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Design of transcription template suitable for circularization. ( A ) Efficient in vitro transcription with <t>T7</t> RNA polymerase requires minimal T7 promoter and GGG at the transcription start site. Linear precursor RNA is generated by addition of the polymerase and nucleotides to <t>the</t> <t>DNA</t> template at 37°C for 2–16 h. ( B ) RNAfold modeled structures of circRNAs generated from unpermuted transcription template with extra GGG were different from circRNAs with no extra Gs, where transcription was initiated from an internal GGG site (permuted transcription template). ( C ) Schematic representation of the permutation strategy, showing that 5′ and 3′ ends were different between linear RNAs from unpermuted and permuted transcription templates. Subsequent circularization produced contiguous circRNAs that were ligated at different junctions. ( D ) Treatment with XRN1 which degrades 5′-monophosphorylated RNAs showed that addition of excess GMP to GTP in in vitro transcription reactions resulted in higher amounts of 5′-monophosphorylated linear precursors than other methods. Representative data are from a mean of n = 3 technical replicates with standard error of the mean (SEM). ( E ) Tapestation analysis of transcribed RNA showed that in vitro transcription reactions incubated at 37°C for 2 h had fewer shorter contaminating RNAs compared to incubations for 16 h. ( F ) in vitro transcription with only GTP had higher yields at 16 h than 2 h. Addition of 5× or 10× GMP reduced the yields for 16 h reactions which was comparable to the yields at 2 h. 10× GMP at 2 h had the lowest yields among all of the conditions. Representative data are from a mean of n = 3 technical replicates. ( G ) Efficiency of 5′-monophosphosphate transcripts was comparable between 5× GMP and 10× GMP in vitro transcription reactions. Representative data are from a mean of n = 3 technical replicates with SEM.
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Design of transcription template suitable for circularization. ( A ) Efficient in vitro transcription with <t>T7</t> RNA polymerase requires minimal T7 promoter and GGG at the transcription start site. Linear precursor RNA is generated by addition of the polymerase and nucleotides to <t>the</t> <t>DNA</t> template at 37°C for 2–16 h. ( B ) RNAfold modeled structures of circRNAs generated from unpermuted transcription template with extra GGG were different from circRNAs with no extra Gs, where transcription was initiated from an internal GGG site (permuted transcription template). ( C ) Schematic representation of the permutation strategy, showing that 5′ and 3′ ends were different between linear RNAs from unpermuted and permuted transcription templates. Subsequent circularization produced contiguous circRNAs that were ligated at different junctions. ( D ) Treatment with XRN1 which degrades 5′-monophosphorylated RNAs showed that addition of excess GMP to GTP in in vitro transcription reactions resulted in higher amounts of 5′-monophosphorylated linear precursors than other methods. Representative data are from a mean of n = 3 technical replicates with standard error of the mean (SEM). ( E ) Tapestation analysis of transcribed RNA showed that in vitro transcription reactions incubated at 37°C for 2 h had fewer shorter contaminating RNAs compared to incubations for 16 h. ( F ) in vitro transcription with only GTP had higher yields at 16 h than 2 h. Addition of 5× or 10× GMP reduced the yields for 16 h reactions which was comparable to the yields at 2 h. 10× GMP at 2 h had the lowest yields among all of the conditions. Representative data are from a mean of n = 3 technical replicates. ( G ) Efficiency of 5′-monophosphosphate transcripts was comparable between 5× GMP and 10× GMP in vitro transcription reactions. Representative data are from a mean of n = 3 technical replicates with SEM.
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Design of transcription template suitable for circularization. ( A ) Efficient in vitro transcription with <t>T7</t> RNA polymerase requires minimal T7 promoter and GGG at the transcription start site. Linear precursor RNA is generated by addition of the polymerase and nucleotides to <t>the</t> <t>DNA</t> template at 37°C for 2–16 h. ( B ) RNAfold modeled structures of circRNAs generated from unpermuted transcription template with extra GGG were different from circRNAs with no extra Gs, where transcription was initiated from an internal GGG site (permuted transcription template). ( C ) Schematic representation of the permutation strategy, showing that 5′ and 3′ ends were different between linear RNAs from unpermuted and permuted transcription templates. Subsequent circularization produced contiguous circRNAs that were ligated at different junctions. ( D ) Treatment with XRN1 which degrades 5′-monophosphorylated RNAs showed that addition of excess GMP to GTP in in vitro transcription reactions resulted in higher amounts of 5′-monophosphorylated linear precursors than other methods. Representative data are from a mean of n = 3 technical replicates with standard error of the mean (SEM). ( E ) Tapestation analysis of transcribed RNA showed that in vitro transcription reactions incubated at 37°C for 2 h had fewer shorter contaminating RNAs compared to incubations for 16 h. ( F ) in vitro transcription with only GTP had higher yields at 16 h than 2 h. Addition of 5× or 10× GMP reduced the yields for 16 h reactions which was comparable to the yields at 2 h. 10× GMP at 2 h had the lowest yields among all of the conditions. Representative data are from a mean of n = 3 technical replicates. ( G ) Efficiency of 5′-monophosphosphate transcripts was comparable between 5× GMP and 10× GMP in vitro transcription reactions. Representative data are from a mean of n = 3 technical replicates with SEM.
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Design of transcription template suitable for circularization. ( A ) Efficient in vitro transcription with <t>T7</t> RNA polymerase requires minimal T7 promoter and GGG at the transcription start site. Linear precursor RNA is generated by addition of the polymerase and nucleotides to <t>the</t> <t>DNA</t> template at 37°C for 2–16 h. ( B ) RNAfold modeled structures of circRNAs generated from unpermuted transcription template with extra GGG were different from circRNAs with no extra Gs, where transcription was initiated from an internal GGG site (permuted transcription template). ( C ) Schematic representation of the permutation strategy, showing that 5′ and 3′ ends were different between linear RNAs from unpermuted and permuted transcription templates. Subsequent circularization produced contiguous circRNAs that were ligated at different junctions. ( D ) Treatment with XRN1 which degrades 5′-monophosphorylated RNAs showed that addition of excess GMP to GTP in in vitro transcription reactions resulted in higher amounts of 5′-monophosphorylated linear precursors than other methods. Representative data are from a mean of n = 3 technical replicates with standard error of the mean (SEM). ( E ) Tapestation analysis of transcribed RNA showed that in vitro transcription reactions incubated at 37°C for 2 h had fewer shorter contaminating RNAs compared to incubations for 16 h. ( F ) in vitro transcription with only GTP had higher yields at 16 h than 2 h. Addition of 5× or 10× GMP reduced the yields for 16 h reactions which was comparable to the yields at 2 h. 10× GMP at 2 h had the lowest yields among all of the conditions. Representative data are from a mean of n = 3 technical replicates. ( G ) Efficiency of 5′-monophosphosphate transcripts was comparable between 5× GMP and 10× GMP in vitro transcription reactions. Representative data are from a mean of n = 3 technical replicates with SEM.
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thermopol buffer  (New England Biolabs)
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Design of transcription template suitable for circularization. ( A ) Efficient in vitro transcription with <t>T7</t> RNA polymerase requires minimal T7 promoter and GGG at the transcription start site. Linear precursor RNA is generated by addition of the polymerase and nucleotides to <t>the</t> <t>DNA</t> template at 37°C for 2–16 h. ( B ) RNAfold modeled structures of circRNAs generated from unpermuted transcription template with extra GGG were different from circRNAs with no extra Gs, where transcription was initiated from an internal GGG site (permuted transcription template). ( C ) Schematic representation of the permutation strategy, showing that 5′ and 3′ ends were different between linear RNAs from unpermuted and permuted transcription templates. Subsequent circularization produced contiguous circRNAs that were ligated at different junctions. ( D ) Treatment with XRN1 which degrades 5′-monophosphorylated RNAs showed that addition of excess GMP to GTP in in vitro transcription reactions resulted in higher amounts of 5′-monophosphorylated linear precursors than other methods. Representative data are from a mean of n = 3 technical replicates with standard error of the mean (SEM). ( E ) Tapestation analysis of transcribed RNA showed that in vitro transcription reactions incubated at 37°C for 2 h had fewer shorter contaminating RNAs compared to incubations for 16 h. ( F ) in vitro transcription with only GTP had higher yields at 16 h than 2 h. Addition of 5× or 10× GMP reduced the yields for 16 h reactions which was comparable to the yields at 2 h. 10× GMP at 2 h had the lowest yields among all of the conditions. Representative data are from a mean of n = 3 technical replicates. ( G ) Efficiency of 5′-monophosphosphate transcripts was comparable between 5× GMP and 10× GMP in vitro transcription reactions. Representative data are from a mean of n = 3 technical replicates with SEM.
Thermopol Buffer, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Design of transcription template suitable for circularization. ( A ) Efficient in vitro transcription with T7 RNA polymerase requires minimal T7 promoter and GGG at the transcription start site. Linear precursor RNA is generated by addition of the polymerase and nucleotides to the DNA template at 37°C for 2–16 h. ( B ) RNAfold modeled structures of circRNAs generated from unpermuted transcription template with extra GGG were different from circRNAs with no extra Gs, where transcription was initiated from an internal GGG site (permuted transcription template). ( C ) Schematic representation of the permutation strategy, showing that 5′ and 3′ ends were different between linear RNAs from unpermuted and permuted transcription templates. Subsequent circularization produced contiguous circRNAs that were ligated at different junctions. ( D ) Treatment with XRN1 which degrades 5′-monophosphorylated RNAs showed that addition of excess GMP to GTP in in vitro transcription reactions resulted in higher amounts of 5′-monophosphorylated linear precursors than other methods. Representative data are from a mean of n = 3 technical replicates with standard error of the mean (SEM). ( E ) Tapestation analysis of transcribed RNA showed that in vitro transcription reactions incubated at 37°C for 2 h had fewer shorter contaminating RNAs compared to incubations for 16 h. ( F ) in vitro transcription with only GTP had higher yields at 16 h than 2 h. Addition of 5× or 10× GMP reduced the yields for 16 h reactions which was comparable to the yields at 2 h. 10× GMP at 2 h had the lowest yields among all of the conditions. Representative data are from a mean of n = 3 technical replicates. ( G ) Efficiency of 5′-monophosphosphate transcripts was comparable between 5× GMP and 10× GMP in vitro transcription reactions. Representative data are from a mean of n = 3 technical replicates with SEM.

Journal: Nucleic Acids Research

Article Title: Generation of precise and accurate engineered circRNAs using enzymatic ligation

doi: 10.1093/nar/gkag405

Figure Lengend Snippet: Design of transcription template suitable for circularization. ( A ) Efficient in vitro transcription with T7 RNA polymerase requires minimal T7 promoter and GGG at the transcription start site. Linear precursor RNA is generated by addition of the polymerase and nucleotides to the DNA template at 37°C for 2–16 h. ( B ) RNAfold modeled structures of circRNAs generated from unpermuted transcription template with extra GGG were different from circRNAs with no extra Gs, where transcription was initiated from an internal GGG site (permuted transcription template). ( C ) Schematic representation of the permutation strategy, showing that 5′ and 3′ ends were different between linear RNAs from unpermuted and permuted transcription templates. Subsequent circularization produced contiguous circRNAs that were ligated at different junctions. ( D ) Treatment with XRN1 which degrades 5′-monophosphorylated RNAs showed that addition of excess GMP to GTP in in vitro transcription reactions resulted in higher amounts of 5′-monophosphorylated linear precursors than other methods. Representative data are from a mean of n = 3 technical replicates with standard error of the mean (SEM). ( E ) Tapestation analysis of transcribed RNA showed that in vitro transcription reactions incubated at 37°C for 2 h had fewer shorter contaminating RNAs compared to incubations for 16 h. ( F ) in vitro transcription with only GTP had higher yields at 16 h than 2 h. Addition of 5× or 10× GMP reduced the yields for 16 h reactions which was comparable to the yields at 2 h. 10× GMP at 2 h had the lowest yields among all of the conditions. Representative data are from a mean of n = 3 technical replicates. ( G ) Efficiency of 5′-monophosphosphate transcripts was comparable between 5× GMP and 10× GMP in vitro transcription reactions. Representative data are from a mean of n = 3 technical replicates with SEM.

Article Snippet: in vitro transcription reactions of 20 μL included 100 ng of DNA templates, 7.5 mM of each of the NTPs, 1× of T7 reaction buffer, and 2 μL of T7 RNA polymerase enzyme at 37°C for either 2 or 16 h (NEB #E2040S).

Techniques: In Vitro, Generated, Produced, Incubation