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EDIT

Methods

DNA Strands, Fluorescent Dyes and Enzymes

All the strands were purchased from Integrated DNA Technologies, Coralville, IA. Strand K1 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).

Fluorometry

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 uL. Reactions were run at 30 °C in Starna Fluorimeter Cell Sub-Micro Quartz cuvettes (Starna, 16.45F/Q)?.

Example: 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

Transcription Protocol

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) 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.

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). Samples were__________________. 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)

2.3 Bound Aptamer-Kleptamer Interactions: Gel Electrophoresis

For the first gel, the concentration of R1 and K1 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, K1 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	K1 Strand (23 base pair)
4	K1 Strand (38 base pair)
5	R1 aptamer + SP6 RNA Polymerase
6	R1 aptamer +SP6 RNA Polymerase +K1 (23 base pair)
7	R1 aptamer +SP6 RNA Polymerase +K1 (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

2.4 Unbound Aptamer-Kleptamer Interactions: Gel Electrophoresis

For this gel, the concentrations used for R1 aptamer and K1 strands are all 1 μM. The R1 and K1 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	K1 strand (38 base pair)
4	K1 strand (23 base pair)
5	R1 aptamer + K1 strand (38 base pair)
6	R1 aptamer + K1 strand (23 base pair)

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. EDIT

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 37^oC 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 42^oC for 20 hours. The supernatant is removed and 100% -20^oC ethanol, from Sigma-Aldrich (E7023-500mL), and glycogen, from ThermoScientific (R0561) ), was added to the supernatant. The supernatant is incubated at -20^oC for 20 hours. The sample is spun at 135000 rpm at 4^oC 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 4^oC for 5 minutes using Z 216 MK Refrigerated Microcentrifuge. All supernatant was removed and the precipitate was dried at 35^oC for 10 minutes using the Vacufuge Concentrator 5301 from Eppendorf. The precipitate is resuspended with nuclease-free water from Ambion (AM9938).

Simulations

Topology Design

Building on previous research, which suggests the possibility of complex, regulatory circuits, simulations were run to determine the existence of conditions necessary for oscillations and bistable systems using RNA aptamers. Stability analyses were performed using MATLAB, a computing software, to find stable equilibrium conditions for the bistable system and stable, yet dynamic conditions for the oscillator. A program was designed to search for parameters representing the conditions necessary for these systems to function.

Modelling and Tuning the Oscillator

MATLAB was used to model and simulate the oscillator. The chemical reactions involved in the schematic were translated into ODEs that represented the rate of synthesis and consumption of corresponding chemical components. The computing software was programmed to solve these equations and display a graphical representation of the behaviors of these ODES. This graph was designed to display the rates of change of the ODEs analogous to the reporters of the systems. A program searched for parameters and relationships suspected to promote oscillatory behavior. Further analysis compared the effects of changing different parameters on the simulations and provided more information on ideal, yet realistic conditions for strong and stable oscillations. Mathematical analysis of the ODES using matrices and phase plots served as compelling evidence for the existence of these conditions.

Modelling and Tuning the Bistable System

In addition to MATLAB, further extensive mathematical analysis was used to linearize the multidimensional and complex system. The ODEs were manipulated and analyzed to determine the two stable equilibrium points representing a toggle between two states. The third equilibrium point represented an unstable and unrealistic condition. Euler's formula, matrices, and phase plots all provided further evidence.

  <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>

</ul> </div> </body> </html>

@@ Line 28: / Line 28: @@
 </font>
-<p> <font size="3.5"> 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).
+<h3><u><font size="4.5">General Fluorometry Protocol</font></u></h3>
+<p> <font size="3.5"> 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 uL.  Reactions were run at 30 °C in Starna Fluorimeter Cell Sub-Micro Quartz cuvettes (Starna, 16.45F/Q)?.
 </font></p>
+<h3><u><font size="4.5">Example: Bound Aptamer-Kleptamer Interactions</font></u></h3>
+For the T7 RNA Polymerase reactivation, Malachite Green was employed as the reporter dye. The transcriptional solution contained the following components:
+::{| border = "1" class="wikitable" style="margin:auto;"
+! scope="col" | Component
+! scope="col" | Concentration
+|- align="center"
+| Malachite Green Dye|| 16 &mu;M
+|- align ="center"
+| Malachite Green Gene|| 100 nM
+|- align ="center"
+| MgCl<sub>2</sub>|| 0.024 M
+|- align ="center"
+|NTPs|| 7.5 mM
+|}
+The solution also contains RNA Polymerase transcription buffer and RNase free water. 1.3 &mu;L of the T7 RNAP enzyme was added to solution along with 0.7 &mu;L of phase to prevent inhibition due to diphosphate waste. The transcription reactions took place at 30&deg;C. For the experimental cuvette, 800 nM of R3 was added as well as 1.2 &mu;M of R2. The total volume used in each cuvette was 60 &mu;L SP6 RNA Polymerase
+<br />
+<font size="4.5">
+<h1>Transcription Protocol</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>
 <br />
@@ Line 44: / Line 71: @@
 <p> <font size="3.5">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 MgCl<sub>2</sub> buffer. 10x TAE buffer was purchased from Ambion (AM9869). Samples were__________________. 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)</font></p>
+<h3><u><font size="4.5">2.3 Bound Aptamer-Kleptamer Interactions: Gel Electrophoresis</font></u></h3>
+For the first gel, the concentration of R1 and K1 used are 1 &mu;M and 2 &mu;M and the T7 RNA Polymerase volume used was 1 &mu;L. The total volume in each well was 7 &mu;L. R1 and SP6 RNAP were mixed together and incubated for 20 minutes at 30°C. Next, K1 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:
+::{| border = "1" class="wikitable" style="margin:auto;"
+! scope="col" | Lane
+! scope="col" | Components
+|- align="center"
+| 1|| 10 base pair ladder
+|- align ="center"
+| 2|| R1 aptamer
+|- align ="center"
+| 3|| K1 Strand (23 base pair)
+|- align ="center"
+|4|| K1 Strand (38 base pair)
+|-align ="center"
+|5|| R1 aptamer + SP6 RNA Polymerase
+|-align ="center"
+|6|| R1 aptamer +SP6 RNA Polymerase +K1 (23 base pair)
+|-align ="center"
+|7|| R1 aptamer +SP6 RNA Polymerase +K1 (38 base pair)
+|}
+For the second gel, the concentrations of R1 and R4 used were both 0.7 &mu;L and the SP6 RNA Polymerase volume used was 1 &mu;L. The total volume used in each well was 7 &mu;L. R1 and SP6 RNA Polymerase were mixed together and incubated for 15 minutes at 30&deg;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:
+::{| border = "1" class="wikitable" style="margin:auto;"
+! scope="col" | Lane
+! scope="col" | Components
+|- align="center"
+| 1|| 10 base pair ladder
+|- align ="center"
+| 2|| R1 aptamer
+|- align ="center"
+| 3|| R4 Strand
+|- align ="center"
+|4|| R1 aptamer + SP6 RNA Polymerase
+|-align ="center"
+|5|| R1 aptamer + SP6 RNA Polymerase + R4 aptamer
+|}
+<h3><u><font size="4.5">2.4 Unbound Aptamer-Kleptamer Interactions: Gel Electrophoresis</font></u></h3>
+For this gel, the concentrations used for R1 aptamer and K1 strands are all 1 &mu;M. The R1 and K1 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:
+::{| border = "1" class="wikitable" style="margin:auto;"
+! scope="col" | Lane
+! scope="col" | Components
+|- align="center"
+| 1|| 10 base pair ladder
+|- align ="center"
+| 2|| R1 aptamer
+|- align ="center"
+| 3|| K1 strand (38 base pair)
+|- align ="center"
+|4|| K1 strand (23 base pair)
+|-align ="center"
+|5|| R1 aptamer + K1 strand (38 base pair)
+|-align ="center"
+|6|| R1 aptamer + K1 strand (23 base pair)
+|}
+<h3><u><font size="4.5">2.5 Bistable Mechanisms</font></u></h3>
+<h3><u><font size="4.5">2.6 Oscillatory Mechanisms</font></u></h3>
+For the inhibition of T7 RNA polymerase with gene G3, 2 &mu;L 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:
+::{| border = "1" class="wikitable" style="margin:auto;"
+! scope="col" | Component
+! scope="col" | Concentration
+|- align="center"
+| Malachite Green Dye|| 16 &mu;M
+|- align ="center"
+| Malachite Green Gene|| 250 nM
+|- align ="center"
+| MgCl<sub>2</sub>|| 0.024 M
+|- align ="center"
+|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&deg;C.
+For the reactivation of T7 RNA Polymerase attempt with G3 and G2, 2 &mu;L 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:
+::{| border = "1" class="wikitable" style="margin:auto;"
+! scope="col" | Component
+! scope="col" | Concentration
+|- align="center"
+| Malachite Green Dye|| 16 &mu;M
+|- align ="center"
+| Malachite Green Gene|| 100 nM
+|- align ="center"
+| MgCl<sub>2</sub>|| 0.024 M
+|- align ="center"
+|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&deg;C.
+[http://openwetware.org/index.php?title=Biomod/2014/UCR/Breaking_RNA/Acknowledgements&action=edit EDIT]
 <br />

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