We describe the engineering of a novel DNA-tethered T7 RNA polymerase to regulate in vitro transcription reactions. We discuss the steps for protein synthesis and characterization, validate proof-of-concept transcriptional regulation, and discuss its applications in molecular computing, diagnostics, and molecular information processing.
We propose a method for regulating polymerase activity using artificial nucleic acid transcription factors. This gene regulatory architecture can serve as a building block for future in vitro genetic devices. By tethering a DNA binding domain to a T7 RNA polymerase, this technique combines the scalability of DNA-based circuits, with the functionality of transcriptional circuits.
Begin by making nine dilutions of single-stranded BG oligonucleotide to a final volume of 50 microliters of double distilled water, from five to one to one to five ratios, as indicated in the table. Prepare one reaction mixture for each dilution by mixing two microliters of SNAP buffer with four microliters of BG oligonucleotide, and four microliters of SNAP T7 RNAP. Prepare the RNAP control by replacing the oligonucleotide with double distilled water, and the DNA control by replacing the SNAP T7 RNAP with double distilled water.
Set up 11 reactions by adding two microliters of each sample to four microliters of SNAP buffer, and two microliters of protein loading dye per sample. Heat the reactions for 10 minutes at 70 degrees Celsius, before loading two microliters of each sample onto a four to 12%vitreous protein gel for 35 minutes of gel electrophoresis on ice at 200 volts. At the end of the run, wash the gel with three water exchanges on a shaker for at least 10 minutes per wash, followed by nucleic acid staining with cyanine dye for 15 minutes.
Image the gel on an appropriate gel imaging system, and stain the gel again with 20 milliliters of Coomassie blue. After one hour, de-stain with double distilled water for at least one hour before imaging the gel again. For oligonucleotide-tethered SNAP T7 purification, first, prepare elution buffer by mixing 50 microliters of one molar trace, with 100 microliters of five molar sodium chloride, with 850 microliters of double distilled water.
Next, place one ion exchange column per sample into individual two milliliter centrifuge tubes, and wash the columns with purification buffer by centrifugation until all of the buffer has been eluted from each column. Dilute each sample with purification buffer at a three to one purification buffer to sample ratio, and load 400 microliters of sample into each column. When all of the buffer has been eluted as demonstrated, collect and label the flow through for each sample.
Wash the columns three times with 400 microliters of purification buffer per wash until all the buffer has been eluted per wash, collecting and labeling the flow throughs as appropriate. After the last wash, collect the eluted protein by adding 50 microliters of elution buffer into the center of the column, and centrifuging the column three times until all 50 microliters of buffer has been eluted. Collect and label the elute after each centrifugation, pooling the elute into a single tube after the last centrifugation, while leaving a small volume of each elute for gel analysis.
Measure the absorbance of the remaining elute aliquots at 260 and 280 nanometers, and add glycerol to the samples at a one-to-one ratio for minus 20 degrees storage until their use. Use a 500 microliter 30 kilodalton centrifical filter unit to buffer exchange the total elute product with 2x storage buffer. And measure the absorbance as demonstrated.
Then add glycerol to the product at a one-to-one ratio for minus 20 degrees Celsius storage. To assess the elutes by SDS-PAGE, run the samples in an appropriate protein ladder on a four to 12%vitreous gel for 35 minutes at 200 volts, or until the dye front migrates to the end of the gel. To demonstrate the on-demand control activity of the tethered RNAP, mix 2.4 microliters of each template with five microliters of 5x annealing buffer, and 14.2 microliters of double distilled water.
Incubate the resulting one micromolar double-stranded DNA CAGE at 75 degrees Celsius for two minutes. And anneal the sense and anti-sense strains of the promoter and malachite green aptamer DNA template in the same manner as indicated in the table. At the end of the incubations, add the tethered SNAP T7 RNAP to the double stranded DNA CAGE at a one to five molar ratio to a final concentration of 500 nanomolar RNAP.
After 15 minutes at room temperature, place the sample on ice, and mix 2.5 microliters of 10x in vitro transcription buffer with one microliter of 25 millimolar ribonucleoside triphosphate, one microliter of one millimolar malachite green, 2.5 microliters of the RNAP CAGE mixture, 2.5 microliters each of one micromolar transcription factors A and B oligonucleotides strands, and three microliters of one millimolar malachite green aptamer template in 10 microliters of double distilled water. In 15 microliters of double distilled water, set up a second reaction containing 2.5 microliters of 10x in vitro transcription buffer, one microliter of 25 millimolar ribonucleoside triphosphate mix, one microliter of one millimolar malachite green, 2.5 microliters of the RNAP CAGE mixture, and three microliters of one millimolar malachite green aptamer template. To set up a reaction containing buffer only, mix 2.5 microliters of 10x in vitro transcription buffer, one microliter of 25 millimolar ribonucleoside triphosphate mix, one microliter of one millimolar malachite green, and three microliters of one millimolar malachite green aptamer template in 17.5 microliters of double distilled water.
When all of the reactions have been prepared, transfer each reaction to a 384-well plate, and monitor the malachite green aptamer transcription on a fluorescence plate reader for two hours at 37 degrees Celsius at a 610 nanometer excitation, and a 655 nanometer emission. At the end of the reading, place the plate on ice, and run the RNA product from each well in a denaturing 12%TBE-urea polyacrylic gel in 70 degrees Celsius TBE buffer at 280 volts for 20 minutes, or until the dye front reaches the end of the gel. Then, stain the gel with cyanine dye nucleic acid stain for 10 minutes on an orbital shaker, before imaging as demonstrated.
A successful expression and purification of the recombinant SNAP T7 RNAP protein can be confirmed using SDS-PAGE. Following SDS-PAGE, the enzymatic activity can be validated by in vitro transcription reaction. Successful coupling of BG functionalized oligonucleotides can be verified via 18%denaturing TBE-urea PAGE.
Compared to the unmodified oligonucleotide, the addition of the BG moiety to the oligonucleotide increases its molecular weight. As observed in this representative cyanine dye stain gel to confirm DNA-tethered T7 RNAP purification from excess BG oligonucleotides, the initial flow through fraction contained mostly excess BG oligonucleotide, as well as a small portion of the DNA-tethered polymerase that did not bind to the cation exchange resin. The pool dilution fractions contained only the single band of RNAP oligo caused by the cyanine dye binding to the oligonucleotide tether, with the up-concentrated product lane exhibiting the same, but darker band signifying a successful up-concentration.
The same patterns are observed by Coomassie blue staining, with a small gel mobility shift observed in the non conjugated RNAP control. Here, fluorescent signal produced at the end of the in vitro transcription in real-time kinetics are shown. In this analysis, a 336 fold activation in fluorescence signal was observed, demonstrating a robust control of polymerase activity.
When attempting this protocol, remember to thoroughly wash SDS off the gel prior to nucleic acid staining. This is because STS will trap the dye in the gel, resulting in high background signal. Applying our method to a large gene circuit with multiple cascades will show the scalability and composability of the tethered transcription system.
This technique is currently being used to develop gene circuits capable of neural network style computation.
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