Non-Enzymatic Template-Directed Recombination of RNAs

RNA non-enzymatic recombination reactions are of great interest within the hypothesis of the “RNA world”, which argues that at some stage of prebiotic life development proteins were not yet engaged in biochemical reactions and RNA carried out both the information storage task and the full range of catalytic roles necessary in primitive self-replicating systems. Here we report on the study of recombination reaction occuring between two 96 nucleotides (nts) fragments of RNAs under physiological conditions and governed by a short oligodeoxyribonucleotide template, partially complementary to sequences within each of the RNAs. Analysis of recombination products shows that ligation is predominantly template-directed, and occurs within the complementary complex with the template in “butt-to-butt” manner, in 1- or 3- nts bulges or in 2–3 nts internal loops. Minor recombination products formed in the template-independent manner are detected as well.     

Designed RNAs with Two Peptide‐Binding Units as Artificial Templates for Native Chemical Ligation of RNA‐Binding Peptides

The template effect plays important roles not only in modern synthetic and enzymatic catalysis but also in the ancient “RNA‐polypeptide (RNP) world,” which has been postulated to be a crucial stage in the origin of life. To mimic primitive template catalysis of peptide ligations by RNAs, we previously reported the design and synthesis of a ternary RNP complex in which the ligation of two peptides was significantly facilitated by a template RNA with two peptide‐binding units. However, RNA molecules also promoted the ligation reaction in a nonspecific manner through electrostatic interactions between RNA and basic peptides. In this study, we suppressed this effect by reducing the length of the original template derived from the Tetrahymena intron RNA. This modification, however, decreased the template ability for the specific reaction. As an alternative RNA that was as effective as the original template, we found that a self‐dimerizing RNA was a promising template for peptide ligation without a nonspecific effect.     

An Efficient Approach for Immobilizing the Oxo‐Vanadium Schiff Base onto Polymer Supports using Staudinger Ligation

The present paper describes the use of Staudinger ligation as an efficient and high‐yielding approach for the immobilization of oxo‐vanadium Schiff base on polymeric supports via covalent attachment under mild conditions. The described strategy is simple in use, versatile, highly efficient with respect to better catalyst loading and proceeds under mild, metal‐free conditions. The catalytic potential of the prepared polymer immobilized oxo‐vanadium Schiff bases was tested for the oxidation of sulfides using aqueous tert‐butyl hydroperoxide (TBHP) as oxidant. The polymer‐supported catalysts could easily be recovered from the reaction mixture and reused for four runs without loss in activity and no metal leaching was observed during this course.     

Towards Efficient Nonenzymatic DNA Ligation: Comparing Key Parameters for Maximizing Ligation Rates and Yields with Carbodiimide Activation**

Chemical ligation is an important tool for the generation of synthetic DNA structures, which are used for a wide range of applications. Surprisingly, reported chemical ligation yields can range from 30 to 95 % for the same chemical activating agent and comparable DNA structures. We report a systematic study of DNA ligation by using a well-defined bimolecular test system and a water-soluble carbodiimide (EDC) as a phosphate-activating agent. Our results emphasize the interplay between template-substrate complex stability and the rates of the chemical steps of ligation, with 3′ phosphate substrates providing yields near 100 % after 24 hours for particularly favorable reaction conditions. Ligation rates are also shown to be sensitive to the identity of the base pairs flanking a nick site, with as much as threefold variation. Finally, the observation that DNA substrates are modified by EDC at rates that can be comparable with ligation rates emphasizes the importance of considering side reactions when designing protocols to maximize ligation yields.     

The Emerging Role of Tetrazines in Drug-Activation Chemistries

Traditionally, prodrug activation has been limited to enzymatic triggers or gross physiological aberrations, such as pH, that offer low selectivity and control over dosage. In recent years, the field of prodrug activation chemistry has been transformed by the use of bioorthogonal reactions that can be carried out under biological conditions at sub-millimolar concentrations, with the tetrazine-mediated inverse electron demand Diels–Alder reaction amongst the most recognised. Their high reaction rates, chemoselectivity and excellent biocompatibility make tetrazines ideal small molecules for activating prodrugs. Recently the tetrazine moiety has been used as a prodrug for a pyridazine thus broadening the scope of prodrug systems. This article discusses the concept of using tetrazines as small-molecule activators for prodrugs, and provides an overview of tetrazine-based prodrug systems, with a particular focus on the recently reported prodrug–prodrug activation strategy.     

Quadruplex-Templated and Catalyzed Ligation of Nucleic Acids

Template-guided chemical reactions between nucleic acid strands are an important process in biomedical research. However, almost all of these reactions employ an oligonucleotide-templated approach that is based on the double-helix alignment. The moderate stability of the double helix makes this approach unsuitable for many chemical reactions, so alternative nucleic acid alignment mechanisms, demonstrating higher thermal and chemical stability, are desirable. Earlier, we described a noncovalent coupling mechanism between DNA strands through a quadruplex-and-Mg²⁺ connection (QMC). QMC is based on G-quadruplexes and allows unusually stable and specific interactions. Herein, a novel catalytic nucleic acid reaction, based on QMC, is described. This approach uses G-tetrads as a structural and recognition element without employing Watson-Crick complementarity rules at any stage of substrate/catalyst formation or interaction between them. Quadruplex-templated ligation can be achieved through the self-ligation of two nucleic acid strands, or through a quadruplex catalyst, which forms a G-triplex and specifically connects the strands. The process is extraordinarily robust and efficient. For instance, the ligation of carbodiimide-activated substrates can proceed in boiling solutions, and complete ligation is demonstrated within a minute. The quadruplex-templated and catalyzed reactions will create new opportunities for chemical reactions requiring harsh experimental conditions.     

Additive-Free Enzymatic Phosphorylation and Ligation of Artificial Oligonucleotides with C-Nucleosides at the Reaction Points

We report enzymatic phosphorylation and additive-free ligation of DNAs containing unnatural C-nucleotide residues through the action of T4 polynucleotide kinase and T4 DNA ligase. The artificial units are each made up of an alkynyl deoxyribose component and one of the unnatural nucleobases D * , T * , G * , and C * , corresponding—from a viewpoint of hydrogen-bonding patterns—to natural A, T, G, and C, respectively. Phosphorylation progressed quantitatively at the 5′-end in the cases of all of the artificial units in the chimeric DNAs. Ligation also smoothly progressed at the 5′-end in the cases of the D* and G* nucleotide residues, but only negligibly in those of their T* and C* counterparts. Chemical redesign of the last two units successfully improved the ligation efficiency, so that enzymatic ligation worked well for all of the artificial units in every 3′-natural ⋅ 5′-artificial, 3′-artificial ⋅ 5′-natural, and 3′-artificial ⋅ 5′-artificial terminal combination at the nicks.     

Switchable Fluorescence of Doxorubicin for Label-Free Imaging of Bioorthogonal Drug Release

Monitoring the release and activation of prodrug formulations provides essential information about the outcome of a therapy. While the prodrug delivery can be confirmed by using different imaging techniques, confirming the release of active payload by using imaging is a challenge. Here, we have discovered that the switchable fluorescence of doxorubicin can validate drug release upon its uncaging reaction with a highly specific chemical partner. We have observed that the conjugation of doxorubicin with a trans-cyclooctene (TCO) diminishes its fluorescence at 595 nm. This quenched fluorescence of the doxorubicin prodrug is recovered upon its bond-cleaving reaction with tetrazine. Clinically assessed iron oxide nanoparticles were used to formulate a doxorubicin nanodrug. The release of doxorubicin from the nanodrug was studied under various experimental conditions. A fivefold increase in doxorubicin fluorescence is observed after complete release. The studies were carried out in vitro in MDA-MB-231 breast cancer cells. An increase in Dox signal was observed upon tetrazine administration. This switchable fluorescence mechanism of Dox could be employed for fundamental studies, that is, the reactivity of various tetrazine and TCO linker types under different experimental conditions. In addition, the system could be instrumental for translational research where the release and activation of doxorubicin prodrug payloads can be monitored by using optical imaging systems.     

Considerations on Probe Design for Affinity-Guided Protein Conjugation

The plethora of methods developed for the creation of protein conjugates often differs significantly with regard to the heterogeneity of the resulting products, in the degree of genetic manipulation of the protein required, and in the technical skills required to perform the conjugation procedure. Affinity-guided protein conjugation is a protein labeling methodology based on noncovalent binding interactions between a labeling probe and the protein of interest. These interactions increase the local concentration of a reactive group in the probe on the protein surface thus facilitating the conjugation in proximity of the complexation site. The ability to produce high-quality conjugates from nongenetically modified proteins both in vitro, but also in cells, demonstrates the power of affinity-guided protein conjugation. Here, we present the progress of affinity-guided protein conjugation in relation to selective protein labeling in living systems and the formation of high-quality protein conjugates. Furthermore, the probe design will be discussed in relation to the utility of the probe for labeling in vitro or in living systems.     

Chemical Reactions Directed Peptide Self-Assembly

Fabrication of self-assembled nanostructures is one of the important aspects in nanoscience and nanotechnology. The study of self-assembled soft materials remains an area of interest due to their potential applications in biomedicine. The versatile properties of soft materials can be tuned using a bottom up approach of small molecules. Peptide based self-assembly has significant impact in biology because of its unique features such as biocompatibility, straight peptide chain and the presence of different side chain functionality. These unique features explore peptides in various self-assembly process. In this review, we briefly introduce chemical reaction-mediated peptide self-assembly. Herein, we have emphasised enzymes, native chemical ligation and photochemical reactions in the exploration of peptide self-assembly.