Masahiko Sisido

Incorporation of non-natural amino acids into proteins

Chemical and biological diversity of protein structures and functions can be widely expanded by position-specific incorporation of non-natural amino acids carrying a variety of specialty side groups. After the pioneering works of Schultzs group and Chamberlins group in 1989, noticeable progress has been made in expanding types of amino acids, in finding novel methods of tRNA aminoacylation and in extending genetic codes for directing the positions. Aminoacylation of tRNA with non-natural amino acids has been achieved by directed evolution of aminoacyl-tRNA synthetases or some ribozymes. Codons have been extended to include four-base codons or non-natural base pairs. Multiple incorporation of different non-natural amino acids has been achieved by the use of a different four-base codon for each tRNA. The combination of these novel techniques has opened the possibility of synthesising non-natural mutant proteins in living cells.

FRET analysis of protein conformational change through position-specific incorporation of fluorescent amino acids.

We designed and synthesized new, fluorescent, non-natural amino acids that emit fluorescence of wavelengths longer than 500 nm and are accepted by an Escherichia coli cell-free translation system. We synthesized p-aminophenylalanine derivatives linked with BODIPY fluorophores at the p-amino group and introduced them into streptavidin using the four-base codon CGGG in a cell-free translation system. Practically, the incorporation efficiency was high enough for BODIPYFL, BODIPY558 and BODIPY576. Next, we incorporated BODIPYFL-aminophenylalanine and BODIPY558-aminophenylalanine into different positions of calmodulin as a donor and acceptor pair for fluorescence resonance energy transfer (FRET) using two four-base codons. Fluorescence spectra and polarization measurements revealed that substantial FRET changes upon the binding of calmodulin-binding peptide occurred for the double-labeled calmodulins containing BODIPY558 at the N terminus and BODIPYFL at the Gly40, Phe99 and Leu112 positions. These results demonstrate the usefulness of FRET based on the position-specific double incorporation of fluorescent amino acids for analyzing conformational changes of proteins.

Random insertion and deletion of arbitrary number of bases for codon-based random mutation of DNAs

A general method was developed for the construction of a library of mutant genes. The method, termed random insertion/deletion (RID) mutagenesis, enables deletion of an arbitrary number of consecutive bases at random positions and, at the same time, insertion of a specific sequence or random sequences of an arbitrary number into the same position. The applicability of the RID mutagenesis was demonstrated by replacing three randomly selected consecutive bases by the BglII recognition sequence (AGATCT) in the GFPUV gene. In addition, the randomly selected three bases were replaced by a mixture of 20 codons. These mutants were expressed in Escherichia coli, and those that showed fluorescence properties different from the wild-type GFP were selected. A yellow fluorescent protein and an enhanced green fluorescent protein, neither of which could be obtained by error-prone PCR mutagenesis, were found among the six mutants selected. Several mutants of the DsRed protein that show different fluorescence properties were also obtained.

A non‐natural amino acid for efficient incorporation into proteins as a sensitive fluorescent probe

A small and highly fluorescent non‐natural amino acid that contains an anthraniloyl group (atnDap) was incorporated into various positions of streptavidin. The positions were directed by a CGGG/CCCG four‐base codon/anticodon pair. The non‐natural mutants were obtained in excellent yields and some of them retained strong biotin‐binding activity. The fluorescence wavelength as well as the intensity of the anthraniloyl group at position 120 were sensitive to biotin binding. These unique properties indicate that the atnDap is the most suitable non‐natural amino acid for a position‐specific fluorescent labeling of proteins that is highly sensitive to microenvironmental changes.

Position-specific incorporation of dansylated non-natural amino acids into streptavidin by using a four-base codon

Novel non‐natural amino acids carrying a dansyl fluorescent group were designed, synthesized, and incorporated into various positions of streptavidin by using a CGGG four‐base codon in an Escherichia coli in vitro translation system. 2,6‐Dansyl‐aminophenylalanine (2,6‐dnsAF) was found to be incorporated into the protein more efficiently than 1,5‐dansyl‐lysine, 2,6‐dansyl‐lysine, and 1,5‐dansyl‐aminophenylalanine. Fluorescence measurements indicate that the position‐specific incorporation of the 2,6‐dnsAF is a useful technique to probe protein structures. These results also indicate that well‐designed non‐natural amino acids carrying relatively large side chains can be accepted as substrates of the translation system.

Photoswitching of peroxidase activity by position-specific incorporation of a photoisomerizable non-natural amino acid into horseradish peroxidase

Horseradish peroxidase mutants containing L‐p‐phenylazophenylalanine (azoAla) at various positions were synthesized by using an Escherichia coli in vitro translation system. Among the 15 mutants examined, four mutants containing a single azoAla unit at the 6th, 68th, 142nd, and 179th positions, respectively, retained the peroxidase activity. The activity of the Phe68azoAla mutant was higher when the azobenzene group was in the cis form than in the trans form. On the contrary, the activity of the Phe179azoAla mutant disappeared when the azobenzene group was photoisomerized to the cis form, but recovered in the trans form. In the latter mutant, therefore, an on/off photoswitching of the peroxidase activity was attained.

Multiple incorporation of non‐natural amino acids into a single protein using tRNAs with non‐standard structures

The ability to introduce non‐natural amino acids into proteins opens up new vistas for the study of protein structure and function. This approach requires suppressor tRNAs that deliver the non‐natural amino acid to a ribosome associated with an mRNA containing an expanded codon. The suppressor tRNAs must be absolutely protected from aminoacylation by any of the aminoacyl‐tRNA synthetases in the protein synthesizing system, or a natural amino acid will be incorporated instead of the non‐natural amino acid. Here, we found that some tRNAs with non‐standard structures could work as efficient four‐base suppressors fulfilling the above orthogonal conditions. Using these tRNAs, we successfully demonstrated incorporation of three different non‐natural amino acids into a single protein.

Site-specific incorporation of photofunctional nonnatural amino acids into a polypeptide through in vitro protein biosynthesis

Nonnatural amino acids with photofunctional groups were incorporated site‐specifically into a polypeptide by using in vitro protein synthesizing system. The nonnatural amino acids were attached to tRNACCU through chemical misacylation method, and added to the in vitro system with a mRNA containing a single AGG codon. l‐p‐Phenylazophenylalanine, l‐2‐anthrylalanine, l‐1‐naphthylalanine, l‐2‐naphthylalanine and l‐p‐biphenylalanine were successfully incorporated into a polypeptide, but l‐1‐pyrenylalanine was not. The polypeptides containing the nonnatural amino acids showed photofunctionalities.

Spatial regulation of specific gene expression through photoactivation of RNAi

In this study we describe the spatial regulation of RNA interference (RNAi) using an RNA-carrier protein labeled with a fluorescent dye and a light source to trigger the RNAi. We demonstrate photo-dependent gene silencing using several dyes with different excitation wavelengths. Additionally, we use light from a halogen lamp and a photomask to produce photopatterned RNAi, and laser light to trigger single-cell RNAi on cell culture plates.

Photoreversible antigen—antibody reactions

A monoclonal antibody (Z1H01) for an oligopeptide carrying an azobenzene group, was prepared under conditions where the azobenzene group is in the trans form. The antibody bound the hapten peptide effectively when the hapten peptide is in the trans form (K=5 × 107 M−1), but the antibody released the hapten under irradiation with UV light where the hapten is in the cis form. The antibody bound the hapten again, when the hapten reverted to the trans form after irradiation with visible light.