

0 50 100 150 200 250

FU

t(min)

C

ATGT

C CC

A

G

CGA

G AT

C CG

GC

C

A

TTC

GTGA

GAA

ACAA

TGA

CACA

TTGA

A TG C

T

G

A

CAATG

CGA

CTG

TTTG

T

AT

TCTTT

G A

CA C T

TTG

A

TA

CACT

IB

IC

IA

T

A

TA

CACTF

TrA

IA AND IB AND NOT IC

OF

TA

CACT

F

TrAG

AG

AGAG

R3'

Triple Input Elements: ANDANDNOT (INHIBIT)

0 00 0

I2I2I1I1

1

I1

I3 I3 I3

0

I2I1

I3

0

I2

0

I3

Triple Input Elements: ANDAND

Harvey Lederman

TA

CA

CT

F

TrA

GG

AA

GA

GR

C

ATG

T

CCCA

CC

GA

G AT

GA

T

C

GC

A

TT

C

GT

GA

GA

A

T

GCAGA

A

CAGA

AT

T TA

TGC

T

G

G

TT

AGA

C

GAA

AGAT

GCC

T

A

C

TA

TAC

A

C A GA

AA

AC

TA

CA

CT

T

A

C

C

C

A

AT

CT

TT

TC

TAC

GG

G AT G

T GA

GC

AT T

A CG A

T GC A

T TA T

A AT A

T GC A

T GC T

A AC C

G CG A

T

TAG

CGCA

TTA

GCT

TC

C

ATGTC

CCA

CCGA

G A T GA

T

CG

C

A

TT

C

GTGA

GA

A

T

GC

AG

A

A

CAG

A

ATT

T

A

T

G

C

TG

G

TT

AGACG

AA

AGAT

GCC

TA

C

TA

TAC

A

C A GA

AA

ACTA

CA C T

T

A

TC

TG

CGTC

T

ATAA

AT

GT

TTG

TCTA

TA

T

G

T

i1ANDi2ANDi3

i1, i2, i3

c3

c3 i3

i2

i1

i1

i2

c3

2

1

3

0 0 0 0 0 0 1

F

1

c3 c3

2

3

0

c3

2

1 1

3

2

3

0

10000

20000

30000

40000

0 50 100 150

t(min)

FU12

13

123

23

1

2

3

0

Before we give examples of gates, a reminder:

CT

CTTC

TAGTGA

C

A

CG

G

GC

A GC

TC

GAA

T

Deoxyribozyme

TA

CACT

F

TrAG

AG

AGAG

R

+


Catalytic core

S


TA

CACTF

TrA

OF

CC

A

CG

G

GC

A GC

TC

GAA

T

TAT

ATGCTAGCAT

GA TA TG CA TG C

rAG

F

R

Cleavage GAAGAG

G

R


First deoxyribozyme

P1

P2

kcat

F

Q

F

QE*S

P1

P2

kcat

T

Q

T

QSecond deoxyribozyme

Let’s add A (0 or 1) and X (0 or 1) and get O (0 or 1, or 2):

A + X = O

1 1

01 01

0 0

00 00

0 1 2

100100

i1 i2

GR GRGR

First deoxyribozyme

P1

P2

kcat

F

Q

F

QE*S

P1

P2

kcat

T

Q

T

QSecond deoxyribozyme

F

T

Ti1

i1

i1

i1

i1

i1

i1

i2i2

i2

i1

i2i2

i2

i2i2

i1

i2

i1 i2

i2

i1

00

SC

0 1

10

01

C S

F T

C S

Stojanovic & Stefanovic, J. Am. Chem. Soc. 2003

0+1

-2000

0

2000

4000

6000

8000

10000

0 100 200 300

1+0

-5000

0

5000

10000

15000

20000

0 100 200 300

1+1

-5000

0

5000

10000

15000

20000

25000

30000

35000

0 100 200 300

Implement addition with gates:

10 012 1

0+0

-5000

-3000

-1000

1000

3000

5000

7000

9000

0 100 200 300

Now, a twist:

CT

CTTC

TAGTGA

C

A

CG

G

GC

A GC

TC

GAA

T

Deoxyribozyme

TA

CACT

F

TrAG

AG

AGAG

R

+


Catalytic core

S


TA

CACTF

TrA

OF

CC

A

CG

G

GC

A GC

TC

GAA

T

TAT

ATGCTAGCAT

GA TA TG CA TG C

rAG

F

R

Cleavage GAAGAG

G

R


1

d6

d4

d9

d5

d1

d2

d3

d8

d7

A B C D E F G

1

2

3

4

5

6

7

8

9

A

F

G

D

B

E C

A

F

G

D

B

E C

A

F

G

D

B

E C

Segments required

J. Macdonald, Columbia University

Here is 7-segment display:

Test by constructing simple 2+2-bit adder

1 0 1 02+2: + =

A1 A0 X1 X0+

0 1 0 11+1: + =

Binary inputs

0 1 1 01+2: + =

1 0 0 12+1: + =

Non generalDisplay output

Expected display

Adder result “hard-wired” into

decoder

Designate 4 oligonucleotides as binary inputs


More arithmetical operations:

• Determine logic gates required, and layout in wells (via Microsoft Excel) Non-general adder

output gates

Segment AYes A0Yes X0

Segment BYes A0Yes A1

Segment CYes X1Yes A1

Segment DYes A0Yes X0

Segment EA0 AND X0

Segment FA1 AND X1

Segment GYes A1Yes A0

Non-general adder output gates





Segment EA0 AND X0

Segment FA1 AND X1






Segment EA0 AND X0

Segment FA1 AND X1


Very simple logic gate arrangement required: • 4 YES gates • 2 AND gates

Fully constructible from reagents in freezer

Tested with all input combinations….

1 0 1 02+2: + =

A1 A0 X1 X0+

0 1 0 11+1: + =

Binary inputs

0 1 1 01+2: + =

1 0 0 12+1: + =


1 0 1 02+2: + =

A1 A0 X1 X0+

0 1 0 11+1: + =

Binary inputs

0 1 1 01+2: + =

1 0 0 12+1: + =



Adding 2+2 with visual display:

Adding 2+2 with visual display:

Perfect digital behaviorConstructed &

tested in a single day

(A1A0 + X1X0 display)

01+01= 01+10= 10+01= 10+10= No inputs

(1+1=2) (1+2=3) (2+1=3) (2+2=4) Background

Binary inputs:

7-segmentDisplay

Arithmetic

B&W imagerwithin ~1hr

Color photograph~5hrs

Fluorescence reader~20-30mins

J. Macdonald, Columbia University; DNA 13, 2007

2x2 multiplier with visual display:

Segment AA0 AND X1A1 AND X0

Segment BA0 and X0

A0 and X1 not A1A1 and X0 not X1A1 not A0 not X0

Segment CA1 and A0X1 and X0A1 and X1A0 and X0Segment D

A1 and A0X1 and X0A0 and X1A1 and X0

Segment EA1 and X0 not A0A0 and X1 not X0

Segment FA1 and X1

Segment GYes A1Yes X1

Segment AA0 AND X1A1 AND X0

Segment BA0 and X0

A0 and X1 not A1A1 and X0 not X1A1 not A0 not X0

Segment CA1 and A0X1 and X0A1 and X1A0 and X0Segment D

A1 and A0X1 and X0A0 and X1A1 and X0

Segment EA1 and X0 not A0A0 and X1 not X0

Segment FA1 and X1

Segment GYes A1Yes X1

Gate arrangement

19 gates required


(A1A0 × X1X0 display)

01 × 01 =

(1×1=1)Arithmetic

Binary inputs:

7-segmentDisplay

01 × 10 = 01 × 11 = 10 × 01 = 10 × 10 =

10 × 11 = 11 × 01 = 11 × 10 = 11 × 11 = (00 × 00)

(1×2=2) (1×3=3) (2×1=2) (2×2=4)

(2×3=6)Arithmetic (3×1=3) (3×2=6) (3×3=9) no inputcontrol

Binary inputs:

7-segmentDisplay

Color photography


2x2 multiplier with visual display:

(A1A0 × X1X0 display)

i11 i12 i13 i14

i11 i11

i11

i12 i12

i12

i13 i13

i13

i14 i14

i14

i22 i23 i21 i23

i24 i24

i21 i22

i24

i21 i22

i23

i11 i12 i13 i14

i11 i11

i11

i12 i12

i12

i13 i13

i13

i14 i14

i14

i22 i23 i21 i23

i24 i24

i21 i22

i24

i21 i22

i23

i11 i12 i13 i14

i11 i11

i11

i12 i12

i12

i13 i13

i13

i14 i14

i14

i22 i23 i21 i23

i24 i24

i21 i22

i24

i21 i22

i23

i11 i12 i13 i14

i11 i11

i11

i12 i12

i12

i13 i13

i13

i14 i14

i14

i22 i23 i21 i23

i24 i24

i21 i22

i24

i21 i22

i23

Silicomimetic automata project (MAYA):

Macdonald et al. Nano Lett. 2006Stojanovic&Stefanovic. Nat.Biotech. 2003

MAYA: MAYA-II: MAYA-III:

Pei, Matamoros, et al. in testing

Human designed

Goal: Demonstrate power of molecular computing!

Computer designed Human designed

Goal: What does it take to coordinate large number of gates?

Goal: Field programmable arrays of gates!-Teaching “by example”-Selecting strategy (“carving”)

Implementation of tic-tac-toe playing algorithm with deoxiribozymes: Molecular Array of YES and ANDANDNOT Gates (MAYA)

Stojanovic, Stefanovic, Nat. Biotech. 2003

MAYA vs. Milan: losing game

Mg2+

From Sci. Am. 2008

Is it possible to have serial circuits?

oi1U D

communication component(Upstream enzyme)

(downstream enzyme)

Intergate Communication : Ligase-Cleavase

Stanka Semova, Dmitry Kolpashchikov

AT

AT

GC

TA

GC

TA

TA

CG

T TT

CG G

C GA

AC

GTA

GC

AT

GC

CG T

A

AT

TA

GC G

C

PIMHO

GC

TT

TT

Ligase E47

GA

A A CT

C

A

GC

GA

C A C T

C T C T T C T A G TC

A

CG

GG

CAG C

T CGA AT

A G A A G AA

C A AT

G

A

CA

GA

T T GA

TA

C

TA C A C T FTrAG

AG

AGAGR

Cleavage

UnionS1

S2

P1

S3

YESP1

0

1

2

3

4

5

0 50 100 150 200 250

time (min)

Ligase-cleaves XOR circuit:

JACS 2005

F

2

o1

O1

1

12

0

2

4

6

8

10

0 50 100 150 200 250

t(min)

Rn

1

2

1,2

P

O

OIA

IB

XOR

IA IB O

1

1

1 1

1

1

0

0

0

0

00

What about cleavase-cleavase?

U D

oa.

U D

+o

b.

U

+D

od.

a. Substrate as inhibitor

c. Substrate as activator

b. product as activator

d. product as inhibitor

o

DU

c.

Cf. Uri Alon, Nat. Rev. Genetics 2007, 450 “Network Motifs: Theory and Experimental Approaches”

Ben-Tov, Tamburi

0

500000

1000000

1500000

2000000

2500000

0 1 2 3 4 5 6Time (h)

F

+ Enzyme

- Enzyme

- inhibitor

+inhibitor

+inhibitorU D

oa.

Connection type I: Substrate as inhibitor

U D

o

Connection type II: Product as activator

U D

+o

b.

50nM d-8-17, 500nM d-sub, 500nM stem-loop, 50nM up-8-17, 500nM input

0

10

20

30

0 80 160 240 320t(min)

FU

+MB

cont

+U

+i,p

MB

U D

+o

Renjun Pei

Connection type III: Substrate as activator

o

DU

c.

100nM E6, 500nM d-sub, 500nM activator, 50nM 8-17

4

12

20

28

36

0 100 200 300 400t(min)

FU +act

+U

cont

Renjun Pei, Aihua Shen

oD

U

Connection type IV: Product as inhibitor

U

+D

od.

100nM E6, 500nM d-sub, 250nM stem-loop, 50nM 8-17

3

6

9

12

15

0 80 160 240 t(mins)

FUP

+MB

+U

Renjun Pei, Aihua Shen

U

+D

o

AND cascade:

U2D

oU1

S1

S2

100nM E6, 500nM d-sub, 350nM inh-1, 350nM inh-2, 50nM 8-17-1, 50nM 8-17-2

3

5

7

9

0 40 80 120 160t(mins)

FUp

E-1

2 Es

2 inhsE-2

0

5

10

15

20

25

30

0 40 80 120 160 200concentration of i1 (nM)

FU

Now we can do signal threshold:

oi1U D

i1

< Di1 >D+Ui1<D+Ui1

o

D-loop U-loop>100nM D, 500nM d-sub, 20nM U, 200nM inhat time 180 minutes.

one eq.

In

Circuits by Winfree’s group:

Inputs Input Translation Computational subcircuit Signal restoration

dna computing and robotics based on deoxyribozymes

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