Novel ceftazidime-resistance β-lactamases generated by a codon-based mutagenesis method and selection

Four known and nine new ceftazidime-resistance β-lactamases were generated by a novel, contaminating codon-based mutagenesis approach. In this method, wild-type codons are spiked with a set of mutant codons during oligonucleotide synthesis, generating random combinatorial libraries of primers that contain few codon replacements per variant. Mutant codons are assembled by tandem addition of a diluted mixture of five Fmoc-dimer amidites to the growing oligo and a mixture of four DMTr-monomer amidites to generate 20 trinucleotides that encode a set of 18 amino acids. Wild-type codons are assembled with conventional chemistry and the whole process takes place in only one synthesis column, making its automation feasible. The random and binomial behavior of this approach was tested in the polylinker region of plasmid pUC19 by the synthesis of three oligonucleotide libraries mutagenized at different rates and cloned as mutagenic cassettes. Additionally, the method was biologically assessed by mutating six contiguous codons that encode amino acids 237–243 (ABL numbering) of the TEM_pUC19 β-lactamase, which is functionally equivalent to the clinically important TEM-1 β-lactamase. The best ceftazidime-recognizing variant was a triple mutant, R164H:E240K: R241A, displaying a 333-fold higher resistance than the wild-type enzyme.     

Orthogonal combinatorial mutagenesis: a codon-level combinatorial mutagenesis method useful for low multiplicity and amino acid-scanning protocols

We describe here a method to generate combinatorial libraries of oligonucleotides mutated at the codon-level, with control of the mutagenesis rate so as to create predictable binomial distributions of mutants. The method allows enrichment of the libraries with single, double or larger multiplicity of amino acid replacements by appropriate choice of the mutagenesis rate, depending on the concentration of synthetic precursors. The method makes use of two sets of deoxynucleoside-phosphoramidites bearing orthogonal protecting groups [4,4′-dimethoxytrityl (DMT) and 9-fluorenylmethoxycarbonyl (Fmoc)] in the 5′ hydroxyl. These phosphoramidites are divergently combined during automated synthesis in such a way that wild-type codons are assembled with commercial DMT-deoxynucleoside-methyl-phosphoramidites while mutant codons are assembled with Fmoc-deoxynucleoside-methyl-phosphoramidites in an NNG/C fashion in a single synthesis column. This method is easily automated and suitable for low mutagenesis rates and large windows, such as those required for directed evolution and alanine scanning. Through the assembly of three oligonucleotide libraries at different mutagenesis rates, followed by cloning at the polylinker region of plasmid pUC18 and sequencing of 129 clones, we concluded that the method performs essentially as intended.     

Novel ceftazidime-resistance beta-lactamases generated by a codon-based mutagenesis method and selection

Four known and nine new ceftazidime-resistance beta-lactamases were generated by a novel, contaminating codon-based mutagenesis approach. In this method, wild-type codons are spiked with a set of mutant codons during oligonucleotide synthesis, generating random combinatorial libraries of primers that contain few codon replacements per variant. Mutant codons are assembled by tandem addition of a diluted mixture of five Fmoc-dimer amidites to the growing oligo and a mixture of four DMTr-monomer amidites to generate 20 trinucleotides that encode a set of 18 amino acids. Wild-type codons are assembled with conventional chemistry and the whole process takes place in only one synthesis column, making its automation feasible. The random and binomial behavior of this approach was tested in the polylinker region of plasmid pUC19 by the synthesis of three oligonucleotide libraries mutagenized at different rates and cloned as mutagenic cassettes. Additionally, the method was biologically assessed by mutating six contiguous codons that encode amino acids 237-243 (ABL numbering) of the TEM(pUC19) beta-lactamase, which is functionally equivalent to the clinically important TEM-1 beta-lactamase. The best ceftazidime-recognizing variant was a triple mutant, R164H:E240K: R241A, displaying a 333-fold higher resistance than the wild-type enzyme.     

A New Approach to the Synthesis of Trinucleotide Phosphoramidites - Synthons for the Generation of Oligonucleotide/Peptide Libraries

Abstract

Trinucleotide phosphoramidites that correspond to the codons of all 20 amino acids were synthesized in high yield in 5g scale. Precursors of those amidites - trinucleotide phosphotriesters - have been prepared using the phosphotriester approach without protection of the 3′-hydroxyl function. More than 10 oligonucleotides up to 90 bases long have been synthesized by a phosphite-triester approach using new synthons. The 67-mer (12 random codons) has been used to generate a display library of 2 × 10⁸ complexity.     

The synthesis of blocked triplet-phosphoramidites and their use in mutagenesis.

A general approach for the synthesis of oligonucleotide-triplet phosphoramidites and the synthesis of four such blocks are described. A strategy was devised to minimize the number of dimer precursors needed for synthesis of a complete set of triplet-amidite blocks encoding all 20 amino acids. Whereas synthesis of 20 triplet-amidite blocks consisting of codon sequences requires 16 dimer blocks, just seven dimer blocks are required to synthesize all required antisense sequences. The antisense sequences are then converted to codons in template mediated replication. Using a mixture of four triplet-amidites and conventional automated solid-phase DNA synthesis, short (6mer) and medium length (30mer) oligonucleotide mixtures were synthesized and analyzed. The latter was replicated in vitro and used as a mutagenic cassette to produce four mutants of Asp 221 in the enzyme thymidylate synthase. The method establishes the direction and utility for the production and use of triplet-amidite blocks in DNA synthesis.     

Combinatorial codon-based amino acid substitutions

Twenty Fmoc-protected trinucleotide phosphoramidites representing a complete set of codons for the natural amino acids were chemically synthesized for the first time. A pool of these reagents was incorporated into oligonucleotides at substoichiometric levels to generate two libraries of variants that randomly carry either few or many codon replacements on a region encoding nine amino acids of the bacterial enzyme TEM-1 β-lactamase. Assembly of the libraries was performed in a completely automated mode through a simple modification of ordinary protocols. This technology eliminates codon redundancy, stop codons and enables complete exploration of sequence space for single, double and triple mutations throughout a protein region spanning several residues. Sequence analysis of many non-selected clones revealed a good incorporation of the trinucleotides, producing combinations of mutations quite different from those obtained using conventional degenerate oligonucleotides. Ceftazidime-selection experiments yielded several never before reported variants containing novel amino acid combinations in the β-lactamase omega loop region.     

Trinucleotide phosphoramidites: ideal reagents for the synthesis of mixed oligonucleotides for random mutagenesis.

Trinucleotide phosphoramidites representing codons for all 20 amino acids have been prepared and used in automated, solid-phase DNA synthesis. In contrast to an earlier report, we show that these substances can be used to introduce entire codons into oligonucleotides in excess of 98% yield, and are ideal reagents for the synthesis of mixed oligonucleotides for random mutagenesis.     

Modified base compositions at degenerate positions of a mutagenic oligonucleotide enhance randomness in site-saturation mutagenesis.

Site-saturation mutagenesis, using degenerate oligonucleotide primers, is a frequently used method in introducing various mutations in a selected target codon. Oligonucleotides that are synthesized using equimolar concentrations of nucleoside phosphoramidites (dA, dC, dG, dT) in the positions to be saturated, result in a mutant population that is biased towards the original nucleotides. We found that this bias could be eliminated by modifying the concentrations of nucleoside phosphoramidites during the oligonucleotide synthesis. We synthesized eight degenerate oligonucleotides to saturate eight different codons, and sequenced a total of 344 mutagenized codons. In six of these eight oligonucleotides, we reduced to varying extents the concentrations of those nucleotides in the target positions that would form base pairs with the template. From the data, we analyzed the effects of different base compositions in the oligonucleotides when mutagenizing different codons, the influence of the positions of mismatches, and the significance of different non-Watson-Crick base pairs. Based on these results, we suggest levels to which different phosphoramidites should be reduced when synthesizing oligonucleotides for site-saturation mutagenesis.     

Tandem oligonucleotide synthesis using linker phosphoramidites

Multiple oligonucleotides of the same or different sequence, linked end-to-end in tandem can be synthesized in a single automated synthesis. A linker phosphoramidite [R. T. Pon and S. Yu (2004) Nucleic Acids Res., 32, 623–631] is added to the 5′-terminal OH end of a support-bound oligonucleotide to introduce a cleavable linkage (succinic acid plus sulfonyldiethanol) and the 3′-terminal base of the new sequence. Conventional phosphoramidites are then used for the rest of the sequence. After synthesis, treatment with ammonium hydroxide releases the oligonucleotides from the support and cleaves the linkages between each sequence. Mixtures of one oligonucleotide with both 5′- and 3′-terminal OH ends and other oligonucleotides with 5′-phosphorylated and 3′-OH ends are produced, which are deprotected and worked up as a single product. Tandem synthesis can be used to make pairs of PCR primers, sets of cooperative oligonucleotides or multiple copies of the same sequence. When tandem synthesis is used to make two self-complementary sequences, double-stranded structures spontaneously form after deprotection. Tandem synthesis of oligonucleotide chains containing up to six consecutive 20mer (120 bases total), various trinucleotide codons and primer pairs for PCR, or self-complementary strands for in situ formation of double-stranded DNA fragments has been demonstrated.     

Analysis of progressive deletions of the transmembrane and cytoplasmic domains of influenza hemagglutinin

Site-directed oligonucleotide mutagenesis has been used to introduce chain termination codons into the cloned DNA sequences encoding the carboxy-terminal transmembrane (27 amino acids) and cytoplasmic (10 amino acids) domains of influenza virus hemagglutinin (HA). Four mutant genes were constructed which express truncated forms of HA that lack the cytoplasmic domain and terminate at amino acids 9, 14, 17, or 27 of the wild-type hydrophobic domain. Analysis of the biosynthesis and intracellular transport of these mutants shows that the cytoplasmic tail is not needed for the efficient transport of HA to the cell surface; the stop-transfer sequences are located in the hydrophobic domain; 17 hydrophobic amino acids are sufficient to anchor HA stably in the membrane; and mutant proteins with truncated hydrophobic domains show drastic alterations in transport, membrane association, and stability.     

A convenient approach to the synthesis of trinucleotide phosphoramidites--synthons for the generation of oligonucleotide/peptide libraries.

Trinucleotide phosphoramidites that correspond to the codons of all 20 amino acids were synthesized in high yield in 5g scale. Precursors of those amidites--trinucleotide phosphotriesters--have been prepared using the phosphotriester approach without protection of the 3'-hydroxyl function. The structures of trinucleotide phosphotriesters and intermediates were confirmed by 1H- and 31P-NMR spectra, mass-spectra and by analysis of SPDE-hydrolysates of deprotected preparations. Purity of the target products has been confirmed by test reactions. The synthons have been used for automated synthesis of oligonucleotides and corresponding libraries by a phosphite-triester approach. A 54mer, containing 12 randomized internal bases, and a 72mer with 24 internal randomized bases have been synthesized.     

Genetic Code-guided Protein Synthesis and Folding in Escherichia coli

Universal genetic codes are degenerated with 61 codons specifying 20 amino acids, thus creating synonymous codons for a single amino acid. Synonymous codons have been shown to affect protein properties in a given organism. To address this issue and explore how Escherichia coli selects its “codon-preferred” DNA template(s) for synthesis of proteins with required properties, we have designed synonymous codon libraries based on an antibody (scFv) sequence and carried out bacterial expression and screening for variants with altered properties. As a result, 342 codon variants have been identified, differing significantly in protein solubility and functionality while retaining the identical original amino acid sequence. The soluble expression level varied from completely insoluble aggregates to a soluble yield of ∼2.5 mg/liter, whereas the antigen-binding activity changed from no binding at all to a binding affinity of > 10⁻⁸ m. Not only does our work demonstrate the involvement of genetic codes in regulating protein synthesis and folding but it also provides a novel screening strategy for producing improved proteins without the need to substitute amino acids.     

Variation of Mutagenic Action on Nonsense Mutants at Different Sites in the Iso-1-Cytochrome c Gene of Yeast

Three ochre and two amber mutants in yeast have been definitively identified by the amino acid replacements in iso-1-cytochromes c from intragenic revertants. Except for rare and sometimes unusual changes, all of the replacements were single amino acids whose codons differed from UAA or UAG by one base. These assignments, which were based on the absence of tryptophan replacements in ochre revertants, could be corroborated from the studies of two groups of suppressors that were shown to act on either the ochre or amber mutants. All five nonsense mutants are located at different sites in the cyc1 gene and all are at sites that can be occupied by amino acids having a wide range of structures. The relative frequencies of the amino acid replacements indicate that identical codons located at different sites may respond differently to a mutagenic agent. Notably glutamine replacements occurred almost exclusively in UV-induced revertants of only one ochre mutant cyc1–9, but not at all or at reduced proportions in the others. Similarly, lysine replacements occurred almost exclusively in the NA-induced revertants of only the ochre mutant cyc1–72, but not at all in the others. These and other results reveal that mutation of A·T base pairs by UV and nitrous acid are dependent upon the location of the codon within the gene as well as the location of the base pair within the codon. From these findings, it appears as if the type of base-pair changes induced by UV and nitrous acid are strongly influenced by adjacent nucleotide sequences.     

Formation of 4,4'-dimethoxytrityl-C-phosphonate oligonucleotides

Incomplete sulfurization during solid-phase synthesis of phosphorothioate oligonucleotides using phosphoramidite chemistry was identified as the cause of formation of two new classes of process-related oligonucleotide impurities containing a DMTr-C-phosphonate (DMTr=4,4'-dimethoxytrityl) moiety. Phosphite triester intermediates that failed to oxidize (sulfurize) to the corresponding phosphorothioate triester react during the subsequent acid-induced (dichloroacetic acid) detritylation with the DMTr cation or its equivalent in an Arbuzov-type reaction. This leads to formation of DMTr-C-phosphonate mono- and diesters resulting in oligonucleotides modified with a DMTr-C-phosphonate moiety located internally or at the 5'terminal hydroxy group. DMTr-C-phosphonate derivatives are not detected when optimized sulfurization conditions are employed.     

Production by quantitative photolithographic synthesis of individually quality checked DNA microarrays

For DNA chip analyses, oligonucleotide quality has immense consequences for accuracy, sensitivity and dynamic range. The quality of chips produced by photolithographic in situ synthesis depends critically on the efficiency of photo-deprotection. By means of base-assisted enhancement of this process using 5′-[2-(2-nitrophenyl)-propyloxycarbonyl]-2′-deoxynucleoside phosphoramidites, synthesis yields improved by at least 12% per condensation compared to current chemistries. Thus, the eventual total yield of full-length oligonucleotide is increased more than 10-fold in the case of 20mers. Furthermore, the quality of every individual array position was checked quantitatively after synthesis. Subsequently, the quality tested chips were used in successive hybridisation experiments.     

Activities of herpes simplex virus type 1 (HSV-1) ICP4 genes specifying nonsense peptides.

Synthetic oligonucleotide linkers containing translational termination codons in all possible reading frames were inserted at various positions in the cloned gene encoding the herpes simplex virus type 1 (HSV-1) immediate-early regulatory protein, ICP4. It was determined that the amino-terminal 60 percent of the ICP4 gene was sufficient for trans-induction of a thymidine kinase promoter-CAT chimera (pTKCAT) and negative regulation of an ICP4 promoter-CAT chimera (pIE3CAT); however, it was relatively inefficient in complementing an ICP4 deletion mutant. The amino-terminal ninety amino acids do not appear to be required for infectivity as reflected by the replication competence of a mutant virus containing a linker insertion at amino acid 12. The size of the ICP4 molecule expressed from the mutant virus was consistent with translational restart at the next methionine codon corresponding to amino acid 90 of the deduced ICP4 amino acid sequence.     

Enzymatic activity of poliovirus RNA polymerase mutants with single amino acid changes in the conserved YGDD amino acid motif.

RNA-dependent RNA polymerases contain a highly conserved region of amino acids with a core segment composed of the amino acids YGDD which have been hypothesized to be at or near the catalytic active site of the molecule. Six mutations in this conserved YGDD region of the poliovirus RNA-dependent RNA polymerase were made by using oligonucleotide site-directed DNA mutagenesis of the poliovirus cDNA to substitute A, C, M, P, S, or V for the amino acid G. The mutant polymerase genes were expressed in Escherichia coli, and the purified RNA polymerases were tested for in vitro enzyme activity. Two of the mutant RNA polymerases (those in which the glycine residue was replaced with alanine or serine) exhibited in vitro enzymatic activity ranging from 5 to 20% of wild-type activity, while the remaining mutant RNA polymerases were inactive. Alterations in the in vitro reaction conditions by modification of temperature, metal ion concentration, or pH resulted in no significant differences in the activities of the mutant RNA polymerases relative to that of the wild-type enzyme. An antipeptide antibody directed against the wild-type core amino acid segment containing the YGDD region of the poliovirus polymerase reacted with the wild-type recombinant RNA polymerase and to a limited extent with the two enzymatically active mutant polymerases; the antipeptide antibody did not react with the mutant RNA polymerases which did not have in vitro enzyme activity. These results are discussed in the context of secondary-structure predictions for the core segment containing the conserved YGDD amino acids in the poliovirus RNA polymerase.     

Optimized syntheses of Fmoc azido amino acids for the preparation of azidopeptides

The rise of CuI‐catalyzed click chemistry has initiated an increased demand for azido and alkyne derivatives of amino acid as precursors for the synthesis of clicked peptides. However, the use of azido and alkyne amino acids in peptide chemistry is complicated by their high cost. For this reason, we investigated the possibility of the in‐house preparation of a set of five Fmoc azido amino acids: β‐azido l ‐alanine and d ‐alanine, γ‐azido l ‐homoalanine, δ‐azido l ‐ornithine and ω‐azido l ‐lysine. We investigated several reaction pathways described in the literature, suggested several improvements and proposed several alternative routes for the synthesis of these compounds in high purity. Here, we demonstrate that multigram quantities of these Fmoc azido amino acids can be prepared within a week or two and at user‐friendly costs. We also incorporated these azido amino acids into several model tripeptides, and we observed the formation of a new elimination product of the azido moiety upon conditions of prolonged couplings with 2‐(1H‐benzotriazol‐1‐yl)‐1,1,3,3‐tetramethyluronium hexafluorophosphate/DIPEA. We hope that our detailed synthetic protocols will inspire some peptide chemists to prepare these Fmoc azido acids in their laboratories and will assist them in avoiding the too extensive costs of azidopeptide syntheses. Experimental procedures and/or analytical data for compounds 3 – 5 , 20 , 25 , 26 , 30 and 43 – 47 are provided in the supporting information. © 2017 The Authors Journal of Peptide Science published by European Peptide Society and John Wiley & Sons Ltd.     

In vitro incorporation of nonnatural amino acids into protein using tRNACys-derived opal,ochre, and amber suppressor tRNAs

Amber suppressor tRNAs are widely used to incorporate nonnatural amino acids into proteins to serve as probes of structure, environment, and function. The utility of this approach would be greatly enhanced if multiple probes could be simultaneously incorporated at different locations in the same protein without other modifications. Toward this end, we have developed amber, opal, and ochre suppressor tRNAs derived from Escherichia coli, and yeast tRNA^Cys that incorporate a chemically modified cysteine residue with high selectivity at the cognate UAG, UGA, and UAA stop codons in an in vitro translation system. These synthetic tRNAs were aminoacylated in vitro, and the labile aminoacyl bond was stabilized by covalently attaching a fluorescent dye to the cysteine sulfhydryl group. Readthrough efficiency (amber > opal > ochre) was substantially improved by eRF1/eRF3 inhibition with an RNA aptamer, thus overcoming an intrinsic hierarchy in stop codon selection that limits UGA and UAA termination suppression in higher eukaryotic translation systems. This approach now allows concurrent incorporation of two different modified amino acids at amber and opal codons with a combined apparent readthrough efficiency of up to 25% when compared with the parent protein lacking a stop codon. As such, it significantly expands the possibilities for incorporating nonnative amino acids for protein structure/function studies.     

Reselection of a Genomic Upstream Open Reading Frame in Mouse Hepatitis Coronavirus 5′-Untranslated-Region Mutants

An AUG-initiated upstream open reading frame (uORF) encoding a potential polypeptide of 3 to 13 amino acids (aa) is found within the 5′ untranslated region (UTR) of >75% of coronavirus genomes based on 38 reference strains. Potential CUG-initiated uORFs are also found in many strains. The AUG-initiated uORF is presumably translated following genomic 5′-end cap-dependent ribosomal scanning, but its function is unknown. Here, in a reverse-genetics study with mouse hepatitis coronavirus, the following were observed. (i) When the uORF AUG-initiating codon was replaced with a UAG stop codon along with a U112A mutation to maintain a uORF-harboring stem-loop 4 structure, an unimpaired virus with wild-type (WT) growth kinetics was recovered. However, reversion was found at all mutated sites within five virus passages. (ii) When the uORF was fused with genomic (main) ORF1 by converting three in-frame stop codons to nonstop codons, a uORF-ORF1 fusion protein was made, and virus replicated at WT levels. However, a frameshifting G insertion at virus passage 7 established a slightly 5′-extended original uORF. (iii) When uAUG-eliminating deletions of 20, 30, or 51 nucleotides (nt) were made within stem-loop 4, viable but debilitated virus was recovered. However, a C80U mutation in the first mutant and an A77G mutation in the second appeared by passage 10, which generated alternate uORFs that correlated with restored WT growth kinetics. In vitro, the uORF-disrupting nondeletion mutants showed enhanced translation of the downstream ORF1 compared with the WT. These results together suggest that the uORF represses ORF1 translation yet plays a beneficial but nonessential role in coronavirus replication in cell culture.