Biomod/2014/UCR/Breaking RNA/Results
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Results
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.
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
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
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.
RNA Polymerase Activation Reactions
Bound Aptamer-Kleptamer Interactions
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.
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
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
- 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 |
- 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 |
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<li><a href='http://openwetware.org/wiki/Biomod/2014/UCR/Breaking_RNA'><span>Home</span></a></li> <li><a href='http://openwetware.org/wiki/Biomod/2014/UCR/Breaking_RNA/Project'><span>Motivation & Objectives</span></a></li> <li><a href='http://openwetware.org/wiki/Biomod/2014/UCR/Breaking_RNA/Methods'><span>Results</span></a></li> <li><a href='http://openwetware.org/wiki/Biomod/2014/UCR/Breaking_RNA/Results'><span>Methods</span></a></li> <li><a href='http://openwetware.org/wiki/Biomod/2014/UCR/Breaking_RNA/Members'><span>Supplement</span></a></li> <li><a href='http://openwetware.org/wiki/Biomod/2014/UCR/Breaking_RNA/Achievements'><span>Team</span></a></li>
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