rna strand displacement for sensing, information ...2012.igem.org/files/presentation/mit.pdf · rna...
TRANSCRIPT
RNA Strand Displacement for
Sensing, Information Processing, and Actuation in Mammalian Cells
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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
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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
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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
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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
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
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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
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Design Test
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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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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
+
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Design objectives and constraints: • orthogonality • three-letter code • accessibility
Downstream Input
Sensor
Input mRNA
+
Fuel
Sensing mRNA for Cellular Interfacing
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Software Rendered eBFP2 mRNA
Design objectives and constraints: orthogonality, three-letter code, accessibility
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
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
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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
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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?
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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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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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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
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Cellular-RNA-Compatible Actuation: Hammerheads
Hef1A mKate Hef1A Hammerhead mKate Hef1A Hammerhead mKate
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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.
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
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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