Abhishek Chatterjee

A Bacterial Strain with a Unique Quadruplet Codon Specifying Non‐native Amino Acids

The addition of noncanonical amino acids to the genetic code requires unique codons not assigned to the 20 canonical amino acids. Among the 64 triplet codons, only the three nonsense “stop” codons have been used to encode non‐native amino acids. Use of quadruplet “frame‐shift” suppressor codons provides an abundant alternative but suffers from low suppression efficiency as a result of competing recognition of their first three bases by endogenous host tRNAs or release factors. Deletion of release factor 1 in a genomically recoded strain of E. coli (E. coli C321), in which all endogenous amber stop codons (UAG) are replaced with UAA, abolished UAG mediated translation termination. Here we show that a Methanocaldococcus jannaschii‐derived frame‐shift suppressor tRNA/aminoacyl‐tRNA synthetase pair enhanced UAGN suppression efficiency in this recoded bacterial strain. These results demonstrate that efficient quadruplet codons for encoding non‐native amino acids can be generated by eliminating competing triplet codon recognition at the ribosome.

A Precise Chemical Strategy To Alter the Receptor Specificity of the Adeno‐Associated Virus

The ability to target the adeno-associated virus (AAV) to specific types of cells, by altering the cell-surface receptor it binds, is desirable to generate safe and efficient therapeutic vectors. Chemical attachment of receptor-targeting agents onto the AAV capsid holds potential to alter its tropism, but is limited by the lack of site specificity of available conjugation strategies. The development of an AAV production platform is reported that enables incorporation of unnatural amino acids (UAAs) into specific sites on the virus capsid. Incorporation of an azido-UAA enabled site-specific attachment of a cyclic-RGD peptide onto the capsid, retargeting the virus to the αv β3 integrin receptors, which are overexpressed in tumor vasculature. Retargeting ability was site-dependent, underscoring the importance of achieving site-selective capsid modification. This work provides a general chemical approach to introduce various receptor binding agents onto the AAV capsid with site selectivity to generate optimized vectors with engineered infectivity.

Expanding the genetic code of mammalian cells

In the last two decades, unnatural amino acid (UAA) mutagenesis has emerged as a powerful new method to probe and engineer protein structure and function. This technology enables precise incorporation of a rapidly expanding repertoire of UAAs into predefined sites of a target protein expressed in living cells. Owing to the small footprint of these genetically encoded UAAs and the large variety of enabling functionalities they offer, this technology has tremendous potential for deciphering the delicate and complex biology of the mammalian cells. Over the last few years, exciting progress has been made toward expanding the toolbox of genetically encoded UAAs in mammalian cells, improving the efficiency of their incorporation and developing innovative applications. Here, we provide our perspective on these recent developments and highlight the current challenges that must be overcome to realize the full potential of this technology.

A unique genetically encoded FRET pair in mammalian cells.

Förster resonance energy transfer (FRET) between two suitable fluorophores is a powerful tool to monitor dynamic changes in protein structure in vitro and in vivo. The ability to genetically encode a FRET pair represents a convenient “labeling‐free” strategy to incorporate them into target protein(s). Currently, the only genetically encoded FRET pairs available for use in mammalian cells use fluorescent proteins. However, their large size can lead to unfavorable perturbations, particularly when two are used at the same time. Additionally, fluorescent proteins are largely restricted to a terminal attachment to the target, which might not be optimal. Here, we report the development of an alternative genetically encoded FRET pair in mammalian cells that circumvents these challenges by taking advantage of a small genetically encoded fluorescent unnatural amino acid as the donor and enhanced green fluorescent protein (EGFP) as the acceptor. The small size of Anap relative to fluorescent proteins, and the ability to co‐translationally incorporate it into internal sites on the target protein, endows this novel FRET pair with improved versatility over its counterparts that rely upon two fluorescent proteins.

Production and Chemoselective Modification of Adeno-Associated Virus Site-Specifically Incorporating an Unnatural Amino Acid Residue into Its Capsid

The ability to modify the capsid proteins of human viruses is desirable both for installing probes to study their structure and function, and to attach retargeting agents to engineer viral infectivity. However, the installation of such capsid modifications currently faces two major challenges: (1) The complex and delicate capsid proteins often do not tolerate large modifications, and (2) capsid proteins are composed of the 20 canonical amino acids, precluding site-specific chemical modification of the virus. Here, we describe a technology for generating adeno-associated virus (AAV) while incorporating an unnatural amino acid (UAA) into specific sites of the virus capsid. Incorporation of this UAA is generally tolerated well, presumably due to its small structural footprint. The resulting virus can be precisely functionalized at the site of UAA incorporation using chemoselective conjugation strategies targeted toward the azido side chain of this UAA. This technology provides a powerful way to modify AAV with unprecedented precision to both probe and engineer its entry process.