Biomod/2014/UCR/Breaking RNA/Methods: Difference between revisions

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<h1>DNA Strands, Fluorescent Dyes and Enzymes</h1>
<h1>DNA Strands, Fluorescent Dyes and Enzymes</h1>
</font>
</font>
<p> <font size="3.5">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, and E. coli RNase H from Ambion (#2292). DFHBI  dye, used for Spinach genelet, was purchased from Lucerna technologies (#400-1). Malachite green dye was purchased from Sigma (#32745).
<p> <font size="3.5">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).
</font></p>
</font></p>


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<h1>Fluorometry</h1>
<h1>Fluorometry</h1>
</font>
</font>
<br />
<font size="4.5">
<h1>Transcription</h1>
</font>
<br>
<p>The DNA strands were 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. The genes are in a solution with 10% (v/v), 10× transcription buffer, 7.5 mM each NTP, 24 mM MgCl<sub>2</sub> 4% (v/v) T7 RNA polymerase, 4-6% (v/v) SP6 RNA Polymerase, 1.2%(v/v) Pyrophosphatase (Ppase), and 3.3% (v/v) E. coli RNase H. Each transcription experiment for fluorescence spectroscopy was prepared for a total target volume of 60 µl. </p>


<h3><u><font size="4.5">General Fluorometry Protocol</font></u></h3>
<h3><u><font size="4.5">General Fluorometry Protocol</font></u></h3>
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The solution also contains RNA Polymerase transcription buffer and RNase free water. 1 &mu;L of the T7 RNAP enzyme was added to solution along with 0.7 &mu;L 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&deg;C. The total volume used in each cuvette was 60 &mu;L.
The solution also contains RNA Polymerase transcription buffer and RNase free water. 1 &mu;L of the T7 RNAP enzyme was added to solution along with 0.7 &mu;L 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&deg;C. The total volume used in each cuvette was 60 &mu;L.
<br />
<font size="4.5">
<h1>Transcription</h1>
</font>
<br>
<p>The DNA strands were 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. The genes are in a solution with 10% (v/v), 10× transcription buffer, 7.5 mM each NTP, 24 mM MgCl<sub>2</sub> 4% (v/v) T7 RNA polymerase, 4-6% (v/v) SP6 RNA Polymerase, 1.2%(v/v) Ppase, and 3.3% (v/v) E. coli RNase H. Each transcription experiment for fluorescence spectroscopy was prepared for a total target volume of 60 µl. </p>


<br />
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Revision as of 18:45, 25 October 2014

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Methods

UNDER CONSTRUCTION

EDIT


DNA Strands, Fluorescent Dyes and Enzymes

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).


Fluorometry


Transcription


The DNA strands were 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. The genes are in a solution with 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), and 3.3% (v/v) E. coli RNase H. Each transcription experiment for fluorescence spectroscopy was prepared for a total target volume of 60 µl.

General Fluorometry Protocol

Fluorometry experiments are performed using Jasco Spectrofluorometer FP-8500 machine. The solution necessary for transcription for our system consisted of nuclease-free water from Ambion(9914G), Ribonucleoside-5´-Triphosphate Solutions purchased from Epicentre(RN02825), RNA Polymerase Transcriptional Buffer from New England Biolabs(B9012S). Standard reactions contained 7.5 mM NTP, 24 mM MgCl2, and 1X RNA Polymerase Transcriptional Buffer, with a total volume of 60 μL. Solution was covered with 40 μL hexadecane oil to prevent evaporation. Reactions were run at 30 °C in 50 microliter-chamber Fluorimeter Cell cuvettes manufactured by Starna (model 16.50F-Q-10/Z15).

Fluorometry Protocol for Specific Experiments

A. Enzyme Inhibition with RNA Aptamers

For the 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 the 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.

B. Bound Aptamer-Kleptamer Interactions

For the 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 phase 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

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

For the 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.

D. Molecular Beacons as Reporters

For the molecular Beacon experiment, D1 strand was employed as the reporter of the system. 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)

Gel Electrophoresis Protocols for Specific Experiments

A. Bound Aptamer-Kleptamer Interactions

For the first gel, the concentration of R1 and D1 used are 1 μM and 2 μM and the T7 RNA Polymerase volume used was 1 μL. The total volume in each well was 7 μL. R1 and SP6 RNAP were mixed together and incubated for 20 minutes at 30°C. Next, D1 was added to the mixture and the solution was incubated for another 10 minutes at 30°C. A list of the components in each well is given below:

Lane Components
1 10 base pair ladder
2 R1 aptamer
3 D1 Strand (23 base pair)
4 D1 Strand (38 base pair)
5 R1 aptamer + SP6 RNA Polymerase
6 R1 aptamer +SP6 RNA Polymerase + D1 (23 base pair)
7 R1 aptamer +SP6 RNA Polymerase + D1 (38 base pair)

For the second gel, the concentrations of R1 and R4 used were both 0.7 μL and the SP6 RNA Polymerase volume used was 1 μL. The total volume used in each well was 7 μL. R1 and SP6 RNA Polymerase were mixed together and incubated for 15 minutes at 30°C. Next, R4 was added to the mixture and the solution was incubated for another 15 minutes. The composition of each lane is given below:

Lane Components
1 10 base pair ladder
2 R1 aptamer
3 R4 Strand
4 R1 aptamer + SP6 RNA Polymerase
5 R1 aptamer + SP6 RNA Polymerase + R4 aptamer


B. Unbound Aptamer-Kleptamer Interactions

For this gel, the concentrations used for R1 aptamer and D1 strands are all 1 μM. The R1 and D1 aptamers were mixed into solution together at the same concentration and incubated at 30°C for 10 minutes. The composition of each lane is given below:

Lane Components
1 10 base pair ladder
2 R1 aptamer
3 D1 strand (38 base pair)
4 D1 strand (23 base pair)
5 R1 aptamer + D1 strand (38 base pair)
6 R1 aptamer + D1 strand (23 base pair)


C. Comparison of Transcription Rates between SP6 RNAP and T7 RNAP

For this denaturing gel, the concentrations used for G1 gene, R1 aptamer, G2 gene, and R2 aptamer are all 1 μM. The transcription protocol of T7 RNAP transcribing G1 gene and SP6 RNAP transcribing G2 gene is as follows:

Component Concentration
G1 gene 10nM
MgCl2 0.024 M
NTPs 7.5 mM

The solution also contains RNA Polymerase transcription buffer and RN are free water. 2μL of T7 RNAP is added to the solution. For SP6 RNAP transcription of G2 gene, the components of the solution is as follows:

Component Concentration
G2 gene 100-200nM
MgCl2 0.024 M
NTPs 7.5 mM

The solution also contains RNA Polymerase transcription buffer and RN are free water. 2μL of SP6 RNAP was added. Both solutions was incubated at 30°C for 45 minutes. The samples are then diluted at a 1:4 ratio using Gel Loading Buffer II. The compositions of each well is given below:

Lane Components
1 10 base pair ladder
2 G1 gene
3 R1 aptamer
4 G2 gene
5 R2 aptamer
6 T7 RNAP transcription of G1 gene [10nM]
6 SP6 RNAP transcription of G2 gene [100nM]
6 SP6 RNAP transcription of G2 gene [150nM]
6 SP6 RNAP transcription of G2 gene [200nM]


2.5 Bistable Mechanisms

2.6 Oscillatory Mechanisms

For the inhibition of T7 RNA polymerase with gene G3, 2 μL of T7 ran Polymerase was used in this experiment. The transcriptional solution has Malachite Green as the reporter and the solution composition is listed below:

Component Concentration
Malachite Green Dye 16 μM
Malachite Green Gene 250 nM
MgCl2 0.024 M
NTPs 7.5 mM

The solution also contains RNA Polymerase transcription buffer and RN are free water. The gene G3 was added at 500 nM. Experiment was incubated at 30°C.

For the reactivation of T7 RNA Polymerase attempt with G3 and G2, 2 μL of both, T7 and SP6 RNA Polymerase were used. The transcriptional solution has Malachite Green as the reporter and the solution composition is listed below:

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

The solution also contained RNA Polymerase transcription buffer along with RNase free water. An addition of 750 nM of G3 was added to the experimental cuvette along with 200 nM of G2 approximately 2 hours later. Experiment was incubated at 30°C.



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 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 Z 216 MK Refrigerated Microcentrifuge. All supernatant was removed and the precipitate was dried at 35oC for 10 minutes using the Vacufuge Concentrator 5301 from Eppendorf. The precipitate is resuspended with nuclease-free water from Ambion (AM9938).


Oscillation Reactions

The fluorometry was performed using protocols given in 'General Fluorometry Protocols' section above. The reaction temperature was, 30◦C. The reaction mix was made as follows,

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 [math]\displaystyle{ \dot x = f(x) }[/math], and its equilibrium is [math]\displaystyle{ \bar x }[/math] such that [math]\displaystyle{ f(\bar x)=0 }[/math], then nearby point [math]\displaystyle{ \bar x }[/math] we can approximate the system as:

[math]\displaystyle{ \dot \xi = \frac{\partial f}{\partial x}(\bar x) \xi, }[/math]

where [math]\displaystyle{ \xi = x-\bar x }[/math]. The partial derivative [math]\displaystyle{ \frac{\partial f}{\partial x}(\bar x) }[/math] is evaluated at the equilibrium [math]\displaystyle{ \bar x }[/math], 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 [math]\displaystyle{ J= \frac{\partial f}{\partial x}(\bar x) }[/math] 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.

EDIT


References

  1. Franco E, Friedrichs E, Kim J, Jungmann R, Murray R, Winfree E, and Simmel FC. Timing molecular motion and production with a synthetic transcriptional clock. Proc Natl Acad Sci U S A. 2011 Oct 4;108(40):E784-93. DOI:10.1073/pnas.1100060108 | PubMed ID:21921236 | HubMed [p1]
  2. Zhang DY, Turberfield AJ, Yurke B, and Winfree E. Engineering entropy-driven reactions and networks catalyzed by DNA. Science. 2007 Nov 16;318(5853):1121-5. DOI:10.1126/science.1148532 | PubMed ID:18006742 | HubMed [p2]

All Medline abstracts: PubMed | HubMed


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