Giulio Alighieri, Eta Atolia, Katie Bodner, Jonathan Elzur, Keren Greenbaum, Divya Israni, Kristjan Eerik Kaseniit, Nathan Kipniss, Jenna Klein, Robert Learsch, Wilson Louie, Ala’a Siam, Felix Sun, Chelsea Voss, Linh Vuong, Lealia Xiong

Overview

•  Qian and Winfree (Science 2011) showed DNA strand displacement as a mechanism for complex digital logic in vitro. •  The basic technique of strand displacement involves three single stranded DNA molecules, or in our case, RNA

molecules. The gate and output strands exist as a complex Gate:Output formed through complementary Watson-Crick base-pairing. The gate strand also contains an open, unbound domain called a toehold. An input strand, a free signal, with a complementary toehold can bind to the toehold on the gate strand, and subsequently displace the output strand through branch migration to yield a gate:input complex as shown above. The output strand is now a free signal and can go on to interact with downstream gates, which makes these reactions cascadable.

•  Once we decided on toehold-mediated strand displacement as our logic processing mechanism, we chose RNA, rather than DNA, because RNA can be continuously produced in cells to produce dynamic circuits and can be utilized for its enzymatic capabilities. We implemented our RNA strand displacement circuits in mammalian cells due to the fact that mammalian cells divide more slowly than bacterial cells and will have a lower dilution rate for our

RNA circuits.

The complexity of engineered genetic circuits in eukaryotic systems is limited by the availability of regulatory components and further hampered by the inability to assemble and deliver large DNA constructs. In contrast, in vitro synthetic DNA circuits utilizing strand displacement have demonstrated complex digital logic with reliable and scalable behaviors in a small base-pair footprint. The possible adoption of such technology into cellular environments can amplify the scale and sophistication of biological circuits, broadening synthetic biology’s application space. Our project focused on the feasibility of RNA strand displacement circuits for sensing, information processing, and actuation. We demonstrated the key reaction behind all such circuits in mammalian cells, as well as designed and tested methods of sensing mRNAs, inverting low signals using our NOT gate, and transcribing short RNAs to produce strand displacement circuits. We envision RNA strand displacement in mammalian cells as a new foundation for rapidly scaling up sophistication in engineered biological systems, with applications in biosynthesis, biomedical diagnostics and therapeutics.

Abstract 1. Free Signal

Input Strand

Strand of RNA

Bound Signal

Gate:Output

Toehold Binding Branch Migration Bound Signal

Gate:Input

Free Signal

Output Strand

Our Aim: Develop RNA strand displacement as a mechanism to build in vivo synthetic circuits

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At the core of synthetic biology is making larger and more sophisticated circuits:

•  Traditionally the focus has been on making DNA easier to synthesize, assemble and deliver

•  Our approach creates and uses ~10x smaller parts which allows for delivery of larger circuits in smaller footprint and a lower operational energy cost for cell

•  Our approach uses RNA strand displacement in vivo to perform information processing, interfaced with sensing and actuating cellular signals

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Qian et al., Science 2011

Moon et al., Nature 2012

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The growth of circuit complexity in synthetic biology has been roughly linear, while strand displacement circuits have grown nearly exponentially in size. Source: Purnick et al., Nature MCB 2009; analysis of publications by Pierce, Winfree, and Yurke groups

•  Our approach enables us to use the features behind the much smaller nucleotide footprint, large combinatorial design space, orthogonal parts and tunable logic gates of strand displacement

Motivation 2.

Input Strand

S6 T T

T* S6*

S6

Fluorescent Complex High Red

+ + S6

T* S6*

Reporter High Green,

Low Red

S6

Waste High

Green

Figure 1A. (Left) RNA reporter design. T=toehold, S= binding domain. This design takes the reporting mechanism described by Qian et. al (Science, 2011) and implements it using 2’-OMe RNA. We added a green fluorophore to track the gate:output complex for in vivo studies, while the red fluorophore indicates strand displacement.

Figure 2A. (Right) RNA reporter design iteration two. Our first reporter design worked in vitro, but not well in vivo. Therefore, we set out to redesign our reporter and take into account orthogonality to the HEK293 transcriptome, protection from proteins in the RNAi pathway and increased toehold and domain lengths for increased reaction kinetics and reporter stability.

Figure 2B. (Above) Testing the improved reporter in vivo with transient transfection. The graph on the left is flow cytometry data from an experiment testing the key strand displacement reaction in HEK293 cells using transient transfection. A 200-point moving average was taken to reduce noise in the data. The x-axis shows relative fluorescence in the FITC channel (green) and the y-axis shows relative fluorescence in the Texas-Red channel. The No Transfection well received only cells. The No Input (negative control) well received the improved reporter (from Fig. 2A; no bulge) and demonstrates that for higher levels of green fluorescence (transfection efficiency), the reporter stays annealed post-transfection. The Scrambled Input well (negative control for strand displacement) received the reporter and an RNA strand with the correct toehold but an incorrect hybridization domain, which will not displace the reporter. The Input well (which received the reporter and a correct input strand), shows higher levels of green fluorescence correlate with higher levels of red fluorescence, which indicates that for higher transfection efficiency, we see more strand displacement occurring. *All data collected by iGEM students

The toehold-mediated strand displacement reaction is the basis for all our circuit components. It is thus crucial to validate this reaction can happen in a cell using RNA, our choice of nucleic acid. We performed both in vitro and in vivo experiments using a reporter (1A, below) and arrived at an affirmative result.

Figure 1B. (Left) Testing strand displacement in vitro. In this experiment, we tested the strand displacement reporting reaction (1A) on a plate reader. The negative control received a scrambled input with the reporter, whereas the positive control only contained the fluorescent complex. The experimental well contained the correct input strand with the reporter. Over time, levels of red fluorescence increased in this well, indicating loss of quenching. This, along with the negative control indicates that strand displacement occurred. *Data collected by iGEM students.

Figure 2C. (Above) Testing the improved reporter in vivo with nucleofection. The graph to the right is flow cytometry data. The dsROX population of cells received the fluorescent complex (Fig. 1A) to act as a positive control. The No Input population received only the reporter (Fig. 2A; no bulge) and shifts right on the FITC (green) axis because of the added Alexa fluorophore. The Input well received the input (Fig. 1A) and reporter (Fig. 2A; no bulge) and the population shifts up ~6 fold along the Texas-Red axis, indicating that strand displacement occurred. *All data collected by iGEM students

The Key Reaction 3.

We designed a strand displacement module to sense cellular mRNA.

We simulated our design for verification, and successfully demonstrated it in vitro on multiple mRNA target sites.

Figure 3. (Left) Simulations reveal the mRNA sensor will detect the presence of an mRNA. Green curve: 9 nanomolar (i.e. 90%) input strand. Red curve: 1 nM (10%) input strand. Both run with 100nM fuel strand, 12 nM ROX:quencher reporter complex, 10 nM gate:output complex. *Simulation using Visual GEC

Figure 4. (Below) Testing various domains of the eBFP2 mRNA as sensing targets. In vitro experiments were carried out using a sensor made of DNA strands. Graphed to the right is the fold increase in fluorescence before and after adding input, relative to baseline. *All data collected by iGEM students

Figure 5. (Above) Illustration of an mRNA sensor. A toehold region (orange) in the mRNA can bind to the toehold on a gate:output complex. Branch migration can then occur on the sensing domain (red), resulting in a free output signal strand. *Designed by iGEM students

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Cellular Interfacing 5.

NOT Gate: (Left) To build a universal logic set, we needed to implement a NOT gate using strand displacement. We designed, built and tested a fully functioning NOT gate.

Figure 8. (Left) In this figure the simulated transfer function when the dynamic gate (A) is in higher concentration with respect the other molecules. New experiments to replicate in vitro this result have already been planned.

Figure 7. (Left) NOT gate in vitro and by simulation using Visual DSD. This figure shows the overlay of the simulated transfer function and the in vitro transfer function, subtracting the basal fluorescence. The in vitro modeling accurately predicted the behavior of the NOT gate. Note the negative slope, characteristic of NOT logic. Rate constants for this simulation were based on Qian et. al (Science, 2011)

Figure 6. (Left) In vitro NOT gate. With no input, downstream input (B) can bind reversibly with dynamic gate (A) and reversibly with buffer (C). When B binds with C, the fuel (D) frees B, that consequently is able to bind with other C molecules increasing the level of output. Instead, when the input strand is present in high concentration, B and the input strand bind irreversibly with the dynamic gate (A). Since B is not free, it cannot bind with C. Therefore the output level will be low. *Designed by iGEM students

Case: Input present low output signal

Irreversible

Trapped!

Case: No input present high output signal Reversible

Downstream Input (B)

Downstream Input (B)

Input Strand

Dynamic Gate (A)

Input Strand

Dynamic Gate (A)

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Processing

AND OR NOT

Qian et al. 2011 Our Contribution

4. Human Practices 7.

Input Strand

Experimental results:

Contribution to the Synthetic Biology Community:

40 MammoBlocks

37 Biobricks

•  37 logic parts for strand displacement •  10 regulatory composite parts •  2 transcriptional regulators •  10 generators

•  3 promoters •  4 Hammerhead Ribozyme coding sequences •  13 reporters

"  INFORMATION PROCESSING: designed, simulated and tested a novel, strand displacement NOT gate

"   CIRCUIT PRODUCTION: characterized the U6 promoter to transcribe short RNAs in vivo, designs for using Hammerhead ribozymes to create double stranded gates in vivo

"   CELLULAR INTERFACING: designed, built and tested a novel mRNA sensor using strand displacement

Summary 8.

"   KEY REACTION: through multiple design iterations, we built and tested a system which shows that RNA strand displacement can occur in vivo. This reaction had never been shown in vivo before. Armed with this technology, we can build circuits with a smaller nucleic acid footprint, which enables more complex, circuits to be delivered to cells. Strand displacement circuits can also interface with traditional transcriptional and translational regulatory synthetic biology circuits.

Instructor:  Ron  Weiss      Advisors:  Deepak  Mishra,  Jonathan  Babb,  Nevin  Summers,  Lulu  Qian  

Acknowledgements 9. *See  wiki  or  talk  for  addi/onal  acknowledgements  

FF1 shRNA (67nt hairpin)

U6 TetO FF1 Hef1A eYFP-4xFF1

Figure 9. This experiment shows our ability to produce short hairpin RNA-s akin to gate:output complexes in strand displacement. Regulation of eYFP furthermore demonstrates RNA actuation. In this circuit, HEK293 cells were transfected with Hef1A:eYFP-4xFF1, where a constitutive promoter drives the expression of eYFP-4xFF1, a yellow fluorescent protein with four FF1 binding sites. However, when co-transfected with U6 TetO: FF1 plasmid, FF1 miRNAs block the expression of yellow fluorescent proteins via binding to FF1 sites on Hef1A:eYFP-4xFF1. As the ratio of mirFF1 increases from 0 to 8X, there is a corresponding decrease in the yellow fluorescent signal, indicating gene knockdown. The histograms above-left show the population of cells shifting from the yellow FITC region on the right towards the non-fluorescent region on the left. The above-right graph shows the population mean averages. *U6 TetO promoter design based on Henriksen et. al (Nucleic Acid Research, 2007). Experiments ran by iGEM students.

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

In Vivo RNA Production 6.

•  Developing hands-on synthetic biology course for college students during MIT's January term

•  Most of the current team took a similar course previous last IAP •  “Engineer Your Own Bacteria” – 2 week lecture, wet lab course •  Will be organized, fundraised and taught by MIT iGEMers

•  Collaboration with Boston University and Wellesley College iGEM teams

•  Use case for Wellesley HCI software •  Bridging the gap between tool designer and end-user

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

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