Biomod/2014/UCR/Breaking RNA/Methods

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Breaking RNA

Contents

Methods

DNA strands, enzymes, dyes and buffers Transcription Fluorometry Gel Electrophoresis RNA Extraction Oscillator Reactions Modeling and Simulations


DNA strands, enzymes, dyes and buffers

All the strands were purchased from Integrated DNA Technologies, Coralville, IA. Strand D1 was labeled with Texas Red at the 5′ end and IOWA Black RQ-Sp quencher at the 3′ end. For transcription experiments, we used the SP6 RNA Polymerase (#EP0133), which was purchased from ThermoScientific, T7 RNA Polymerase (C-T7300K) purchased from CellScript, E. coli RNase H from Ambion (#2292) and Pyrophosphatase from New England Biolabs (#M0296L). DFHBI dye, used for Spinach genelet, was purchased from Lucerna technologies (#400-1). Malachite green dye was purchased from Sigma (#32745). Transcription buffer was purchased from Epicentre Biotechnologies (#E7525), and transcription buffer was purchased from New England Biolabs (#E8590.)


Transcription


The genes were first annealed with 10% (v/v) 10× transcription buffer from 90°C to 37°C for 1 h 30 min at a target concentration of 10 μM. Then genes are mixed to a transcription mix composed of 10% (v/v), 10× transcription buffer, 7.5 mM each NTP, 24 mM MgCl2 4% (v/v) T7 RNA polymerase, 4-6% (v/v) SP6 RNA Polymerase, 1.2%(v/v) Pyrophosphatase (Ppase). When needed, we used 3.3% (v/v) E. coli RNase H. Each transcription experiment for fluorescence spectroscopy was prepared for a total target volume of 60 µl, unless noted otherwise.


Fluorometry

General Fluorometry Protocol

Fluorometry experiments are performed using a Jasco Spectrofluorometer FP-8500. Reactions were run at 30 °C in 50 μL chamber cuvettes manufactured by Starna (model 16.50F-Q-10/Z15). Samples were covered with 40 μL hexadecane oil to prevent evaporation.

Fluorometry Protocol for Specific Experiments

A. Transcription of Spinach gene by SP6 RNA polymerase

The following transcription solution was prepared with the following components, along with DHFBI Dye as the fluorescent reporter:

Component Concentration
DHFBI Dye 5 μM
Spinach Gene 300 nM
MgCl2 0.024 M
NTPs 7.5 mM

Solution also contained nuclease-free water and RNA polymerase reaction buffer. 2 μL of SP6 RNA polymerase was added to initiate transcription. Reaction took place at 30°C, with a total reaction volume of 60 μL.

B. Enzyme Inhibition with RNA Aptamers

For experiments testing T7 RNA Polymerase inhibition, Malachite Green was employed as the reporter dye. The transcriptional solution contained the following components:

Component Concentration
Malachite Green Dye 25 μM
Malachite Green Gene 50 nM
Gene G2 150 nM
MgCl2 0.024 M
NTPs 7.5 mM

The solution also contains RNA Polymerase transcription buffer and RNase free water. 3 μL of the T7 RNAP enzyme and 3 μL of SP6 RNAP enzyme was added. The transcription reactions took place at 30°C.

For experiments testing SP6 RNA Polymerase inhibition, DHFBI was employed as the reporter dye. The transcriptional solution contained the following components:

Component Concentration
DHFBI Dye 25 μM
Spinach Gene 50 nM
Gene G1 150 nM
MgCl2 0.024 M
NTPs 7.5 mM

The solution also contains RNA Polymerase transcription buffer and RNase free water. 3 μL of the T7 RNAP enzyme and 3 μL of SP6 RNAP enzyme was added. The transcription reactions took place at 30°C.

C. Bound Aptamer-Kleptamer Interactions

For experiments testing T7 RNA Polymerase reactivation, Malachite Green was employed as the reporter dye. The transcriptional solution contained the following components:

Component Concentration
Malachite Green Dye 16 μM
Malachite Green Gene 100 nM
MgCl2 0.024 M
NTPs 7.5 mM

The solution also contains RNA Polymerase transcription buffer and RNase free water. 1.3 μL of the T7 RNAP enzyme was added to solution along with 0.7 μL of Ppase to prevent inhibition due to diphosphate waste. The transcription reactions took place at 30°C. For the experimental cuvette, 800 nM of R3 was added as well as 1.2 μM of R2. The total volume used in each cuvette was 60 μL SP6 RNA Polymerase

D. Re-activation of inhibited SP6 RNA polymerase using a kleptamer

For experiments testing SP6 RNA Polymerase reactivation, DHFBI was the reporter dye. The transcriptional solution contained the following components:

Component Concentration
DHFBI Dye 25 μM
Spinach Gene 100 nM
MgCl2 0.024 M
NTPs 7.5 mM

The solution also contains RNA Polymerase transcription buffer and RNase free water. 3 μL of the SP6 RNAP enzyme was added to solution. The transcription reactions took place at 30°C. For the experimental cuvette, 1.77μM of R1 was added as well as 2.67μM D1. The total volume used in the cuvette was 67.421 μL.

E. Molecular Beacons as Reporters

For the experiments on the molecular beacon D1 (which is the main reporter in our systems), the transcriptional solution contained the following components:

Component Concentration
D1 100nM
R1 aptamer 200 nM
MgCl2 0.014 M
NTPs 5 mM

The solution also contains RNA Polymerase transcription buffer and RNase free water. 1 μL of the T7 RNAP enzyme was added to solution along with 0.7 μL of Ppase to prevent inhibition due to diphosphate waste and 2μL of RNase H to degrade the RNA bound to D1. The transcription reactions took place at 30°C. The total volume used in each cuvette was 60 μL.


Gel Electrophoresis

Denaturing Polyacrylamide Gels

Denaturing polyacrylamide gels (10% 19:1 acrylamide:bis and 6.93 M urea in 1x TBE buffer, 0.089M Tris, 0.089M boric acid, 0.002M EDTA) were run at 22 °C for 60-90 min with 10 V/cm in 1x TBE buffer. 10x TBE buffer was purchased from Ambion (AM9863). Samples were loaded with Gel Loading Buffer II from Ambion (AM8546G). A 10-base DNA ladder (Invitrogen, Carlsbad, CA; #1082- 015) was used as a reference. For imaging and quantifying, Denaturing gels were stained with SYBR Gold (Molecular Probes, Eugene, OR; #S-11494). Gels were scanned using the ChemiDoc XRS+Imager (Biorad, Hercules, CA) and analyzed using the Image Lab software (Biorad, Hercules, CA).


Non-Denaturing Polyacrylamide Gels

Non-Denaturing polyacrylamide gels (10% 19:1 acrylamide:bis and TAE buffer, 0.04M Tris, 0.004M Acetate, 0.001M EDTA) were run at 4 °C for 60-90 min with 15 V/cm in 1x TAE/12.5mM MgCl2 buffer. 10x TAE buffer was purchased from Ambion (AM9869). A 10-base DNA ladder (Invitrogen, Carlsbad, CA; #1082- 015) was used as a reference. For imaging and quantifying, Denaturing gels were stained with SYBR Gold (Molecular Probes, Eugene, OR; #S-11494). Gels were scanned using the ChemiDoc XRS+Imager (Biorad, Hercules, CA) and analyzed using the Image Lab software (Biorad, Hercules, CA)


RNA Extraction

AmpliScribe-T7-Flash Transcription Kit, from Epicentre (ASF3257), and MEGAscript SP6 Transcription Kit, from Ambion (AM1330), were used to perform rapid transcription of genelets for RNA Extractions. The samples are incubated at 37oC for 4 hours. Denaturing polyacrylamide gels (10% 19:1 acrylamide:bis and 6.93 M urea in 1x TBE buffer, 0.089M Tris, 0.089M boric acid, 0.002M EDTA) were run at 22 °C for 60-90 min with 10 V/cm in 1x TBE buffer. 10x TBE buffer was purchased from Ambion (AM9863). Samples were loaded with Gel Loading Buffer II from Ambion (AM8546G). Shortwave ultraviolet light was used to illuminate RNA strand of interest. The RNA strands were cut and submerged in 0.3M of sodium acetate (pH 5.3). The samples are incubated at 42oC for 20 hours. The supernatant is removed and 100% -20oC ethanol, from Sigma-Aldrich (E7023-500mL), and glycogen, from ThermoScientific (R0561), was added to the supernatant. The supernatant is incubated at -20oC for 20 hours. The sample is spun at 135000 rpm at 4oC for 15 minutes using a Z 216 MK Refrigerated Microcentrifuge from Hermle Labnet. The supernatant was removed to keep the precipitate pellet. The precipitate pellet is washed twice with 70% ethanol and spun at 135000 rpm at 4oC for 5 minutes using a Z 216 MK Refrigerated Microcentrifuge. All supernatant was removed and the precipitate was dried at 35oC for 10 minutes using a Vacufuge Concentrator 5301 from Eppendorf. The precipitate is resuspended with nuclease-free water from Ambion (AM9938).


Oscillator Reactions

Oscillator Experiment 1

The experiment shown in Fig. 13 in the results section was done as follows. Fluorometry was performed as described in 'General Fluorometry Protocols' section above. The reaction temperature was, 30°C. The reaction conditions are listed below:

Oscillator conditions
Oscillator conditions


Oscillator Experiment 2: RNaseH titration

The experiment shown in Fig. 14 in the results section was done as follows. Fluorometry was performed as described in 'General Fluorometry Protocols' section above. The reaction temperature was, 30°C. The reaction conditions are listed below:

Oscillator conditions
Oscillator conditions

Modeling and Simulations


To build our models, we first wrote all the reactions designed to occur in each system. Then, we used the law of mass action to derive ordinary differential equations (ODEs). The complete set of reactions and the ODEs are shown in the Supplement. ODE models were numerically integrated using MATLAB's ode23s solver. The nucleic acid binding rates and enzyme transcription and degradation rates were chosen to be in a physically plausible range based on the literature [1, 2]. All the simulation parameters are reported in Tables 1 and 2 in the Supplement.

Because both models are nonlinear, to gain insights into their behavior we performed a local linear analysis. Local linear analysis means that we approximated each nonlinear model with its linearized version around each equilibrium point. In practice, if our initial system is written as  \dot x = f(x), and its equilibrium is \bar x such that  f(\bar x)=0 , then nearby point \bar x we can approximate the system as:

 \dot \xi = \frac{\partial f}{\partial x}(\bar x) \xi,

where  \xi = x-\bar x. The partial derivative  \frac{\partial f}{\partial x}(\bar x) is evaluated at the equilibrium  \bar x, so in our case it is a constant matrix, known as the Jacobian of the system. The formula above is a direct application of Taylor series expansion stopped at the first order. First, for both models we found closed form expressions for their equilibria, by setting the ODEs to zero. These expressions, which are shown in the Supplement, are complex and had to be solved numerically to find the actual value of the equilibria. This was done again writing MATLAB scripts. Then, we found expressions for the Jacobian matrices  J= \frac{\partial f}{\partial x}(\bar x) at each equilibrium point. To derive the plots shown in Figures 2 and 4 of the Results section, we wrote a script to find the eigenvalues of the Jacobian. Based on the nature of the eigenvalues, we classified the equilibrium as stable (eigenvalues have negative real part) or unstable (eigenvalues have positive real part). If there is a pair of complex conjugate eigenvalues with positive real part, then the point presents local oscillations. We also manipulated the models by re-ordering their variables, so that their Jacobians would clearly reveal the overall feedback interconnection of the systems. The matrices are shown in the Supplement.

References

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All Medline abstracts: PubMed HubMed

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