While DNA-templated ligations typically rely on the sequence-specific hybridization of complementary strands to bring reactive groups into close proximity, charge-transfer assisted ligation (CTAL) is proposed as a fundamentally different strategy. Rather than depending on a DNA template for spatial organization, CTAL exploits charge-transfer interactions between reactants to promote bond formation between two noncomplementary DNA strands. The strategic placement of donor and acceptor units at the termini of DNA strands drives the colocalization of reactive functional groups, enabling efficient reactions at sub-micromolar concentrations. The versatility of the CTAL is demonstrated on CuAAC and SPAAC click reactions, allowing the formation of conjugates with 3 '-5 ', 3 '-3 ', or 5 '-5 ' junctions, with outstanding apparent rate constants in the 104-105 M-1s-1 range, while the nonassisted reactions are inoperative in such dilute conditions (0.5 & micro;M). Furthermore, the potential of the CTAL is extended to RNA-RNA and siRNA-DNA aptamer ligations. Finally, a traceless strategy based on a cleavable linker is developed, which allows the removal of aromatic units after ligation under basic conditions.

In the search for effective and low-toxicity anticoagulant agents, the G-quadruplex-forming thrombin-binding aptamer (TBA) with sequence 5'-GGTTGGTGTGGTTGG-3', able to selectively recognize the fibrinogen-binding exosite I of the thrombin enzyme, emerged as a promising therapeutic and surgical tool. In this frame, we recently synthesized and evaluated a library of TBA analogues carrying a naphthalene diimide (N) moiety and a 3-hydroxypropylphosphate (p) either at the 5'- or 3'-end of the TBA sequence. Interestingly, N-TBA-p and p-TBA-N analogues, having the same pendant groups at 5'- or 3'-end but in reversed position, showed very different behavior in terms of thermal stability, nuclease resistance in serum, and anticoagulant activity. N-TBA-p showed enhanced properties compared to both p-TBA-N and the parent TBA and thus emerged as a very promising candidate for future in vivo studies. Here, by in-depth molecular dynamics-based analyses, we disclosed the structural features determining the higher thermal stability and nuclease resistance as well as the higher anticoagulant activity due to thrombin recognition, experimentally observed for N-TBA-p than p-TBA-N and TBA.

A phthalimide or 3‐formylindole functionality was incorporated into a tris(hydroxymethyl)ethane (THME) scaffold, and the resulting compounds were subsequently transformed into phosphoramidites and solid supports. These building blocks were then used to synthesise oligonucleotides containing amine or aldehyde groups at various positions within their sequences. The resulting modified oligonucleotides were further conjugated through either amide bond formation or oxime ligation. These novel building blocks expand the repertoire of phosphoramidites and solid supports available for modifying oligonucleotides.

Inspired by automated DNA synthesis, electron-rich dialkoxynaphthalene (DAN) donor and electron-deficient naphthalene-tetracarboxylic diimide (NDI) acceptor phosphodiester-linked homohexamers were synthesized by the phosphoramidite method. Two types of hexamers were prepared, one with only one phosphodiester between the aromatics (i.e., DAN or NDI) and a second with two phosphodiesters around a propanediol between the aromatics, leading to the latter more flexible and more hydrophilic hexamers. The folding properties of these homohexamers alone or mixed together, in water only, were studied by UV-visible absorption spectroscopy and atomic force microscopy (AFM). AFM imaging revealed that a 1:1 mixture of hexaDAN and hexaNDI formed fibers by charge transfer donor-acceptor recognition leading to a hydrogel after drying. The organization of the resulting structures is strongly dependent on the nature of the complementary partner, leading to the formation of mono- or multilayer hydrogel networks with different compactness.

The limitations associated with the in vivo use of the thrombin binding aptamer (TBA or TBA15) have dramatically stimulated the search of suitable chemically modified analogues in order to discover effective and reversible inhibitors of thrombin activity. In this context, we previously proposed cyclic and pseudo-cyclic TBA analogues with improved stability that proved to be more active than the parent aptamer.Herein, we have investigated a novel library of TBA derivatives carrying naphthalene diimide (NDI) moieties at the 3 '- or 5 '-end. In a subset of the investigated oligonucleotides, additional 3-hydroxypropylphosphate (HPP) groups were introduced at one or both ends of the TBA sequence. Evaluation of the G-quadruplex thermal stability, serum nuclease resistance and in vitro anticoagulant activity of the new TBA analogues allowed rationalizing the effect of these appendages on the activity of the aptamer on the basis of their relative position. Notably, most of the different TBA analogues tested were more potent thrombin inhibitors than unmodified TBA. Particularly, the analogue carrying an NDI group at the 5 '-end and an HPP group at the 3 '-end, named N-TBA-p, exhibited enhanced G-quadruplex thermal stability (Delta Tm + 14 degrees C) and ca. 10-fold improved nuclease resistance in serum compared to the native aptamer. N-TBA-p also induced prolonged and dose-dependent clotting times, showing a ca. 11-fold higher anticoagulant activity compared to unmodified TBA, as determined by spectroscopic methods. Overall, N-TBA-p proved to be in vitro a more efficient thrombin inhibitor than all the best ones previously investigated in our group. Its interesting features, associated with its easy preparation, make it a very promising candidate for future in vivo studies.

Electron-rich 1,5-dialkoxynaphthalene (DAN) and electron-deficient 1,8,4,5-naphthalenetetracarboxylic diimide (NDI) are known to interact through the formation of charge-transfer complexes. The introduction of DAN and NDI into various DNA duplexes and hairpins was investigated by ultraviolet (UV) melting curve analysis. The positioning of the DAN:NDI pair was found to strongly influence the stability of DNA duplex and hairpins. In particular, while the introduction of one DAN/NDI pair in front of each other in the center of a DNA duplex led to a decrease of the thermal stability (ΔTm - 6 °C), the addition of a second pair restored or even increased the stability. In contrast, the introduction of DAN:NDI pairs at the end of a duplex always induced a strong stabilization (ΔTm up to +20 °C). Finally, a DAN:NDI pair positioned in the loop of a hairpin induced a stronger stabilization than a T4 loop (ΔTm + 10 °C). Based on charge-transfer interactions, the strong stabilizations observed allow the preparation of highly stabilized DNA nanostructures opening the way to numerous applications in nanotechnology.

N,O ‐Diacetyl tyrosine and 3‐(4‐hydroxy‐phenyl)‐propanoic acid were converted into phosphoramidite derivatives and introduced to the 5′‐end of oligonucleotides. The resulting oligonucleotides exhibiting a 4‐hydroxyphenyl alkyl group were conjugated with 4‐phenyl‐1,2,4‐triazoline‐3,5‐dione as a model of a Y‐click reaction. The reaction is fast (<1 h) and efficient (>90 %). A partial side reaction occurred at the C8 position of deoxyguanosine in single‐stranded oligonucleotides. This side reaction does not take place on the G quadruplex and double‐stranded oligonucleotides and when N2 position of guanine is protected. This Y‐click reaction could be applied to 1,2,4‐triazoline‐3,5‐dione derivatives displaying a fluorescent dye, a carbohydrate or orthogonal functions (alkyne, azide, ketone or maleimide) for the labelling of oligonucleotides.

How cells respond to mechanical forces by converting them into biological signals underlie crucial cellular processes. Our understanding of mechanotransduction has been hindered by technical barriers, including limitations in our ability to effectively apply low range piconewton forces to specific mechanoreceptors on cell membranes without laborious and repetitive trials. To overcome these challenges we introduce the Nano-winch, a robust, easily assembled, programmable DNA origami-based molecular actuator. The Nano-winch is designed to manipulate multiple mechanoreceptors in parallel by exerting fine-tuned, low- piconewton forces in autonomous and remotely activated modes via adjustable single- and double-stranded DNA linkages, respectively. Nano-winches in autonomous mode can land and operate on the cell surface. Targeting the device to integrin stimulated detectable downstream phosphorylation of focal adhesion kinase, an indication that Nano-winches can be applied to study cellular mechanical processes. Remote activation mode allowed finer extension control and greater force exertion. We united remotely activated Nano-winches with single-channel bilayer experiments to directly observe the opening of a channel by mechanical force in the force responsive gated channel protein, BtuB. This customizable origami provides an instrument-free approach that can be applied to control and explore a diversity of mechanotransduction circuits on living cells.

Among the numerous molecular diagnostic methods, isothermal reverse transcription recombinase polymerase amplification (RT-RPA) is a simple method that has high sensitivity and avoids the use of expensive instruments. However, detection of amplified genomes often requires a fluorescence readout on costly readers or migration on a lateral flow strip with a subjective visual reading. Aiming to establish a new approach to rapidly and sensitively detect viruses, we combined RT-RPA with a magnetic field-enhanced agglutination (MFEA) assay and assessed the ability of this method to detect the dengue virus (DENV). Magnetization cycles accelerated the capture of amplified DENV genomes between functionalized magnetic nanoparticles by a fast chaining process to less than 5 min; the agglutination was quantified by simple turbidimetry. A total of 37 DENV RNA+ and 30 DENV RNA− samples were evaluated with this combined method. The sensitivity and specificity were 89.19% (95% CI, 72.75–100.00%) and 100% (95% CI, 81.74–100.00%), respectively. This approach provides a solution for developing innovative diagnostic assays for the molecular detection of emerging infections.

Despite their unquestionable properties, oligonucleotide ap-tamers display some drawbacks that continue to hinder their applications. Several strategies have been undertaken to over-come these weaknesses, using thrombin binding aptamers as proof-of-concept. In particular, the functionalization of a thrombin exosite I binding aptamer (TBA) with aromatic moi-eties, e.g., naphthalene dimides (N) and dialkoxynaphthalenes (D), attached at the 50 and 30 ends, respectively, proved to be highly promising. To obtain a molecular view of the effects of these modifications on aptamers, we performed a crystallo-graphic analysis of one of these engineered oligonucleotides (TBA-NNp/DDp) in complex with thrombin. Surprisingly, three of the four examined crystallographic structures are ternary complexes in which thrombin binds a TBA-NNp/ DDp molecule at exosite II as well as at exosite I, highlighting the ability of this aptamer, differently from unmodified TBA, to also recognize a localized region of exosite II. This novel abil-ity is strictly related to the solvophobic behavior of the terminal modifications. Studies were also performed in solution to examine the properties of TBA-NNp/DDp in a crystal-free environment. The present results throw new light on the importance of appendages inducing a pseudo-cyclic charge -transfer structure in nucleic acid-based ligands to improve the interactions with proteins, thus considerably widening their potentialities.

In the search for optimized thrombin binding aptamers (TBAs), we herein describe the synthesis of a library of TBA analogues obtained by end-functionalization with the electron-rich 1,5-dialkoxy naphthalene (DAN) and the electron-deficient 1,8,4,5-naphthalenetetra-carboxylic diimide (NDI) moieties. Indeed, when these G-rich oligonucleotides were folded into the peculiar TBA G-quadruplex (G4) structure, effective donor–acceptor charge transfer interactions between the DAN and NDI residues attached to the extremities of the sequence were induced, providing pseudo-cyclic structures. Alternatively, insertion of NDI groups at both extremities produced TBA analogues stabilized by π–π stacking interactions. All the doubly-modified TBAs were characterized by different biophysical techniques and compared with the analogues carrying only the DAN or NDI residue and unmodified TBA. These modified TBAs exhibited higher nuclease resistance, and their G4 structures were markedly stabilized, as evidenced by increased Tm values compared to TBA. These favorable properties were also associated with improved anticoagulant activity for one DAN/NDI-modified TBA, and for one NDI/NDI-modified TBA. Our results indicated that TBA pseudo-cyclic structuring by ad hoc designed end-functionalization represents an efficient approach to improve the aptamer features, while pre-organizing and stabilizing the G4 structure but allowing sufficient flexibility to the aptamer folding, which is necessary for optimal thrombin recognition.

Arbovirus diagnostics on blood from donors and travelers returning from endemic areas is increasingly important for better patient management and epidemiological surveillance. We developed a flexible approach based on a magnetic field-enhanced agglutination (MFEA) readout to detect either genomes or host-derived antibodies. Dengue viruses (DENVs) were selected as models. For genome detection, a pan-flavivirus amplification was performed before capture of biotinylated amplicons between magnetic nanoparticles (MNPs) grafted with DENV probes and anti-biotin antibodies. Magnetization cycles accelerated this chaining process to within 5 min while simple turbidimetry measured the signal. This molecular MFEA readout was evaluated on 43 DENV RNA(+) and 32 DENV RNA(−) samples previously screened by real-time RT-PCR. The sensitivity and the specificity were 88.37% (95% CI, 78.76%–97.95%) and 96.87% (95% CI, 90.84%–100%), respectively. For anti-DENV antibody detection, 103 plasma samples from donors were first screened using ELISA assays. An immunological MFEA readout was then performed by adding MNPs grafted with viral antigens to the samples. Anti-DENV antibodies were detected with a sensitivity and specificity of 90.62% (95% CI, 83.50%–97.76%) and 97.44% (95% CI, 92.48%–100%), respectively. This adaptable approach offers flexibility to platforms dedicated to the screening of emerging infections.

Inspired by the automated synthesis of DNA on a solid support, the electron-rich dialkoxynaphthalene (DAN) donor and the electron-deficient naphthalene-tetracarboxylic diimide (NDI) acceptor, amphiphilic foldamers have been synthesised from their respective phosphoramidite building blocks. The folding of the phosphodiester-linked hexamer (DAN-NDI)3 revealed the formation of regular supramolecular nanotubes in water resulting from the self-assembly of multiple hexamers stabilized by donor/acceptor interactions and the solvophobic effect.

The Thrombin Binding Aptamer or TBA (5′-GGTTGGTGTGGTTGG-3′) is a 15-mer G-rich oligonucleotide able to inhibit the thrombin-catalysed fibrinogen-fibrin conversion after specific binding to its exosite I. TBA entered clinical trials but its evaluation was halted after phase I studies due to suboptimal dosing profiles. Aiming at obtaining TBA analogues better performing in vivo, a large number of chemically modified TBA variants have been proposed. In this frame, we prepared a series of cyclic TBA analogues by linking its 5′ and 3′-ends with a variety of flexible linkers. The first derivative was realized introducing a 20-atom long linker. Compared to native TBA, it exhibited a G4 structure with exceptionally improved stability and nuclease resistance. However, these favourable properties were associated with reduced biological activity, suggesting that higher flexibility in the linker structure was necessary. Therefore, a mini-library of second generation cyclic TBAs (cycTBA I-IV) was prepared, carrying circularizing linkers overall spanning from 22 to 48 atoms. Among these derivatives, cycTBA II showed improved anticoagulant activity, associated with a dramatically stabilized G4 structure and enhanced enzymatic resistance in serum compared to the native TBA. Current studies are focused on pseudocyclic TBA analogues, where the cyclic structure is obtained not through covalent bonds but via p-p stacking or charge-transfer interactions of different aromatic probes inserted at the termini of the oligonucleotide. Among ten different TBA derivatives, we identified a promising candidate in this pseudocyclic series showing improved anticoagulant activity compared to native TBA, also having higher nuclease resistance and G-quadruplex thermal stability.

Galacto- and fuco-clusters conjugated with one to three catechol or hydroxamate motifs were synthesised to target LecA and LecB lectins ofPseudomonas aeruginosa(PA) localised in the outer membrane and inside the bacterium. The resulting glycocluster-pseudosiderophore conjugates were evaluated as Trojan horses to cross the outer membrane of PA by iron transport. The data suggest that glycoclusters with catechol moieties are able to hijack the iron transport, whereas those with hydroxamates showed strong nonspecific interactions. Mono- and tricatechol galactoclusters (G1CandG3C) were evaluated as inhibitors of infection by PA in comparison with the free galactocluster (G0). All of them exhibited an inhibitory effect between 46 to 75 % at 100 mu M, with a higher potency thanG0. This result shows that LecA localised in the outer membrane of PA is involved in the infection mechanism.

NU172—a 26-mer oligonucleotide able to bind exosite I of human thrombin and inhibit its activity—was the first aptamer to reach Phase II clinical studies as an anticoagulant in heart disease treatments. With the aim of favoring its functional duplex-quadruplex conformation and thus improving its enzymatic stability, as well as its thrombin inhibitory activity, herein a focused set of cyclic NU172 analogues—obtained by connecting its 5′- and 3′-extremities with flexible linkers—was synthesized. Two different chemical approaches were exploited in the cyclization procedure, one based on the oxime ligation method and the other on Cu(I)-assisted azide-alkyne cycloaddition (CuAAC), affording NU172 analogues including circularizing linkers with different length and chemical nature. The resulting cyclic NU172 derivatives were characterized using several biophysical techniques (ultraviolet (UV) and circular dichroism (CD) spectroscopies, gel electrophoresis) and then investigated for their serum resistance and anticoagulant activity in vitro. All the cyclic NU172 analogues showed higher thermal stability and nuclease resistance compared to unmodified NU172. These favorable properties were, however, associated with reduced—even though still significant—anticoagulant activity, suggesting that the conformational constraints introduced upon cyclization were somehow detrimental for protein recognition. These results provide useful information for the design of improved analogues of NU172 and related duplex-quadruplex structures.

The detection of DNA molecules by agglutination assays has suffered from a lack of specificity. The specificity can be improved by introducing a hybridization step with a specific probe. We developed a setting that captured biotinylated DNA targets between magnetic nanoparticles (MNPs) grafted with tetrathiolated probes and anti-biotin antibodies. The agglutination assay was enhanced using a series of magnetization cycles. This setting allowed to successfully detect a synthetic single stranded DNA with a sensitivity as low as 9 pM. We next adapted this setting to the detection of PCR products. We first developed an asymmetric pan-flavivirus amplification. Then, we demonstrated its ability to detect dengue virus with a limit of detection of 100 TCID50/mL. This magnetic field-enhanced agglutination assay is an endpoint readout, which benefits from the advantages of using nanoparticles that result in particular from a very reduced duration of the test; in our case it lasts less than 5 min. This approach provides a solution to develop new generation platforms for molecular diagnostics.

Methylthioethanol and 2,2′‐thiodiethanol were derivatized into cyanoethyl‐phosphoramidites and solid support and were used to synthesize 5′‐, 3′‐monophosphate or 5′‐, 3′‐monothiophosphate oligonucleotides by thermolytic treatment followed by ammonia. The corresponding 2,2′‐thiodiethanol solid support was also used to release fully protected oligonucleotides from solid support without ammonia treatment, to monitor the oligonucleotide elongation on solid support by MALDI‐TOF mass spectrometry without any prior chemical treatment or to synthesize 3′‐pentynyl oligonucleotides in combination with a modified phosphoramidite where the cyanoethyl group was replaced by a pentynyl one.

Nucleic acid testing during the preseroconversion viremic phase is required to differentially diagnose arboviral infections. The continuing emergence of arboviruses, such as Zika virus (ZIKV), dengue virus (DENV), and chikungunya virus (CHIKV), necessitates the development of a flexible diagnostic approach. Similar clinical signs and the priority to protect pregnant women from ZIKV infection indicate that the differential diagnosis of arboviruses is essential for effective patient management, clinical care, and epidemiologic surveillance. We describe an innovative diagnostic approach that combines generic RT-PCR amplification and identification by hybridization to specific probes. Original tetra-thiolated probes were designed for the robust, sensitive, and specific detection of amplified arboviral genomes. The limit of detection using cultured and quantified stocks of whole viruses was 1 TCID50/mL for DENV-1, DENV-3, and CHIKV and 10 TCID50/mL for DENV-2, DENV-4, and ZIKV. The assay had 100% specificity with no false-positive results. The approach was evaluated using 179 human samples that previously tested as positive for the presence of ZIKV, DENV, or CHIKV genomes. Accordingly, the diagnostic sensitivity for ZIKV, DENV, and CHIKV was 87.88% (n = 58/66), 96.67% (n = 58/60), and 94.34% (n = 50/53), respectively. This method could be easily adapted to include additional molecular targets. Moreover, this approach may also be adapted to develop highly specific, sensitive, and easy to handle platforms dedicated to the multiplex screening and identification of emerging viruses.