RNA Strand Displacement for Information Processing in Mammalian Cells

World Jamboree 11.04.2012

The Importance of Smaller Parts

SynBio Challenge: Make more sophisticated circuits to control cell behavior
~10X Smaller Parts: Number of gates
Circuits with 10X smaller parts

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 Current Transcriptional Parts:

Circuits with typical sized parts

1 2 3

DNA required to encode logic circuits

Greater sophistication per unit DNA Better delivery with payload limits Less energy cost
2

Motivation

Mechanism

Processing

Production

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Conclusion

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Progression of Circuit Sophistication Over Time

In vitro = In solution In vivo = In cells

Number of Promoters / dsGates

40 35 30 25 20 15 10 5 0 2000
Moon et al., Nature 2012

Transcription-translational circuits In vitro strand displacement circuits

Qian et al., Science 2011

AIM: Demonstrate strand displacement computation in cells

2002

2004

2006

2008

2010

2012

Year
Adapted from Purnick et al., Nature MCB 2009 Analysis of strand displacement publications from Pierce, Winfree, and Yurke groups 3

Motivation

Mechanism

Processing

Production

Interfacing

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Conclusion

Human Practices

Signals and Computation

BUFFER

NOT

OR

AND

In traditional transcriptional circuits:

Low activator Input signal = 0

Low protein Output signal = 0

High activator Input signal = 1

High protein Output signal = 1

1
BUFFER Motivation Mechanism Processing

1
Production Interfacing Key Reaction Conclusion Human Practices

Signals and Computation: Strand Displacement

High signal (single-stranded DNA): Low signal (double-stranded DNA): (We use fluorescence as a proxy to indicate signal level) Input signal = 0 Output signal = 0

1 0

0
Low signal Input signal = 1 Output signal = 1

1
BUFFER Motivation Mechanism Processing

1
High signal
Production Interfacing Key Reaction Conclusion Human Practices

Mechanism of Toehold-Mediated Strand Displacement

S1

Input Signal

S2 S3

15 nt

S2

Output Signal

S2*

Gate

BUFFER Motivation Mechanism Processing Production Interfacing Key Reaction Conclusion

Human Practices

Mechanism of Toehold-Mediated Strand Displacement

S2 T S1

S3

Output Signal

Input Signal

S2

S2 S1 S3 T S2 S3

15 nt

Input Signal

T Output Output

Signal Signal

T*

S2*

T*

Gate
5 nt Qian et al. 2011
BUFFER Motivation Mechanism Processing Production Interfacing Key Reaction Conclusion
7

Human Practices

Mechanism of Signal Cascading

S2

Input Signal

S3

S1

Input Signal

S2

T*

S2*

T*

Gate
Qian et al. 2011
BUFFER Motivation BUFFER Mechanism Processing Production Interfacing Key Reaction Conclusion
8

Human Practices

Mechanism of Signal Cascading

S3 S2 T T S3

S4

Input Signal

Output Signal

S2

S4 T S3 S3 T

Input Signal
T*

Output Signal

2*

T*

S3*

T*

ate

Gate
Qian et al. 2011
BUFFER BUFFER Mechanism Processing Production Interfacing Key Reaction Conclusion
9

Motivation

Human Practices

Overview of In Vivo Strand Displacement Systems

Sensing Inputs

Processing Information

Actuating Responses

Producing Circuits
10

Processing Information
Qian et al. 2011 Nothing compatible

?
AND True if both inputs true OR True if at least one input is true NOT Inverts a signal

11

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Our NOT Gate Using Strand Displacement

Dynamic Gate (A)
Input Strand Output (B) Buffer (C) Fuel / Catalyst (D)

Operation: Low Input

Dynamic Gate (A)

High Output

Output (B) Output (B)

0
NOT Motivation

1
Mechanism Processing

B is free to act downstream! C is displaced.

12

Production

Interfacing

Key Reaction

Conclusion

Human Practices

Our NOT Gate Using Strand Displacement

Dynamic Gate (A)
Input Strand Output (B) Buffer (C) Fuel / Catalyst (D)

Operation: High Input

Dynamic Gate (A) Input Strand

Low Output

Output (B)

1
NOT Motivation

0
Mechanism

B is trapped, cannot act downstream! C is stable.

13

Processing

Production

Interfacing

Key Reaction

Conclusion

Human Practices

Our NOT Gate In Vitro

Design Simulate
Visual DSD Using ODEs 9 reactions per NOT gate

Test
Plate reader studies Measuring fluorescence 100 uL reactions

Cooperative Hybridization Initial Simulation at Low Concentrations

Initial Transfer Function NOT Gate Transfer Function

Output

Simulations using Visual DSD; experiment by Jon, Giulio Motivation Mechanism Processing Production

Input
Interfacing Key Reaction

Output

Input Conclusion

14

Human Practices

Producing Circuits

Sensing Inputs

Processing Information

Actuating Responses

Producing Circuits
15

In Vivo Implementation Choices

T Tl* S Sl* In vitro In vivo

Utilize RNA

Mammalian Cells
1 cell cycle 24 hours

Can be produced continuously Provide for dynamic circuit operation

Slow dilution rate Natural RNAi pathway RNA more stable

16

Motivation

Mechanism

Processing

Production

Interfacing

Key Reaction

Conclusion

Human Practices

Transcription of Short RNAs In Vivo

67nt shRNA

U6 TetO Mammalian HEK293 cell

shRNA FF1
Hef1A TagBFP Transfection marker

Hef1A

eYFP-4xFF1

Knockdown of Hef1A:eYFP-4xFF1 using U6-TetO:FF1

U6 TetO Sequence: Henriksen et al., NAR 2007 Motivation Mechanism Processing

Built by Lealia, Transfection by Linh, FACS by Nathan Interfacing Key Reaction Conclusion

17

Production

Human Practices

Interfacing With the Cell

Sensing Inputs

Processing Information

Actuating Responses

Producing Circuits
18

Sensing mRNA Inputs

Input mRNA

+
Output Sensor Gate Fuel

Design rules orthogonality three-letter code accessibility

Gate

Short RNA Output Signal

Production Interfacing

Downstream Input Signal

19

Motivation

Mechanism

Processing

Key Reaction

Conclusion

Human Practices

Sensing mRNA for Cellular Interfacing

NUPACK Rendering of eBFP2 mRNA

20

Motivation

Mechanism

Processing

Production

Interfacing

Key Reaction

Conclusion

Human Practices

Sensing mRNA In Vitro Using DNA-Based Sensor

2.03 2.62 2.28 3.18

Baseline mRNA Input

Presence of mRNA yields fluorescence 1 2 3 4

IVT by Lealia, Data by Eerik and Chelsea Motivation Mechanism Processing Production Interfacing Key Reaction Conclusion

21

Human Practices

Using Strand Displacement: Feasible

Sensing Inputs

Processing Information

Actuating Responses

Producing Circuits
22

The Key Reaction Behind It All

1
REPORTER BUFFER Input Strand

Output Strand

Gate High green / Low red

23

Motivation

Mechanism

Processing

Production

Interfacing

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The Key Reaction Behind It All

1
REPORTER Input Strand Output Strand

Input Strand Output Strand

Gate High green / High red

24

Motivation

Mechanism

Processing

Production

Interfacing

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Human Practices

The Key Reaction: In Vitro with RNA

T S1 S6 S6

+
T* S6* Reporter

S6

S6

+
T* S6* Fluorescent Complex Waste

Incorrect Input Input Strand Strand

1
25

REPORTER Data collected by Eerik/Chelsea/Felix Motivation Mechanism Processing Production Interfacing Key Reaction Conclusion

Human Practices

In Vivo RNA Delivery

T = 0h
HEK293 cell

T = 2h

T = 3h

T = 4h

Vesicle Tagged RNA

Video Here

Experiment by Katie

The Key Reaction with RNA in Cells: Iteration 1

T S Input Strand S

+
T* S* Reporter

T* S* Fluorescent Complex

Waste

1
27

REPORTER Transfection by Katie, FACS by Nathan Motivation Mechanism Processing Production Interfacing Key Reaction Conclusion

Human Practices

Design

Test

28

Motivation

Mechanism

Processing

Production

Interfacing

Key Reaction

Conclusion

Human Practices

The Key Reaction with RNA in Cells: Iteration 2

Tlong Slong

+
Tlong*

Slong Slong* Reporter

Tlong

Slong

Slong

Input Strand

Tlong* Slong* Fluorescent Complex

Waste

We got RNA Strand Displacement to work in mammalian cells!

~6 fold increase in red
Longer toehold, hybridization domain Orthogonal sequences
Nucleofection by Giulio, FACS by Rob Motivation Mechanism Processing Production Interfacing Key Reaction Conclusion
29

Human Practices

Experimental Accomplishments

PROCESSING
AND

TOEHOLD-MEDIATED RNA STRAND DISPLACEMENT

OR NOT

CIRCUIT PRODUCTION

Input Strand

NOT Gate Transfer Function

Output Strand

CELL INTERFACING

Gate

KEY REACTION
30

Motivation

Mechanism

Processing

Production

Interfacing

Key Reaction

Conclusion

Human Practices

An Example Future Cancer Detect-and-Destroy Circuit Enabled by Our Results

Sensing
ANXA2

G1:1,8
m1 T1*

S8 T

Processing
Th8,9:9
S9 s8* T* S9* S10 T T*

Actuation

m1* T* m2 S8 T

CD-55

G2:2,8
T2*

m2* T* m3 S8 T

CEACAM-6 SEL1L

G3:3,8
T3*

G8:8,9
S8 T* S8*

S9 T T*

G9:9,10
S9 T* S9*

m3* T*

Th4:8,7
BRCA2
T4*

w9,f
m4 m4* S8* S8 S7 m5 m5* m6 m6* S8* S8 S8* S8 T* s7* T* s7* T T* s7* S9

Sf T

Killer protein production

Th5:8,7
T5*

w7,8
S8

Th6:8,7
INK4A
Motivation
T6*

ANXA2 CD-55 CEACAM-6 SEL1L BRCA2 INK4A

Pancreatic Cancer (PDAC)

31

Mechanism

Processing

Production

Interfacing

Key Reaction

Conclusion

Human Practices

New MammoBlocks (RFC 65) / BioBricks

37
Biobricks
Best 22 Parts Submitted To Registry

40
MammoBlocks
37 Logic Parts for Strand Displacement
BBa_K779500 BBa_K779501 BBa_K779502 BBa_K779503 BBa_K779504 BBa_K779100 BBa_K779101 BBa_K779102 BBa_K779103 BBa_K779104 BBa_K779105 BBa_K779106 BBa_K779107 BBa_K779108 BBa_K779109 BBa_K779110 BBa_K779111 BBa_K779112 BBa_K779113 BBa_K779114 BBa_K779115 BBa_K779116 BBa_K779117 BBa_K779118 BBa_K779119 BBa_K779120 BBa_K779121 BBa_K779122 BBa_K779123 BBa_K779124 BBa_K779125 BBa_K779126 BBa_K779127 BBa_K779128 BBa_K779129 BBa_K779130 BBa_K779131

10 Regulatory Composite Parts

BBa_K779400 BBa_K779401 BBa_K779402 BBa_K779403 BBa_K779404 BBa_K779405 BBa_K779406 BBa_K779407 BBa_K779408 BBa_K779409

3 Promoters
BBa_K779200 BBa_K779201 BBa_K779202

4 Hammerhead Ribozyme Coding Sequences

BBa_K779315 BBa_K779316 BBa_K779317 Bba_K779318

2 Transcriptional Regulators
BBa_K779305 BBa_K779308

13 Reporters
BBa_K779300 BBa_K779307 BBa_K779301 BBa_K779309 BBa_K779302 BBa_K779310 BBa_K779303 BBa_K779311 BBa_K779304 BBa_K779312 BBa_K779306 BBa-K779313 BBa_K779314

10 Generators
BBa_K779600 BBa_K779602 BBa_K779604 BBa_K779606 BBa_K779608 BBa_K779601 BBa_K779603 BBa_K779605 BBa_K779607 BBa_K779609

Outreach: Middle School, High School, College

Summer HSSP: Educating local middle school students Biology Lecture Series: Synthetic Biology Focus on applications, practices, and opportunities in synthetic biology

Splash: Educating local high school students MIT Educational Studies Program Coming soon! November 17, 2012

MIT: January Term Synthetic Biology Class Engineer Your Own Bacteria 2 week lecture, wet lab Wellesley: Building multi-university communities Use case for Wellesley HCI software Bridging gap between tool designer and end-user
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Acknowledgments
MIT iGEM 2012 Team
Felix Sun, Giulio Alighieri, Eta Atolia, Katie Bodner, Jonathan Elzur, Keren Greenbaum, Divya Israni, Lealia Xiong, Chelsea Voss, Kristjan Eerik Kaseniit, Nathan Kipniss, Jenna Klein, Robert Learsch, Wilson Louie, Alaa Siam, Linh Vuong

Coordinators:
Ron Weiss (faculty) Jonathan Babb Deepak Mishra

Lab Shift Monitors:

Jameel Zayed Kenneth H. Hu Leanna S. Morishini Mariya Barch Mark Andrew Keibler Nathan S. Lachenmyer Sebastien Lemire

Thanks to our sponsors for their generous support!

Additional thanks to:

Christopher Voigt Lulu Qian Peter Andrew Carr Feng Zhang Nevin M. Summers Jacquin Niles Kristala L. Jones Timothy Lu Prather Domitilla Del Rahul Sarpeshkar Vecchio Alice M. Rushforth Narendra Maheshri Natalie Kuldell Roger Kamm BU-Wellesley iGEM Team

NOT Gate Transfer Function

PROCESSING

CIRCUIT PRODUCTION

Thank you! Questions?

CELL INTERFACING KEY REACTION

Highly Scalable Parts Comparison

Transcriptional:
Hef1A 1174 bp Cag 1718 bp

Strand Displacement:
U6 187 bp

Hef1A: TAL Effector TAL-VP16 w/ 17bp DNA recognition 1174 + 2477 = 3651bp

HH Gate:Output HH 216 bp

Poly A / Terminator 527 bp

U6 terminator 6 bp

3651+527 = 4178bp

187+216+6 = 409bp

Strand Displacement Reactions

Qian et al. 2011

38

AND, OR Logic Using Strand Displacement

39

Qian et al. 2011

Full NOT Gate Reaction Diagram

Case: No input present high output signal
Dynamic Gate (A) No Input Strand Downstream Input (B) reacts: Downstream Input (B) Buffer (C) Downstream Input (B)

Reversible

Signal!
Fuel / Catalyst (D)

Case: Input present low output signal

Dynamic Gate (A) Input Strand B

Trapped! Irreversible

40

Digital NOT Gate Behavior

RFC65 Review: Recombination Cloning

Kan pENTR L4 Prom R1 L1

Ori

Kan pENTR Gene

Ori

L2

R4

Cm Amp

ccdB pDEST

R2

Term

Marker
LR

B4

Prom

B1

Gene pEXPR

B2

Term
42

Amp

Marker

Actuation: Controlling Hammerhead Activity

cleaves

+
cleaves

cleaves

c.f. Hammerhead-Stem (inactive) Input Strand Active Hammerhead

43

Design by Divya & Eerik, Hammerhead adapted from Yen et al. 2004

Transduction of Nucleic Acids to Protein Outputs

Input

Output

Protein

None

GFP

mRNA is stable, protein produced

GFP

cleaves

mRNA unstable, no protein expression

Actuation: Testing Hammerhead Cleavage

Hef1A

mKate

Hef1A Hammerhead mKate

Hef1A

mKate Hammerhead

45

Producing Components Using Transcription and Hammerheads

Hammerhead

Initial RNA Transcript T*

Output S2 S2* Gate

S3 T T* Hammerhead

RNA Folds Goal:

Hammerhead Cleaves
46

In Vitro Strand Displacement: Iteration 2

Tlong Slong

+
Tlong*

Slong/bulge Slong* Reporter

Tlong

Slong

Slong/bulge

Input Strand

Tlong* Slong* Fluorescent Complex

Waste

47

In Vivo Strand Displacement: Iteration 2

Tlong Slong

+
Tlong*

Slong Slong* Reporter

Tlong

Slong

Slong

Input Strand

Tlong* Slong* Fluorescent Complex

Waste

48

The Key Reaction: In Vivo Iteration 2 (Bulge)

Tlong Slong

+
Tlong*

Slong Slong* Reporter

Tlong

Slong

Slong

Input Strand

Tlong* Slong* Fluorescent Complex

Waste

Longer toehold Longer hybridization domain Incorporation of bulge region Orthogonal sequences

1
Nucleofection by Giulio, FACS by Rob REPORTER
49

Optimizing Transfection of 2-O-Me dsRNA

50

Inducible Expression Systems

Cellular-RNA-Compatible Actuation: Relieving miRNA Repression with Decoys

Antisense to miRNA

miRNA

Hef1A

mKate-Intronic miR-FF4

Hef1A-LacO

eYFP-4xFF4

U6-TetO

Decoy FF4 TuD FF4

1:0:1 Reporter:miRNA:Decoy 1:1:1 Reporter:miRNA:Decoy 1:1:2 Reporter:miRNA:Decoy

Slight Relief of miRNA Knock-Down of Reporter via Antisense Decoy RNA

Outreach: International
Internationalization Project of Synthetic Biology Launching a collaboration between MIT and Tel-Aviv University. Bringing together high school Palestinians and Israelis for three years to work on an iGEM technical and entrepreneurial projects. One-year pilot program to be launched this summer. A college component and an incubator to follow.

Synthetic Biology Policy Research One student conducted synthetic biology policy research, with Prof. Kenneth Oye of MITs Engineering Systems Division. Work presented in SynBERC retreat and at a conference in the Woodrow Wilson International Center for Scholars.
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Production

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Key Reaction

Conclusion

Human Practices