Stop codon suppression using dedicated tRNA/aminoacyl-tRNA synthetase (aaRS) pairs allows for genetically encoded, site-specific incorporation of non-canonical amino acids (ncAAs) as chemical handles for protein labeling and modification. Here, we demonstrate that piggyBac-mediated genomic integration of archaeal pyrrolysine tRNA (tRNA)/pyrrolysyl-tRNA synthetase (PylRS) or bacterial tRNA/aaRS pairs, using a modular plasmid design with multi-copy tRNA arrays, allows for homogeneous and efficient genetically encoded ncAA incorporation in diverse mammalian cell lines. We assess opportunities and limitations of using ncAAs for fluorescent labeling applications in stable cell lines. We explore suppression of ochre and opal stop codons and finally incorporate two distinct ncAAs with mutually orthogonal click chemistries for site-specific, dual-fluorophore labeling of a cell surface receptor on live mammalian cells.
Download full-text PDF |
Source |
|---|---|
| http://www.ncbi.nlm.nih.gov/pmc/articles/PMC10694491 | PMC |
| http://dx.doi.org/10.1016/j.crmeth.2023.100626 | DOI Listing |
Publication Analysis
Top Keywords
Similar Publications
When "loss-of-function" means proteostasis burden: Thinking again about coding DNA variants.
Am J Hum Genet
January 2025
Department of Medicine, Indiana University School of Medicine, Indianapolis, IN, USA.
Each human genome has approximately 5 million DNA variants. Even for complete loss-of-function variants causing inherited, monogenic diseases, current understanding based on gene-specific molecular function does not adequately predict variability observed between people with identical mutations or fluctuating disease trajectories. We present a parallel paradigm for loss-of-function variants based on broader consequences to the cell when aberrant polypeptide chains of amino acids are translated from mutant RNA to generate mutated proteins.
View Article and Find Full Text PDFGenetically Recoding Respiratory Syncytial Virus to Visualize Nucleoprotein Dynamics and Virion Assembly.
ACS Infect Dis
January 2025
Department of Biomedical Engineering, Washington University in St. Louis, St. Louis, Missouri 63130, United States.
RNA viruses possess small genomes encoding a limited repertoire of essential and often multifunctional proteins. Although genetically tagging viral proteins provides a powerful tool for dissecting mechanisms of viral replication and infection, it remains a challenge. Here, we leverage genetic code expansion to develop a recoded strain of respiratory syncytial virus (RSV) in which the multifunctional nucleoprotein is site-specifically modified with a noncanonical amino acid.
View Article and Find Full Text PDFEngineered tRNAs efficiently suppress CDKL5 premature termination codons.
Sci Rep
December 2024
Department of Medical Biotechnology and Translational Medicine, University of Milan, Segrate (Milan), 20054, Italy.
The CDKL5 deficiency disorder (CDD) is a severe neurodevelopmental disorder characterized by early-onset epilepsy, intellectual disability, motor and visual dysfunctions. The causative gene is CDKL5, which codes for a kinase required for brain development. There is no cure for CDD patients; treatments are symptomatic and focus mainly on seizure control.
View Article and Find Full Text PDFLight-Activatable Ubiquitin for Studying Linkage-Specific Ubiquitin Chain Formation Kinetics.
Adv Sci (Weinh)
December 2024
Department of Chemistry and Chemical Biology, TU Dortmund University, Otto-Hahn Str. 4a, 44227, Dortmund, Germany.
Ubiquitination is a dynamic post-translational modification governing protein abundance, function, and localization in eukaryotes. The Ubiquitin protein is conjugated to lysine residues of target proteins, but can also repeatedly be ubiquitinated itself, giving rise to a complex code of ubiquitin chains with different linkage types. To enable studying the cellular dynamics of linkage-specific ubiquitination, light-activatable polyubiquitin chain formation is reported here.
View Article and Find Full Text PDFA frameshift mutation in resolves the growth versus defense dilemma in rice.
Proc Natl Acad Sci U S A
December 2024
State Key Laboratory of Rice Biology and Breeding, Zhejiang Key Laboratory of Biology and Ecological Regulation of Crop Pathogens and Insects, Institute of Insect Sciences, Zhejiang University, Hangzhou 310058, China.
CRISPR-Cas9 genome editing systems have revolutionized plant gene functional studies by enabling the targeted introduction of insertion-deletions (INDELs) via the nonhomologous end-joining (NHEJ) pathway. Frameshift-inducing INDELs can introduce a premature termination codon and, in other instances, can lead to the appearance of new proteins. Here, we found that mutations in the rice jasmonate (JA) signaling gene by CRISPR-Cas9-based genome editing did not affect canonical JA signaling.
View Article and Find Full Text PDFWant AI Summaries of new PubMed Abstracts delivered to your In-box?
Enter search terms and have AI summaries delivered each week - change queries or unsubscribe any time!