Biomod/2014/UCR/Breaking RNA/Motivation & Objectives

From OpenWetWare

Jump to: navigation, search

Breaking RNA

Contents

Motivation & Objectives

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. Synthetic biologists have taken advantage of this concept to engineer cellular activity and expand its 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. In this project we use a 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 that recognize and alter the conformation of the promoter region of another gene, thus modulating its activity [4]. This approach is effective, but because new transcription factors (proteins) are hard to design, it is difficult to create arbitrary regulatory interactions between genes. We take an alternative approach: we use RNA aptamers to modulate the activity of enzymes, rather than targeting the promoters. Thus, aptamers create the feedback loops necessary to build our circuits. RNA is easier to engineer than transcription factors: therefore, aptamers could be a promising method to build quickly arbitrary links among genes. 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 (RNAP) 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 will be an active part of the final circuits, and at the same time, act as a reporter.

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

The most important design principle to follow when building oscillators and switches is the presence of feedback. For switches, it is typically required to have a positive feedback loop; for oscillators, it is required to have a negative feedback loop (see, for instance [10, 11]). Following this general guideline, we identified RNA/enzyme interaction schemes that generate a positive and a negative loop. We then wrote ordinary differential equation (ODE) models for the two candidate circuits, and used parameters derived from the literature to explore their ability to behave as desired. The systems are nonlinear, so we checked their local behavior near equilibria. The models helped us improve our designs, in particular the oscillator. Also, the models provided useful guidelines for our preliminary experiments testing the behavior of the circuits.

Switch

To make a bistable switch, we want to generate an overall positive loop. A positive loop results from two consecutive inhibition reactions (see figure above, panel A). In our scheme, T7 RNAP transcribes an aptamer R1, which inhibits SP6 RNAP. In turn, SP6 RNAP transcribes aptamer R2, which inhibits T7 RNAP. These two inhibition reactions are straightforward to achieve using recently published aptamer sequences [6, 7]. For the system to work, the polymerases must spontaneously re-activate. This general scheme is shown in the figure above, panel B. We decided to implement re-activation reactions by designing DNA strands that peel off the aptamers from their target enzymes. These reactions are described more in detail below and in the Results section.

Clock

To make an oscillator, we want to generate an overall negative loop (figure above, panel A). However, this is not trivial using our two enzymes T7 RNAP and SP6 RNAP and their inhibiting aptamers. We designed one inhibition reaction as in the bistable switch, where T7 RNAP transcribes an aptamer inhibiting SP6 RNAP. Then, we assumed we could inactivate T7 with its aptamer, and use the RNA transcribed by SP6 to peel off the inhibiting aptamer of T7 RNAP. This idea is summarized in the figure above, panel B. The implementation details are discussed below and in the Results section.

2. Inhibition and activation reactions, and fluorescent reporters.

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. 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 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 restore transcription. RNA kleptamers are possible as well and are discussed in more detail in the Results section.


A kelptamer bound to an inhibiting aptamer can be recycled using RNaseH, which specifically breaks apart RNA bound to a 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, all the inhibiting RNA will bind directly to the kleptamers instead of inhibiting the enzyme, and the circuit will not function at all.

Fluorescent Reporters

In order to be able to watch the changes in the components of the circuit in real time, we need a robust reporter system. For testing and characterizing individual components of the circuits we aim to use fluorescent "light-up" aptamers like Spinach and Malachite Green aptamers. For tracking the dynamics of the bistable switch and the oscillator, we hope to develop a molecular beacon reporter that will exert only minimal strain on the circuit. We aim to characterize the behavior of these reporter systems to identify any underlying biases or other disadvantages.

3. Assembly of Circuits

In order to put together the circuits from individual components, we first need to establish the initial concentrations of different interacting components. The approximate relative concentrations of different component can be estimated based on model predictions. We also need to run experiments for characterizing the relative kinetics of different components. For example, the rates of transcription of T7 RNA polymerase and SP6 RNA polymerase are very different, so we need to establish proper concentrations at which both of the enzymes yield similar quantities of transcripts.

Once we have an estimate of initial concentrations, we can put the components together and test the operation of the circuits. Each parameter of the circuits can be varied one at a time to tune the circuits to desired operational point. The mathematical models will then be updated using experimentally obtained parameters.

References

Error fetching PMID 13718526:
Error fetching PMID 10659856:
Error fetching PMID 10659857:
Error fetching PMID 21921236:
Error fetching PMID 1697402:
Error fetching PMID 23650601:
Error fetching PMID 22426482:
Error fetching PMID 21283141:
Error fetching PMID 21283142:
Error fetching PMID 18971947:
Error fetching PMID 14647386:
  1. Error fetching PMID 13718526: [p1]
  2. Error fetching PMID 10659856: [p2]
  3. Error fetching PMID 10659857: [p3]
  4. Error fetching PMID 21921236: [p4]
  5. Error fetching PMID 1697402: [p5]
  6. Error fetching PMID 23650601: [p6]
  7. Error fetching PMID 22426482: [p7]
  8. Error fetching PMID 21283141: [p8]
  9. Error fetching PMID 21283142: [p9]
  10. Error fetching PMID 14647386: [p11]
  11. Error fetching PMID 18971947: [p10]
All Medline abstracts: PubMed HubMed

Personal tools