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RNA Strand Displacement for Sensing, Information Processing, and - - PowerPoint PPT Presentation

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Hello all! Weve tried to minimize the overlap but several slides are best viewed animated! - MIT iGEM RNA Strand Displacement for Sensing, Information Processing, and Actuation in Mammalian Cells Americas East Regional 10.13.2012 1

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SLIDE 1

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
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SLIDE 2

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

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SLIDE 3

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

Number of Promoters / dsGates

Purnick et al., Nature MCB 2009

Moon et al., Nature 2012 Qian et al., Science 2011

Trancription-translational circuits Strand displacement circuits

3

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SLIDE 4

4

Increased Complexity / Smaller Footprint

Much smaller nucleotide footprint!

Transcriptional-translational circuit: ~13000 bp

Moon et al., Nature 2012

Strand displacement circuit: 100 bp

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SLIDE 5

Large Sophisticated Circuits Enabled By:

  • Decreased Size
  • Simple Combinatorial Design Space
  • Ease of Composition
  • Tunability

5

Increased Complexity / Smaller Footprint

Much smaller nucleotide footprint!

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SLIDE 6

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

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SLIDE 7

Toehold-Mediated Strand Displacement

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Input Strand Gate Input Strand Output Strand Output Strand

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SLIDE 8

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

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SLIDE 9

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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

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SLIDE 10

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

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SLIDE 11

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

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SLIDE 12

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

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SLIDE 13

Our NOT Gate In Vitro

Test Simulate Design x5

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SLIDE 14

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

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SLIDE 15

Video Here

HEK293 cell Vesicle Tagged RNA

In Vivo RNA Delivery

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

Experiment by Katie

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SLIDE 16

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

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SLIDE 17

17

Design Test

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SLIDE 18

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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

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SLIDE 19

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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

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SLIDE 20

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

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SLIDE 21

Sensing mRNA for Cellular Interfacing

Short RNA Output

+

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Design objectives and constraints:

  • orthogonality
  • three-letter code
  • accessibility

Downstream Input Sensor Input mRNA

+

Fuel

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SLIDE 22

Sensing mRNA for Cellular Interfacing

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Software Rendered eBFP2 mRNA

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

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SLIDE 23

Sensing mRNA In Vitro Using DNA

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1 3 2 4

2.03 2.62 2.28 3.18

Data by Eerik and Chelsea

mRNA + Sensor + Reporter = Fluorescence

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SLIDE 24

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

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SLIDE 25

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

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SLIDE 26

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

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SLIDE 27

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

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SLIDE 28

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

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SLIDE 29

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

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SLIDE 30

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!

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SLIDE 31

Thank you!

Questions?

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SLIDE 32
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SLIDE 33

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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!

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AND and OR Logic

Qian et al. 2011

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Strand Displacement Reactions

Qian et al. 2011

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SLIDE 36

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Optimizing Transfection of 2’-O-Me dsRNA

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In Vitro Strand Displacement: Iteration 2

Input Strand Slong Tlong Tlong Tlong* Slong* Slong Fluorescent Complex

+ +

Slong/bulge Tlong* Slong* Slong/bulge Waste Reporter

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SLIDE 38

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In Vivo Strand Displacement: Iteration 2

+

Slong Tlong* Slong* Reporter Input Strand Slong Tlong Tlong Tlong* Slong* Slong Fluorescent Complex

+

Slong Waste

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SLIDE 39

Inducible Expression Systems

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SLIDE 40

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

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Cellular-RNA-Compatible Actuation: Hammerheads

Hef1A mKate Hef1A Hammerhead mKate Hef1A Hammerhead mKate

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SLIDE 42

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

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

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SLIDE 43

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

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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