Biomod/2014/UCR/Breaking RNA/Acknowledgements
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Supplementary
1.1 Oscillator Genes and Strands
DNA Oligonucleotides Base Pair 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' k1 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'
1.2 Bistable Genes and Strands
DNA Oligonucleotides Base Pair 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' k1 100 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' 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'
2.1 Enzyme Inhibition RNA Aptamers: Fluorometry
2.2 Bound Aptamer-Kleptamer Interactions: Fluorometry
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 uL of the T7 RNAP enzyme was added to solution along with 0.7 uL 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.
SP6 RNA Polymerase
