“IPTG induces T7 RNA polymerase to transcribe target sequences in both directions, producing complementary RNA strands that hybridize into double-stranded RNA.”

We describe here a previously unreported property of transcription by SP6 and T7 RNA polymerases, which is the synthesis of extraneous RNA transcripts from promoter-containing DNA templates with 3' overhanging ends. Transcription on these templates proceeds from both strands of DNA, leading to production of vector sequences and RNA which is complementary to the expected runoff transcripts. … Significant amounts of extraneous transcripts were synthesized in vitro by SP6 or T7 RNA polymerase only when DNA templates with the respective promoter had 3' protruding ends.DISCUSSION We describe here a previously unreported property of transcription by SP6 and T7 RNA polymerases, which is the synthesis of extraneous RNA transcripts from promoter-containing DNA templates with 3' overhanging ends. Transcription on these templates proceeds from both strands of DNA, leading to production of vector sequences and RNA which is complementary to the expected runoff transcripts. Significant amounts of extraneous transcripts were synthesized in vitro by SP6 or T7 RNA polymerase only when DNA templates with the respective promoter had 3' protruding ends. The background of spurious transcription was less than 17 from all DNA templates with blunt or 5' overhanging ends. This observation is in agreement with the 0.27 background previously reported for SP6 RNA polymerase (2). The presence of RNA transcribed by SP6 RNA polymerase from both DNA strands was revealed in RNase protection studies. Self-annealing of the probe produced a high background which interfered with RNase-protection or mapping analyses. …

T7 RNA polymerase has been shown to generate unintended dsRNA byproducts by using its newly made transcript as a template to extend it into stem-loops (Cazenave and Uhlenbeck 1994; Triana-Alonso et al. 1995; Gholamalipour et al. 2018; Wu et al. 2020) or by using the coding DNA strand as a template to produce antisense transcripts that hybridize with sense transcripts (Konarska and Sharp 1989; Biebricher and Luce 1996; Mu et al. 2018). … To determine whether antisense transcripts are produced in the T7 RNA polymerase reaction, we performed northern blots using radiolabeled probes targeting antisense or sense transcripts. We detected antisense transcripts in RNA produced by T7 RNA polymerase with UTP (Fig. 5 I), which was decreased in samples treated with RNase III. Notably, no antisense transcripts were found in RNA synthesized with m1ΨTP (Fig. 5 I).dsRNA byproducts from T7 RNA polymerase reactions trigger eIF2α phosphorylation and stress granule assembly T7 RNA polymerase has been shown to generate unintended dsRNA byproducts by using its newly made transcript as a template to extend it into stem-loops (Cazenave and Uhlenbeck 1994; Triana-Alonso et al. 1995; Gholamalipour et al. 2018; Wu et al. 2020) or by using the coding DNA strand as a template to produce antisense transcripts that hybridize with sense transcripts (Konarska and Sharp 1989; Biebricher and Luce 1996; Mu et al. 2018). Since dsRNAs are potent activators of PKR (Kerr et al. 1974; Nanduri et al. 1998), we asked whether microinjected mRNAs triggered PKR and eIF2α phosphorylation due to dsRNA byproducts. To test this, we first treated ftz mRNA produced with T7 RNA polymerase with the dsRNA-selective endonuclease RNase III (Lamontagne et al. 2001; Mu et al. 2018; Wu et al. 2020) prior to in vitro capping and polyadenylation. … 1991; Bonin et al. 2000). As expected, this antibody strongly recognized annealed dsRNA but not plasmid DNA (Fig. 5 H). J2 signal was higher in ftz RNA produced by T7 RNA polymerase with UTP (“U”) compared to ftz RNA produced with m1ΨTP (“m1Ψ”) (Fig. 5 H) and was reduced with RNase III treatment. The total amount of RNA in each sample was similar, as judged by methylene blue staining. This suggests that m1ΨTP inhibited dsRNA production. To determine whether antisense transcripts are produced in the T7 RNA polymerase reaction, we performed northern blots using radiolabeled probes targeting antisense or sense transcripts. We detected antisense transcripts in RNA produced by T7 RNA polymerase with UTP (Fig. 5 I), which was decreased in samples treated with RNase III. Notably, no antisense transcripts were found in RNA synthesized with m1ΨTP (Fig. 5 I). The levels of sense transcripts remained relatively similar across all four samples. From these observations, we conclude that m1ΨTP inhibits the production of dsRNA and antisense transcripts by T7 RNA polymerase, which is consistent with published findings (Mu et al. 2018; Baiersdörfer et al. 2019). Excess exogenous mRNAs trigger stress granules independently of PKR activation or eIF2α phosphorylation

We here showed that the immune-stimulatory activity of T7 transcript is contributed by its aberrant activity to initiate transcription from a promoter-less DNA end. This activity results in the production of an antisense RNA that is fully complementary to the intended sense RNA product, and consequently a long doublestranded RNA (dsRNA) that can robustly stimulate a cytosolic pattern recognition receptor, MDA5. … Briefly, the protein was expressed in BL21(DE3) at 20 • C for 16-20 h following induction with 0.5 mM IPTG. … The dsRNA is formed by hybridization of the intended sense transcript and its fully complementary antisense transcript. The antisense RNA is produced by promoterindependent transcriptional initiation from a DNA end, which differs from previously reported erroneous behaviors of T7 pol, such as 3 -extension of RNA, RNA-dependent RNA polymerization and DNA-primed RNA synthesis (4-7).… While in vitro transcription using the bacteriophage T7 RNA polymerase is one such popular method, its transcripts are known to display an immune-stimulatory activity that is often undesirable and uncontrollable. We here showed that the immune-stimulatory activity of T7 transcript is contributed by its aberrant activity to initiate transcription from a promoter-less DNA end. This activity results in the production of an antisense RNA that is fully complementary to the intended sense RNA product, and consequently a long doublestranded RNA (dsRNA) that can robustly stimulate a cytosolic pattern recognition receptor, MDA5. This promoter-independent transcriptional activity of the T7 RNA polymerase was observed for a wide range of DNA sequences and lengths, but can be suppressed by altering the transcription reaction with modified nucleotides or by reducing the Mg 2+ concentration. … ## Plasmids and proteins Mammalian expression plasmids for RIG-I and MDA5 and the bacterial expression plasmid for MDA5 N (residue 287-1025) were described previously (24) . Protocols to express and purify MDA5 N was reported in (25) . Briefly, the protein was expressed in BL21(DE3) at 20 • C for 16-20 h following induction with 0.5 mM IPTG. Cells were lysed by high pressure homogenization using an Emulsiflex C3 (Avestin), and the protein was purified by a combination of Ni-NTA and heparin affinity chromatography and size exclusion chromatography (SEC) in 20 mM HEPES, pH 7.5, 150 mM NaCl and 2 mM DTT. … the identity of the RNA species and its immune-stimulatory mechanism-has been unclear. This study provides an answer to these questions by showing that T7 pol often produces long dsRNA byproducts that can robustly activate the cytosolic sensors RIG-I and MDA5 in 5'ppp-dependent and -independent manners, respectively. The dsRNA is formed by hybridization of the intended sense transcript and its fully complementary antisense transcript. The antisense RNA is produced by promoterindependent transcriptional initiation from a DNA end, which differs from previously reported erroneous behaviors of T7 pol, such as 3 -extension of RNA, RNA-dependent RNA polymerization and DNA-primed RNA synthesis (4-7). Our finding also shows that this promoter-independent transcription is observed with a wide range of DNA templates, and is not limited to the DNA end with 3 overhangs as previously reported (28) . …

Template 4 generated substantial quantities of dsRNA, as ev idenced by the appearance of upper bands on native agarose gels and a strong signal in J2 antibody-based dot blot as says. … These results indicate that dsRNA byproducts primarily origi nate from promoter-independent antisense transcription at the downstream dsDNA terminus. … NiLoT-based IVT significantly reduced dsRNA levels com pared to the fully double-stranded control, as evidenced by decreased upper bands on native gels and reduced J2 anti body signals, without compromising the overall RNA yield.… Template 3 gener ated both full-length transcripts and the smeared products ob served with template 2, suggesting that unpaired downstream regions act as unintended substrates for abnormal initiation. Template 4 generated substantial quantities of dsRNA, as ev idenced by the appearance of upper bands on native agarose gels and a strong signal in J2 antibody-based dot blot as says. Template 3 showed absence of these dsRNA bands, in dicating substantially reduced dsRNA formation. Quantita tive analysis of dot blot intensities revealed a ∼ 80% reduc tion in dsRNA with template 3 relative to template 4, while total RNA yield measured by RiboGreen decreased by ∼ 80%. These results indicate that dsRNA byproducts primarily origi nate from promoter-independent antisense transcription at the downstream dsDNA terminus. Template nicking suppresses dsRNA formation while maintaining RNA output To evaluate whether introducing a nick into the non-template strand could suppress antisense transcription (Fig. 2A), we designed three DNA templates: partially ssDNA (template 1; identical to template 3 in Fig. … Efficient nick formation was confirmed by S1 nuclease digestion, which selectively cleaves SSBs to yield fragmenta tion patterns (Supplementary Fig. S2). NiLoT-based IVT significantly reduced dsRNA levels com pared to the fully double-stranded control, as evidenced by decreased upper bands on native gels and reduced J2 anti body signals, without compromising the overall RNA yield. Notably, the extent of dsRNA reduction achieved with NiLoT Figure 1. Promoter-independent antisense transcription contributes to dsRNA. …

We here showed that the immune-stimulatory activity of T7 transcript is contributed by its aberrant activity to initiate transcription from a promoter-less DNA end. This activity results in the production of an antisense RNA that is fully complementary to the intended sense RNA product, and consequently a long double-stranded RNA (dsRNA) that can robustly stimulate a cytosolic pattern recognition receptor, MDA5. … Briefly, the protein was expressed in BL21(DE3) at 20°C for 16–20 h following induction with 0.5 mM IPTG. … The dsRNA is formed by hybridization of the intended sense transcript and its fully complementary antisense transcript. The antisense RNA is produced by promoter-independent transcriptional initiation from a DNA end, which differs from previously reported erroneous behaviors of T7 pol, such as 3′-extension of RNA, RNA-dependent RNA polymerization and DNA-primed RNA synthesis (4–7).… While in vitro transcription using the bacteriophage T7 RNA polymerase is one such popular method, its transcripts are known to display an immune-stimulatory activity that is often undesirable and uncontrollable. We here showed that the immune-stimulatory activity of T7 transcript is contributed by its aberrant activity to initiate transcription from a promoter-less DNA end. This activity results in the production of an antisense RNA that is fully complementary to the intended sense RNA product, and consequently a long double-stranded RNA (dsRNA) that can robustly stimulate a cytosolic pattern recognition receptor, MDA5. This promoter-independent transcriptional activity of the T7 RNA polymerase was observed for a wide range of DNA sequences and lengths, but can be suppressed by altering the transcription reaction with modified nucleotides or by reducing the Mg 2+ concentration. … ### Plasmids and proteins Mammalian expression plasmids for RIG-I and MDA5 and the bacterial expression plasmid for MDA5ΔN (residue 287–1025) were described previously (24). Protocols to express and purify MDA5ΔN was reported in (25). Briefly, the protein was expressed in BL21(DE3) at 20°C for 16–20 h following induction with 0.5 mM IPTG. Cells were lysed by high pressure homogenization using an Emulsiflex C3 (Avestin), and the protein was purified by a combination of Ni-NTA and heparin affinity chromatography and size exclusion chromatography (SEC) in 20 mM HEPES, pH 7.5, 150 mM NaCl and 2 mM DTT. … the identity of the RNA species and its immune-stimulatory mechanism−has been unclear. This study provides an answer to these questions by showing that T7 pol often produces long dsRNA byproducts that can robustly activate the cytosolic sensors RIG-I and MDA5 in 5’ppp-dependent and -independent manners, respectively. The dsRNA is formed by hybridization of the intended sense transcript and its fully complementary antisense transcript. The antisense RNA is produced by promoter-independent transcriptional initiation from a DNA end, which differs from previously reported erroneous behaviors of T7 pol, such as 3′-extension of RNA, RNA-dependent RNA polymerization and DNA-primed RNA synthesis (4–7). Our finding also shows that this promoter-independent transcription is observed with a wide range of DNA templates, and is not limited to the DNA end with 3′ overhangs as previously reported (28). …

We here showed that the immune-stimulatory activity of T7 transcript is contributed by its aberrant activity to initiate transcription from a promoter-less DNA end. This activity results in the production of an antisense RNA that is fully complementary to the intended sense RNA product, and consequently a long double-stranded RNA (dsRNA) that can robustly stimulate a cytosolic pattern recognition receptor, MDA5. … Briefly, the protein was expressed in BL21(DE3) at 20°C for 16–20 h following induction with 0.5 mM IPTG.Abstract The emergence of RNA-based therapeutics demands robust and economical methods to produce RNA with few byproducts from aberrant activity. While in vitro transcription using the bacteriophage T7 RNA polymerase is one such popular method, its transcripts are known to display an immune-stimulatory activity that is often undesirable and uncontrollable. We here showed that the immune-stimulatory activity of T7 transcript is contributed by its aberrant activity to initiate transcription from a promoter-less DNA end. This activity results in the production of an antisense RNA that is fully complementary to the intended sense RNA product, and consequently a long double-stranded RNA (dsRNA) that can robustly stimulate a cytosolic pattern recognition receptor, MDA5. This promoter-independent transcriptional activity of the T7 RNA polymerase was observed for a wide range of DNA sequences and lengths, but can be suppressed by altering the transcription reaction with modified nucleotides or by reducing the Mg 2+ concentration. The current work thus not only offers a previously unappreciated mechanism by which T7 transcripts stimulate the innate immune system, but also shows that the immune-stimulatory activity can be readily regulated. INTRODUCTION … ### Plasmids and proteins Mammalian expression plasmids for RIG-I and MDA5 and the bacterial expression plasmid for MDA5ΔN (residue 287–1025) were described previously (24). Protocols to express and purify MDA5ΔN was reported in (25). Briefly, the protein was expressed in BL21(DE3) at 20°C for 16–20 h following induction with 0.5 mM IPTG. Cells were lysed by high pressure homogenization using an Emulsiflex C3 (Avestin), and the protein was purified by a combination of Ni-NTA and heparin affinity chromatography and size exclusion chromatography (SEC) in 20 mM HEPES, pH 7.5, 150 mM NaCl and 2 mM DTT. T7 polymerase was expressed in BL21(DE3) using the plasmid pT7–911Q. …

Upon addition of IPTG, the T7RNAP enzyme is produced at high levels and subsequently transcribes the target gene which is cloned downstream of a T7 promoter in the expression plasmid.… The T7 and arabinose promoters have contributed to 80% of all unique MP structures (63% and 17%, respectively) 3. The T7 RNA polymerase (T7RNAP)-based expression system was designed to maximize the production levels of the target protein 4. Bacteriophage lambda was modified by inserting the T7 RNA polymerase gene under the control of the lac L8 UV5 promoter, a variant of the lactose promoter that is insensitive to catabolic repression. Upon addition of IPTG, the T7RNAP enzyme is produced at high levels and subsequently transcribes the target gene which is cloned downstream of a T7 promoter in the expression plasmid. This expression system is powerful but has two major drawbacks. Firstly, the lacUV5 promoter is leaky and proportional expression of the target gene is difficult to achieve with increasing concentrations of its non-metabolized inducer IPTG. Secondly, Dong and coworkers have shown that overproduction of the T7RNAP enzyme triggers ribosome destruction and cell death 5. This intrinsic toxicity of the T7 system was used to isolate C41(DE3) and C43(DE3) mutant hosts 6. …

In addition to the desired full-length mRNA, WT T7RNAP generates double stranded RNA (dsRNA) byproducts through two general mechanisms: antisense RNA products formed during IVT hybridize to the sense mRNA, creating dsRNA duplexes,22 and 3′ extension products longer than the expected runoff transcript are produced when the 3′ end of an mRNA self-hybridizes in cis, and T7RNAP extends via an intrinsic RNA-dependent RNA-polymerase activity to form regions of self-complementary dsRNA.dsRNA contaminants from IVT In addition to the desired full-length mRNA, WT T7RNAP generates double stranded RNA (dsRNA) byproducts through two general mechanisms: antisense RNA products formed during IVT hybridize to the sense mRNA, creating dsRNA duplexes,22 and 3′ extension products longer than the expected runoff transcript are produced when the 3′ end of an mRNA self-hybridizes in cis, and T7RNAP extends via an intrinsic RNA-dependent RNA-polymerase activity to form regions of self-complementary dsRNA.23,24 Because dsRNA byproducts are known to stimulate an adverse immune response, which concomitantly reduces translation efficiency,25 we characterized the dsRNA byproducts produced by the WT and T7-68 polymerases.

In addition to normal transcription, the enzyme can produce anomalous transcripts in the absence of a promoter. … Oligonucleotides that are able to form transient unimolecular loop structures closed by as little as one Watson-Crick base pair between the 3' terminal residue and an internal nucleotide proved to be viable substrates. … This intermittent extension process can be quite efficient. Short DNA or RNA substrates were good substrates for the enzyme (affinities ranged from 2 to 43 microM) and were extended rapidly with apparent catalytic rates of up to 240 min(-1) being observed, only 2-fold slower than the rate of transcriptional initiation.Abstract T7 RNA polymerase is a processive, DNA-dependent RNA polymerase that has a high specificity for its 17 base pair (bp) promoter. In addition to normal transcription, the enzyme can produce anomalous transcripts in the absence of a promoter. We report here the systematic characterization of the transient aspects of this promoter-independent process. Oligonucleotides that are able to form transient unimolecular loop structures closed by as little as one Watson-Crick base pair between the 3' terminal residue and an internal nucleotide proved to be viable substrates. A single nucleotide triphosphate assay system found that incorporation was encoded by the nucleotide 5' to the predicted transient base pair. When this coding nucleotide was identical to the internal nucleotide participating in the transient base pair, multiple nucleotide incorporations were observed and could only be explained by a continuous shifting and resetting of the transient base-pairing required for extension. This intermittent extension process can be quite efficient. Short DNA or RNA substrates were good substrates for the enzyme (affinities ranged from 2 to 43 microM) and were extended rapidly with apparent catalytic rates of up to 240 min(-1) being observed, only 2-fold slower than the rate of transcriptional initiation. Our data suggest a possible mechanism for this promoter-independent extension activity and may add to the understanding of viral RNA replicative strategies.

Overexpression of RNAPs was induced by addition of 0.5 mM isopropyl β-D-1-thiogalactopyranoside and continuous incubation at 16°C for 16 h. … However, if T7 RNAP initiates transcription at the 3′-termini of the DNA template, the antisense GFP transcript would be extended following the extension of the antisense DNA, and the size of the full-length dsRNA annealed by the GFP transcript (initiated at the promoter) and the antisense GFP transcript (initiated at the 3′ DNA terminus) would also increase.Protein expression and purification DNA fragments encoding T7 RNAP variants were inserted into the pQE82L vector and DNA fragments encoding wild-type (WT) SP6 RNAP were inserted into the pET28b vector with N-terminal His tags. The vectors were transformed into Escherichia coli BL21(DE3), and cells were cultured in 1 l LB medium containing 100 mg/ml ampicillin (for T7-RNAP-pQE82L vector) or 50 mg/ml kanamycin (for SP6-RNAP-pET28b vector) at 37°C until the OD 600 reached 1.2. Overexpression of RNAPs was induced by addition of 0.5 mM isopropyl β-D-1-thiogalactopyranoside and continuous incubation at 16°C for 16 h. The cells were collected, resuspended in buffer (50 mM Tris–HCl, pH 7.5, 300 mM NaCl) and lysed by ultrasonication. The supernatants were filtered through the 0.2 μm filters and then loaded into the Ni-NTA-agarose columns (Qiagen) equilibrated with wash buffer containing 50 mM Tris-HCl, pH 7.5, and 300 mM NaCl. … To reveal the origin of the full-length dsRNA in GFP transcripts, we extended the gfp DNA templates with 53-, 290- or 583-bp noncoding sequences at the 5′ termini, upstream of the T7 promoter (Figure 2A). These extensions do not affect the desired GFP ssRNA initiated from the promoter, so the size of the full-length dsRNA would not change if it originated from the self-templated RNA extension as described previously (9–13). However, if T7 RNAP initiates transcription at the 3′-termini of the DNA template, the antisense GFP transcript would be extended following the extension of the antisense DNA, and the size of the full-length dsRNA annealed by the GFP transcript (initiated at the promoter) and the antisense GFP transcript (initiated at the 3′ DNA terminus) would also increase. As shown in Figure 2A, the gel bands corresponding to the GFP ssRNA had the same mobility, despite the template 5′ extension. However, the gel mobilities of the upper bands corresponding to the full-length dsRNA were lower following the template 5′ extensions, confirming that the full-length dsRNA in GFP transcripts originated from promoter-free DNA-terminus-initiated transcription (Figure 2B) but not from RNA-templated self-extension. …

the results unambiguously confirm that product RNA rebinds to the polymerase and self-primes (in cis) generation of a hairpin duplex … the sequence beyond the expected runoff transcript is almost exclusively the reverse complement of a sequence in the upstream region of the encoded RNA. We further confirm that such products are formed predominantly by cis primer extension, in which the runoff RNA primes and transcribes from its own upstream sequence by looping back on itself.Abstract Synthetic RNA is widely used in basic science, nanotechnology and therapeutics research. The vast majority of this RNA is synthesized in vitro by T7 RNA polymerase or one of its close family members. However, the desired RNA is generally contaminated with products longer and shorter than the DNA-encoded product. To better understand these undesired byproducts and the processes that generate them, we analyze in vitro transcription reactions using RNA-Seq as a tool. The results unambiguously confirm that product RNA rebinds to the polymerase and self-primes (in cis) generation of a hairpin duplex, a process that favorably competes with promoter driven synthesis under high yield reaction conditions. While certain priming modes can be favored, the process is heterogeneous, both in initial priming and in the extent of priming, and already extended products can rebind for further extension, in a distributive process. Furthermore, addition of one or a few nucleotides, previously termed 'nontemplated addition,' also occurs via templated primer extension. … ## Product runoff RNA loops back to prime from its own upstream sequence In this study, we have focused on the formation of RNA products longer than the expected runoff. These products have been reported in the past and have been attributed to different mechanistic origins (19, 20, 27) . The RNA-Seq results presented here confirm that longer RNA products arise from RNA primer extension (18) . Specifically, the sequence beyond the expected runoff transcript is almost exclusively the reverse complement of a sequence in the upstream region of the encoded RNA. We further confirm that such products are formed predominantly by cis primer extension, in which the runoff RNA primes and transcribes from its own upstream sequence by looping back on itself. It has been proposed previously that RNA polymerase, at the end of a runoff template, might switch to the nontemplate strand, giving the same reverse complement pattern to the appended sequence. Although transcription here was carried out with 'partially single stranded templates', in which the nontemplate strand is truncated at position +2, the first round of transcription could lay down RNA as a nontemplate strand for subsequent rounds. …

Upon addition of IPTG, LacI activity is inhibited, and the newly produced T7 RNAP strongly transcribes the gene of interest from de-repressed PT7lac (Figure 1A).… subtilis. Our system utilizes the hybrid PT7lac promoter17 to express a gene of interest, and the IPTG-inducible promoter Phy-spank21 to express the T7 RNA Polymerase (T7 RNAP) (Figure 1A). In the absence of inducer, LacI, which is constitutively expressed from the PpenP promoter10, represses both expression of T7 RNAP and transcription from PT7lac. This dual repression is expected to minimize leaky expression of the gene of interest. Upon addition of IPTG, LacI activity is inhibited, and the newly produced T7 RNAP strongly transcribes the gene of interest from de-repressed PT7lac (Figure 1A). Our LacI-T7 design is conceptually similar to some variants of the commercial E. coli pET expression system, where IPTG also induces both expression of T7 RNAP and de-repression of PT7lac17. However, our system uses B. subtilis-specific promoters (other than PT7lac) and ribosome-binding sites (RBSs). Additionally, while a few B. …

the polylinker region is flanked by two modified RNA polymerase promoters such as the T7 RNA promoter, each containing a unique restriction site that has been engineered into the consensus T7 promoter sequence … Selective unidirectional transcription from one promoter is achieved by simply cleaving the other at an internally engineered restriction site … RNA probes corresponding to either strand of a cloned insert can thus be prepared, under identical conditions, using the inexpensive and well-characterized T7 RNA polymerase… - modified RNA polymerase promoters such as the T7 RNA promoter, each containing a unique restriction site that has been engineered into the consensus T7 promoter sequence (FIG. 6). - the polylinker region is flanked by two modified RNA polymerase promoters such as the T7 RNA promoter, each containing a unique restriction site that has been engineered into the consensus T7 promoter sequence (FIG. 6). - the polylinker regions of the vectors of this invention are preferably fused to lacZ ⁇ to facilitate blue-white screening, so the reading frame of each restriction site is chosen to minimize rare E. coli codons. … Selective unidirectional transcription from one promoter is achieved by simply cleaving the other at an internally engineered restriction site. Since efficient labeling of RNA probes requires that the template be linearized at a site downstream from the insert prior to transcription anyway (Melton, supra), cutting at the site within the undesired promoter performs both functions in a single step. RNA probes corresponding to either strand of a cloned insert can thus be prepared, under identical conditions, using the inexpensive and well-characterized T7 RNA polymerase. This bypasses problems associated with other phage-encoded RNA polymerases (e.g., SP6 or T3), which have different optimum reaction conditions, and are often more expensive, more dilute, or less pure. Introduction of an internal restriction site into each promoter necessitated altering the consensus promoter sequence. …

Analysis of the reaction indicated that the product resulted from initial DNA-directed run-off transcription followed by RNA template-directed RNA synthesis. This reaction occurred because the RNA made from the DNA template displayed self-complementarity at its 3' end and therefore could form an intra- or intermolecular primed template.Abstract In an attempt to synthesize an oligoribonucleotide by run-off transcription by bacteriophage T7 RNA polymerase, a major transcript was produced that was much longer than expected. Analysis of the reaction indicated that the product resulted from initial DNA-directed run-off transcription followed by RNA template-directed RNA synthesis. This reaction occurred because the RNA made from the DNA template displayed self-complementarity at its 3' end and therefore could form an intra- or intermolecular primed template. In reactions containing only an RNA template, the rate of incorporation of NTPs was quite comparable to DNA-dependent transcription. RNA template-directed RNA synthesis has been found to occur with a great number of oligoribonucleotides, even with primed templates that are only marginally stable. In one instance, we observed a multistep extension reaction converting the oligonucleotide into a final product longer than twice its original length. …