Biology subjects

Costello, A.

Publications and source records attributed to Costello, A..

3 recordsLinked to original sources

bioRxiv2026

Structural adaptations for enhanced translation kinetics in evolved ribosomes

The ribosomal RNA sequence governs translation dynamics, yet understanding how changes beyond the conserved catalytic centers influence kinetics and protein yield remains limited. Using orthogonal ribosome phage-assisted continuous evolution (oRibo-PACE), we recently reported chimeric ribosomes derived from Escherichia coli, Pseudomonas aeruginosa, and Vibrio cholerae endowed with elevated orthogonal translation activity as compared to their starting counterparts. Here, we structurally characterize these kinetically enhanced ribosomes using cryo-electron microscopy and uncover a potential relationship between 16S rRNA stability and translation efficiency. Compared to their naive starting points, evolved ribosomes exhibit extensive RNA structural adaptation, often introduced by mismatches at key helical junctions, which leads to local RNA-protein rearrangements and destabilizes non-canonical base pairs. Compensatory mutations that restore base-pairing stability and eliminate flexibility reduced translational activity to wild-type levels. Across trajectories, increased translational output correlates with subtle, localized changes in the 16S rRNA sequence that introduce limited structural destabilization at specific elements. Taken together, our work provides new insights into rRNA structural malleability and establishes principles for engineering ribosomes with altered translation properties. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=74 SRC="FIGDIR/small/706023v2_ufig1.gif" ALT="Figure 1"> View larger version (16K): org.highwire.dtl.DTLVardef@8678bdorg.highwire.dtl.DTLVardef@60cde5org.highwire.dtl.DTLVardef@16db00dorg.highwire.dtl.DTLVardef@413ed5_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2024

Efficient Genetic Code Expansion Without Host Genome Modifications

Supplementing translation with non-canonical amino acids (ncAAs) can yield protein sequences with new-to-nature functions, but existing ncAA incorporation strategies suffer from low efficiency and context dependence. We uncover codon usage as a previously unrecognized contributor to efficient genetic code expansion using non-native codons. Relying only on conventional E. coli strains with native ribosomes, we develop a novel plasmid-based codon compression strategy that minimizes context dependence and improves ncAA incorporation at quadruplet codons. We confirm that this strategy is compatible with all known genetic code expansion resources, which allows us to identify 12 mutually orthogonal tRNA-synthetase pairs. Enabled by these findings, we evolve and optimize five tRNA-synthetase pairs to incorporate a broad repertoire of ncAAs at orthogonal quadruplet codons. Finally, we extend these resources to an in vivo biosynthesis platform that can readily create >100 new-to-nature peptide macrocycles bearing up to three unique ncAAs. Given the generality of our approach and streamlined resources, our findings will accelerate innovations in multiplexed genetic code expansion and enable the discovery of chemically diverse biomolecules for researcher-defined applications.

synthetic biology↗

bioRxiv2019

Sub physiological temperature induces pervasive alternative splicing in Chinese hamster ovary cells

RNA sequencing (RNASeq) has been widely used to associate alterations in Chinese hamster ovary (CHO) cell gene expression with bioprocess phenotypes, however alternative mRNA splicing, has thus far, received little attention. In this study, we utilised RNASeq for transcriptomic analysis of a monoclonal antibody producing CHOK1 cell line subjected to a temperature shift. More than 2,365 instances of differential splicing were observed 24hrs after the reduction of cell culture temperature. 1,163 of these alternative splicing events were identified in genes where no changes in abundance were detected by standard differential expression analysis. Ten examples of alternative splicing were selected for independent validation using qPCR in the monoclonal antibody producing CHOK1 line used for RNASeq and a further 2 CHOK1 cell lines. This analysis provided evidence that exon skipping and mutually exclusive splicing events occur in genes linked to the cellular response to changes in temperature and mitochondrial function. While further work is required to determine the impact of changes in mRNA sequence on cellular phenotype, this study demonstrates that alternative splicing analysis can be utilised to gain a deeper understanding of post-transcriptional regulation in CHO cells during biopharmaceutical production.

cell biology↗