scholarly journals Optical Control of DNA Helicase Function through Genetic Code Expansion

ChemBioChem ◽  
2017 ◽  
Vol 18 (5) ◽  
pp. 466-469 ◽  
Author(s):  
Ji Luo ◽  
Muwen Kong ◽  
Lili Liu ◽  
Subhas Samanta ◽  
Bennett Van Houten ◽  
...  
2021 ◽  
Author(s):  
Zhiyan Xi ◽  
Lloyd Davis ◽  
Kieran Baxter ◽  
Ailish Tynan ◽  
Angeliki Goutou ◽  
...  

Genetic code expansion in multicellular organisms is currently limited to the use of repurposed amber stop codons. Here we introduce a system for the use of quadruplet codons to direct incorporation of non-canonical amino acids in vivo in an animal, the nematode worm Caenorhabditis elegans. We develop hybrid pyrrolysyl tRNA variants to incorporate non-canonical amino acids in response to the quadruplet codon UAGA. We demonstrate the efficiency of the quadruplet decoding system by incorporating photocaged amino acids into two proteins widely used as genetic tools. We use photocaged lysine to express photocaged Cre recombinase for the optical control of gene expression and photocaged cysteine to express photo-activatable caspase for light inducible cell ablation. Our approach will facilitate the routine adoption of quadruplet decoding for genetic code expansion in eukaryotic cells and multicellular organisms.


2017 ◽  
Vol 139 (27) ◽  
pp. 9100-9103 ◽  
Author(s):  
Jihe Liu ◽  
James Hemphill ◽  
Subhas Samanta ◽  
Michael Tsang ◽  
Alexander Deiters

Author(s):  
Tatsuo Yanagisawa ◽  
Mitsuo Kuratani ◽  
Eiko Seki ◽  
Nobumasa Hino ◽  
Kensaku Sakamoto ◽  
...  

Amino Acids ◽  
2020 ◽  
Author(s):  
Thomas L. Williams ◽  
Debra J. Iskandar ◽  
Alexander R. Nödling ◽  
Yurong Tan ◽  
Louis Y. P. Luk ◽  
...  

AbstractGenetic code expansion is a powerful technique for site-specific incorporation of an unnatural amino acid into a protein of interest. This technique relies on an orthogonal aminoacyl-tRNA synthetase/tRNA pair and has enabled incorporation of over 100 different unnatural amino acids into ribosomally synthesized proteins in cells. Pyrrolysyl-tRNA synthetase (PylRS) and its cognate tRNA from Methanosarcina species are arguably the most widely used orthogonal pair. Here, we investigated whether beneficial effect in unnatural amino acid incorporation caused by N-terminal mutations in PylRS of one species is transferable to PylRS of another species. It was shown that conserved mutations on the N-terminal domain of MmPylRS improved the unnatural amino acid incorporation efficiency up to five folds. As MbPylRS shares high sequence identity to MmPylRS, and the two homologs are often used interchangeably, we examined incorporation of five unnatural amino acids by four MbPylRS variants at two temperatures. Our results indicate that the beneficial N-terminal mutations in MmPylRS did not improve unnatural amino acid incorporation efficiency by MbPylRS. Knowledge from this work contributes to our understanding of PylRS homologs which are needed to improve the technique of genetic code expansion in the future.


2021 ◽  
Vol 120 (3) ◽  
pp. 338a
Author(s):  
Matthew L. Rook ◽  
Tyler A. Couch ◽  
Jackson Hernandez ◽  
Alison J. Frontier ◽  
David M. MacLean

2018 ◽  
Vol 46 ◽  
pp. 146-155 ◽  
Author(s):  
Yonatan Chemla ◽  
Eden Ozer ◽  
Itay Algov ◽  
Lital Alfonta

2021 ◽  
Author(s):  
Thomas Williams ◽  
Yu-Hsuan Tsai ◽  
Louis Luk

Abstract Here, incorporation of secondary amine by genetic code expansion was used to expand the potential protein templates for artificial enzyme design. Pyrrolysine analogue containing a D-proline could be stably incorporated into proteins, including the multidrug-binding LmrR and nucleotide-binding dihydrofolate reductase (DHFR). Both modified scaffolds were catalytically active, mediating transfer hydrogenation with a relaxed substrate scope. The protein templates played a distinctive role in that, while the LmrR variants were confined to the biomimetic BNAH as the hydride source, the optimal DHFR variant favorably used the pro-R hydride from NADPH for reactions. Due to the cofactor compatibility, the DHFR secondary amine catalysis could also be coupled to an enzymatic recycling scheme. This work has illustrated the unique advantages of using proteins as hosts, and thus the presented concept is expected to find uses in enabling tailored secondary amine catalysis.


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