Biomod/2014/UCR/Breaking RNA/Supplement

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Breaking RNA

Contents

Supplement

Oligonucleotide Sequences Characterization of Spinach and Malachite Green aptamer Fluorometer Experiments On Circuit Components Gel Experiments On Circuit Components Modeling

Oligonucleotide Sequences


Bistable switch

DNA Oligonucleotides Length Sequences
G1 Non-Template 83 5'-TAA TAC GAC TCA CTA TAG GAT GGC AGC GGA GAG TTG CTT GGA ATG CGT TAT AGT CTC TTA GGT GTG TTC GCA CAC CAC TCT CC-3'
G1 Template 83 5'-GGA GAG TGG TGT GCG AAC ACA CCT AAG AGA CTA TAA CGC ATT CCA AGC AAC TCT CCG CTG CCA TCC TAT AGT GAG TCG TAT TA-3'
G2 Non-Template 57 5'-ATT TAG GTG ACA CTA TAG AGG CGA GCG TAA GTC AAT TCC ACT ATC ATT GCT GCA AGC-3'
G2 Template 57 5'-GCT TGC AGC AAT GAT AGT GGA ATT GAC TTA CGC TCG CCT CTA TAG TGT CAC CTA AAT-3'
D1 61 5'-TexRd-CGT CGC CTC TCA ACG AAC CTT ACG CAA TAT CAG AGA ATC CAC ACA AGC GTG TGG TGA GAG G-IowaBlack-3'
D2* 57 5'-GCT TGC AGC AAT GAT AGT GGA ATT GAC TTA CGC TCG CCT CTA TAG TGT CAC CTA AAT-3'
Spinach Non-Template 100 5'-ATT TAG GTG ACA CTA TAG AGG ACG CGA CCG AAA TGG TGA AGG ACG GGT CCA GTG CTT CGG CAC TGT TGA GTA GAG TGT GAG CTC CGT AAC TGG TCG CGT C-3'
Spinach Template 100 5'-GAC GCG ACC AGT TAC GGA GCT CAC ACT CTA CTC AAC AGT GCC GAA GCA CTG GAC CCG TCC TTC ACC ATT TCG GTC GCG TCC TCT ATA GTG TCA CCT AAA T-3'
Malachite Green Non-Template 68 5'-ACT ATG ATA ATA CGA CTC ACT ATA GGG AGA GGA TCC CGA CTG GCG AGA GCC AGG TAA CGA ATG GAT CC-3'
Malachite Green Template 68 5'-GGA TCC ATT CGT TAC CTG GCT CTC GCC AGT CGG GAT CCT CTC CCT ATA GTG AGT CGT ATT ATC ATA GT-3'


Oscillator

DNA Oligonucleotides Length Sequences
G1 Non-Template 83 5'-TAA TAC GAC TCA CTA TAG GAT GGC AGC GGA GAG TTG CTT GGA ATG CGT TAT AGT CTC TTA GGT GTG TTC GCA CAC CAC TCT CC-3'
G1 Template 83 5'-GGA GAG TGG TGT GCG AAC ACA CCT AAG AGA CTA TAA CGC ATT CCA AGC AAC TCT CCG CTG CCA TCC TAT AGT GAG TCG TAT TA-3'
G2 Non-Template 56 5'-ATT TAG GTG ACA CTA TAG AGG CGA GAA GCA GCA ATG ATA GTG GAA TTG ACT TAC GC-3'
G2 Template 56 5'-GCG TAA GTC AAT TCC ACT ATC ATT GCT GCT TCT CGC CTC TAT AGT GTC ACC TAA AT-3'
G3 Non-Template 52 5'-TTC TAA TAC GAC TCA CTA TAG CGT AAG TCA ATT CCA CTA TCA TTG CTG CTT C-3'
G3 Template 52 5'-GAA GCA GCA ATG ATA GTG GAA TTG ACT TAC GCT ATA GTG AGT CGT ATT AGA A-3'
G4 Non-Template 83 5'-ATT TAG GTG ACA CTA TAG AGG CGA CGT CGC CTC TCA ACG AAC CTT ACG CAA TAT CAG AGA ATC CAC ACA AGC GTG TGG TGA GAG G-3'
G4 Template 83 5'-CCT CTC ACC ACA CGC TTG TGT GGA TTC TCT GAT ATT GCG TAA GGT TCG TTG AGA GGC GAC GTC GCC TCT ATA GTG TCA CCT AAA T-3'
D1 61 5'-TexRd-CGT CGC CTC TCA ACG AAC CTT ACG CAA TAT CAG AGA ATC CAC ACA AGC GTG TGG TGA GAG G-IowaBlack-3'


*This strand is equivalent to the template strand of G2 in the bistable system. There are extra bases in the D2 strand; however, that does not diminish from its effectiveness to act as a kleptamer and remove the RNA aptamer from T7 RNAP.

Characterization of Spinach and Malachite Green aptamer

Aptamer-Dye Titrations

Figure 1:  Fluorometry readings of Spinach aptamer (A) and Malachite Green aptamer (B) titrations.  Aptamer concentration was increased as indicated.  More than 10 hours after Spinach aptamer addition was needed before stable fluorescence was reached, while only approximately 2 hours was needed after the Malachite Green aptamer addition.
Figure 1: Fluorometry readings of Spinach aptamer (A) and Malachite Green aptamer (B) titrations. Aptamer concentration was increased as indicated. More than 10 hours after Spinach aptamer addition was needed before stable fluorescence was reached, while only approximately 2 hours was needed after the Malachite Green aptamer addition.

Aptamer-dye titration experiments were performed to correlate fluorescent intensity levels to dye-aptamer levels. Two types of titrations were performed: one with aptamers as the titrant and another with fluorescent dye as the titrant. Aside from the original premise, these experiments uncovered some challenges in using Malachite Green and Spinach as reporters.

For the aptamer titrations, RNA Polymerase Transcription Buffer, Nuclease Free Water, and excess DHFBI and Malachite Green dye (20 μM each) were combined in a 0.5 mL tube and transferred to a cuvette for measurement in a spectrofluorometer. Varying amounts of extracted Spinach and Malachite Green aptamer were added to the cuvette and stirred for approximately 20 seconds. Fluorescence was measured until intensity remained relatively constant. This data shows that both the Spinach and Malachite Green aptamers do not bind to their respective dyes quickly after being added. Spinach took significantly longer than Malachite Green to reach a stable fluorescent intensity after the concentration of both aptamers in the solution was increased from 2 μM to 3 μM (Figure 1). These results suggest Spinach is unsuitable as a reporter due to the large time lag. However, Malachite Green still may be a viable, albeit not as effective as molecular beacons, as described in the results.

Figure 2: Mapping and comparison of DHFBI dye titration done on 9/2/14 and 9/9/14.  Data indicates that DHFBI fluorescence levels are not consistent at high concentrations of dye.
Figure 2: Mapping and comparison of DHFBI dye titration done on 9/2/14 and 9/9/14. Data indicates that DHFBI fluorescence levels are not consistent at high concentrations of dye.

Dye titrations were also performed with DHFBI dye. Prior to the titration, a 60 μL transcription was prepared by combining nuclease-free water, 7.5 μM NTP, 24 mM MgCl2, 200 nM spinach gene, 1x NEB RNA Polymerase Transcription Buffer, and 4 μL SP6 RNAP. Solution was incubated at 30°C for 24 hours to produce an abundance of Spinach aptamer. Ten μL of this transcript solution was then diluted to 60 uL and transferred into a cuvette and placed in a spectrofluorometer. Varying known concentrations of DHFBI was added to the solution. After each Spinach dye addition, fluorescent intensity was measured for 15 minutes after fluorescent intensity stabilized. Intensities for each dye addition were averaged. Intensity and dye concentration were plotted and mapped (figure 2). The results of the dye titration experiments indicate that intensities are consistent at low dye concentrations up to 3 μM, but vary greatly with higher concentrations. This is further evidence that Spinach may not be a good reporter to use for our systems. .

Fluorometer Experiments On Circuit Components

Reactivation of T7 RNA Polymerase with Genelets
In this case, the genes- G3 and G2 –were used instead of the aptamers. There is obvious inhibition of the enzyme with the addition of varying concentrations of G3. After a few hours, G2 (750 nM) was added to the mixture to be transcribed into the reactivator, R2. Unfortunately, there was no reactivation of the enzyme. It’s possible this may be because the transcription of G3 is outcompeting the transcription of G2. This would prevent any reactivation since T7 RNAP would immediately re-inhibit itself.

Figure 3: Reactivation attempt of 7 RNA Polymerase using g2.
Figure 3: Reactivation attempt of 7 RNA Polymerase using g2.

Reactivation of SP6 RNA polymerase with 38-base length complimentary kleptamer

Figure 4: Reactivation of SP6 RNAP.  A) Reaction scheme of an aptamer-inhibited enzyme, such as aptamer-bound SP6, being reactivated with the addition of a complimentary kleptamer strand.  B) Fluorometry transcription data showing transcription, inhibition, and reactivation of SP6.  C) Secondary structure of the SP6 aptamer with 38-base length complimentary kleptamer outlined in blue.
Figure 4: Reactivation of SP6 RNAP. A) Reaction scheme of an aptamer-inhibited enzyme, such as aptamer-bound SP6, being reactivated with the addition of a complimentary kleptamer strand. B) Fluorometry transcription data showing transcription, inhibition, and reactivation of SP6. C) Secondary structure of the SP6 aptamer with 38-base length complimentary kleptamer outlined in blue.

In order to verify that enzymes could be reactivated by kleptamers after being inhibited by aptamers, we tested the reactivation of SP6 using kleptamers that were complimentary to the SP6 aptamer to varying degrees. Fully complimentary, 38-base length complimentary, and 23-base length complimentary kleptamers were tested.

The 38-base length kleptamer was tested using fluorometry. Our standard fluorometry transcription conditions were prepared, with 25 uM DHFB1 dye and 100 nM Spinach gene. After about 1 hour of fluorescence measurement, 3 μL SP6 RNAP was added to start transcription. 2 %mu;M of the SP6 aptamer was added to inhibit SP6 after about 0.5 hour of transcription. This significantly reduced the transcription rate of the Spinach gene. The 38-base length kleptamer was then added to remove the aptamer 1 hour later. Transcription levels increased, and were surprisingly faster than the initial rate. The reason for the increased transcription rate is still unknown. However, this experiment shows that the 38-base length kleptamer can successfully remove the aptamer from inhibited SP6 and consequently reactivate it.


Gel Experiments On Circuit Components

Gel Electrophoresis Protocols for Specific Experiments

A. Bound Aptamer-Kleptamer Interactions

Figure 5: Reactivation of SP6 RNA Polymerase. The first lane represents the 10 base pair ladder. Lanes 2-4 represent the aptamer, and D1 (23 base length and 38 base length), respectively. Lane 5 represents the aptamer bound to SP6 RNA Polymerase. Finally, lanes 6 and 7 have bound aptamer interacting with the kleptamer.
Figure 5: Reactivation of SP6 RNA Polymerase. The first lane represents the 10 base pair ladder. Lanes 2-4 represent the aptamer, and D1 (23 base length and 38 base length), respectively. Lane 5 represents the aptamer bound to SP6 RNA Polymerase. Finally, lanes 6 and 7 have bound aptamer interacting with the kleptamer.

For figure 5, 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 length)
4 D1 Strand (38 base length)
5 R1 aptamer + SP6 RNA Polymerase
6 R1 aptamer +SP6 RNA Polymerase + D1 (23 base length)
7 R1 aptamer +SP6 RNA Polymerase + D1 (38 base length)

The amount of aptamer bound to the enzyme decrease significantly when the kleptamer is introduced into the system. This shows the kleptamer performs it function properly, allowing for reactivation of the enzyme. The aptamer becomes free in solution once it is removed.

Figure 6: Reactivation of SP6 RNA Polymerase. The first lane represents the 10 base pair ladder. Lanes 2 and 3 represent the aptamer and G4 strand, respectively. Lane 4 represents the aptamer bound to SP6 RNA Polymerase. Finally, lane 5 shows the interaction between the klepatmer, R4, with aptamer bound RNA Polymerase.
Figure 6: Reactivation of SP6 RNA Polymerase. The first lane represents the 10 base pair ladder. Lanes 2 and 3 represent the aptamer and G4 strand, respectively. Lane 4 represents the aptamer bound to SP6 RNA Polymerase. Finally, lane 5 shows the interaction between the klepatmer, R4, with aptamer bound RNA Polymerase.

For figure 6, 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

The second kleptamer used in this system was also tested for the proper interactions with the aptamer. Once the kleptamer is introduced into the system, a greater concentration of the aptamer is found in solution, suggesting it is functioning properly.

The non-denaturing gel in Figure 7 in Supplement section, the concentration of R1 aptamer, R2 kleptamer are 1μM. For the two samples that required SP6 RNAP to transcribe G2 gene, the solution mix is given below:

Component Concentration
G2 500nM
MgCl2 0.024 M
NTPs7. 5 mM

The solution also contains RNA Polymerase transcription buffer and RNase free water. 1 μL of the SP6 RNAP enzyme was added to both solutions, as well as 1μL of T7 RNAP was added to one of the samples. All solutions were incubated at 30°C for 30 minutes. The solutions were then diluted 1:2 ratio before being placed in each well. The composition of each well is listed below:

Figure 7: Possible binding interaction between R2 kleptamer and R3 kleptamer.
Figure 7: Possible binding interaction between R2 kleptamer and R3 kleptamer.

Lane Component
1 R2 +T7 RNAP+R3
2 SP6 RNAP transcribing G2+T7 RNAP+R3
3 SP6 RNAP transcribing G2+R3
4 R2+R3
5 T7 RNAP+R3
6 R3
7 R2
8 DNA Ladder 10bp

This gel helped determine that there is possible binding between R2 kleptamer and R3 aptamer.


Unbound Aptamer-Kleptamer Interaction
An important factor in the oscillatory system is to ensure the R1 and D1 strands are not strongly interacting before the inhibition of SP6 RNAP. In figure 8, the R1 and D1 aptamers were mixed into solution together at the same concentration and incubated. For both variations of the D1 strand (23&38 base length), there are two distinct bands in lanes 4 and 6. This indicates that there are little interactions occurring between these two strands before R1 binds to the enzyme. This can be explained by a difference in secondary structure between the bound and unbound forms of R1.

Figure 8: Non-denaturing gel electrophoresis of aptamer and kleptamer interactions. Lanes 2-4 represent the aptamer, D1 (38 base pair), and D1 (23 base pair), respectively. Lanes 5 and 6 correspond to the aptamer along with D1 (23 base pair) and D1 38 base pair, respectively.
Figure 8: Non-denaturing gel electrophoresis of aptamer and kleptamer interactions. Lanes 2-4 represent the aptamer, D1 (38 base pair), and D1 (23 base pair), respectively. Lanes 5 and 6 correspond to the aptamer along with D1 (23 base pair) and D1 38 base pair, respectively.

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 length)
4 D1 strand (23 base length)
5 R1 aptamer + D1 strand (38 base length)
6 R1 aptamer + D1 strand (23 base length)

In this case, we wanted to view the interactions between the aptamer free in solution and the kleptamer. When added together the bands representing the free aptamer and kleptamer are clearly visible. This suggests the interactions are minimal for aptamer free in solution. This is important so that the aptamer will become bound solely to the RNAP before interacting with the kleptamer.

Comparison of Transcription Rates between SP6 RNAP and T7 RNAP
In order to establish the proper concentrations of enzymes and genes for assembling a dynamic circuit, we first need to know how the transcription rates of both T7 RNA polymerase and SP6 RNA polymerase compare. To find this out we ran series of gel electrophoresis experiments to compare the transcription yield of both polymerases at same buffer and reaction conditions.

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 following transcription yield gel in Figure 9 shows, titration of different concentrations of a gene with SP6 promoter, to that of a fixed concentration of a gene with T7 promoter.

Figure 9: Denaturing gel comparing the transcription rates of SP6 and T7 RNA Polymerase. Lane 9 represents the 10 bp ladder. Lanes 5-8 represent aptamer R2, gene G2, aptamer R1, and gene G1, respectively. Lane 4 represents T7 RNA Polymerase transcription of G1 (10 nM) to produce the aptamer, R1. Lanes 1-3 represent the SP6 RNA Polymerase transcription of G2 at 100 nM, 150 nM, and 200 nM, respectively to produce the aptamer, R2. All transcription experiments are incubated at 30°C for 1 hour. This provides evidence that T7 RNA polymerase transcribes the aptamer approximately 10 times faster than SP6 RNA Polymerase, a critical consideration in the development of accurate computational models.
Figure 9: Denaturing gel comparing the transcription rates of SP6 and T7 RNA Polymerase. Lane 9 represents the 10 bp ladder. Lanes 5-8 represent aptamer R2, gene G2, aptamer R1, and gene G1, respectively. Lane 4 represents T7 RNA Polymerase transcription of G1 (10 nM) to produce the aptamer, R1. Lanes 1-3 represent the SP6 RNA Polymerase transcription of G2 at 100 nM, 150 nM, and 200 nM, respectively to produce the aptamer, R2. All transcription experiments are incubated at 30°C for 1 hour. This provides evidence that T7 RNA polymerase transcribes the aptamer approximately 10 times faster than SP6 RNA Polymerase, a critical consideration in the development of accurate computational models.

The composition of each well is given below:

Lane Components
1 T7 RNAP transcription of G1 gene [10nM]
2 SP6 RNAP transcription of G2 gene [100nM]
3 SP6 RNAP transcription of G2 gene [150nM]
4 SP6 RNAP transcription of G2 gene [200nM]
5 R2 aptamer
6 G2 gene
7 R1 aptamer
8 G1 gene
9 10 base pair ladder


We found that T7 RNA polymerase is much faster than SP6 RNA polymerase at our desired conditions. As you can see even at 1:20 ratio of T7:SP6 promoter genes, the yield of transcript from SP6 promoter gene is lower than T7 RNAP.

Modeling

Bistable switch


Scheme showing the reactions for the bistable system. Dashed arrows indicate enzymatic reactions.
Scheme showing the reactions for the bistable system. Dashed arrows indicate enzymatic reactions.

Table 1 displays the parameters that were used to simulate the bistable switch. These nominal parameters (used for Figure 2 in the Results section) were chosen to be in a realistic range based on the literature [1, 2], after performing a randomized search.

Oscillator


Scheme showing the reactions for the bistable system. Dashed arrows indicate enzymatic reactions.
Scheme showing the reactions for the bistable system. Dashed arrows indicate enzymatic reactions.

Table 2 displays the parameters that were used to simulate the oscillator. These nominal parameters (used for Figure 4 in the Results section) were chosen to be in a realistic range based on the literature [1, 2], after performing a randomized search.

References

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All Medline abstracts: PubMed HubMed

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