An integrated computational pipeline for designing high-affinity nanobodies with expanded genetic codes.

Protein engineering and design principles employing the 20 standard amino acids have been extensively used to achieve stable protein scaffolds and deliver their specific activities. Although this confers some advantages, it often restricts the sequence, chemical space, and ultimately the functional diversity of proteins. Moreover, although site-specific incorporation of non-natural amino acids (nnAAs) has been proven to be a valuable strategy in protein engineering and therapeutics development, its utility in the affinity-maturation of nanobodies is not fully explored. Besides, current experimental methods do not routinely employ nnAAs due to their enormous library size and infinite combinations. To address this, we have developed an integrated computational pipeline employing structure-based protein design methodologies, molecular dynamics simulations and free energy calculations, for the binding affinity prediction of an nnAA-incorporated nanobody toward its target and selection of potent binders. We show that by incorporating halogenated tyrosines, the affinity of 9G8 nanobody can be improved toward epidermal growth factor receptor (EGFR), a crucial cancer target. Surface plasmon resonance (SPR) assays showed that the binding of several 3-chloro-l-tyrosine (3MY)-incorporated nanobodies were improved up to 6-fold into a picomolar range, and the computationally estimated binding affinities shared a Pearson's r of 0.87 with SPR results. The improved affinity was found to be due to enhanced van der Waals interactions of key 3MY-proximate nanobody residues with EGFR, and an overall increase in the nanobody's structural stability. In conclusion, we show that our method can facilitate screening large libraries and predict potent site-specific nnAA-incorporated nanobody binders against crucial disease-targets.

Incorporation of Halogenated Amino Acids into Antibody Fragments at Multiple Specific Sites Enhances Antigen Binding.

Expansion of the amino-acid repertoire with synthetic derivatives introduces novel structures and functionalities into proteins. In this study, we improved the antigen binding of antibodies by incorporating halogenated tyrosines at multiple selective sites. Tyrosines in the Fab fragment of an anti-EGF-receptor antibody 059-152 were systematically replaced with 3-bromo- and 3-chlorotyrosines, and simultaneous replacements at four specific sites were found to cause a tenfold increase in the affinity toward the antigen. Structure modeling suggested that this effect was due to enhanced shape complementarity between the antigen and antibody molecules. On the other hand, we showed that chlorination in the constant domain, far from the binding interface, of Rituximab Fab also increased the affinity significantly (up to 17-fold). Our results showed that antigen binding is tunable with the halogenation in and out of the binding motifs.

Engineering an Automaturing Transglutaminase with Enhanced Thermostability by Genetic Code Expansion with Two Codon Reassignments.

In the present study, we simultaneously incorporated two types of synthetic components into microbial transglutaminase (MTG) from Streptoverticillium mobaraense to enhance the utility of this industrial enzyme. The first amino acid, 3-chloro-l-tyrosine, was incorporated into MTG in response to in-frame UAG codons to substitute for the 15 tyrosine residues separately. The two substitutions at positions 20 and 62 were found to each increase thermostability of the enzyme, while the seven substitutions at positions 24, 34, 75, 146, 171, 217, and 310 exhibited neutral effects. Then, these two stabilizing chlorinations were combined with one of the neutral ones, and the most stabilized variant was found to contain 3-chlorotyrosines at positions 20, 62, and 171, exhibiting a half-life 5.1-fold longer than that of the wild-type enzyme at 60 °C. Next, this MTG variant was further modified by incorporating the α-hydroxy acid analogue of Nε-allyloxycarbonyl-l-lysine (AlocKOH), specified by the AGG codon, at the end of the N-terminal inhibitory peptide. We used an Escherichia coli strain previously engineered to have a synthetic genetic code with two codon reassignments for synthesizing MTG variants containing both 3-chlorotyrosine and AlocKOH. The ester bond, thus incorporated into the main chain, efficiently self-cleaved under alkaline conditions (pH 11.0), achieving the autonomous maturation of the thermostabilized MTG. The results suggested that synthetic genetic codes with multiple codon reassignments would be useful for developing the novel designs of enzymes.

Engineering the elongation factor Tu for efficient selenoprotein synthesis.

Selenocysteine (Sec) is naturally co-translationally incorporated into proteins by recoding the UGA opal codon with a specialized elongation factor (SelB in bacteria) and an RNA structural signal (SECIS element). We have recently developed a SECIS-free selenoprotein synthesis system that site-specifically--using the UAG amber codon--inserts Sec depending on the elongation factor Tu (EF-Tu). Here, we describe the engineering of EF-Tu for improved selenoprotein synthesis. A Sec-specific selection system was established by expression of human protein O(6)-alkylguanine-DNA alkyltransferase (hAGT), in which the active site cysteine codon has been replaced by the UAG amber codon. The formed hAGT selenoprotein repairs the DNA damage caused by the methylating agent N-methyl-N'-nitro-N-nitrosoguanidine, and thereby enables Escherichia coli to grow in the presence of this mutagen. An EF-Tu library was created in which codons specifying the amino acid binding pocket were randomized. Selection was carried out for enhanced Sec incorporation into hAGT; the resulting EF-Tu variants contained highly conserved amino acid changes within members of the library. The improved UTu-system with EF-Sel1 raises the efficiency of UAG-specific Sec incorporation to >90%, and also doubles the yield of selenoprotein production.

Stepwise regulation of hole transport in DNA by control of triplex formation.

A functionality for regulating hole transport efficiency is a prerequisite for the utilization of DNA duplexes as nanodevices. Herein, we report the regulation of hole transport in anthraquinone-tethered DNA with dual triplex forming sites. Long-range photooxidation experiments showed that hole transport was effectively suppressed by the formation of triplex at low temperature, while it was recovered by dissociation to the duplex at higher temperature. Variation of temperature induced the formation and dissociation of the third strand at each triplex region individually, leading to the stepwise regulation of hole transport in DNA.

Photoelectrochemical evaluation of pH effect on hole transport through triplex-forming DNA immobilized on a gold electrode.

We characterized pH effect on hole transport through DNA duplexes possessing a partial triplex-forming region. Direct electrochemical measurement of the current response of photosensitizer-tethered DNA immobilized on a gold electrode revealed that the partial triplex formation under acidic conditions suppressed photocurrent due to hole transport, while dissociation of the triplex into the duplex as occurred upon increasing pH values recovered the photocurrent efficiency. Reversible conversion between duplex and triplex induced upon cyclic alternation of pH values resulted in a rise and fall of photocurrent responses, indicating that pH change may feature in the switching function of hole transport in DNA. These electrochemical behaviors could be correlated to the results obtained in long-range photo-oxidative DNA cleavage experiments, in which DNA cleavage at the hole trapping site beyond the triplex region was significantly suppressed under triplex-forming acidic conditions.

Development of novel oxygen-independent photosensitizers.

We proposed a strategy of photooxidizer-reduction activated alkylator (P-A) hybrid molecule to develop novel oxygen-independent photosensitizers. Two prototypes of such photosensitizers camptothecin-indolequinone (CPT-IQ) and camptothecin-nitrofuryl (CPT-NF) was designed and prepared. A mechanism of photo-induced oxidation and alkylation of 2'-deoxyguanosine by CPT-IQ was investigated. CPT-NF was confirmed to effectively induce DNA cleavage via 365-nm UV irradiation both under normaxia and hypoxia.

Effects of structural modification on the DNA binding properties and photo-induced cleavage reactivity of propargylic sulfones conjugated with an anthraquinone structure.

Propargylic sulfones are known as pH-dependent DNA cleaving agents. We have designed a novel propargylic sulfone conjugated with an anthraquinone structure and evaluated its DNA binding and cleavage characteristics. The propargylic sulfone 3 showed high intercalating ability attributable to anthraquinone chromophore, leading to the efficient alkylation of DNA. The anthraquinone chromophore in 3 also acted as a photosensitizer, and photoirradiation of 3 with DNA induced one-electron oxidation, resulting in the further DNA cleavage. Evaluation of the effect of 3 against EMT6/KU cells revealed that 3 exhibited potent cytotoxicity, even without photoirradiation.

Electrochemical evaluation of alternating duplex-triplex conversion effect on the anthraquinone-photoinjected hole transport through DNA duplex immobilized on a gold electrode.

Amperometry was employed to characterize the anthraquinone (AQ)-photoinjected hole transport through a 20-mer oligodeoxynucleotide (ODN) duplex, as immobilized on the surface of a gold electrode, and its triplex forms converted by association with several third oligopyrimidine (OPD) short strands. While the cathodic photocurrent was observed upon irradiation at 365 nm of the AQ photosensitizer linked to the end of DNA duplex, a marked lowering of the current density was identified to occur by the triplex formation of a duplex with a given third OPD short strand. The photocurrent through the DNA duplex showed a reversible fall-rise response concomitant with alternating association-dissociation cycle of the OPD short-strand, as regulated by temperature change around the corresponding melting temperature of the DNA triplex. Both the switched photoirradiation and the thermally alternating duplex-triplex conversion could provide tools of regulating the DNA hole transport.

Switching of charge transport in DNA by regulated triplex formations at the dual sites.

A functionality of on/off switching to regulate conductivity should be among the prerequisites for the utilization of DNA duplex as a conducting material. We have reported that variation of temperatures can regulate the triplex formation and dissociation, thereby regulating charge transport in DNA. Based on this result, the on/off switching of conductivity at dual triplex forming sites in DNA was attempted, using two types of oligonucleotide (ODNs) with different strand lengths. Variation of temperatures produced DNA duplexes without any triplex site and with single and double triplex sites, by which the intramolecular charge transport in DNA duplex could be regulated at the respective triplex-forming sites. This may promote the utility of DNA duplex as a potential constituent of electronic nanodevices.

The effect of triple helix forming on charge transport in DNA.

The charge transport efficiency in DNA duplex could be regulated by thermal conversion between the formation and dissociation of partial triplex structure with a short strand oligonucleotide (ODN). Photoinduced long-distance one-electron oxidation in duplex ODNs with partial triplex structural region was examined at various temperatures to evaluate the charge transport efficiency. Under these conditions, the apparent charge transport was suppressed dramatically at 0 degrees C relative to 25 degrees C. These results strongly indicate that variation of temperatures could regulate the triplex formation and dissociation, resulting in the regulation of charge transport in DNA. Thus, triplex formation is expected to be a constituent of new well regulated biomaterial that will applicable to nano-scale electronic devices.

Photo-induced DNA cleavage reaction characteristics of propargylic sulfones possessing anthraquinone chromophore.

DNA cleavage potency of propargylic sulfones possessing anthraquinone chromophore 1 under UV-irradiation was evaluated in comparison with the dark reaction. 1 showed inefficient DNA cleavage activity, while having considerably strong DNA binding ability. This result is accounted for by spatial conditions that the activated alkylating allenic site of intercalated 1 could not effectively approach to DNA bases, most probably guanine moiety, and thereby led to insufficient DNA strand cleavage. In contrast, the DNA cleavage activity of 1 was notably enhanced upon UV-irradiation (lambda(ex)=365 nm) followed by incubation. Under UV-irradiation, further DNA cleavage were occurred primary at 5'-G of GG steps within DNA. A DNA cleavage mechanism for 1, by which photo-induced one-electron oxidation of 5'-G of GG steps may occur along with ordinary alkylation, has been proposed.

Photo-induced DNA cleavage reaction characteristics of propargylic sulfones possessing an anthraquinone chromophore.

Propargylic sulfones (PS) are known as pH dependent DNA cleaver. DNA cleavage by PS was considered to proceed by alkylation of G base to allenic sulfones formed from PS in basic condition. We designed PS posessing naphthalene and anthraquinone (AQ) unit and investigated DNA cleavage characteristics. Although these compounds showed high intercalating abilities, this high intercalating ability did not lead to DNA cleaving activity. This result indicates that spatial arrangement of activated allene against guanine base is very important in DNA cleavage by PS. In addition, UV-irradiation to PS possessing AQ unit leads to efficient DNA cleavage at 5'-G of GG sequence. This cleavage pattern exhibited typical cleavage of one-electron oxidation of B-form DNA. Therefore, this result suggests that PS possessing AQ unit cleave DNA by both the alkylation mechanism and the photooxidaization mechanism.