Revisiting T7 RNA polymerase transcription in vitro with the Broccoli RNA aptamer as a simplified real-time fluorescent reporter

Methods for rapid and high-throughput screening of transcription in vitro to examine reaction conditions, enzyme mutants, promoter variants, and small molecule modulators can be extremely valuable tools. However, these techniques may be difficult to establish or inaccessible to many researchers. To develop a straightforward and cost-effective platform for assessing transcription in vitro, we used the “Broccoli” RNA aptamer as a direct, real-time fluorescent transcript readout. To demonstrate the utility of our approach, we screened the effect of common reaction conditions and components on bacteriophage T7 RNA polymerase (RNAP) activity using a common quantitative PCR instrument for fluorescence detection. Several essential conditions for in vitro transcription by T7 RNAP were confirmed with this assay, including the importance of enzyme and substrate concentrations, covariation of magnesium and nucleoside triphosphates, and the effects of several typical additives. When we used this method to assess all possible point mutants of a canonical T7 RNAP promoter, our results coincided well with previous reports. This approach should translate well to a broad variety of bacteriophage in vitro transcription systems and provides a platform for developing fluorescence-based readouts of more complex transcription systems in vitro. Methods for rapid and high-throughput screening of transcription in vitro to examine reaction conditions, enzyme mutants, promoter variants, and small molecule modulators can be extremely valuable tools. However, these techniques may be difficult to establish or inaccessible to many researchers. To develop a straightforward and cost-effective platform for assessing transcription in vitro, we used the “Broccoli” RNA aptamer as a direct, real-time fluorescent transcript readout. To demonstrate the utility of our approach, we screened the effect of common reaction conditions and components on bacteriophage T7 RNA polymerase (RNAP) activity using a common quantitative PCR instrument for fluorescence detection. Several essential conditions for in vitro transcription by T7 RNAP were confirmed with this assay, including the importance of enzyme and substrate concentrations, covariation of magnesium and nucleoside triphosphates, and the effects of several typical additives. When we used this method to assess all possible point mutants of a canonical T7 RNAP promoter, our results coincided well with previous reports. This approach should translate well to a broad variety of bacteriophage in vitro transcription systems and provides a platform for developing fluorescence-based readouts of more complex transcription systems in vitro. Early methods to monitor transcription in vitro largely relied on the incorporation of radioactive nucleotides or detection of transcripts by hybridization-based methods (1Rohde W. Sanger H.L. Detection of complementary RNA intermediates of viroid replication by northern blot hybridization.Biosci. Rep. 1981; 1: 327-336Crossref PubMed Scopus (39) Google Scholar, 2Green M.H. Strand selective transcription of T4 DNA in vitro.Proc. Natl. Acad. Sci. U. S. A. 1964; 52: 1388-1395Crossref PubMed Scopus (11) Google Scholar, 3Diaz G.A. Raskin C.A. McAllister W.T. Hierarchy of base-preference in the binding domain of the bacteriophage T7 promoter.J. Mol. Biol. 1993; 229: 805-811Crossref PubMed Scopus (35) Google Scholar, 4Imburgio D. Rong M. Ma K. McAllister W.T. Studies of promoter recognition and start site selection by T7 RNA polymerase using a comprehensive collection of promoter variants.Biochemistry. 2000; 39: 10419-10430Crossref PubMed Scopus (121) Google Scholar). 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Implementation of a straightforward screening approach that uses common laboratory equipment would enable cost-effective screening of focused small chemical libraries. Inspired by this need, we designed a simple, rapid throughput assay for monitoring transcriptional output in real-time with a fluorescent RNA aptamer reporter transcript. To test and develop this method as a potentially useful platform, we chose to characterize in vitro T7 RNA polymerase (RNAP) transcription, a common laboratory enzyme with well-studied promoter preferences and established reaction conditions. For nearly half a century the mechanisms and applications of minimal viral RNA polymerases, such as those from the lambda, T4, T7, SP6, and SP8 bacteriophages, have served as a paradigm for transcription and RNA research (2Green M.H. Strand selective transcription of T4 DNA in vitro.Proc. Natl. Acad. Sci. U. S. A. 1964; 52: 1388-1395Crossref PubMed Scopus (11) Google Scholar, 15Marmur J. Greenspan C.M. Transcription in vivo of DNA from bacteriophage SP8.Science. 1963; 142: 387-389Crossref PubMed Scopus (10) Google Scholar, 16Roberts J.W. Promoter mutation in vitro.Nature. 1969; 223: 480-482Crossref PubMed Scopus (29) Google Scholar, 17Chamberlin M. Ring J. Characterization of T7-specific ribonucleic acid polymerase. 1. General properties of the enzymatic reaction and the template specificity of the enzyme.J. Biol. Chem. 1973; 248: 2235-2244Abstract Full Text PDF PubMed Google Scholar, 18Melton D.A. Krieg P.A. Rebagliati M.R. Maniatis T. Zinn K. Green M.R. Efficientin vitrosynthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter.Nucleic Acids Res. 1984; 12: 7035-7056Crossref PubMed Scopus (4005) Google Scholar, 19Milligan J.F. Groebe D.R. Witherell G.W. Uhlenbeck O.C. Oligoribonucleotide synthesis using T7 RNA polymerase and synthetic DNA templates.Nucleic Acids Res. 1987; 15: 8783-8798Crossref PubMed Scopus (1819) Google Scholar, 20Palmer A.C. Ahlgren-Berg A. Egan J.B. Dodd I.B. Shearwin K.E. Potent transcriptional interference by pausing of RNA polymerases over a downstream promoter.Mol. Cell. 2009; 34: 545-555Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 21Gholamalipour Y. Karunanayake Mudiyanselage A. Martin C.T. 3′ End additions by T7 RNA polymerase are RNA self-templated, distributive and diverse in character-RNA-Seq analyses.Nucleic Acids Res. 2018; 46: 9253-9263Crossref PubMed Scopus (20) Google Scholar). Early experiments used bacteriophage transcription to help unravel the basic mechanisms of transcription and virulence (2Green M.H. Strand selective transcription of T4 DNA in vitro.Proc. Natl. Acad. Sci. U. S. A. 1964; 52: 1388-1395Crossref PubMed Scopus (11) Google Scholar, 15Marmur J. Greenspan C.M. Transcription in vivo of DNA from bacteriophage SP8.Science. 1963; 142: 387-389Crossref PubMed Scopus (10) Google Scholar, 16Roberts J.W. Promoter mutation in vitro.Nature. 1969; 223: 480-482Crossref PubMed Scopus (29) Google Scholar, 17Chamberlin M. Ring J. Characterization of T7-specific ribonucleic acid polymerase. 1. General properties of the enzymatic reaction and the template specificity of the enzyme.J. Biol. Chem. 1973; 248: 2235-2244Abstract Full Text PDF PubMed Google Scholar). These systems were subsequently reduced to their simplest components and co-opted for synthesis of RNA in the laboratory for biochemistry, molecular biology, and structural biology investigations (17Chamberlin M. Ring J. Characterization of T7-specific ribonucleic acid polymerase. 1. General properties of the enzymatic reaction and the template specificity of the enzyme.J. Biol. Chem. 1973; 248: 2235-2244Abstract Full Text PDF PubMed Google Scholar, 18Melton D.A. Krieg P.A. Rebagliati M.R. Maniatis T. Zinn K. Green M.R. Efficientin vitrosynthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter.Nucleic Acids Res. 1984; 12: 7035-7056Crossref PubMed Scopus (4005) Google Scholar, 19Milligan J.F. Groebe D.R. Witherell G.W. Uhlenbeck O.C. Oligoribonucleotide synthesis using T7 RNA polymerase and synthetic DNA templates.Nucleic Acids Res. 1987; 15: 8783-8798Crossref PubMed Scopus (1819) Google Scholar, 22Milligan J.F. Uhlenbeck O.C. [5] Synthesis of small RNAs using T7 RNA polymerase.Methods Enzymol. 1989; 180: 51-62Crossref PubMed Scopus (980) Google Scholar, 23Raskin C.A. Diaz G.A. McAllister W.T. T7 RNA polymerase mutants with altered promoter specificities.Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3147-3151Crossref PubMed Scopus (46) Google Scholar, 24Lukavsky P.J. Puglisi J.D. Large-scale preparation and purification of polyacrylamide-free RNA oligonucleotides.RNA. 2004; 10: 889-893Crossref PubMed Scopus (94) Google Scholar). The bacteriophage RNA polymerases, especially T7, have proven to be reliable work horses that continue to offer new insights into transcription as well as generate large quantities of RNA for research. The further optimization by affordable and high-throughput methods might be justified by the growing demand for pure and maximal transcript synthesis by modern molecular biology, structural biology applications, and even future mRNA therapeutics (21Gholamalipour Y. Karunanayake Mudiyanselage A. Martin C.T. 3′ End additions by T7 RNA polymerase are RNA self-templated, distributive and diverse in character-RNA-Seq analyses.Nucleic Acids Res. 2018; 46: 9253-9263Crossref PubMed Scopus (20) Google Scholar, 24Lukavsky P.J. Puglisi J.D. Large-scale preparation and purification of polyacrylamide-free RNA oligonucleotides.RNA. 2004; 10: 889-893Crossref PubMed Scopus (94) Google Scholar, 25Zangi L. Lui K.O. von Gise A. Ma Q. Ebina W. Ptaszek L.M. Spater D. Xu H. Tabebordbar M. Gorbatov R. Sena B. Nahrendorf M. Briscoe D.M. Li R.A. Wagers A.J. et al.Modified mRNA directs the fate of heart progenitor cells and induces vascular regeneration after myocardial infarction.Nat. Biotechnol. 2013; 31: 898-907Crossref PubMed Scopus (368) Google Scholar). Thus, T7 RNAP is an appropriate system to revisit and use as a benchmark for a rapid fluorescence-based screening method. For in vitro T7 transcription reactions, the T7 RNAP is purified and combined with a DNA template that contains its cognate promoter and a downstream sequence encoding the RNA to be synthesized. A common consensus promoter is TAATACGACTCACTATA followed by one to three guanine nucleotides before the desired sequence for synthesis (19Milligan J.F. Groebe D.R. Witherell G.W. Uhlenbeck O.C. Oligoribonucleotide synthesis using T7 RNA polymerase and synthetic DNA templates.Nucleic Acids Res. 1987; 15: 8783-8798Crossref PubMed Scopus (1819) Google Scholar). In this study, we inserted the sequence encoding the Broccoli RNA aptamer (26Filonov G.S. Moon J.D. Svensen N. Jaffrey S.R. Broccoli: rapid selection of an RNA mimic of green fluorescent protein by fluorescence-based selection and directed evolution.J. Am. Chem. Soc. 2014; 136: 16299-16308Crossref PubMed Scopus (308) Google Scholar, 27Paige J.S. Wu K.Y. Jaffrey S.R. RNA mimics of green fluorescent protein.Science. 2011; 333: 642-646Crossref PubMed Scopus (753) Google Scholar) to allow monitoring of RNA synthesis in real time by fluorescence detection. Upon proper folding and binding to the small molecule DFHBI-1T (3,5-difluoro-4-hydroxy-benzylidene imidazolinone), the short 49-nt Broccoli RNA aptamer fluoresces green with similar excitation and emission peaks as green fluorescent protein. This approach is similar to that taken by Hofer and colleagues previously, who fused the Spinach aptamer separated by a self-cleaving ribozyme to transcripts of interest (28Hofer K. Langejurgen L.V. Jaschke A. Universal aptamer-based real-time monitoring of enzymatic RNA synthesis.J. Am. Chem. Soc. 2013; 135: 13692-13694Crossref PubMed Scopus (49) Google Scholar). However, our focus was on low-cost synthetic DNA templates, high reproducibility, and robust fluorescence readout to characterize reaction conditions, polymerase properties, or promoter sequence. This led us to utilize only the short Broccoli RNA aptamer sequence itself in a hybrid template that is single stranded but possesses a double-stranded promoter (19Milligan J.F. Groebe D.R. Witherell G.W. Uhlenbeck O.C. Oligoribonucleotide synthesis using T7 RNA polymerase and synthetic DNA templates.Nucleic Acids Res. 1987; 15: 8783-8798Crossref PubMed Scopus (1819) Google Scholar). The Broccoli RNA aptamer has a very low dependence on magnesium and an increased thermostability compared with similar aptamers like Spinach (26Filonov G.S. Moon J.D. Svensen N. Jaffrey S.R. Broccoli: rapid selection of an RNA mimic of green fluorescent protein by fluorescence-based selection and directed evolution.J. Am. Chem. Soc. 2014; 136: 16299-16308Crossref PubMed Scopus (308) Google Scholar, 28Hofer K. Langejurgen L.V. Jaschke A. Universal aptamer-based real-time monitoring of enzymatic RNA synthesis.J. Am. Chem. Soc. 2013; 135: 13692-13694Crossref PubMed Scopus (49) Google Scholar). The Broccoli RNA forms a G-quadruplex that relies on potassium or sodium ions to help stabilize structure and DFHBI-1T binding (29Ageely E.A. Kartje Z.J. Rohilla K.J. Barkau C.L. Gagnon K.T. Quadruplex-flanking stem structures modulate the stability and metal ion preferences of RNA mimics of GFP.ACS Chem. Biol. 2016; 11: 2398-2406Crossref PubMed Scopus (15) Google Scholar) (Fig. 1A). As a result of the RNA folded structure, the brightness and stability of Broccoli RNA fluorescence is also partially dependent on temperature, as will be the case for all folded aptamers (26Filonov G.S. Moon J.D. Svensen N. Jaffrey S.R. Broccoli: rapid selection of an RNA mimic of green fluorescent protein by fluorescence-based selection and directed evolution.J. Am. Chem. Soc. 2014; 136: 16299-16308Crossref PubMed Scopus (308) Google Scholar, 29Ageely E.A. Kartje Z.J. Rohilla K.J. Barkau C.L. Gagnon K.T. Quadruplex-flanking stem structures modulate the stability and metal ion preferences of RNA mimics of GFP.ACS Chem. Biol. 2016; 11: 2398-2406Crossref PubMed Scopus (15) Google Scholar). Using 96-well plate screening, we confirmed the core buffer components necessary for efficient in vitro T7 transcription and explored the effect of several typical additives on transcriptional output. Fluorescence readout was correlated with denaturing polyacrylamide gel electrophoresis (PAGE) of selected samples, transcription of two additional longer RNAs, and comparison across several common buffer conditions. We also performed saturation mutagenesis of the T7 RNAP promoter with this method and found that our results generally coincided well with previous mutagenesis studies. The results of this study provide a resource to researchers interested in the properties of T7 RNAP or that use T7 RNAP for routine synthesis of RNA in the laboratory. The Broccoli RNA aptamer did possess some shortcomings and may have introduced some sequence-dependent biases in template transcription. However, other aptamers with distinct fluorescent properties and folding are now available that may address some of Broccoli’s weaknesses, including Mango, Pepper, and o-Coral (30Autour A. Jeng S.C.Y. Cawte A.D. Abdolahzadeh A. Galli A. Panchapakesan S.S.S. Rueda D. Ryckelynck M. Unrau P.J. Fluorogenic RNA mango aptamers for imaging small non-coding RNAs in mammalian cells.Nat. Commun. 2018; 9: 656Crossref PubMed Scopus (94) Google Scholar, 31Bouhedda F. Fam K.T. Collot M. Autour A. Marzi S. Klymchenko A. Ryckelynck M. A dimerization-based fluorogenic dye-aptamer module for RNA imaging in live cells.Nat. Chem. Biol. 2020; 16: 69-76Crossref PubMed Scopus (30) Google Scholar, 32Chen X. Zhang D. Su N. Bao B. Xie X. Zuo F. Yang L. Wang H. Jiang L. Lin Q. Fang M. Li N. Hua X. Chen Z. Bao C. et al.Visualizing RNA dynamics in live cells with bright and stable fluorescent RNAs.Nat. Biotechnol. 2019; 37: 1287-1293Crossref PubMed Scopus (56) Google Scholar). The ability of this method to recapitulate and confirm many of the commonly known properties of in vitro T7 RNAP transcription suggests that this approach should translate well to a variety of other bacteriophage RNA polymerases. It should also serve as an initial platform for developing screens for more complex eukaryotic in vitro transcription systems. To enable cost-effective, rapid, and direct readout of transcription, we selected the Broccoli RNA aptamer for its compact size, relative thermal stability, low magnesium dependence, and bright green fluorescence. Our design is similar to that reported previously by Jäschke and co-workers (28Hofer K. Langejurgen L.V. Jaschke A. Universal aptamer-based real-time monitoring of enzymatic RNA synthesis.J. Am. Chem. Soc. 2013; 135: 13692-13694Crossref PubMed Scopus (49) Google Scholar). However, we focused on a generalizable assay amenable to most laboratories without the potential shortcomings of the Spinach RNA aptamer or complexity of ribozyme modules. We used a simple hybrid ssDNA template structure comprising a single-stranded antisense sequence encoding Broccoli RNA with only the T7 RNAP promoter region being double stranded. This template structure was originally described by the Uhlenbeck laboratory and is widely used for efficient transcription of small RNAs from synthetic DNA oligonucleotides (19Milligan J.F. Groebe D.R. Witherell G.W. Uhlenbeck O.C. Oligoribonucleotide synthesis using T7 RNA polymerase and synthetic DNA templates.Nucleic Acids Res. 1987; 15: 8783-8798Crossref PubMed Scopus (1819) Google Scholar). Upon initiation of the reaction in the presence of DFHBI-1T, green fluorescence is observed within the first few minutes. Fluorescence was typically measured every minute in a standard quantitative PCR (qPCR) instrument in small 10-μl reactions in a 96-well format. By this method, Broccoli RNA aptamer synthesis and fluorescence are observed in real time (Fig. 1B), which provides a relative measurement of total RNA production over time. In most experiments, RNA transcripts from fluorescent reaction replicates were pooled and resolved by denaturing PAGE to compare staining on a gel to fluorescence-based detection. Resolution of RNA transcripts by denaturing PAGE sometimes resulted in multiple bands or higher-molecular-weight smearing. It is possible that the strong quadruplex nature of the Broccoli RNA aptamer (29Ageely E.A. Kartje Z.J. Rohilla K.J. Barkau C.L. Gagnon K.T. Quadruplex-flanking stem structures modulate the stability and metal ion preferences of RNA mimics of GFP.ACS Chem. Biol. 2016; 11: 2398-2406Crossref PubMed Scopus (15) Google Scholar) resulted in varying degrees of denaturation across experiments. Based on standard curves, the concentration of RNA produced can be estimated by the relative amount of fluorescence observed or the intensity of band staining by PAGE (Fig. S1, A–B). The overall concentration of MgCl2 and ribonucleoside triphosphates (rNTPs), as well as their concentrations relative to one another, has been known to impact in vitro T7 RNAP transcription efficiency (22Milligan J.F. Uhlenbeck O.C. [5] Synthesis of small RNAs using T7 RNA polymerase.Methods Enzymol. 1989; 180: 51-62Crossref PubMed Scopus (980) Google Scholar, 33Brunelle J.L. Green R. In vitro transcription from plasmid or PCR-amplified DNA.Methods Enzymol. 2013; 530: 101-114Crossref PubMed Scopus (13) Google Scholar). The T7 RNAP enzyme itself requires a certain concentration of free magnesium to function, and the triphosphate groups on rNTPs can stoichiometrically chelate magnesium ions. It has been suggested that MgCl2 should be 6 mM above the concentration of rNTPs for optimal efficiency (22Milligan J.F. Uhlenbeck O.C. [5] Synthesis of small RNAs using T7 RNA polymerase.Methods Enzymol. 1989; 180: 51-62Crossref PubMed Scopus (980) Google Scholar, 33Brunelle J.L. Green R. In vitro transcription from plasmid or PCR-amplified DNA.Methods Enzymol. 2013; 530: 101-114Crossref PubMed Scopus (13) Google Scholar). Thus, for standard transcription reactions the concentration of each rNTP is usually set at 4 mM (16 mM total rNTPs) while MgCl2 is set at a final concentration of 20 mM (19Milligan J.F. Groebe D.R. Witherell G.W. Uhlenbeck O.C. Oligoribonucleotide synthesis using T7 RNA polymerase and synthetic DNA templates.Nucleic Acids Res. 1987; 15: 8783-8798Crossref PubMed Scopus (1819) Google Scholar). We decided to revisit this fundamental aspect of in vitro T7 transcription. We varied the MgCl2 concentration widely across total rNTP concentrations of 10, 20, and 30 mM under otherwise standard conditions (Fig. 1C). Maximal RNA synthesis at each rNTP concentration appeared to be centered on a MgCl2 concentration that was approximately 10 mM higher than the total rNTP concentration. This was observed at all three rNTP concentrations, suggesting that aptamer reporter dependence on Mg2+ does not contribute significantly to this effect. Nonetheless, although the Broccoli RNA aptamer was demonstrated to only require very low millimolar concentrations of Mg2+ (26Filonov G.S. Moon J.D. Svensen N. Jaffrey S.R. Broccoli: rapid selection of an RNA mimic of green fluorescent protein by fluorescence-based selection and directed evolution.J. Am. Chem. Soc. 2014; 136: 16299-16308Crossref PubMed Scopus (308) Google Scholar, 29Ageely E.A. Kartje Z.J. Rohilla K.J. Barkau C.L. Gagnon K.T. Quadruplex-flanking stem structures modulate the stability and metal ion preferences of RNA mimics of GFP.ACS Chem. Biol. 2016; 11: 2398-2406Crossref PubMed Scopus (15) Google Scholar), its fluorescence may be influenced by increasing Mg2+ in our reactions and therefore may not translate to typical T7 RNAP transcription reactions. Although 30 mM rNTPs should support greater RNA synthesis, the conditions that produced the most consistent yield appeared to be 20 mM rNTPs with 30 mM MgCl2 (Fig. 1D, Fig. S2). These results suggest that both limiting and excessive MgCl2 can inhibit the activity of T7 RNAP. To confirm this, we instead held MgCl2 constant at 20 mM and titrated rNTPs from 10 mM up to 35 mM (Fig. S3A). The RNA yield was severely reduced when rNTP concentrations exceeded MgCl2 concentration. In addition, 50 mM MgCl2 was inhibitory in transcription reactions with 20 mM rNTPs when transcribing 7SK-Broccoli, a ∼450-nt template that encodes the human 7SK RNA sequence fused to the Broccoli RNA aptamer (Fig. S4A). Precipitates of Mg2+ and pyrophosphate are common during in vitro transcription reactions and can potentially alter the effective free Mg2+ concentration available for T7 RNAP. However, precipitation might be neutral or beneficial since the reduction of free Mg2+ would conceivably be proportional to consumption of rNTPs and would also remove potentially inhibitory pyrophosphate from solution. We included inorganic pyrophosphatase (PPase) in fluorescence reactions to prevent cloudiness and maintain accurate reading. Therefore, we investigated the effect of PPase at three different rNTP concentrations. When transcription products were resolved by denaturing PAGE, we did not observe any increase in transcription yield by the addition of PPase (Fig. S3B). Thus, we concluded that PPase or the formation of magnesium pyrophosphate precipitates is unlikely to alter the interpretation of results or cause unforeseen issues in fluorescence-based transcription screens. Together, these results support the importance of balancing and properly covarying rNTP and MgCl2 concentrations. These results are similar to previous reports but suggest that a slightly elevated concentration of Mg2+ during in vitro T7 transcription reactions could be beneficial. We decided to test other conditions fundamental to in vitro T7 RNAP transcription, which included the concentration of DNA template, T7 RNAP, monovalent ions, and polycations, as well as temperature and pH. Beginning with our standard conditions, we titrated the ssDNA hybrid template for Broccoli RNA transcription. As expected, the amount of DNA template proportionally increased the overall production of RNA transcript when the template was limiting (Fig. 2A). Transcription plateaued around 1.25 μM of template. Of importance, the amount of RNA made at each individual concentration of template also plateaued, indicating that longer incubations will not necessarily result in greater amounts of RNA when the template is limiting. Conversely, excess template did not seem to inhibit the reaction. When transcribing 7SK-Broccoli from a linearized plasmid template we observed a similar proportional increase in transcriptional output up to the final amount of 2 μg (0.2 μg/μl) at which point the total output appeared to begin plateauing (Fig. S4B). This is similar to the concentration of 1 to 3 μg of linearized plasmid per 20 μl reaction that is recommended in common commercial T7 transcription kits, such as the Ampliscribe T7-Flash Transcription Kit (Epicentre/Lucigen). In contrast to the synthetic DNA template encoding Broccoli, transcription of the longer 7SK-Broccoli from a linearized plasmid increased steadily over time at low template concentrations until reaching 1 μg of template (0.1 μg/μl) (Fig. S4B), suggesting that longer incubations may be required to produce more RNA product when plasmid DNA templates are limiting or when longer RNAs are transcribed. The amount of T7 RNAP affected not only the amount of RNA produced but also the rate at which it was produced (Fig. 2B). The transcription reaction reached completion much sooner when higher concentrations of T7 RNAP were present. For example, the reaction appears to have produced its maximum amount of RNA product at 30 min with 5 μM T7 RNAP while the product continues to increase steadily up to 60 min with 2.5 μM T7 RNAP. We also noted that Broccoli fluorescence was quenched at the highest concentrations of T7 RNAP, even though increasing amounts of transcript appeared to be synthesized when visualized by denaturing PAGE. Since Broccoli RNA aptamer fluorescence is sensitive to salts and DFHBI-1T interaction, it is possible that elevated concentrations of protein can partially titrate these or other components in the reaction. Overall, greater amounts of enzyme appear to proportionally increase transcript production independent of other factors. Nonetheless, exceeding 10 μM T7 RNAP under our standard conditions did not produce substantially greater yields and excess RNA polymerase has been reported to inhibit large-scale transcriptions (22Milligan J.F. Uhlenbeck O.C. [5] Synthesis of small RNAs using T7 RNA polymerase.Methods Enzymol. 1989; 180: 51-62Crossref PubMed Scopus (980) Google Scholar). Thus, suboptimal concentrations of either template or T7 RNAP will limit overall synthesis of RNA, although in vitro transcription efficiency appears much more sensitive to T7 RNAP concentrations. When we tested transcription as a function of temperature using a temperature gradient setting on our qPCR instrument, we observed reduced fluorescence with increasing temperature (Fig. 3A). This was not surprising given that Broccoli RNA, like other folded RNA structures, is more stable at lower temperatures and therefore is expected to bind DFHBI-1T more stably to produce greater fluorescence. When pooled reactions were resolved by denaturing PAGE, transcript production was greatest around the 37.8 and 41.4 °C temperature points. Thus, it appears that transcription is most efficient