An official website of the United States government
Abstract Using genetic code expansion (GCE) to encode bioorthogonal chemistry has emerged as a promising method for protein labeling, both in vitro and within cells. Here, we demonstrate that tetrazine amino acids incorporated into proteins are highly tunable and have extraordinary potential for fast and quantitative bioorthogonal ligations. We describe the synthesis and characterize reaction rates of 29 tetrazine amino acids (20 of which are new) and compare their encoding ability into proteins using evolved Tet ncAA encoding tRNA/RS pairs. For these systems, we characterized on-protein Tet stability, reaction rates, and ligation extents, as the utility of a bioorthogonal labeling group depends on its stability and reactivity when encoded into proteins. By integrating data on encoding efficiency, selectivity, on-protein stability, and in-cell labeling for Tet tRNA/RS pairs, we developed the smallest, fastest, and most stable Tet system to date. This was achieved by introducing fluorine substituents to Tet4, resulting in reaction rates at the 10⁶ M⁻¹s⁻¹ level while minimizing degradation. This study expands the toolbox of bioorthogonal reagents for Tet-sTCO-based, site-specific protein labeling and demonstrates that the Tet-ncAA is a uniquely tunable, highly reactive, and encodable bioorthogonal functional group. These findings provide a foundation to further explore Tet-ncAA encoding and reactivity.
more »
« less
Enhanced Directed Evolution in Mammalian Cells Yields a Hyperefficient Pyrrolysyl tRNA for Noncanonical Amino Acid Mutagenesis
Abstract Heterologous tRNAs used for noncanonical amino acid (ncAA) mutagenesis in mammalian cells typically show poor activity. We recently introduced a virus‐assisted directed evolution strategy (VADER) that can enrich improved tRNA mutants from naïve libraries in mammalian cells. However, VADER was limited to processing only a few thousand mutants; the inability to screen a larger sequence space precluded the identification of highly active variants with distal synergistic mutations. Here, we report VADER2.0, which can process significantly larger mutant libraries. It also employs a novel library design, which maintains base‐pairing between distant residues in the stem regions, allowing us to pack a higher density of functional mutants within a fixed sequence space. VADER2.0 enabled simultaneous engineering of the entire acceptor stem ofM. mazeipyrrolysyl tRNA (tRNAPyl), leading to a remarkably improved variant, which facilitates more efficient incorporation of a wider range of ncAAs, and enables facile development of viral vectors and stable cell‐lines for ncAA mutagenesis.
more »
« less
- Award ID(s):
- 1817893
- PAR ID:
- 10535013
- Publisher / Repository:
- Wiley
- Date Published:
- Journal Name:
- Angewandte Chemie International Edition
- ISSN:
- 1433-7851
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
While the centrality of posttranscriptional modifications to RNA biology has long been acknowledged, the function of the vast majority of modified sites remains to be discovered. Illustrative of this, there is not yet a discrete biological role assigned for one of the most highly conserved modifications, 5-methyluridine at position 54 in tRNAs (m5U54). Here, we uncover contributions of m5U54 to both tRNA maturation and protein synthesis. Our mass spectrometry analyses demonstrate that cells lacking the enzyme that installs m5U in the T-loop (TrmA inEscherichia coli, Trm2 inSaccharomyces cerevisiae) exhibit altered tRNA modification patterns. Furthermore, m5U54-deficient tRNAs are desensitized to small molecules that prevent translocation in vitro. This finding is consistent with our observations that relative to wild-type cells,trm2Δcell growth and transcriptome-wide gene expression are less perturbed by translocation inhibitors. Together our data suggest a model in which m5U54 acts as an important modulator of tRNA maturation and translocation of the ribosome during protein synthesis.more » « less
-
Abstract BackgroundFolate is an essential B-group vitamin and a key methyl donor with important biological functions including DNA methylation regulation. Normal neurodevelopment and physiology are sensitive to the cellular folate levels. Either deficiency or excess of folate may lead to neurological disorders. Recently, folate has been linked to tRNA cytosine-5 methylation (m5C) and translation in mammalian mitochondria. However, the influence of folate intake on neuronal mRNA m5C modification and translation remains largely unknown. Here, we provide transcriptome-wide landscapes of m5C modification in poly(A)-enriched RNAs together with mRNA transcription and translation profiles for mouse neural stem cells (NSCs) cultured in three different concentrations of folate. ResultsNSCs cultured in three different concentrations of folate showed distinct mRNA methylation profiles. Despite uncovering only a few differentially expressed genes, hundreds of differentially translated genes were identified in NSCs with folate deficiency or supplementation. The differentially translated genes induced by low folate are associated with cytoplasmic translation and mitochondrial function, while the differentially translated genes induced by high folate are associated with increased neural stem cell proliferation. Interestingly, compared to total mRNAs, polysome mRNAs contained high levels of m5C. Furthermore, an integrative analysis indicated a transcript-specific relationship between RNA m5C methylation and mRNA translation efficiency. ConclusionsAltogether, our study reports a transcriptome-wide influence of folate on mRNA m5C methylation and translation in NSCs and reveals a potential link between mRNA m5C methylation and mRNA translation.more » « less
-
While the centrality of post-transcriptional modifications to RNA biology has long been acknowledged, the function of the vast majority of modified sites remains to be discovered. Illustrative of this, there is not yet a discrete biological role assigned for one the most highly conserved modifications, 5-methyluridine at position 54 in tRNAs (m5U54). Here, we uncover contributions of m5U54 to both tRNA maturation and protein synthesis. Our mass spectrometry analyses demonstrate that cells lacking the enzyme that installs m5U in the T-loop (TrmA inE. coli, Trm2 inS. cerevisiae) exhibit altered tRNA modifications patterns. Furthermore, m5U54 deficient tRNAs are desensitized to small molecules that prevent translocationin vitro.This finding is consistent with our observations that, relative to wild-type cells,trm2Δ cell growth and transcriptome-wide gene expression are less perturbed by translocation inhibitors. Together our data suggest a model in which m5U54 acts as an important modulator of tRNA maturation and translocation of the ribosome during protein synthesis.more » « less
-
Summary Alfalfa (Medicago sativaL.) is a perennial flowering plant in the legume family that is widely cultivated as a forage crop for its high yield, forage quality and related agricultural and economic benefits. Alfalfa is a photoperiod sensitive long‐day (LD) plant that can accomplish its vegetative and reproductive phases in a short period of time. However, rapid flowering can compromise forage biomass yield and quality. Here, we attempted to delay flowering in alfalfa using multiplex CRISPR/Cas9‐mediated mutagenesis ofFLOWERING LOCUS Ta1(MsFTa1), a key floral integrator and activator gene. Four guide RNAs (gRNAs) were designed and clustered in a polycistronic tRNA–gRNA system and introduced into alfalfa byAgrobacterium‐mediated transformation. Ninety‐six putative mutant lines were identified by gene sequencing and characterized for delayed flowering time and related desirable agronomic traits. Phenotype assessment of flowering time under LD conditions identified 22 independent mutant lines with delayed flowering compared to the control. Six independentMsfta1lines containing mutations in all four copies ofMsFTa1accumulated significantly higher forage biomass yield, with increases of up to 78% in fresh weight and 76% in dry weight compared to controls. Depending on the harvesting schemes, many of these lines also had reduced lignin, acid detergent fibre (ADF) and neutral detergent fibre (NDF) content and significantly higher crude protein (CP) and mineral contents compared to control plants, especially in the stems. These CRISPR/Cas9‐editedMsfta1mutants could be introduced in alfalfa breeding programmes to generate elite transgene‐free alfalfa cultivars with improved forage biomass yield and quality.more » « less
- Free Publicly Accessible Full Text
-
Accepted Manuscript
- Journal Article:
- https://doi.org/10.1002/anie.202316428
