Biomod/2014/UCR/Breaking RNA/Results

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Results


EDIT

Design and Modeling

Bistable switch

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Oscillator

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Experimental Characterization


 The oligonucleotide sequences are specified in the Supplementary section. Once the necessary genelets and strands for our RNA clocks and switches systems are designed, it is important to characterize and verify that our DNA sequences are designed correctly.

Spinach and Malachite Green Reporters

For experimental characterization of inhibition and reactivation RNA polymerases, we used genes that produce fluorescent RNA aptamers. The RNA transcript from these genes bind the corresponding dyes, giving rise to fluorescence. The we can track the activity of the RNA polymerases using fluorometer. We designed a gene with SP6 promoter which produces Spinach aptamer[1] and another gene with T7 promoter which produces Malachite green aptamers[2] . In the following figure, section B illustrates increase in fluorescence with accumulation of Spinach aptamer due to activity of SP6 RNA polymerase.

Figure 1: Characterization of reporter system. A) Reaction scheme. RNA Polymerase transcribes a gene encoding an aptamer that interacts with the dye in order to activate fluorescence. Since the dye is in excess, we can monitor the activity of RNA polymerase by monitoring the increase or decrease in fluorescence. B) Fluorometer data of SP6 RNA Polymerase transcription of Spinach aptamer to produce fluorescence.

These reporters while invaluable in characterization of inhibition/activation of RNA polymerases, were found not to be ideal for use as a reporter for bistable switch or oscillator circuit. Further characterization of these reporters and their disadvantages can be found in the supplementary material section.

Molecular Beacons as Reporters

Figure 2: Molecular beacon reporter system: (A) Schematic diagram showing operation of the molecular beacon. When the RNA of interest is present, the fluorescence is ON and when it the RNA gets degraded, the fluorescence is turned OFF. (B) This fluorescence plot shows experimental verification of molecular beacon operation. When the RNA of interest is added to D1 strand (DNA) the fluorescence is high and when RNAseH is added to degrade the RNA bound to DNA, the fluorescence goes back down.

In order to address the need to measure the transcription rates of our systems, we explored alternatives to Spinach and Malachite green aptamer reporter systems. Ideally, addition of a reporter system in to a dynamic circuit like an oscillator, should not overload or perturb the circuit in any way. To achieve this we designed a reporter out of one of the components of our circuits, the DNA strand D1. The original function of the strand D1 is to reactivate inhibited enzymes by removing the inhibiting RNA aptamer (can be seen in this schematic diagram). In other words, it is a 'kleptamer'. Using RNA folding prediction softwares, we found that the single strand D1 is expected to form a hairpin like structure naturally, bringing the 5' end and 3' end closer.

Based on our oscillator design and models, D1 is expected to cycle between two states - (1) In double stranded form bound to RNA (R1), (2) In single stranded hairpin form. By placing a fluorophore and quencher at the 5' and 3' ends, respectively, we can monitor D1 switching from state (1) to state (2). This provides us a neat way to keep track of the RNA concentration (of R1) during operation of the oscillator. The same is true for the bistable switch circuit - 'ON' and 'OFF' states of D1 will represent the flipping between equilibrium states in the bistable switch.

The use of molecular beacons such as D1 as reporters creates a simple yet effective reporter system. Other reporter systems can potentially affect or complicate the main system, such as by competing with RNAP or by requiring the use of additional genes. Molecular beacons can function successfully without many components. This minimizes the clutter and complications associated with other reporter systems.

RNA Polymerase Inhibition Reactions


Figure 3: Inhibtion of T7 RNA Polymerase. A) Reaction scheme. SP6 RNA Polymerase transcribes the inhibiting aptamer, which will interact and inhibit T7 RNA Polymerase. B) The plot shows fluorometry data on the successful inhibition of T7 RNA Polymerase via the reaction pathway illustrated in A. The fluorescence is produced by the Malachite Green aptamer, which is transcribed by T7 RNA polymerase from a T7 promoter containing gene. Inhibition of the enzyme results in a flat line in the fluorescence plot.

The topologies of our RNA clocks and switches rely on the idea that inhibition of a module is possible, whether it is self-mediated or caused by another module. Fluorescent RNA aptamer genes, Malachite Green and Spinach, were used to characterize and quantify different components of our system. The fluorescence from these genes give us a quantitative and visual readout mechanism for activity of the RNA polymerases. Figure 3 and Figure 4 successfully illustrate the inhibition of T7 and SP6 RNA polymerases by the respective aptamers.

Figure 4: Inhibition of SP6 RNA Polymerase. A) Reaction scheme. T7 RNA Polymerase transcribes the inhibiting aptamer, which will interact and inhibit with SP6 RNA Polymerase. B) The plot shows fluorometry data on the successful inhibition of SP6 RNA Polymerase via the reaction pathway illustrated in A. The fluorescence is produced by the Spinach aptamer, which is transcribed by SP6 RNA polymerase from a SP6 promoter containing gene. Inhibition of the enzyme results in a flat line in the fluorescence plot. Experimental conditions are provided in detail in the supplementary material section.


RNA Polymerase Activation Reactions

Bound Aptamer-Kleptamer Interactions

Reactivation of T7 RNA Polymerase. A) Reaction schematic. T7 RNA Polymerase is inhibited by the aptamer. The kleptamer will interact with the aptamer and remove it from the enzyme, forming a waste complex. B) Flourometry results showing successful reactivation of RNAP after inhibition using the extracted RNA aptamer and kleptamer. C) Non-denaturing gel electrophoresis indicating the proper interactions are taking place.


Inhibition of the modules is not sufficient for RNA clocks and switches. It must also be possible for the enzymes to regain transcriptional activity after the addition of the kleptamer. The following experiments show unequivocally that the kleptamers can successfully undermine inhibition. The extent of reactivation varies for both, T7 and SP6, RNA Polymerase systems. T7 RNAP does not seem to reactivate completely but this may be sufficient for our purposes. SP6 RNA Polymerase is reactivated to completion and is very efficient. Both gels show the interactions between all components are taking place as expected. The amount of aptamer binding to the enzyme is greatly diminished after the addition of the kleptamer in lanes 6 and 7.

Reactivation of SP6 RNA Polymerase. A) Reaction Scheme. SP6 Polymerase is reactivated by the interactions of the kleptamer with the bound aptamer. B) Fluorometry data representing the inhibition with R1 and reactivation with D1. C) Non-denaturing gel. Lanes 2-4 represent the aptamer, kleptamer (23 base pair), and kleptamer (38 base pair). Lane five represents the interaction between the aptamer and enzyme RNA Polymerase. Lanes 6 and 7 represent the addition of D1, 23 base pair and 38 base pair, respectively.

For both enzyme transcriptional systems, the fluorometer data shows inhibition of enzyme activity evident by the sharp decline in the fluorescence intensity rate. With the addition of the kleptamer, enzymatic activity is restored and the transcription of either Spinach or Malachite Green is continued.

Inhibition of T7 RNA Polymerase with Genelets

Reactivation of T7 RNA Polymerase. A) Reaction scheme. T7 RNA Polymerase will transcribe the gene, g3 to produce the inhibition aptamer for itself. B) Fluorometry data verifying this mechanism. T7 RNA Polymerase activity is completely suppressed upon the addition of g3. See supplement for experimental details. C) Denaturing gel electrophoresis data. When comparing the lanes that contain only Malachite Green transcription to the lanes that contain Malachite Green and gene g3, there is less intensity of the Malachite Green RNA transcript band in the lanes that contain gene g3, than in the lanes that contain only Malachite Green. Experimental conditions are in detail in the Supplement Section ___.

By transcribing G3, T7 RNAP will become inhibited by the transcribed RNA aptamer. This can be seen using fluorometry by measuring the transcription rate of Malachite Green. The blue trace represents our negative control in which there is no G3. The orange trace represent the solution with G3 added. The gene was added around 1 hour into the experiment at 500 nM. It’s evident that the activity has been completely inhibited.



Assembling the Circuits



References

  1. Paige JS, Wu KY, and Jaffrey SR. RNA mimics of green fluorescent protein. Science. 2011 Jul 29;333(6042):642-6. DOI:10.1126/science.1207339 | PubMed ID:21798953 | HubMed [p1]
  2. Babendure JR, Adams SR, and Tsien RY. Aptamers switch on fluorescence of triphenylmethane dyes. J Am Chem Soc. 2003 Dec 3;125(48):14716-7. DOI:10.1021/ja037994o | PubMed ID:14640641 | HubMed [p2]

All Medline abstracts: PubMed | HubMed

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