Journal: Nucleic Acids Research
Article Title: Design and application of cotranscriptional non-enzymatic RNA circuits and signal transducers
Figure Lengend Snippet: An entirely RNA-based CHA circuit operation and fluorimetric detection. ( a ) CHA circuit components (hairpins H1B and H2 and catalyst C1) and the RNA reporter Spinach.ST1 were separately transcribed by T7 RNA polymerase from 500 ng of PCR-generated duplex DNA transcription templates. H1B, H2 and C1 transcription templates were amplified using primers complementary to the exact ends of the cloned inserts (H1B.amp.F:H1B.amp.R, H2.amp.F:H2.amp.R and C1.amp.F:C1.amp.R, respectively) rather than the flanking plasmid. Spinach.ST transcription templates were amplified using primers specific to the flanking plasmid sequence at the 5′-end (pCR2.1.F) and the primer sphT.U.R specific to the 3′-end sequence of Spinach.ST. Transcription reactions were filtered through Sephadex G25 columns before circuit assembly. Three microliters of H1B, H2, C1 and Spinach.ST1 transcripts was mixed in indicated combinations and incubated in 1× TNaK buffer containing 70 µM DFHBI and 20 U of RNaseOUT. Circuit output was measured as increasing fluorescence intensity over time at 37°C. ( b–d ) Performance of DNA reporter duplex H1BF:H1BQ (b) versus Spinach.ST1 (c) in measuring RNA CHA circuit output. Indicated concentrations of gel-purified RNA hairpins H1B and H2 were incubated with equal concentration of H1BF:H1BQ or gel-purified Spinach.ST1 (+ 70 µM DFHBI) in the presence of titrating concentrations of pure C1 RNA. All circuits were operated in 1× TNaK buffer containing 20 U of RNaseOUT at 37°C, and average data from triplicate experiments are represented. Signal-to-noise ratio of H1BF:H1BQ versus Spinach.ST1 over the time course of RNA CHA detection is plotted in (d).
Article Snippet: In all, 100 pg to 1000 ng of double-stranded DNA transcription templates was transcribed using 100 U of T7 RNA polymerase (NEB) in 50 µl reactions containing 40 mM Tris–HCl, pH 7.9, 30 mM MgCl2 , 10 mM DTT, 2 mM spermidine, 4 mM ribonucleotide (rNTP) mix and 20 U of the recombinant ribonuclease inhibitor RNaseOUT (Life Technologies).
Techniques: Polymerase Chain Reaction, Generated, Amplification, Clone Assay, Plasmid Preparation, Sequencing, Incubation, Fluorescence, Purification, Concentration Assay