Making Sense of “Nonsense” and More: Challenges and Opportunities in the Genetic Code Expansion, in the World of tRNA Modifications

Abstract

The evolutional development of the RNA translation process that leads to protein synthesis based on naturally occurring amino acids has its continuation via synthetic biology, the so-called rational bioengineering. Genetic code expansion (GCE) explores beyond the natural translational processes to further enhance the structural properties and augment the functionality of a wide range of proteins. Prokaryotic and eukaryotic ribosomal machinery have been proven to accept engineered tRNAs from orthogonal organisms to efficiently incorporate noncanonical amino acids (ncAAs) with rationally designed side chains. These side chains can be reactive or functional groups, which can be extensively utilized in biochemical, biophysical, and cellular studies. Genetic code extension offers the contingency of introducing more than one ncAA into protein through frameshift suppression, multi-site-specific incorporation of ncAAs, thereby increasing the vast number of possible applications. However, different mediating factors reduce the yield and efficiency of ncAA incorporation into synthetic proteins. In this review, we comment on the recent advancements in genetic code expansion to signify the relevance of systems biology in improving ncAA incorporation efficiency. We discuss the emerging impact of tRNA modifications and metabolism in protein design. We also provide examples of the latest successful accomplishments in synthetic protein therapeutics and show how codon expansion has been employed in various scientific and biotechnological applications.

EpisomiR, a New Family of miRNAs, and Its Possible Roles in Human Diseases

“…Thus, the miRNA family is expanding [ 88 ]. In this article, we describe episomiRs as the miRNAs with sequence modifications that cannot be identified by standard methods, such as Sanger’s and Illumina sequencing, but which might be responsible for their functioning, similar to the role of tRNA modifications and metabolism in protein design [ 89 ]. The modifications of miRNAs include N6-methyladenosine (m6A) [ 90 ], 5-methylcytosine (5mC) [ 91 ], and 7-methylguanosine (m7G) [ 92 ].…”

SynBio 2.0, a new era for synthetic life: Neglected essential functions for resilience

“…This goes hand in hand with orthogonal implementation of specific replication/transcription and translation pathways. Various experiments have modified the (almost) universal codon usage table to allow the introduction of non‐canonical amino acids into proteins, limited to a fraction of the genome chassis (de la Torre & Chin, 2021; Lateef et al, 2022; Tang et al, 2022). Exploration of the codon usage table modification has also led to experiments where the table has been consistently reduced throughout a genome, thus representing a global recoding of living cells (Fredens et al, 2019).…”

Posttranslational Chemical Mutagenesis Methods to Insert Posttranslational Modifications into Recombinant Proteins

“…However, in vitro enzymatic modification of isolated proteins is challenging, as it often results in a heterogeneous product due to insufficient specificity and a lack of control over the degree of modification. Other biological approaches, such as genetic code expansion technology, allow site-specific incorporation of unnatural amino acids into any protein of interest in cells using engineered orthogonal aminoacyl-tRNA synthetase/tRNA [ 5 , 6 ]. Importantly, this strategy has enabled selective insertion of several PTMs into recombinant proteins, including methylation, acetylation, nitration, and phosphorylation.…”

“…Importantly, this strategy has enabled selective insertion of several PTMs into recombinant proteins, including methylation, acetylation, nitration, and phosphorylation. Despite the enormous potential of this approach, this method relies on the availability of an orthogonal tRNA synthetase for desired modification, and it is difficult to apply for transferring multiple PTMs into the same protein [ 5 , 6 ].…”

“…Molecules 2022, 27, x FOR PEER REVIEW 2 of 16 potential of this approach, this method relies on the availability of an orthogonal tRNA synthetase for desired modification, and it is difficult to apply for transferring multiple PTMs into the same protein [5,6]. Chemical protein synthesis provides a powerful means to prepare native and modified proteins in an effective and controlled manner [7,8].…”