Genetically Expanded Fluorescent SSBT7 RNA Polymerase in Action: Intriguing Photophysics with Enhanced RNA Production
- 1Indian Institute of Technology Guwahati , , ,
Abstract
T7 RNA polymerase (T7-RNAP) is a highly efficient transcriptional enzyme. However, it is prone to mutation at the functionally important Y571 residue, which strongly influences its functional properties. Here, we demonstrate a strategy to modulate the T7-RNAP enzyme by replacing the Y571 residue with a fluorescent triazolyl unnatural amino acid (FTUAA), TCNBAlaAc, via genetic code expansion (GCE) technology using two orthogonal aminoacyl-tRNA synthetase/tRNA pairs. Thus, TCNBAlaAc-encoded variants, herein termed SSBT7-RNAP, were successfully expressed, purified, and characterized by SDS-PAGE, MALDI-TOF mass spectrometry, CD, UV–vis, and fluorescence spectroscopy. Incorporation of TCNBAlaAc introduced distinct and enhanced fluorescence signatures while largely preserving the overall secondary structures of the engineered enzymes. Notably, these novel TCNBAlaAc-encoded variants exhibited higher relative RNA yields than those of wild-type T7-RNAP under identical in vitro transcription conditions. Molecular docking further supported the structural compatibility of the modification with the promoter-bound enzyme. These findings establish GCE-based, site-specific incorporation of an FTUAA into a functionally relevant site of T7 RNA polymerase as a promising strategy for generating spectroscopically traceable, transcriptionally competent T7-RNAP variants for future enzyme engineering and mechanistic studies.
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BibTeX
@article{bag2026genetically,
title = {{Genetically Expanded Fluorescent SSBT7 RNA Polymerase in Action: Intriguing Photophysics with Enhanced RNA Production}},
author = {Subhendu Sekhar Bag and Aniket Banerjee and Sinchini Barman},
journal = {ACS Omega},
year = {2026},
month = oct,
doi = {10.1021/acsomega.6c07950},
url = {https://pubs.acs.org/acsodf/article/doi/10.1021/acsomega.6c07950/5451899/Genetically-Expanded-Fluorescent-SSBT7-RNA},
note = {CC BY 4.0},
}
RIS
TY - JOUR
TI - Genetically Expanded Fluorescent SSBT7 RNA Polymerase in Action: Intriguing Photophysics with Enhanced RNA Production
AU - Subhendu Sekhar Bag
AU - Aniket Banerjee
AU - Sinchini Barman
PY - 2026
DA - 2026/10/15
JO - ACS Omega
DO - 10.1021/acsomega.6c07950
UR - https://pubs.acs.org/acsodf/article/doi/10.1021/acsomega.6c07950/5451899/Genetically-Expanded-Fluorescent-SSBT7-RNA
AB - T7 RNA polymerase (T7-RNAP) is a highly efficient transcriptional enzyme. However, it is prone to mutation at the functionally important Y571 residue, which strongly influences its functional properties. Here, we demonstrate a strategy to modulate the T7-RNAP enzyme by replacing the Y571 residue with a fluorescent triazolyl unnatural amino acid (FTUAA), TCNBAlaAc, via genetic code expansion (GCE) technology using two orthogonal aminoacyl-tRNA synthetase/tRNA pairs. Thus, TCNBAlaAc-encoded variants, herein termed SSBT7-RNAP, were successfully expressed, purified, and characterized by SDS-PAGE, MALDI-TOF mass spectrometry, CD, UV–vis, and fluorescence spectroscopy. Incorporation of TCNBAlaAc introduced distinct and enhanced fluorescence signatures while largely preserving the overall secondary structures of the engineered enzymes. Notably, these novel TCNBAlaAc-encoded variants exhibited higher relative RNA yields than those of wild-type T7-RNAP under identical in vitro transcription conditions. Molecular docking further supported the structural compatibility of the modification with the promoter-bound enzyme. These findings establish GCE-based, site-specific incorporation of an FTUAA into a functionally relevant site of T7 RNA polymerase as a promising strategy for generating spectroscopically traceable, transcriptionally competent T7-RNAP variants for future enzyme engineering and mechanistic studies.
SN - 2470-1343
ER -