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RNA Strand Displacement for Sensing, Information Processing,
and Actuation in Mammalian Cells

Background and Motivation

In the near future, biological circuits will be much more modular and sophisticated than they are now, with a ten-fold smaller nucleotide footprint.

The Enabling Technology: Toehold-Mediated Strand Displacement

A gate strand and output strand exist as a complex that is partially bound through complementary Watson-Crick base-pairing within the S2 binding domain. The gate strand also contains an open, unbound domain called a toehold region, T*. An input strand with a free complementary toehold region, T, can bind to the toehold region on the gate strand, and subsequently displace the output strand to yield an input-gate complex. This is called a toehold-mediated strand displacement reaction. The output strand is used as an input for a downstream gate-output complex,enabling sophisticated interactions which yield full logic circuits.

Background

Qian and Winfree (Science 2011) utilized DNA computation to create AND and OR logic gates in vitro. They constructed a sophisticated binary square root circuit using these gates: Each of these:

undergoes the same toehold-mediated strand displacement reaction. These reactions are fully modular and can be scaled to circuits of any degree of sophistication.

Motivation for Bringing Strand Displacement to Mammalian Synthetic Biology

• More sophisticated circuits with smaller nucleotide footprint
  
  Sophistication of traditional transcription-translational circuits has grown linearly over the past 10 years, while sophistication of strand-displacement circuits has grown nearly exponentially. The most complex traditional circuit is a 4-input AND gate which includes 11 promoters, whereas the most complex strand-displacement circuit incorporates
• Simple combinatorial design space
  With 4 nucleotides, we can create a nearly infinite number of orthogonal sequences leading to orthogonal parts.
• Ease of composition
  The input motif matches the output motif allowing for modular cascading reactions.
• Tunability
  We can set arbitrary digital signal thresholds by varying the concentration of circuit species. We can also achieve signal amplification by including a fuel molecule.

RNA versus DNA

An information processing system is of limited use without dynamic production. RNA is a good medium because it can be continually produced from a few initial DNA parts. As in nature, DNA acts as the information-storage medium, and RNA acts as the information-processing medium. We can transfect DNA parts into mammalian cells to co-opt existing cellular machinery to produce our RNA parts.
Our RNA parts can then interact with the cell through sensing and actuation. Endogenous cellular RNAs can act as inputs, and we can actuate by knocking down endogenous cellular RNAs.