RNA Strand Displacement for

Sensing, Information Processing, and Actuation in Mammalian Cells

1

America’s East Regional 10.13.2012

Hello all! We’ve tried to minimize the overlap but several slides are best viewed animated! - MIT iGEM

Brief Overview of Nucleic Acid Technology

Rothemund, Nature 2006

Static

Omabegho et al., Science 2009

Dynamic

DNA Structure

Zhang et al., Science 2007

Signal Amplification

A

A

Digital Logic

A AND B AND (C OR D) AND (E OR F)

Seelig et al, Science 2006

DNA Computing

AIM: Develop strand displacement as a mechanism to build in vivo synthetic circuits

0

5

10

15

20

25

30

35

40

2000 2002 2004 2006 2008 2010 2012

Year

Attractiveness of Nucleic Acid Computation

Analysis of publications of the Winfree group

Nu

mb

er

of

Pro

mo

ters

/ d

sGat

es

Purnick et al., Nature MCB 2009

Moon et al., Nature 2012

Qian et al., Science 2011

Trancription-translational circuits

Strand displacement circuits

3

4

Increased Complexity / Smaller Footprint

Much smaller nucleotide footprint!

Transcriptional-translational circuit:

~13000 bp

Moon et al., Nature 2012

Strand displacement circuit:

100 bp

Large Sophisticated Circuits Enabled By:

• Decreased Size • Simple Combinatorial Design Space

• Ease of Composition

• Tunability

5

Increased Complexity / Smaller Footprint

Much smaller nucleotide footprint!

Implementation Strategies

Utilize RNA

Nucleic Acid Computing Strategy

Mammalian Cells

Tlong* Slong*

Tlong Slong

In vitro In vivo

24 hours

1 cell cycle

72 cell cycles

vs.

Toehold Hybridization domain

Toehold-Mediated Strand Displacement

7

Input Strand

Gate

Input Strand Output Strand

Output Strand

Design & Test NOT Gate

Demonstrate In Vitro RNA Strand Displacement

Deliver RNA

Demonstrate In Vivo RNA Strand

Displacement

Produce Short RNAs In Vivo

Design & Test mRNA Sensor

9

In Vitro Strand Displacement Using RNA

Input Strand

S6 T T

T* S6*

S6

Fluorescent Complex

+ + S6

T* S6*

S6

Waste Reporter Incorrect Input

Strand

S1

Data collected by Eerik

Processing Using Strand Displacement

? AND OR NOT

True if both inputs true True if at least one input is true Inverts a signal

Qian et al. 2011 Nothing compatible

10

Inversion Using Strand Displacement

Dynamic Gate (A)

Dynamic Gate (A)

Input Strand

Buffer (C)

Downstream Input (B)

Fuel / Catalyst (D)

Downstream Input (B)

Downstream Input (B)

B is free to act downstream! C is displaced.

Design:

Operation: Low Input

Inversion Using Strand Displacement

Dynamic Gate (A)

Dynamic Gate (A)

Input Strand

Buffer (C)

Downstream Input (B)

Fuel / Catalyst (D)

Design:

Operation: High Input

B is trapped, cannot act downstream! C is ‘stable’

Downstream Input (B)

Input Strand

Our NOT Gate In Vitro

Test Simulate Design

x5

Designed & Tested NOT Gate

Demonstrated In Vitro RNA Strand Displacement

Deliver RNA

Demonstrated In Vivo RNA Strand

Displacement

Produced Short RNAs In Vivo

Designed & Tested mRNA Sensor

Video Here

HEK293 cell

Vesicle

Tagged RNA

In Vivo RNA Delivery

T = 0h T = 2h T = 3h T = 4h

Experiment by Katie

Designed & Tested NOT Gate

Demonstrated In Vitro RNA Strand Displacement

Delivered RNA

Demonstrate In Vivo RNA Strand

Displacement

Produced Short RNAs In Vivo

Designed & Tested mRNA Sensor

17

Design Test

18

In Vivo Strand Displacement: Iteration 1

Input Strand

S T + +

T * S*

S

Reporter T* S*

T S

Fluorescent Complex

S

Waste

Transfection by Katie, FACS by Nathan

19

In Vivo Strand Displacement: Iteration 2

Input Strand

Slong Tlong + + Tlong* Slong*

Slong

Reporter Tlong* Slong*

Tlong Slong

Fluorescent Complex

Slong

Waste

~6 fold increase in red

Nucleofection by Giulio, FACS by Rob

In Vivo Strand Displacement Summary

Designed & Tested NOT Gate

Demonstrated In Vitro RNA Strand Displacement

Delivered RNA

Demonstrated In Vivo RNA Strand

Displacement

Sensors

Short RNA Transcription

Sensing mRNA for Cellular Interfacing

Short RNA Output

+

21

Design objectives and constraints: • orthogonality • three-letter code • accessibility

Downstream Input

Sensor

Input mRNA

+

Fuel

Sensing mRNA for Cellular Interfacing

22

Software Rendered eBFP2 mRNA

Design objectives and constraints: orthogonality, three-letter code, accessibility

Sensing mRNA In Vitro Using DNA

23

1 3 2 4

2.03 2.62 2.28 3.18

Data by Eerik and Chelsea

mRNA + Sensor + Reporter = Fluorescence

Designed & Tested NOT Gate

Demonstrated In Vitro RNA Strand Displacement

Delivered RNA

Demonstrated In Vivo RNA Strand

Displacement

Produce Short RNAs In Vivo

Design & Test mRNA Sensor

Producing Short RNAs In Vivo

U6 TetO FF1 Hef1A eYFP-4xFF1

25

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

67bp hairpin

Hef1A TagBFP Transfection marker

Transfection by Linh, FACS by Nathan

Summary of Achievements

Demonstrated In Vitro RNA Strand Displacement

Delivered RNA

Demonstrated In Vivo RNA

Strand Displacement

Designed & Tested NOT Gate

Design & Test mRNA Sensor

Produce Short RNAs In Vivo

40 MammoBlocks

New MammoBlocks / BioBricks

Best 22 Parts Submitted To Registry

10 Regulatory Composite Parts

BBa_K779400 BBa_K779405 BBa_K779401 BBa_K779406 BBa_K779402 BBa_K779407 BBa_K779403 BBa_K779408 BBa_K779404 BBa_K779409

37 Biobricks

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

3 Promoters BBa_K779200 BBa_K779201 BBa_K779202

4 Hammerhead Ribozyme Coding Sequences

BBa_K779315 BBa_K779316 BBa_K779317 Bba_K779318

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

BBa_K779305 BBa_K779308

2 Transcriptional Regulators

10 Generators

BBa_K779600 BBa_K779601 BBa_K779602 BBa_K779603 BBa_K779604 BBa_K779605 BBa_K779606 BBa_K779607 BBa_K779608 BBa_K779609

Long Term Impact on Society

Sophisticated Circuits in Mammalian Cells

In Vivo RNA Strand Displacement

Smart Immune System

Control Circuitry For Mammalian Protein

Production

Cancer Detection

Organ Regeneration

Outreach: Middle School, High School, College

Splash: Educating local high school students

MIT Educational Studies Program Coming soon! November 17, 2012 Inform soon-to-be college students about a potential field

Wellesley: Building multi-university communities

Use case for Wellesley HCI software Bridging gap between tool designer and end-user MIT: IAP Synthetic Biology Class “Engineer Your Own Bacteria” 2 week lecture, wet lab Fundraised, Organized, Taught by MIT iGEMers

Summer HSSP: Educating local middle school students

Biology Lecture Series: Synthetic Biology, organized and taught by iGEM students

Educate the public about applications, practices, and opportunities in synthetic biology

29

Acknowledgments MIT iGEM 2012 Team

Keren Greenbaum, Giulio Alighieri, Divya Israni, Lealia Xiong, Jenna Klein, Katie Bodner, Nathan Kipniss, Felix Sun, Ala’a Siam,

Kristjan Eerik Kaseniit, Robert Learsch, Linh Vuong, Chelsea Voss, Wilson Louie, Jonathan Elzur, Eta Atolia

Ron Weiss (faculty) Jonathan Babb Deepak Mishra

Coordinators:

Lab Shift Monitors:

Additional thanks to:

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

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

generous support!

Thank you!

Questions?

33

Full NOT Gate Reaction Diagram

Case: Input present low output signal

Irreversible

Trapped!

Case: No input present high output signal

Reversible

Downstream Input (B)

B

No Input Strand

Dynamic Gate (A)

Input Strand

Dynamic Gate (A)

Downstream Input (B) reacts:

Downstream Input (B) Buffer (C)

Fuel / Catalyst (D) Signal!

34

AND and OR Logic

Qian et al. 2011

35

Strand Displacement Reactions

Qian et al. 2011

36

Optimizing Transfection of 2’-O-Me dsRNA

37

In Vitro Strand Displacement: Iteration 2

Input Strand

Slong Tlong Tlong

Tlong* Slong*

Slong

Fluorescent Complex

+ + Slong/bulge

Tlong* Slong*

Slong/bulge

Waste Reporter

38

In Vivo Strand Displacement: Iteration 2

+ Slong

Tlong* Slong* Reporter Input Strand

Slong Tlong Tlong

Tlong* Slong*

Slong

Fluorescent Complex

+ Slong

Waste

Inducible Expression Systems

Output Strand

T S2 S3

T*

Hammerhead

Hammerhead

Spacer RNA

Output Strand

T S2 S3

T* T*

Gate S2*

Initial RNA transcript

RNA Folds

Hammerhead Cleaves

Gate S2* T*

Producing Components Using Transcription and Hammerheads

41

Cellular-RNA-Compatible Actuation: Hammerheads

Hef1A mKate Hef1A Hammerhead mKate Hef1A Hammerhead mKate

42

Cellular-RNA-Compatible Actuation: Controlling Hammerhead Activity Using Strand Displacement

Hammerhead-Stem (inactive)

+ Input Strand

(from Strand Displacement) Active Hammerhead

c.f.

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

Hef1A-LacO eYFP-4xFF4 Hef1A mKate-Intronic

miR-FF4

U6-TetO Decoy FF4

TuD FF4

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

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

43

Antisense to miRNA

miRNA

44

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 MIT’s Engineering Systems Division.

Work presented in SynBERC retreat and at a conference in the Woodrow Wilson International Center for Scholars.

Outreach: International