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Motivation and approach

Many cellular processes behave like circuits. Since the discovery of the Lac Operon in 1961 [1], a myriad of circuit-like systems have been shown to exist in a wide variety of organisms. Many synthetic biologists have taken advantage of this concept to engineer cellular activity and achieve new functions [2, 3]. However, most mechanisms involve thousands of unknown regulatory pathways. This complexity hampers our ability to predict and program new biological functions. Our project focuses on designing two canonical biological circuits, a switch and a clock, in an in vitro environment. In contrast to in vivo systems, in vitro circuits can be better controlled and understood using mathematical models, and can be tuned more easily to produce desired effects. We use few biological components (DNA, RNA and few proteins) to design a switch and a clock that are regulated through aptamers, and we characterize the main reactions required for their operation.

In other existing transcriptional synthetic circuits, regulatory interactions between genes were built using proteins or RNA that recognize and alter the conformation of the promoter region of another gene, thus modulating its activity [4]. We take an alternative approach: we use RNA aptamers modulate the activity of enzymes, rather than targeting promoters. Thus, aptamers create the feedback loops necessary to build our circuits. RNA is unique in that it can be an information-carrying polymer (such as mRNA) or a functional molecule (such as ribozymes and aptamers). RNA functions are mediated by its ability to fold into tertiary structures, of which aptamers are an example[5]. For our project, we use RNA aptamers that were evolved to inhibit T7 RNA polymerase and SP6 RNA polymerase[6, 7]. However, currently there are no established methods to reverse aptamer binding and inhibition in a rational manner. Reversible inhibition and repression are necessary to build dynamic circuits. Therefore, our first objective is to design and characterize DNA and RNA aptamer complements, which can reactivate the enzymes by removing the bound aptamers. We call these re-activating strands "Kleptamers" (from the Greek verb klepto = 'to steal'; Kleptamer ='strand that steals away the aptamer'). To monitor the dynamics of our circuits we also design and characterize a multifunctional molecular beacon which be will be an active part of the final circuits and at the same time will act as a reporter.

To build our circuits we integrate domain-level strand design, modeling, and experiments in vitro. Only few in vitro toolkits currently exist to program molecular dynamic circuits like clocks and switches [8, 9], and our project aims at expanding these toolkits to include aptamers.

Objectives

To achieve our overall goal of building aptamer-based circuits we pursued these objectives:
1) Design of the circuits and mathematical modeling. 2) Characterization of reporters, inhibition-activation reactions. 3) Assembly of the circuits.

1) Design of the circuits and mathematical modeling
2) Characterization of activation and inhibition reactions
3) Assembly of the circuits.

EDIT

1. Design of the circuits and mathematical modeling

General approach description.

Creating and designing simulations using computing programs, such as MATLAB, are important for testing the various mechanisms involved in the systems. This is particularly useful for coming up with preliminary conditions for in vitro experiments, as well as determining the robustness of the schematic.

Switch

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Clock

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2. Inhibition and activation reactions

Inhibition reactions: Aptamer Design

Specific inhibition of enzymatic activity is of major importance for our system. RNA aptamers that inhibit T7 RNA polymerase and SP6 RNA polymerase have been reported previously[6, 7]. Among various sequence variants published in the original papers, we chose those reported to have higher binding affinity to RNA polymerases. We then designed genes to synthesize RNA aptamer transcripts . Then we proceeded to characterize the effectiveness of aptamer induced RNA polymerase inhibition in each case.

Activation reactions: "Kleptamer" Design

Similar to inhibition, it is also crucial to allow the reactivation of the enzymes for oscillations. In order to reactivate the RNA polymerases, the RNA aptamers must be removed. We can do this by using a DNA strand that is complementary to the aptamer. If the DNA complement has a higher affinity to the RNA aptamer than does the enzyme, the inhibitor will be "peeled" off from the enzyme and restoring transcription.

A kelptamer bound to an inhibiting aptamer can be recycled using the RNaseH, which specifically breaks apart RNA bound to DNA strand. So, in presence of RNaseH, a small amount of kleptamer DNA is sufficient to destroy a much larger quantity of inhibiting aptamers. In other words, the kleptamers-RNaseH combination can reset a system by reactivating the enzymes and breaking apart the inhibiting RNA aptamer. Using small quantities of kleptamers is crucial in dynamic circuits because if there is an excess of kleptamers, then all the inhibiting RNA will bind directly to the kleptamer instead of inhibiting the enzyme and the circuit will not function at all.