Marek Kwiatkowski

Solid-phase synthesis of chelate-labelled oligonucleotides: application in triple-color ligase-mediated gene analysis

Oligonucleotides labelled with detectable groups are essential tools in gene detection. We describe here the synthesis of pyrimidine deoxynucleotide-building blocks, modified at their C-5 position with a protected form of a strongly chelating agent. These reagents can be used to introduce multiple metal ions into oligodeoxynucleotides during standard oligonucleotide synthesis. The chelating functions form strongly fluorescent complexes with europium ions, characterized by a wide separation between the excitation and emission spectra. Moreover, the long decay time of the fluorescence permits sensitive time-resolved fluorescence measurements. The chelates also have the stability required to function in triple-color assays involving europium, samarium, and terbium ions. We demonstrate the application of these reagents for ligase-based gene analysis reactions.

Inefficient delivery but fast peptide bond formation of unnatural l -aminoacyl-tRNAs in translation

Translations with unnatural amino acids (AAs) are generally inefficient, and kinetic studies of their incorporations from transfer ribonucleic acids (tRNAs) are few. Here, the incorporations of small and large, non-N-alkylated, unnatural l-AAs into dipeptides were compared with those of natural AAs using quench-flow techniques. Surprisingly, all incorporations occurred in two phases: fast then slow, and the incorporations of unnatural AA-tRNAs proceeded with rates of fast and slow phases similar to those for natural Phe-tRNA(Phe). The slow phases were much more pronounced with unnatural AA-tRNAs, correlating with their known inefficient incorporations. Importantly, even for unnatural AA-tRNAs the fast phases could be made dominant by using high EF-Tu concentrations and/or lower reaction temperature, which may be generally useful for improving incorporations. Also, our observed effects of EF-Tu concentration on the fraction of the fast phase of incorporation enabled direct assay of the affinities of the AA-tRNAs for EF-Tu during translation. Our unmodified tRNA(Phe) derivative adaptor charged with a large unnatural AA, biotinyl-lysine, had a very low affinity for EF-Tu:GTP, while the small unnatural AAs on the same tRNA body had essentially the same affinities to EF-Tu:GTP as natural AAs on this tRNA, but still 2-fold less than natural Phe-tRNA(Phe). We conclude that the inefficiencies of unnatural AA-tRNA incorporations were caused by inefficient delivery to the ribosome by EF-Tu, not slow peptide bond formation on the ribosome.

A tRNA body with high affinity for EF-Tu hastens ribosomal incorporation of unnatural amino acids

There is evidence that tRNA bodies have evolved to reduce differences between aminoacyl-tRNAs in their affinity to EF-Tu. Here, we study the kinetics of incorporation of L-amino acids (AAs) Phe, Ala allyl-glycine (aG), methyl-serine (mS), and biotinyl-lysine (bK) using a tRNA(Ala)-based body (tRNA(AlaB)) with a high affinity for EF-Tu. Results are compared with previous data on the kinetics of incorporation of the same AAs using a tRNA(PheB) body with a comparatively low affinity for EF-Tu. All incorporations exhibited fast and slow phases, reflecting the equilibrium fraction of AA-tRNA in active ternary complex with EF-Tu:GTP before the incorporation reaction. Increasing the concentration of EF-Tu increased the amplitude of the fast phase and left its rate unaltered. This allowed estimation of the affinity of each AA-tRNA to EF-Tu:GTP during translation, showing about a 10-fold higher EF-Tu affinity for AA-tRNAs formed from the tRNA(AlaB) body than from the tRNA(PheB) body. At ∼1 µM EF-Tu, tRNA(AlaB) conferred considerably faster incorporation kinetics than tRNA(PheB), especially in the case of the bulky bK. In contrast, the swap to the tRNA(AlaB) body did not increase the fast phase fraction of N-methyl-Phe incorporation, suggesting that the slow incorporation of N-methyl-Phe had a different cause than low EF-Tu:GTP affinity. The total time for AA-tRNA release from EF-Tu:GDP, accommodation, and peptidyl transfer on the ribosome was similar for the tRNA(AlaB) and tRNA(PheB) bodies. We conclude that a tRNA body with high EF-Tu affinity can greatly improve incorporation of unnatural AAs in a potentially generalizable manner.

The tetraisopropyldisiloxane-1,3-diyl: a versatile protecting group for the synthesis of adenylyl-(2′→5′)-adenylyl-(2′→5′)-adenosine (2-5a core).

Abstract A2′p5′A2′p5′A has been synthesised in large scale using 3′, 5′- tetraisopropyldisilyl-6- N -benzoyl adenosine as the starting material.

Synthesis of adenosine containing carborane modification

Abstract The carboranyl cage is a new modifying entity for nucleosides and DNA-oligonucleotides. Most of carborane–nucleoside conjugates described so far belong to pyrimidine series. Herein, the first synthesis of adenosine, nucleoside containing purine nucleic base, modified with carborane cluster, is described.

Kinetics of tRNA(Pyl) -mediated amber suppression in Escherichia coli translation reveals unexpected limiting steps and competing reactions.

The utility of ribosomal incorporation of unnatural amino acids (AAs) in vivo is generally restricted by low efficiencies, even with the most widely used suppressor tRNAPyl. Because of the difficulties of studying incorporation in vivo, almost nothing is known about the limiting steps after tRNA charging. Here, we measured the kinetics of all subsequent steps using a purified Escherichia coli translation system. Dipeptide formation from initiator fMet‐tRNAfMet and tRNAPyl charged with allylglycine or methylserine displayed unexpectedly sluggish biphasic kinetics, ∼30‐fold slower than for native substrates. The amplitude of the fast phases increased with increasing EF‐Tu concentration, allowing measurement of Kd values of EF‐Tu binding, both of which were ∼25‐fold weaker than normal. However, binding could be increased ∼30‐fold by lowering temperature. The fast phase rates were limited by the surprisingly ∼10‐fold less efficient binding of EF‐Tu:GTP:AA‐tRNAPyl ternary complex to the ribosomes, not GTP hydrolysis or peptide bond formation. Furthermore, processivity was unexpectedly impaired as ∼40% of the dipeptidyl‐tRNAPyl could not be elongated to tripeptide. Dipeptide formation was slow enough that termination due to misreading the UAG codon by non‐cognate RF2 became very significant. This new understanding provides a framework for improving unnatural AA incorporation by amber suppression. Biotechnol. Bioeng. 2016;113: 1552–1559.

Peptide Formation by N-Methyl Amino Acids in Translation Is Hastened by Higher pH and tRNAPro

Applications of N-methyl amino acids (NMAAs) in drug discovery are limited by their low efficiencies of ribosomal incorporation, and little is known mechanistically about the steps leading to incorporation. Here, we demonstrate that a synthetic tRNA body based on a natural N-alkyl amino acid carrier, tRNA(Pro), increases translation incorporation rates of all three studied NMAAs compared with tRNA(Phe)- and tRNA(Ala)-based bodies. We also investigate the pH dependence of the incorporation rates and find that the rates increase dramatically in the range of pH 7 to 8.5 with the titration of a single proton. Results support a rate-limiting peptidyl transfer step dependent on deprotonation of the N-nucleophile of the NMAA. Competition experiments demonstrate that several futile cycles of delivery and rejection of A-site NMAA-tRNA are required per peptide bond formed and that increasing magnesium ion concentration increases incorporation yield. Data clarify the mechanism of ribosomal NMAA incorporation and provide three generalizable ways to improve incorporation of NMAAs in translation.

Kinetics of Ribosome-Catalyzed Polymerization Using Artificial Aminoacyl-tRNA Substrates Clarifies Inefficiencies and Improvements.

Ribosomal synthesis of polymers of unnatural amino acids (AAs) is limited by low incorporation efficiencies using the artificial AA-tRNAs, but the kinetics have yet to be studied. Here, kinetics were performed on five consecutive incorporations using various artificial AA-tRNAs with all intermediate products being analyzed. Yields within a few seconds displayed similar trends to our prior yields after 30 min without preincubation, demonstrating the relevance of fast kinetics to traditional long-incubation translations. Interestingly, the two anticodon swaps were much less inhibitory in the present optimized system, which should allow more flexibility in the engineering of artificial AA-tRNAs. The biggest kinetic defect was caused by the penultimate dC introduced from the standard, chemoenzymatic, charging method. This prompted peptidyl-tRNA drop-off, decreasing processivities during five consecutive AA incorporations. Indeed, two tRNA charging methods that circumvented the dC dramatically improved efficiencies of ribosomal, consecutive, unnatural AA incorporations to give near wild-type kinetics.

A high-capacity manifold support for the detection of specific IgE antibodies in allergic individuals.

A high-capacity manifold support with immobilized antigen was developed for the analysis of IgE-mediated immune reactivity in allergic subjects. Using this 96-pronged support, specific antibodies were trapped and detected from large sets of serum samples. We describe the binding of large amounts of antigen onto the expanded surface of the manifold support, permitting efficient identification of allergic individuals.