Improving Structural Stability and Anticoagulant Activity of a Thrombin Binding Aptamer by Aromatic Modifications

The thrombin binding aptamer (TBA) is a 15-mer DNA oligonucleotide (5′-GGT TGG TGT GGT TGG-3′), that can form a stable intramolecular antiparallel chair-like G-quadruplex structure. This aptamer shows anticoagulant properties by interacting with one of the two anion binding sites of thrombin, namely the fibrinogen-recognition exosite. Here, we demonstrate that terminal modification of TBA with aromatic fragments such as coumarin, pyrene and perylene diimide (PDI), improves the G-quadruplex stability. The large aromatic surface of these dyes can π-π stack to the G-quadruplex or to each other, thereby stabilizing the aptamer. With respect to the original TBA, monoPDI-functionalized TBA exhibited the most remarkable improvement in melting temperature (ΔT_m≈+18 °C) and displayed enhanced anticoagulant activity.     

Truncated Analogues of a G-Quadruplex-Forming Aptamer Targeting Mutant Huntingtin: Shorter Is Better!

Two analogues of the MS3 aptamer, which was previously shown to have an exquisite capability to selectively bind and modulate the activity of mutant huntingtin (mHTT), have been here designed and evaluated in their physicochemical and biological properties. Featured by a distinctive propensity to form complex G-quadruplex structures, including large multimeric aggregates, the original 36-mer MS3 has been truncated to give a 33-mer (here named MS3-33) and a 17-mer (here named MS3-17). A combined use of different techniques (UV, CD, DSC, gel electrophoresis) allowed a detailed physicochemical characterization of these novel G-quadruplex-forming aptamers, tested in vitro on SH-SY5Y cells and in vivo on a Drosophila Huntington’s disease model, in which these shorter MS3-derived oligonucleotides proved to have improved bioactivity in comparison with the parent aptamer.     

Synthesis of 5-(1,2,3-triazol-4-yl)-2'-deoxyuridines by a click chemistry approach: stacking of triazoles in the major groove gives increased nucleic acid duplex stability

A general protocol for converting alkyl and aryl halides into azides and for converting these in situ into 1,4-disubstituted triazoles was applied with 5-ethynyl-2'-deoxyuridine. This afforded three modified 2'-deoxyuridine analogues with either unsubstituted or 1-phenyl-/1-benzyl-substituted triazoles in their 5-positions. Modelling demonstrates coplanarity of the two heteroaromatic rings, and UV spectroscopy showed the uracil pK(a) values to be almost unchanged. The three nucleosides were introduced into nonamer oligonucleotides by phosphoramidite chemistry. The heteroaromatic triazoles became positioned in the major grooves of the short dsDNA and DNA-RNA duplexes. While single modifications led to decreased duplex stability, the stacking of four consecutive modifications led to enhanced duplex stability, especially for DNA-RNA duplexes. The duplex structures were studied by CD spectroscopy and molecular dynamics simulations, which supported the conjecture that the duplex stabilizing effect is due to efficient stacking of the heteroaromatic triazoles.     

Modification of Different Zirconium Propoxide Precursors by Diethanolamine. Is There a Shelf Stability Issue for Sol-Gel Applications?

Modification of different zirconium propoxide precursors with H₂dea was investigated by characterization of the isolated modified species. Upon modification of zirconium n-propoxide and [Zr(OⁿPr)(OⁱPr)₃(ⁱPrOH)]₂ with ½ a mol equivalent of H₂dea the complexes [Zr₂(OⁿPr)₆(OCH₂CH₂)₂NH]₂ (1) and [Zr₂(OⁿPr)₂(OⁱPr)₄(OCH₂CH₂)₂NH]₂ (2) were obtained. However, ¹H-NMR studies of these tetranuclear compounds showed that these are not time-stable either in solution or solid form. The effect of this time instability on material properties is demonstrated by light scattering and TEM experiments. Modification of zirconium isopropoxide with either ½ or 1 equivalent mol of H₂dea results in formation of the trinuclear complex, Zr{η³μ₂-NH(C₂H₄O)₂}₃[Zr(OⁱPr)₃]₂(iPrOH)₂ (3) countering a unique nona-coordinated central zirconium atom. This complex 3 is one of the first modified zirconium propoxide precursors shown to be stable in solution for long periods of time. The particle size and morphology of the products of sol-gel synthesis are strongly dependent on the time factor and eventual heat treatment of the precursor solution. Reproducible sol-gel synthesis requires the use of solution stable precursors.     

Modulation of Aptamer–Ligand-Binding by Complementary Oligonucleotides: A G-Quadruplex Anti-Ochratoxin A Aptamer Case Study

Short oligonucleotides are widely used for the construction of aptamer-based sensors and logical bioelements to modulate aptamer–ligand binding. However, relationships between the parameters (length, location of the complementary region) of oligonucleotides and their influence on aptamer–ligand interactions remain unclear. Here, we addressed this task by comparing the effects of short complementary oligonucleotides (ssDNAs) on the structure and ligand-binding ability of an aptamer and identifying ssDNAs’ features that determine these effects. Within this, the interactions between the OTA-specific G-quadruplex aptamer 1.12.2 (5′-GATCGGGTGTGGGTGGCGTAAAGGGA GCATCGGACA-3′) and 21 single-stranded DNA (ssDNA) oligonucleotides complementary to different regions of the aptamer were studied. Two sets of aptamer–ssDNA dissociation constants were obtained in the absence and in the presence of OTA by isothermal calorimetry and fluorescence anisotropy, respectively. In both sets, the binding constants depend on the number of hydrogen bonds formed in the aptamer–ssDNA complex. The ssDNAs’ having more than 23 hydrogen bonds with the aptamer have a lower aptamer dissociation constant than for aptamer–OTA interactions. The ssDNAs’ having less than 18 hydrogen bonds did not affect the aptamer–OTA affinity. The location of ssDNA’s complementary site in the aptamer affeced the kinetics of the interaction and retention of OTA-binding in aptamer–ssDNA complexes. The location of the ssDNA site in the aptamer G-quadruplex led to its unfolding. In the presence of OTA, the unfolding process was longer and takes from 20 to 70 min. The refolding in the presence of OTA was possible and depends on the length and location of the ssDNA’s complementary site. The location of the ssDNA site in the tail region led to its rapid displacement and wasn’t affecting the G-qaudruplex’s integrity. It makes the tail region more perspective for the development of ssDNA-based tools using this aptamer.     

Molecular tweezers as synthetic receptors: molecular recognition of cationic substrates; an insight into the mechanism of complexation

The results of ¹H NMR binding studies of mole-cular tweezers 1a with various aliphatic and aromatic cations in organic solvents are described. By the use of the viologene substrates 11 and 12 bearing bulky endgroups and therefore having a dumb-bell topology it could be demonstrated that besides the obvious mechanism of complexation in which the guest molecule enters the tweezer's cavity through the open sides of the host, a second mechanism is available in which the substrate enters the cavity through a gap emerging after a substantial spreading of the tweezer's tips by at least of about 280 pm.     

Expanding the Applicability of the Metal Labeling of Biomolecules by the RIKEN Click Reaction: A Case Study with Gallium‐68 Positron Emission Tomography

Radiolabeled biomolecules with short half‐life times are of increasing importance for positron emission tomography (PET) imaging studies. Herein, we demonstrate an improved and generalized method for synthesizing a [radiometal]‐unsaturated aldehyde as a lysine‐labeling probe that can be easily conjugated into various biomolecules through the RIKEN click reaction. As a case study, ⁶⁸Ga‐PET imaging of U87MG xenografted mice is demonstrated by using the ⁶⁸Ga‐DOTA‐RGDyK peptide, which is selective to α_Vβ₃ integrins.     

Multimeric Lactoside “Click Clusters” as Tools to Investigate the Effect of Linker Length in Specific Interactions with Peanut Lectin,Galectin‐1, and ‐3

Multimeric lactosides based on carbohydrate scaffolds with valencies ranging from 1 to 4 and different linker lengths were synthesized by a copper‐catalyzed azide–alkyne cycloaddition (CuAAC). The binding affinities and crosslinking abilities of the new “click clusters” toward biologically relevant galectins (gal‐1, gal‐3) and peanut lectin were evaluated by fluorescent polarization assay (FPA) and enzyme‐linked lectin assay (ELLA), respectively. FPA indicated that the binding affinities of the synthetic multilactosides towards the galectins increased proportionally with their lactosyl content, without significant differences due to the spacer length. ELLA evidenced a modest cluster effect for the multivalent conjugates, with a relative potency per lactoside ranging from 2.1 to 3.2. Nearly identical binding affinities were recorded for derivatives differing in the length of the linkers, in agreement with the FPA data. These results demonstrate that this parameter does not significantly influence the recognition process when interactions occur at a single lectin site. Molecular dynamics revealed that glycoconjugates adopt a pseudoglobular structure with a random localization of the lactoside residues. These spatial distributions were observed irrespective of the linker length; this explains the virtually equal affinities recorded by ELLA. In contrast, two‐site “sandwich” ELLA clearly revealed that multivalent derivatives bearing the longest spacers were more efficient for crosslinking lectins. Intrinsic affinities, devoid of aggregation effects, and crosslinking capabilities are, therefore, not directly related phenomena that must be taking into consideration in neoglycoconjugate design for specific applications.     

Second-Generation Inhibitors of the Mitochondrial Permeability Transition Pore with Improved Plasma Stability

Excessive mitochondrial matrix Ca²⁺ and oxidative stress leads to the opening of a high-conductance channel of the inner mitochondrial membrane referred to as the mitochondrial permeability transition pore (mtPTP). Because mtPTP opening can lead to cell death under diverse pathophysiological conditions, inhibitors of mtPTP are potential therapeutics for various human diseases. High throughput screening efforts led to the identification of a 3-carboxamide-5-phenol-isoxazole compounds as mtPTP inhibitors. While they showed nanomolar potency against mtPTP, they exhibited poor plasma stability, precluding their use in in vivo studies. Herein, we describe a series of structurally related analogues in which the core isoxazole was replaced with a triazole, which resulted in an improvement in plasma stability. These analogues were readily generated using the copper-catalyzed “click chemistry”. One analogue, N-(5-chloro-2-methylphenyl)-1-(4-fluoro-3-hydroxyphenyl)-1H-1,2,3-triazole-4-carboxamide ( TR001 ), was efficacious in a zebrafish model of muscular dystrophy that results from mtPTP dysfunction whereas the isoxazole isostere had minimal effect.     

Quick Click: The DNA‐Templated Ligation of 3′‐O‐Propargyl‐ and 5′‐Azide‐Modified Strands Is as Rapid as and More Selective than Ligase

The copper(I)‐mediated azide–alkyne cycloaddition (CuAAC) of 3′‐propargyl ether and 5′‐azide oligonucleotides is a particularly promising ligation system because it results in triazole linkages that effectively mimic the phosphate–sugar backbone of DNA, leading to unprecedented tolerance of the ligated strands by polymerases. However, for a chemical ligation strategy to be a viable alternative to enzymatic systems, it must be equally as rapid, as discriminating, and as easy to use. We found that the DNA‐templated reaction with these modifications was rapid under aerobic conditions, with nearly quantitative conversion in 5 min, resulting in a k_obs value of 1.1 min^?1, comparable with that measured in an enzymatic ligation system by using the highest commercially available concentration of T4 DNA ligase. Moreover, the CuAAC reaction also exhibited greater selectivity in discriminating C:A or C:T mismatches from the C:G match than that of T4 DNA ligase at 29 °C; a temperature slightly below the perfect nicked duplex dissociation temperature, but above that of the mismatched duplexes. These results suggest that the CuAAC reaction of 3′‐propargyl ether and 5′‐azide‐terminated oligonucleotides represents a complementary alternative to T4 DNA ligase, with similar reaction rates, ease of setup and even enhanced selectivity for certain mismatches.