Directed Evolution of a Bacterial Leucyl tRNA in Mammalian Cells for Enhanced Noncanonical Amino Acid Mutagenesis.

The Escherichia coli leucyl-tRNA synthetase (EcLeuRS)/tRNAEcLeu pair has been engineered to genetically encode a structurally diverse group of enabling noncanonical amino acids (ncAAs) in eukaryotes, including those with bioconjugation handles, environment-sensitive fluorophores, photocaged amino acids, and native post-translational modifications. However, the scope of this toolbox in mammalian cells is limited by the poor activity of tRNAEcLeu. Here, we overcome this limitation by evolving tRNAEcLeu directly in mammalian cells by using a virus-assisted selection scheme. This directed evolution platform was optimized for higher throughput such that the entire acceptor stem of tRNAEcLeu could be simultaneously engineered, which resulted in the identification of several variants with remarkably improved efficiency for incorporating a wide range of ncAAs. The advantage of the evolved leucyl tRNAs was demonstrated by expressing ncAA mutants in mammalian cells that were challenging to express before using the wild-type tRNAEcLeu, by creating viral vectors that facilitated ncAA mutagenesis at a significantly lower dose and by creating more efficient mammalian cell lines stably expressing the ncAA-incorporation machinery.

Enhanced Directed Evolution in Mammalian Cells Yields a Hyperefficient Pyrrolysyl tRNA for Noncanonical Amino Acid Mutagenesis.

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 of M. mazei pyrrolysyl 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.

Electrochemical labelling of hydroxyindoles with chemoselectivity for site-specific protein bioconjugation.

Electrochemistry has recently emerged as a powerful approach in small-molecule synthesis owing to its numerous attractive features, including precise control over the fundamental reaction parameters, mild reaction conditions and innate scalability. Even though these advantages also make it an attractive strategy for chemoselective modification of complex biomolecules such as proteins, such applications remain poorly developed. Here we report an electrochemically promoted coupling reaction between 5-hydroxytryptophan (5HTP) and simple aromatic amines-electrochemical labelling of hydroxyindoles with chemoselectivity (eCLIC)-that enables site-specific labelling of full-length proteins under mild conditions. Using genetic code expansion technology, the 5HTP residue can be incorporated into predefined sites of a recombinant protein expressed in either prokaryotic or eukaryotic hosts for subsequent eCLIC labelling. We used the eCLIC reaction to site-specifically label various recombinant proteins, including a full-length human antibody. Furthermore, we show that eCLIC is compatible with strain-promoted alkyne-azide and alkene-tetrazine click reactions, enabling site-specific modification of proteins at two different sites with distinct labels.

Virus-assisted directed evolution of biomolecules.

Directed evolution is a powerful technique that uses principles of natural evolution to enable the development of biomolecules with novel functions. However, the slow pace of natural evolution does not support the demand for rapidly generating new biomolecular functions in the laboratory. Viruses offer a unique path to design fast laboratory evolution experiments, owing to their innate ability to evolve much more rapidly than most living organisms, facilitated by a smaller genome size that tolerate a high frequency of mutations, as well as a fast rate of replication. These attributes offer a great opportunity to evolve various biomolecules by linking their activity to the replication of a suitable virus. This review highlights the recent advances in the application of virus-assisted directed evolution of designer biomolecules in both prokaryotic and eukaryotic cells.

Photoredox-Catalyzed Labeling of Hydroxyindoles with Chemoselectivity (PhotoCLIC) for Site-Specific Protein Bioconjugation.

We have developed a novel visible-light-catalyzed bioconjugation reaction, PhotoCLIC, that enables chemoselective attachment of diverse aromatic amine reagents onto a site-specifically installed 5-hydroxytryptophan residue (5HTP) on full-length proteins of varied complexity. The reaction uses catalytic amounts of methylene blue and blue/red light-emitting diodes (455/650 nm) for rapid site-specific protein bioconjugation. Characterization of the PhotoCLIC product reveals a unique structure formed likely through a singlet oxygen-dependent modification of 5HTP. PhotoCLIC has a wide substrate scope and its compatibility with strain-promoted azide-alkyne click reaction, enables site-specific dual-labeling of a target protein.

An Efficient Opal-Suppressor Tryptophanyl Pair Creates New Routes for Simultaneously Incorporating up to Three Distinct Noncanonical Amino Acids into Proteins in Mammalian Cells.

Site-specific incorporation of multiple distinct noncanonical amino acids (ncAAs) into proteins in mammalian cells is a promising technology, where each ncAA must be assigned to a different orthogonal aminoacyl-tRNA synthetase (aaRS)/tRNA pair that reads a distinct nonsense codon. Available pairs suppress TGA or TAA codons at a considerably lower efficiency than TAG, limiting the scope of this technology. Here we show that the E. coli tryptophanyl (EcTrp) pair is an excellent TGA-suppressor in mammalian cells, which can be combined with the three other established pairs to develop three new routes for dual-ncAA incorporation. Using these platforms, we site-specifically incorporated two different bioconjugation handles into an antibody with excellent efficiency, and subsequently labeled it with two distinct cytotoxic payloads. Additionally, we combined the EcTrp pair with other pairs to site-specifically incorporate three distinct ncAAs into a reporter protein in mammalian cells.

Virus-assisted directed evolution of enhanced suppressor tRNAs in mammalian cells.

Site-specific incorporation of unnatural amino acids (Uaas) in living cells relies on engineered aminoacyl-transfer RNA synthetase-tRNA pairs borrowed from a distant domain of life. Such heterologous suppressor tRNAs often have poor intrinsic activity, presumably due to suboptimal interaction with a non-native translation system. This limitation can be addressed in Escherichia coli using directed evolution. However, no suitable selection system is currently available to do the same in mammalian cells. Here we report virus-assisted directed evolution of tRNAs (VADER) in mammalian cells, which uses a double-sieve selection scheme to facilitate single-step enrichment of active yet orthogonal tRNA mutants from naive libraries. Using VADER we developed improved mutants of Methanosarcina mazei pyrrolysyl-tRNA, as well as a bacterial tyrosyl-tRNA. We also show that the higher activity of the most efficient mutant pyrrolysyl-tRNA is specific for mammalian cells, alluding to an improved interaction with the unique mammalian translation apparatus.

d-3-Deoxy-dioctanoylphosphatidylinositol induces cytotoxicity in human MCF-7 breast cancer cells via a mechanism that involves downregulation of the D-type cyclin-retinoblastoma pathway.

Phosphatidylinositol analogs (PIAs) were originally designed to bind competitively to the Akt PH domain and prevent membrane translocation and activation. d-3-Deoxy-dioctanoylphosphatidylinositol (d-3-deoxy-diC8PI), but not compounds with altered inositol stereochemistry (e.g., l-3-deoxy-diC8PI and l-3,5-dideoxy-diC8PI), is cytotoxic. However, high resolution NMR field cycling relaxometry shows that both cytotoxic and non-toxic PIAs bind to the Akt1 PH domain at the site occupied by the cytotoxic alkylphospholipid perifosine. This suggests that another mechanism for cytotoxicity must account for the difference in efficacy of the synthetic short-chain PIAs. In MCF-7 breast cancer cells, with little constitutively active Akt, d-3-deoxy-diC8PI (but not l-compounds) decreases viability concomitant with increased cleavage of PARP and caspase 9, indicative of apoptosis. d-3-Deoxy-diC8PI also induces a decrease in endogenous levels of cyclins D1 and D3 and blocks downstream retinoblastoma protein phosphorylation. siRNA-mediated depletion of cyclin D1, but not cyclin D3, reduces MCF-7 cell proliferation. Thus, growth arrest and cytotoxicity induced by the soluble d-3-deoxy-diC8PI occur by a mechanism that involves downregulation of the D-type cyclin-pRb pathway independent of its interaction with Akt. This ability to downregulate D-type cyclins contributes, at least in part, to the anti-proliferative activity of d-3-deoxy-diC8PI and may be a common feature of other cytotoxic phospholipids.