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Transcription yield of fully 2'-modified RNA can be increased by the addition of thermostabilizing mutations to T7 RNA polymerase mutants.

Meyer AJ, Garry DJ, Hall B, Byrom MM, McDonald HG, Yang X, Yin YW, Ellington AD - Nucleic Acids Res. (2015)

Bottom Line: Mutations conferring new function to a protein often come at the expense of protein folding or stability, reducing overall activity.These modified RNAs have proven useful, especially in vivo, but the transcriptional yields tend to be quite low.The resulting polymerase mutants can be used to generate 2'-O-methyl modified RNA with yields much higher than enzymes currently employed.

View Article: PubMed Central - PubMed

Affiliation: Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, TX 78712, USA andy.ellington@mail.utexas.edu.

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Stabilizing mutations increase the activity of the VRS mutant. (A) Real time measurement of ribonucleotide (rN) transcriptional output. (B) Real time measurement of 2′-fluoropyrimidine (rRfY) transcriptional output. (C) Measurement of ribonucleotide (rN) transcriptional output after 3 h. (D) Measurement of 2′-fluoropyrimidine (rRfY) transcriptional output after 3 h. Fluorescent readings (in Relative Fluorescent Units, RFU) indicate the presence of the fluorescent aptamer, spinach. Data shown in panels (A) and (B) result from single experiments. Data shown in panels (C) and (D) are the average of three independently assembled reactions with error bars representing standard error.
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Figure 1: Stabilizing mutations increase the activity of the VRS mutant. (A) Real time measurement of ribonucleotide (rN) transcriptional output. (B) Real time measurement of 2′-fluoropyrimidine (rRfY) transcriptional output. (C) Measurement of ribonucleotide (rN) transcriptional output after 3 h. (D) Measurement of 2′-fluoropyrimidine (rRfY) transcriptional output after 3 h. Fluorescent readings (in Relative Fluorescent Units, RFU) indicate the presence of the fluorescent aptamer, spinach. Data shown in panels (A) and (B) result from single experiments. Data shown in panels (C) and (D) are the average of three independently assembled reactions with error bars representing standard error.

Mentions: To test this hypothesis, we constructed a derivative of VRS without H772R, a variant we termed ‘VS’. We then assayed purified enzymes for their ability to polymerize RNA composed either of natural NTPs (rN) or of ribo-purines and 2′-F-pyrimidines (rRfY; Figure 1A, B). Real-time polymerase activity was assayed by transcribing the fluorescent aptamer Spinach in the presence of DFHBI (34). Spinach will bind DFHBI and fluoresce irrespective of whether it is transcribed as a purely ribo-aptamer or when substituted with 2′-F-pyrimidines, although the 2′-F-pyrimidine version is only about 30% as fluorescent as the purely ribonucleotide version. 2′-O-methyl substituted Spinach is not detectably fluorescent. As predicted, VS showed a decreased activity for each substrate composition assayed.


Transcription yield of fully 2'-modified RNA can be increased by the addition of thermostabilizing mutations to T7 RNA polymerase mutants.

Meyer AJ, Garry DJ, Hall B, Byrom MM, McDonald HG, Yang X, Yin YW, Ellington AD - Nucleic Acids Res. (2015)

Stabilizing mutations increase the activity of the VRS mutant. (A) Real time measurement of ribonucleotide (rN) transcriptional output. (B) Real time measurement of 2′-fluoropyrimidine (rRfY) transcriptional output. (C) Measurement of ribonucleotide (rN) transcriptional output after 3 h. (D) Measurement of 2′-fluoropyrimidine (rRfY) transcriptional output after 3 h. Fluorescent readings (in Relative Fluorescent Units, RFU) indicate the presence of the fluorescent aptamer, spinach. Data shown in panels (A) and (B) result from single experiments. Data shown in panels (C) and (D) are the average of three independently assembled reactions with error bars representing standard error.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4551944&req=5

Figure 1: Stabilizing mutations increase the activity of the VRS mutant. (A) Real time measurement of ribonucleotide (rN) transcriptional output. (B) Real time measurement of 2′-fluoropyrimidine (rRfY) transcriptional output. (C) Measurement of ribonucleotide (rN) transcriptional output after 3 h. (D) Measurement of 2′-fluoropyrimidine (rRfY) transcriptional output after 3 h. Fluorescent readings (in Relative Fluorescent Units, RFU) indicate the presence of the fluorescent aptamer, spinach. Data shown in panels (A) and (B) result from single experiments. Data shown in panels (C) and (D) are the average of three independently assembled reactions with error bars representing standard error.
Mentions: To test this hypothesis, we constructed a derivative of VRS without H772R, a variant we termed ‘VS’. We then assayed purified enzymes for their ability to polymerize RNA composed either of natural NTPs (rN) or of ribo-purines and 2′-F-pyrimidines (rRfY; Figure 1A, B). Real-time polymerase activity was assayed by transcribing the fluorescent aptamer Spinach in the presence of DFHBI (34). Spinach will bind DFHBI and fluoresce irrespective of whether it is transcribed as a purely ribo-aptamer or when substituted with 2′-F-pyrimidines, although the 2′-F-pyrimidine version is only about 30% as fluorescent as the purely ribonucleotide version. 2′-O-methyl substituted Spinach is not detectably fluorescent. As predicted, VS showed a decreased activity for each substrate composition assayed.

Bottom Line: Mutations conferring new function to a protein often come at the expense of protein folding or stability, reducing overall activity.These modified RNAs have proven useful, especially in vivo, but the transcriptional yields tend to be quite low.The resulting polymerase mutants can be used to generate 2'-O-methyl modified RNA with yields much higher than enzymes currently employed.

View Article: PubMed Central - PubMed

Affiliation: Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, TX 78712, USA andy.ellington@mail.utexas.edu.

Show MeSH