Site-specific incorporation of unnatural amino acids into receptors expressed in Mammalian cells

We describe an approach to achieve unnatural amino acid incorporation into channels and receptors expressed in mammalian cells. We show that microelectroporation provides a general method to deliver DNA, mRNA, and tRNA simultaneously. In both CHO cells and cultured neurons, microelectroporation efficiently delivers an in vitro transcribed, serine amber suppressor tRNA, leading to nonsense suppression in a mutant EGFP gene. In CHO cells, both natural and unnatural amino acids chemically appended to a suppressor tRNA are site specifically incorporated into the nicotinic acetylcholine receptor (nAChR). Electrophysiology confirms the expected functional consequences of the unnatural residue. The microelectroporation strategy described here is more general, less tedious, and less damaging to mammalian neuronal and nonneuronal cells than previous approaches to nonsense suppression in small cells and provides the first example of unnatural amino acid incorporation in mammalian cells using chemically aminoacylated tRNA.     

An efficiently extended class of unnatural base pairs

A third DNA base pair, which is synthesized efficiently and selectively, would have wide ranging applications from synthetic organisms to nucleic acids biotechnology. Hydrophobic unnatural nucleobases offer a promising route to such a pair, but are often limited by inefficient extension, defined as synthesis immediately following the unnatural pair. Here, we describe a simple screen which enables the characterization of large numbers of previously uncharacterized hetero base pairs. From this screen, we identified a class of unnatural base pairs which are extended more efficiently than any unnatural base pair reported to date. Screening, when complemented by further kinetic analysis, can improve the understanding of the determinants of efficient extension as well as identify viable hetero base pairs.     

Site-specific incorporation of a redox-active amino acid into proteins

The redox-active amino acid 3,4-dihydroxy-l-phenylalanine (DHP), which can undergo two-electron oxidation to a quinone, has been incorporated selectively and efficiently into proteins in Escherichia coli in response to a TAG codon. We have demonstrated that DHP can be oxidized electrochemically within the protein. The ability to incorporate a redox-active amino acid site specifically into proteins should facilitate the study of electron transfer in proteins, as well as enable the engineering of redox proteins with novel properties.     

Minor groove hydrogen bonds and the replication of unnatural base pairs

As part of an effort to expand the genetic alphabet, we examined the synthesis of DNA with six different unnatural nucleotides bearing methoxy-derivatized nucleobase analogues. Different nucleobase substitution patterns were used to systematically alter the nucleobase electronics, sterics, and hydrogen-bonding potential. We determined the ability of the Klenow fragment of E. coli DNA polymerase I to synthesize and extend the different unnatural base pairs and mispairs under steady-state conditions. Unlike other hydrogen-bond acceptors examined in the past, the methoxy groups do not facilitate mispairing, implying that they are not recognized by any of the hydrogen-bond donors of the natural nucleobases; however, they do facilitate replication. The more efficient replication results largely from an increase in the rate of extension of primers terminating at the unnatural base pair and, interestingly, requires that the methoxy group be at the ortho position where it is positioned in the developing minor groove and can form a functionally important hydrogen bond with the polymerase. Thus, ortho methoxy groups should be generally useful for the effort to expand the genetic alphabet.     

Optimization of interstrand hydrophobic packing interactions within unnatural DNA base pairs

As part of an effort to expand the genetic alphabet, we have evaluated a large number of predominantly hydrophobic unnatural base pairs. We now report the synthesis and stability of unnatural base pairs formed between simple phenyl rings modified at different positions with methyl groups. Surprisingly, several of the unnatural base pairs are virtually as stable as a natural base pair in the same sequence context. The results show that neither hydrogen-bonding nor large aromatic surface area are required for base pair stability within duplex DNA and that interstrand interactions between small aromatic rings may be optimized for both stability and selectivity. These smaller nucleobases are not expected to induce the distortions in duplex DNA or at the primer terminus that seem to limit replication of larger unnatural base pairs, and they therefore represent a promising approach to the expansion of the genetic alphabet.     

Electronic coupling mediated by stacked [Thymine-Hg-Thymine] base pairs

Very recently it has been shown that stable metal-mediated base pairs [Thymine-Hg-Thymine] can form in DNA. To estimate the effect of such pairs on the efficiency of charge transfer through DNA, we carry out quantum mechanical calculations of double-stranded pi-stacks GXG, GXXG, and GXXXG, where X = [Thymine-Hg-Thymine] and stacks GT(n)G of canonical base pairs. The charge-transfer efficiency in short duplexes GXG and GTG is found to be similar. However, the donor-acceptor coupling in GXXG and GXXXG is stronger by a factor of 2.5-3.0 than that in GT(n)G (n = 2 and 3), respectively. It is shown that the valence orbitals of Hg atoms do not essentially participate in mediating the electronic coupling for hole transfer; however, they may play an important role in excess electron transfer.     

Theoretical investigation on DNA/RNA base pairs mediated by copper, silver, and gold cations

B3LYP density functional based computations were performed in order to characterize the interactions present in some Cu(+), Ag(+), and Au(+) metal ion-mediated DNA and RNA base pairs from both structural and electronic points of view. Examined systems involve as ligands canonical Watson-Crick, Hoogsteen and Wobble base pairs. Two artificial Hoogsteen base pairs were also taken into account. Binding energy values indicate that complexes involving silver cations are less stable than those in which copper or gold are present, and propose a similar behaviour for these two latter ions. The nature of the bond linking metal ions and bases was described by the NBO analysis that suggests metal coordinative interactions to be covalent. An evaluation of the dispersion contributions for the investigated systems was performed with the B3LYP-D3 functional.     

Major groove substituents and polymerase recognition of a class of predominantly hydrophobic unnatural base pairs

Expansion of the genetic alphabet with an unnatural base pair is a long‐standing goal of synthetic biology. We have developed a class of unnatural base pairs, formed between d 5SICS and analogues of d MMO2 that are efficiently and selectively replicated by the Klenow fragment (Kf) DNA polymerase. In an effort to further characterize and optimize replication, we report the synthesis of five new d MMO2 analogues bearing different substituents designed to be oriented into the developing major groove and an analysis of their insertion opposite d 5SICS by Kf and Thermus aquaticus DNA polymerase I (Taq). We also expand the analysis of the previously optimized pair, d NaM –d 5SICS , to include replication by Taq. Finally, the efficiency and fidelity of PCR amplification of the base pairs by Taq or Deep Vent polymerases was examined. The resulting structure–activity relationship data suggest that the major determinants of efficient replication are the minimization of desolvation effects and the introduction of favorable hydrophobic packing, and that Taq is more sensitive than Kf to structural changes. In addition, we identify an analogue (d NMO1 ) that is a better partner for d 5SICS than any of the previously identified d MMO2 analogues with the exception of d NaM . We also found that d NaM –d 5SICS is replicated by both Kf and Taq with rates approaching those of a natural base pair.     

Site-specific incorporation of 5-methylaminomethyl-2-thiouridine and 2-thiouridine(s) into RNA sequences

We present a reproducible protocol for the site-specific incorporation of 5-methylaminomethyl-2-thiouridine (mnm⁵s²U) into a model RNA fragment and, together with 2-methyladenosine (m²A), into the native sequence of the Escherichia coli tRNA^Glu2 anticodon arm (E. coli ASL^Glu2). This approach is also utilized for the synthesis of oligomers modified with multiple 2-thiouridines.     

Monitoring the site-specific incorporation of dual fluorophore-quencher base analogues for target DNA detection by an unnatural base pair system

We developed intramolecular dual fluorophore-quencher base analogues for site-specific incorporation into DNA by an unnatural base pair replication system. An unnatural base pair between 7-(2-thienyl)-imidazo[4,5-b]pyridine (Ds) and 2-nitro-4-propynylpyrrole (Px) exhibits high fidelity in PCR amplification, and the 2-nitropyrrole moiety of Px acts as a quencher. Deoxyribonucleoside triphosphates of Px linked with a fluorophore (Cy3, Cy5 or FAM) were chemically synthesized, and the fluorescent properties and the enzymatic incorporation of the fluorophore-linked dPxTPs into DNA were examined in PCR amplification. The fluorophore-linked dPxTPs were site-specifically incorporated by PCR into DNA, opposite Ds in templates, with high selectivity. Furthermore, we found that the fluorescence of the triphosphates was partially quenched, but increased upon their incorporation into DNA. These dual fluorophore-quencher base analogues would be useful for site-specific DNA labeling and for monitoring the amplification products of target nucleic acid molecules with a specific sequence. We have demonstrated the utility of the fluorophore-linked Px substrates and the Ds-Px pairing in real-time quantitative PCR for target DNA molecule detection.     

Site-specific incorporation of glycosylated serine and tyrosine derivatives into proteins

Glycosylation of proteins can have a dramatic effect on their physical, chemical, and biological properties. Analogues of dihydrofolate reductase and firefly luciferase containing glycosylated amino acids at single, predetermined sites have been elaborated. Misacylated suppressor tRNAs activated with glycosylated serine and tyrosine derivatives were used for suppression of the nonsense codons in a cell-free protein biosynthesizing system, thereby permitting the preparation of the desired glycosylated proteins. In this fashion, it was possible to obtain proteins containing both mono- and diglycosylated amino acids, including glycosylated serine and tyrosine moieties. For the modified firefly luciferases, the effect of these substitutions on the wavelength of the light emitted by firefly luciferase was investigated. The maximum wavelength for mutants containing peracetylated glycosylated serine derivatives at position 284 showed a red shift in the emission spectra. For mutants containing glycosylated tyrosines, the red shift was observed only when the carbohydrate moiety was fully deacetylated.     

Enantioselective synthesis of an unnatural bipyridyl amino acid and its incorporation into a peptide

The synthesis of a bipyridyl amino acid, 2-amino-3-(4′-methyl-2,2′-bipyridin-4-yl) propanoic acid, is described. A short three step synthesis from commercially available 4,4′-dimethyl-2,2′-bipyridine provides the amino acid in 65% enantiomeric excess (ee). An enzyme-mediated chiral resolution increases the ee to 95% in two additional steps. The amino acid was incorporated into a 22 amino acid peptide composed predominantly of alanine. The peptide was found to be 88% α-helical in aqueous solution at 1°C by circular dichroism (CD) spectropolarimetry, indicating a high helical propensity for this amino acid. This amino acid can provide a means to incorporate a metal into structure-forming peptides.