< To RS/tRNA Database

pCNPhe RS1 (pCNF-RS; polyspecific RS)

RS/tRNA Foundational Publication Support

Schultz, Kathryn C, Lubica Supekova, Youngha Ryu, Jianming Xie, Roshan Perera, and Peter G Schultz. (2006) 2006. “A Genetically Encoded Infrared Probe.”. Journal Of The American Chemical Society 128 (43): 13984-5.
Young, Douglas D, Travis S Young, Michael Jahnz, Insha Ahmad, Glen Spraggon, and Peter G Schultz. (2011) 2011. “An Evolved Aminoacyl-Trna Synthetase With Atypical Polysubstrate Specificity.”. Biochemistry 50 (11): 1894-900. doi:10.1021/bi101929e.
Chatterjee, Abhishek, Sophie B Sun, Jennifer L Furman, Han Xiao, and Peter G Schultz. (2013) 2013. “A Versatile Platform For Single- And Multiple-Unnatural Amino Acid Mutagenesis In Escherichia Coli.”. Biochemistry 52 (10): 1828-37. doi:10.1021/bi4000244.
Wilkinson, Henry C, and Paul A Dalby. (2021) 2021. “Fine-Tuning The Activity And Stability Of An Evolved Enzyme Active-Site Through Noncanonical Amino-Acids.”. The Febs Journal 288 (6): 1935-1955. doi:10.1111/febs.15560.

RS/tRNA Usage Publications

Chen, Wan-Na, Kekini Kuppan, Michael Lee, Kristaps Jaudzems, Thomas Huber, and Gottfried Otting. (2015) 2015. “O-Tert-Butyltyrosine, An Nmr Tag For High-Molecular-Weight Systems And Measurements Of Submicromolar Ligand Binding Affinities.”. Journal Of The American Chemical Society 137 (13): 4581-6. doi:10.1021/jacs.5b01918.
Loh, Choy Theng, Luke A Adams, Bim Graham, and Gottfried Otting. (2018) 2018. “Genetically Encoded Amino Acids With Tert-Butyl And Trimethylsilyl Groups For Site-Selective Studies Of Proteins By Nmr Spectroscopy.”. Journal Of Biomolecular Nmr 71 (4): 287-293. doi:10.1007/s10858-017-0157-y.
Jabar, Shereen, Luke A Adams, Yao Wang, Luigi Aurelio, Bim Graham, and Gottfried Otting. (2017) 2017. “Chemical Tagging With Tert-Butyl And Trimethylsilyl Groups For Measuring Intermolecular Nuclear Overhauser Effects In A Large Protein-Ligand Complex.”. Chemistry (Weinheim An Der Bergstrasse, Germany) 23 (53): 13033-13036. doi:10.1002/chem.201703531.
RS/tRNA Pair Development Year
2006

ncAA(s) Incorporated

p-azido-L-phenylalanine (pAzF)

Image showing the ChemDraw Structure of p-azido-l-phenylalanine (pAzF)
ncAA Utility
Used as a photocrosslinker, allowing for crosslinking and bioorthogonal ligation of protein.

p-iodo-L-phenylalanine

p-Iodo-L-phenylalanine
ncAA Utility
Can be used for phasing in protein crystallography.

p-acetyl-L-phenylalanine

p-acetyl-l-phenylalanine
ncAA Utility
Site-directed spin labeling, electron paramagnetic resonance

O-allyl-L-tyrosine

O-Allyl-L-tyrosine
ncAA Utility
in E. coli expressed sfGFP2TAG at 10 mg/L using GMML media

BiphenylAla(BipAla)

BiphenylAla
ncAA Utility
Synthetase intermediate developed on way to BpyAla incorporation
RS Organism of Origin
Methanocaldococcus jannaschii
Parent RS
Tyr RS
RS Mutations
Y32L
L65V
F108W
D158G
I159A
tRNA Organism of Origin
Methanocaldococcus jannaschii
Parent tRNA
Tyr tRNA
tRNA Anticodon
CUA
Other tRNA Mutations
C17A
U17aG
U20C
G37A
U47G
RS/tRNA Availability
pULTRA-CNF plasmid with this RS is available as Addgene Plasmid #48215 (developed in 2013 foundational paper for use in dual encoding)
Used in what cell line?
DH10B
BL21(DE3)
C321.ΔA.exp
RS/tRNA Additional Notes
In presence of 2 mM pCNF, MS confirmed full-fidelity incorporation into Z-domain(7). Also installed into myoglobin(64) yielding 30 mg/L, the pCNF provided a useful infrared probe of ligand binding.

2011 foundational paper showed this RS to be unusually permissive (i.e. polyspecific) and identified 18 ncAAs that were all incorporated into myoglobin(107) at between 43 and 96% efficiency (w/ WT expression being ~16 mg/L). Also reported a crystal structure with bound pCNF.

In 2013, putting into pUltra plasmid led to 1.3-2-fold efficiency gains for various ncAAs.

2021 paper incorporated p-nitro, p-cyano and p-aminoPhe into a transketolase in BL21 and C321.ΔA.exp cells with careful MS analyses indicating both cell lines yielded roughly 80%, 75% and 50% fidelity, respectively.