Transcription yield of fully 2'-modified RNA can be increased by the addition of thermostabilizing mutations to T7 RNA polymerase mutants.
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.
Affiliation: Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, TX 78712, USA email@example.com.Show MeSH
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.
Affiliation: Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, TX 78712, USA firstname.lastname@example.org.